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<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a compound that is capable of binding as a ligand to a folate recognition site. The compound is referred to as a “non-peptide folic acid analog.” The present invention also provides a ligand-agent conjugate capable of binding to a folate recognition site, the ligand-agent conjugate comprising a diagnostic or therapeutic agent in association with a non-peptide folic acid analog. The present invention also provides a ligand-agent conjugate capable of binding to a folate recognition site with high affinity, the ligand-agent conjugate comprising a diagnostic or therapeutic agent in association with a plurality of non-peptide folic acid analogs. The present invention also provides a method for targeting a cell or tissue with a diagnostic or therapeutic agent, comprising the step of administering to a patient an effective amount of a ligand-agent conjugate comprising a diagnostic or therapeutic agent in association with a non-peptide folic acid analog. |
3-structure of a whole integrin $g(a) v$g(b) extracellular region and uses therefor |
The invention features the structural coordinates of a portion of a αVβ3 integrin and the use of the coordinates in methods for identifying molecules which will bind to αVβ3 integrin and, preferably, modulate, e.g., increase or decrease, αVβ3 integrin-mediated adhesion and/or signalling. The identification methods generally involve computer-based structural modelling methods. Such methods can be combined with in vitro or in vivo screening methods to identify candidate therapeutic agent. |
1. A method for evaluating the potential of a chemical entity to associate with a molecule or molecular complex comprising a ligand binding pocket of an αVβ3 extracellular domain, the method comprising: (i) employing computational means to perform a fitting operation between the chemical entity and a binding pocket defined by the structural coordinates described in Table 2; and (ii) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket. 2. A computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises a binding pocket defined by the structural coordinates of Table 2 wherein said computer comprises: (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of the amino acids of the αVβ3 extracellular domain contained in Table 2; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and (d) a display coupled to said central-processing unit for displaying said three-dimensional representation. 3. A method for identifying a potential agonist or antagonist of a molecule comprising a αVβ3 receptor ligand binding pocket comprising the steps of: (a) using the structure coordinates of Table 2 or coordinates having a root mean square deviation from the backbone atoms of amino acids of the αVβ3 extracellular domain coordinates in Table 2 of not more than 1.5 Angstroms, to generate a three-dimensional structure of molecule comprising a αVβ3 receptor ligand binding pocket; (b) employing said three-dimensional structure to design or select said potential agonist or antagonist; (c) providing said agonist or antagonist; and (d) contacting said agonist or antagonist with said receptor to determine the ability of said potential agonist or antagonist to interact with said receptor. 4. The method of claim 3, wherein a three dimensional representation of the ligand binding pocket is generated by a computer program. 5. A method of identifying an compound capable of binding to an αVβ3 extracellular domain, the method comprising: (a) introducing into a suitable computer program information defining at least a portion of the αVβ3 extracellular domain conformation defined by the structural coordinates of Table 2, wherein said program displays the three-dimensional structure thereof; (b) creating a three dimensional structure of a test compound in said computer program; (c) displaying and superimposing the model of said test compound on the model of said active site; and (d) assessing whether said test compound model fits spatially into the ligand binding pocket. 6. A method of using the three-dimensional structure of αVβ3 integrin in a drug screening assay comprising: (a) selecting a candidate drug by performing rational drug design with the structural coordinates in Table 2, wherein the selecting entail computer modeling of the three dimensional structure of αVβ3 integrin; (b) contacting the selected candidate drug with a cell expressing αVβ3 integrin or a αVβ3 integrin extracellular domain in solution; (c) detecting the binding of the candidate drug to the αVβ3 integrin expressed by the cell or the αVβ3 integrin extracellular domain in solution. 7. The method of claim 1 wherein the αVβ3 integrin comprises amino acid residues 31 to 989 of SEQ ID NO:1 and amino acid residues 26 to 718 of SEQ ID:2. |
<SOH> BACKGROUND OR THE INVENTION <EOH>Integrins are a family of cell surface adhesion receptors that establish strong physical linkages between molecules outside of the cell and the actin cytoskeleton within (Burridge and Chrzanowska-Wodnicka (1996), Annu Rev Cell Dev Biol 12: 463-518; Hynes (1992), Cell 69(1): 11-25). Via these linkages, signals are transmitted across the membrane, in both directions. The signals are part of a complex network of events that control the adhesive properties of cells, the reorganization of the actin cytoskeleton, and the overall cell structure. Integrin-mediated signaling also influences cellular migration, survival, proliferation, and differentiation (Giancotti and Ruoslahti (1999), Science 285: 1028-32; Cary et al. (1999), Histol Histopathol 14(3): 1001-9). The fact that integrins are involved in so many critical cellular processes is reflected in the prominent role that they play during normal biological events such as embryonic development, wound healing, angiogenesis, bone remodeling, and the immune response, as well as aberrant events like tumor metastasis and auto-immune disease. Integrins are believed to be the principal class of cell-surface receptors for the molecules that constitute the extracellular matrix (ECM), like laminin, collagen, and fibronectin (Hemler (1999) Integrins , in Guidebook to the Extracellular Matrix and Adhesion Proteins (Kreis and Vale, eds), Sambrook and Tooze Publishers, Oxford University Press; Hynes, supra). Integrins are also capable of interacting with other cell-surface adhesion receptors, including some members of the cell adhesion molecule (CAM) family. Overall, cell-cell adhesion involving integrins is less common than integrin-mediated cell-matrix adhesion, but it is frequently used by lymphocytes when they are responding to infection (Hemler, supra; Hynes, supra). Integrins are heterodimers that are composed of alpha and beta subunits. At least eighteen genes encoding alpha integrin subunits and at least eight genes encoding beta integrin subunits have been identified in vertebrates. Since not all alpha subunits will dimerize with all beta subunits, however, only about twenty-four dimer pairs have been characterized (Hemler, supra). Each of these integrins binds to a specific set of ECM proteins, which is determined by both the interaction between the alpha and beta subunits and their combined interaction with the ligands (Plow et al. (2000), J Biol Chem 275(29): 21785-88; Hynes, supra). αVβ3 integrin binds to many different extracellular proteins, including vitronectin, bone sialoprotein, and matrix metalloproteinase-2 (Plow et al., supra). Through its interactions with its extracellular ligands, and the resulting effects on cell differentiation, migration, and survival, αVβ3 mediates numerous physiologic processes such as angiogenesis and bone remodeling. (αVβ3 is the most abundant integrin displayed by osteoclasts, one of the cell types responsible for bone remodeling. Endothelial cells undergoing angiogenesis in wounds, tumors or inflammatory tissues express high levels of αVβ3. Some invasive tumors, such as metastatic melanoma, breast cancer, and late-stage glioblastoma also express αVβ3. Several studies indicate that αVβ3 over-expression on melanoma cells correlates with the invasive phase of human melanoma. Loss of the αV chain in an experimental cell line leads to a delay in tumor growth in vivo compared with the parental cells, and conversely, re-expression of the αV subunit in αV-defective cells restored tumorigenicity in vivo and survival in vitro. Ligation of αVβ3 integrin in malignant melanoma cells promotes cell survival in vitro, and blockade of αVβ3 triggers apoptosis of melanoma cells. Activation of αVβ3 is also a necessary feature of metastasis in breast cancer. Occupation of αVβ3 integrin by ligands and its activation are important in the normal and pathologic function of αVβ3. Thus, compounds that bind to αVβ3 integrin, and thereby modulate, e.g., antagonize, its activity, could be effective drugs for the treatment of metastatic cancer and osteoporosis. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based, in part, on the determination of the structure of human αVβ3 integrin, which is a complex formed by the extracellular domain of the αV integrin subunit and the mature β3 integrin subunit. The coordinates for the αVβ3 integrin complex, consisting of amino acid residues 31 to 989 of the αv integrin subunit (SEQ ID NO:1) and 26 to 718 of the β3 integrin subunit (SEQ ID NO:2), are presented in Table 2 in standard Brookhaven database format. In one aspect, the invention features a method for modeling the structure of other molecules that are homologous to αVβ33 integrin, e.g., other integrins. The method includes: generating alignments between the sequences of αV and another alpha integrin subunit of interest and between the sequences of β3 and another beta integrin subunit of interest; using the alignments to map the alpha and beta integrin subunits of interest onto the alpha-carbon backbone trace obtained from the αVβ3 integrin structure; and performing energy minimization on the alpha and beta integrin subunits of interest thus mapped, thereby producing a model for the most energetically favorable strucuture(s) of the integrin consisting of the alpha and beta integrin subunits of interest. In a preferred embodiment, the alpha and beta subunits of interest are experimentally known to associate with one another, e.g., αIIb and β3, αV and β1, α5 and β1, or αL and β2. In another aspect, the invention features methods for modeling structural perturbations produced by mutations in an integrin subunit. The method is identical to the method described above, except that the integrin subunit sequences used contain at least one point mutation or a deletion of at least one amino acid. In a preferred embodiment, the mutations are present in an αV or β3 integrin subunit. In another embodiment, the mutations are present in a non-αV alpha integrin subunit or a non-β3 beta integrin subunit. In another aspect, the invention features methods for identifying molecules which will bind to αVβ3 integrin and, preferably, modulate, e.g., increase or decrease, αVβ3 integrin-mediated adhesion and/or signaling. Preferred αVβ3 integrin-binding molecules identified using the method of the invention act as antagonists in one or more in vitro or in vivo assays of αVβ3 integrin-mediated adhesion and/or signaling. Preferred αVβ33 integrin-binding molecules lack peptide bonds. The preferred weight of αVβ3 integrin-binding molecules is less than about 5000, 4000, 3000, 2000, 1000, 750, 500, or even 400 Daltons. In a preferred embodiment, the methods of the invention entail the identification of compounds that occupy a particular structural space. The methods rely upon the use of precise structural information derived from X-ray crystallographic studies of αVβ3 integrin. These crystallographic data permit the determination of the surface topology of the αVβ3 integrin extracellular domain. Molecules that are predicted to have a topology that includes a surface which is complemetary to a subset of the αVβ3 integrin extracellular domain surface, such that the interface between the molecule and a portion of the αVβ3 integrin extracellular domain is closely juxtaposed and the energy of association arising from the association, e.g., van der Waals forces and charge-charge interactions, is minimized, may be capable of binding to the αVβ3 integrin extracellular domain via, e.g., van der Waals forces and charge-charge interactions. In a preferred embodiment, the αVβ3 integrin extracellular domain surface with which molecules are predicted to form an interface includes a subset of the αVβ3 integrin extracellular domain surface defined by residues from both the αV and the β3 subunits. In another embodiment, the αVβ3 integrin extracellular domain surface with which molecules are predicted to form an interface includes a subset of the αVβ3 integrin extracellular domain surface defined exclusively by residues from the αV subunit, or a portion thereof. In yet another embodiment, the αVβ3 integrin extracellular domain surface with which molecules are predicted to form an interface includes a subset of the αVβ3 integrin extracellular domain surface defined exclusively by residues from the β3 subunit, or a portion thereof. In yet another aspect, the invention features methods for identifying molecules which will bind to a non-αVβ3 integrin of interest and modulate adhesion and/or signaling mediated by the integrin. Preferred integrin-binding molecules identified using the method of the invention act as antagonists in one or more in vitro or in vivo assays of integrin-mediated adhesion and/or signaling which involve the integrin. Preferred integrin-binding molecules lack peptide bonds. The preferred molecular weight of αVβ3 integrin-binding molecules is less than about 5000, 4000, 3000, 2000, 1000, 750, 500, or even 400 Daltons. In a preferred embodiment, the method is a combination of two of the methods described above: first, a structural model, based on the structure of αVβ3 integrin, is produced for a non-αVβ3 integrin; second, molecules are identified that are predicted to have a topology that includes a surface which is complementary to a subset of the extracellular domain surface of the structural model of the non-αVβ3 integrin, such that the interface between the molecule and the extracellular domain surface of the structural model of the non-αVβ3 is closely juxtaposed and the energy of association arising from the association, e.g., van der Waals forces and charge-charge interactions, is minimized. In a preferred embodiment, the extracellular domain surface of the model for the non-αVβ3 integrin, with which molecules are predicted to form an interface, includes a subset of the modeled extracellular domain surface defined by residues from both the alpha and beta subunit of said modeled integrin. In another embodiment, the extracellular domain surface of the model for the non-αVβ3 integrin, with which molecules are predicted to form an interface, includes a subset of the modeled extracellular domain surface defined exclusively by residues from the alpha subunit of said modeled integrin, or a portion thereof. In yet another embodiment, the extracellular domain surface of the model for the non-αVβ3 integrin, with which molecules are predicted to form an interface, includes a subset of the modeled extracellular domain surface defined exclusively by residues from the beta subunit of said modeled integrin, or a portion thereof. In another aspect, the invention features a method for using the coordinates of the αVβ3 integrin structure of the invention to assist in the determination of the structure of the extracellular domain of αVβ3 integrin complexed with another molecule. The method involves producing a crystal of αVβ3 complexed with another molecule, gathering X-ray diffraction data from the crystal, and using the coordinates of the αVβ3 integrin structure of the present invention to solve, e.g., by molecular replacement, the structure of the complex. In a preferred embodiment, the molecule that is complexed with the extracellular domain of αVβ3 integrin modulates the activity, e.g., the cellular adhesion and/or signaling activity, of αVβ3 integrin. In an even more preferred embodiment, the molecule that is complexed with the extracellular domain of αVβ3 integrin antagonizes the activity, e.g., the cellular adhesion and/or signaling activity, of αVβ3 integrin. The method used to produce crystals of αVβ3 for the purpose of X-ray diffraction and structural determination may be applicable to the crystallization of non-αVβ3 integrins. The method includes co-expressing an extracellular domain fragment of an alpha integrin subunit comprising a region equivalent to about amino acid residues 31 to 989 of the αV subunit along with an extracellular domain fragment of a beta integrin subunit comprising a region equivalent to about amino acid residues 26 to 718 of the β3 subunit in an eukayotic cell expression system, e.g., a baculovirus expression system, purifying the resulting integrin heterodimer, and forming crystals in an appropriate solution. In a preferred embodiment, the co-expressed alpha and beta subunit fragments are derived from integrin subunits experimentally known to associate with one another, e.g., αIIb and β3, αV and ⊖1, α5 and β1, α4 and β1, or αL and β2. |
Method and apparatus for altering conduction properties in the heart and in adjacent vessels |
Method and apparatus for treating conductive irregularities in the heart, particularly atrial fibrillation and accessory path arrythmias. An ablative catheter is positioned relative to an inter-atria electrical pathway, or a vicinity of accessory paths such as the coronary sinus or fossa ovalis, and actuated to form a lesion that partially or completely blocks electrical conduction in at least one direction along the pathway. |
1. A method of treating atrial fibrillation, comprising: providing a catheter including a distal portion having an arrangement for conductive alteration of an inter-atrial conductive pathway of the heart; positioning the distal portion of the catheter relative to a portion of the inter-atrial conductive pathway; and actuating the conductive alteration arrangement to alter the conduction of the inter-atrial conductive pathway. 2. The method recited in claim 1, wherein the conductive alteration arrangement is an electrode. 3. The method recited in claim 2, wherein the electrode is a mesh electrode. 4. The method recited in claim 1, wherein the portion of the inter-atrial conductive path includes the coronary sinus. 5. The method recited in claim 4, wherein the coronary sinus includes the ostium. 6. The method recited in claim 1, wherein the portion of the inter-atrial conductive pathway includes Bachmann's bundle. 7. The method recited in claim 1, wherein the portion of the inter-atrial conductive pathway includes the inter-atrial septum. 8. The method recited in claim 1, wherein the conductive alteration completely blocks conduction along the inter-atrial conductive pathway. 9. The method recited in claim 1, wherein the conductive alteration partially blocks conduction along the inter-atrial conductive pathway. 10. The method recited in claim 1, wherein the conductive alteration disturbs but does not completely block conduction along the inter-atrial conductive pathway. 11. The method recited in claim 1, wherein the conductive alteration is uni-directional 12. The method recited in claim 11, wherein the uni-directional alteration is in the left to right direction. 13. The method recited in claim 11, wherein the uni-directional alteration is in the right to left direction. 14. The method recited in claim 1, wherein the conductive alteration is bi-directional. 15. A method of treating arrhythmia of an accessory pathway of the heart, comprising: providing a catheter including a distal portion having an arrangement for conductive alteration of a portion of the heart and/or of a vessel in communication with the heart that is in the vicinity of an accessory pathway; positioning the distal portion of the catheter relative to the portion of the heart and/or of the vessel in the vicinity of the accessory pathway; and actuating the conductive alteration arrangement to alter the conduction of the accessory pathway. 16. The method as recited in claim 15, wherein the portion of a vessel in the vicinity of an accessory pathway includes the coronary sinus. 17. The method as recited in claim 16, wherein the coronary sinus includes the ostium. 18. The method recited in claim 15, wherein the conductive alteration arrangement is an electrode. 19. The method recited in claim 18, wherein the electrode is a mesh electrode. 20. The method recited in claim 15, wherein the conductive alteration completely blocks conduction along the accessory path. 21. The method recited in claim 15, wherein the conductive alteration partially blocks conduction along the accessory pathway. 22. The method recited in claim 15, wherein the conductive alteration disturbs but does not completely block conduction along the accessory pathway. 23. The method recited in claim 15, wherein the conductive alteration is uni-directional 24. The method recited in claim 15, wherein the conductive alteration is bi-directional. 25. A method for treating a condition of the heart, comprising acts of: introducing a catheter into the heart, the catheter having a braided conductive member at a distal end thereof; forming a lesion on tissue at a selected location of the heart with the braided conductive member; and measuring the quality of the lesion with the braided conductive member. 26. The method of claim 25, wherein the act of forming a lesion on tissue at a selected location of the heart with the braided conductive member and the act of measuring the quality of the lesion with the braided conductive member are performed concurrently. 27. The method of claim 25, wherein the act of measuring the quality of the lesion with the braided conductive member includes measuring the impedance of the tissue at the selected location. 28. The method of claim 25, wherein the act of measuring the quality of the lesion with the braided conductive member includes measuring an amplitude of an electrical signal at the selected location. 29. A heart catheter having a braided conductive member, the braided conductive member comprising: one or more ablation filaments for applying ablative energy to a surface of a heart; and one or more mapping filaments for measuring an electrical signal at a surface of the heart. 30. The heart catheter of claim 29, wherein the one or more ablation filaments and the one or more mapping filaments may be activated concurrently. 31. The heart catheter of claim 29, wherein the braided conductive member is deployable. 32. The heart catheter of claim 29, wherein the heart catheter further includes means for steering the heart catheter. 33. The heart catheter of claim 32, wherein the means for steering the heart catheter includes means for manipulating a portion of the heart catheter to form a curve that is proximal to the braided conductive member. 34. A method for treating a condition of a heart, comprising an act of: using a catheter having a braided conductive member to create a lesion at a location selected from the group consisting of a wall of the coronary sinus, a wall of the right atrium at the opening of the coronary sinus, a wall of the right atrium at the fossa ovalis, and a wall of the left atrium at the fossa ovalis. 35. The method of claim 34, wherein the act of using a catheter having a braided conductive member to create a lesion includes using a catheter having a braided conductive member to create a lesion at a wall of the coronary sinus. 36. The method of claim 35, further including an act of assessing the conductive alteration of the coronary sinus. 37. The method of claims claim 35 or 36, further including an act of using a catheter having a braided conductive member to create a lesion includes using a catheter having a braided conductive member to create a lesion at the fossa ovalis. 38. The method of claim 34, wherein the act of using a catheter having a braided conductive member to create a lesion includes using a catheter having a braided conductive member to create a lesion at a wall of the right atrium at the opening of the coronary sinus. 39. The method of claim 38, further including an act of assessing the conductive alteration of the coronary sinus. 40. The method of claim 38, further including an act of using a catheter having a braided conductive member to create a lesion includes using a catheter having a braided conductive member to create a lesion at the fossa ovalis. 41. The method of claim 34, wherein the act of using a catheter having a braided conductive member to create a lesion includes using a catheter having a braided conductive member to create a lesion at a wall of the right atrium at the fossa ovalis. 42. The method of claim 34, wherein the act of using a catheter having a braided conductive member to create a lesion includes using a catheter having a braided conductive member to create a lesion at a wall of the left atrium at the fossa ovalis. 43. The method of any of claim 35, further including an act of reducing electrical conduction between the right atrium and left atrium of the heart. 44. The method of claim 43, further including an act of using the catheter to measure the electrical conduction between the right atrium and left atrium of the heart. 45. The method of claim 44, wherein the act of using a catheter having a braided conductive member to create a lesion includes applying RF energy to tissue at the location selected. 46. The method of claim 45, further including an act of using the catheter to measure a quality of the lesion. 47. A method for treating cardiac arrhythmia, comprising acts of: introducing a catheter into the right atrium of a patient, the catheter having a braided conductive member at a distal end thereof; passing the braided conductive member of the catheter through the inter-atrial septum separating the right atrium and the left atrium; expanding the braided conductive member in the left atrium of the patient; positioning the braided conductive member so that the braided conductive member contacts the inter-atrial septum; and applying energy to the inter-atrial septum via the braided conductive member to create a lesion on the inter-atrial septum. 48. The method of claim 47, further including an act of reducing the electrical conductivity of the heart between the right and left atrium via the inter-atrial septum. 49. The method of claim 48, further including an act of using the catheter to measure the electrical conductivity between the right atrium and left atrium via the inter-atrial septum. 50. The method of claim 47, further including an act of using the catheter to measure a quality of the lesion. 51. The method of claim 50, wherein the act of using the catheter to measure a quality of the lesion includes measuring the impedance of the inter-atrial septum at the lesion. 52. The method of claim 50, wherein the act of using the catheter to measure a quality of the lesion includes measuring an amplitude of an electrical signal at the lesion. 53. A method for treating cardiac arrhythmia, comprising acts of: introducing a catheter into the right atrium of a patient, the catheter having a braided conductive member at a distal end thereof; passing the distal end of the catheter through the inter-atrial septum separating the right atrium and the left atrium; expanding the braided conductive member in the right atrium of the patient; positioning the braided conductive member so that the braided conductive member contacts the inter-atrial septum; and applying energy to the inter-atrial septum via the braided conductive member to create a lesion on the inter-atrial septum. 54. The method of claim 53, further including an act of reducing the electrical conductivity of the heart between the right and left atrium via the inter-atrial septum. 55. The method of claim 54, further including an act of using the catheter to measure the electrical conductivity between the right atrium and left atrium via the inter-atrial septum. 56. The method of claim 53, further including an act of using the catheter to measure a quality of the lesion. 57. The method of claim 56, wherein the act of using the catheter to measure a quality of the lesion includes measuring the impedance of the inter-atrial septum at the lesion. 58. The method of claim 56, wherein the act of using the catheter to measure a quality of the lesion includes measuring an amplitude of an electrical signal at the lesion. 59. A method for treating atrial fibrillation in a heart, comprising an act of: using a catheter having a braided conductive member to ablate a region of the heart that serves as an electrical pathway between that left atrium and the right atrium of the heart to alter the conductivity of the electrical pathway. 60. The method of claim 59, wherein the act of using a catheter having a braided conductive member includes using a catheter having a braided conductive member to ablate the coronary sinus. 61. The method of claim 60, wherein the act of using a catheter having a braided conductive member includes using a catheter having a braided conductive member to reduce an electrical conductivity the coronary sinus 62. The method of claim 59, wherein the act of using a catheter having a braided conductive member includes using a catheter having a braided conductive member to ablate the fossa ovalis. 63. The method of claim 62, wherein the act of using a catheter having a braided conductive member includes using a catheter having a braided conductive member to reduce an electrical conductivity the fossa ovalis. 64. A method for treating a condition of the heart, comprising acts of: introducing a catheter into the heart, the catheter having a braided conductive member; forming a lesion in the heart with the braided conductive member; generating a pacing signal at a pacing electrode on the catheter on a first side of the braided conductive member; and detecting a received signal at a detection electrode on the catheter on a second side of the braided conductive member, wherein the received signal is related to a quality of the lesion. 65. The method of claim 64, wherein: the act of generating a pacing signal includes generating a pacing signal at a pacing electrode on the catheter on a distal side of the braided conductive member; and the act of detecting a received signal includes detecting a received signal at a detection electrode on the catheter on a proximal side of the braided conductive member, wherein the received signal is related to a quality of the lesion. 66. The method of claim 65, wherein: the act of generating a pacing signal includes generating a pacing signal at a pacing electrode on the catheter on a proximal side of the braided conductive member; and the act of detecting a received signal includes detecting a received signal at a detection electrode on the catheter on a distal side of the braided conductive member, wherein the received signal is related to a quality of the lesion. 67. A method of treating a condition of a heart, comprising acts of: altering the conductivity of at least one first pathway in at least one direction between the left atrium and the right atrium of the heart; and maintaining at least one second pathway between the left atrium and right atrium such that the heart generates a normal sinus rhythm. 68. The method of claim 67, wherein the act of altering the conductivity of at least one first pathway includes altering the conductivity of the coronary sinus. 69. The method of claim 68, wherein the act of altering the conductivity of at least one first pathway includes electrically disconnecting the coronary sinus. 70. The method of claim 68, wherein the act of altering the conductivity of at least one first pathway includes altering the conductivity of the coronary sinus in a left atrium to right atrium direction. 71. The method of claim 67, wherein the act of altering the conductivity of at least one first pathway includes altering the conductivity of the fossa ovalis. 72. The method of claim 71, wherein the act of altering the conductivity of at least one first pathway includes electrically disconnecting the fossa ovalis. 73. The method of claim 71, wherein the act of altering the conductivity of at least one first pathway includes altering the conductivity of the fossa ovalis in a left atrium to right atrium direction. 74. The method of claim 67, maintaining at least one second pathway includes maintaining an electrical pathway between the left atrium and right atrium via Bachmann's bundle. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The human heart is a very complex organ, which relies on both muscle contraction and electrical impulses to function properly. Electrical impulses travel through the heart in a desired sequence so that the various chambers receive and pump blood in the proper order. With respect to the atria, normal excitation is propagated in a right atrium to left atrium direction via inter-atrial conduction pathways, including the coronary sinus, fossa ovalis, and Bachmann's bundle. Abnormal inter-atrial electric flow, such as left-to-right conduction, may pose serious health risks to a patient including atrial fibrillation. Catheter ablation, that is the application of energy at a distal portion of a catheter positioned within or about the heart, or a vessel in electrical communication with the heart, to form lesions that alter conductive properties in the heart, is known for treating atrial fibrillation. Such techniques have targeted the focal trigger of an atrial arrhythmia as well as reentrant circuits in the myocardium. |
<SOH> SUMMARY OF THE INVENTION <EOH>One illustrative embodiment of the invention is directed to a method of treating atrial fibrillation. The method comprises providing a catheter including a distal portion having an arrangement for conductive alteration of an inter-atrial conductive pathway of the heart, positioning the distal portion of the catheter relative to a portion of the inter-atrial conductive pathway, and actuating the conductive alteration arrangement to alter the conduction of the inter-atrial conductive pathway. Another illustrative embodiment of the invention is directed to a method for treating arrhythmia of an accessory pathway of the heart. The method comprises providing a catheter including a distal portion having an arrangement for conductive alteration of a portion of the heart and/or of a vessel in communication with the heart that is in the vicinity of an accessory pathway, positioning the distal portion of the catheter relative to the portion of the heart and/or of the vessel in the vicinity of the accessory pathway, and actuating the conductive alteration arrangement to alter the conduction of the accessory pathway. A further illustrative embodiment of the invention is directed to a method for treating a condition of the heart. The method comprises acts of introducing a catheter into the heart, the catheter having a braided conductive member at a distal end thereof, forming a lesion on tissue at a selected location of the heart with the braided conductive member, and measuring the quality of the lesion with the braided conductive member. Another illustrative embodiment of the invention is directed to a catheter having a braided conductive member. The braided conductive member comprises one or more ablation filaments for applying ablative energy to a surface of a heart, and one or more mapping filaments for measuring an electrical signal at a surface of the heart. A further illustrative embodiment of the invention is directed to a method for treating a condition of a heart. The method comprises an act of using a catheter having a braided conductive member to create a lesion at a location selected from the group consisting of a wall of the coronary sinus, a wall of the right atrium at the opening of the coronary sinus, a wall of the right atrium at the fossa ovalis, and a wall of the left atrium at the fossa ovalis. Another illustrative embodiment of the invention is directed to a method for treating cardiac arrhythmia. The method comprises acts of introducing a catheter into the right atrium of a patient, the catheter having a braided conductive member at a distal end thereof, passing the braided conductive member of the catheter through the inter-atrial septum separating the right atrium and the left atrium, expanding the braided conductive member in the left atrium of the patient, positioning the braided conductive member so that the braided conductive member contacts the inter-atrial septum, and applying energy to the inter-atrial septum via the braided conductive member to create a lesion on the inter-atrial septum. A further illustrative embodiment of the invention is directed to a method for treating cardiac arrhythmia. The method comprises acts of introducing a catheter into the right atrium of a patient, the catheter having a braided conductive member at a distal end thereof, passing the distal end of the catheter through the inter-atrial septum separating the right atrium and the left atrium, expanding the braided conductive member in the right atrium of the patient, positioning the braided conductive member so that the braided conductive member contacts the inter-atrial septum, and applying energy to the inter-atrial septum via the braided conductive member to create a lesion on the inter-atrial septum. Another illustrative embodiment of the invention is directed to a method for treating atrial fibrillation in a heart. The method comprises an act of using a catheter having a braided conductive member to ablate a region of the heart that serves as an electrical pathway between that left atrium and the right atrium of the heart to alter the conductivity of the electrical pathway. A further illustrative embodiment of the invention is directed to a method for treating a condition of the heart. The method comprises acts of introducing a catheter into the heart, the catheter having a braided conductive member, forming a lesion in the heart with the braided conductive member, generating a pacing signal at a pacing electrode on the catheter on a first side of the braided conductive member, and detecting a received signal at a detection electrode on the catheter on a second side of the braided conductive member, wherein the received signal is related to a quality of the lesion. The features and advantages of the present invention will be more readily understood and apparent from the following detailed description of the invention, which should be read in conjunction with the accompanying drawings, and from the claims which are appended at the end of the Detailed Description. |
System and method for the measurement of the layer thickness of a multi-layer pipe |
System for measuring layer thicknesses of a multi-layer pipe by measuring with a detector array (2) the attenuation of an X-ray transmitted though the pipe. According to the invention the detector array (2) comprises an array of detector elements D1, D2, D3, D4 with a collimator for defining the field of radiation in front of each detector element. The collimator has a narrow diaphragm aperture setting the resolution when the position of the pipe walls is to be determined. The defined field of radiation has an extent sufficient to radiate the four detector elements D1, D2, D3, D4 in parallel. In a suitable signal processing of the output signals from the detector elements D1, D2, D3, D4, eg by using the method of least squares, the thicknesses of the different layers may be fairly accurately determined. |
1. System for the measurement of the layer thicknesses of a multi-layer pipe comprising a detector array (2) for measuring the attenuation of X-ray transmitted through the pipe, the detector array (2) comprising scintillation counters arranged in pairs with a collimator for defining a field of radiation in front of each scintillation counter, a leaded scintillation disc (4) being arranged at one end of each scintillation counter, characterised in that the two detector pairs are juxtaposed, one detector pair, however, being longitudinally displaced in relation to the other detector pair. 2. System according to claim 1 characterised in that the one detector pair is longitudinally displaced by approximately 0.5 mm in relation to the other detector pair. 3. System according to claim 1, characterised in that a lead plate (5) is arranged on the scintillation disc (4), said lead plate (5) serving as a support for an additional detector so as to provide a detector pair. 4. System according to claim 1, characterised in that the collimator has a narrow diaphragm aperture (8) provided in a thin plate (7) of a thickness of 1 mm. 5. System according to claim 4, characterised in that the thin plate (7) being composed of tantalum. 6. System according to claim 4, characterised in that the diaphragm aperture (8) in the thin plate (7) has a gap width of about 50 μm. 7-8. (canceled) 9. Method for measuring layer thicknesses of a multi-layer pipe by measuring with a detector array the attenuation of an X-ray transmitted though the pipe, characterised in that the used detector array comprises detector elements arranged in pairs with a collimator for defining the field of radiation in front of each detector element, the layer thicknesses and/or the densities of each layer of a multi-layer pipe being determined by means of a simulation calculation based on a model, in which the values of the layer thicknesses and/or the densities, respectively, are optimally adjusted; in that fluctuations, such as vibrations in the pipe's position are compensated for at an online measurement of the pipe wall characteristics; and in that the amplitude of the pipe vibration is determined by measuring the deviation between the actual time of detection of a pipe wall and the expected time of detection of the same pipe wall. |
<SOH> BACKGROUND ART <EOH>A system for examining plastic pipes by means of an X-ray transmitted through the pipe is known from EP 216.705. This system is, however not able to render a sufficiently high resolution. Furthermore it is not possible to compensate for pipe fluctuations (vibrations) occurring during such a measurement, eg in connection with an online measurement of a through-going pipe. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>The invention is explained in greater detail below with reference to the accompanying drawings, in which FIG. 1 shows a measuring equipment according to the invention including an X-ray source and a detector array with associated signal analysis means. FIG. 2 shows the detector array with associated diaphragm apertures, FIG. 3 shows an example of measuring signals from the detector array, FIG. 4 is a front and a side view of a bracket for supporting the measuring equipment, FIG. 5 is a model of a multi-layer pipe, FIG. 6 shows the absorption coefficient as a function of the area weight, and FIGS.7-9 shows examples of measured curves. detailed-description description="Detailed Description" end="lead"? |
Acetabular prosthesis assembly |
An acetabular prosthesis assembly (1) comprising an acetabular cup (3) with a generally convex outer surface for engaging acetabular bone and a generally concave inner surface, an insert (10) capable of insertion in the cup (3) for receiving a femoral head component, wherein a wear liner (13) is disposed between the cup (3) and the insert (10), the liner (13) providing a wear inhibiting surface (4a). |
1. An acetabular prosthesis assembly comprising an acetabular cup having a generally convex outer surface for engaging acetabular bone and a generally concave inner surface together defining a cup wall which has an apex and an equator; an insert capable of insertion inside the acetabular cup; the insert including a convex outer surface and a concave recess which receives a femoral head component; wherein, the assembly further includes a liner disposed between the concave inner surface of the cup and the convex outer surface of the insert; the liner providing wear inhibiting surface. 2. An acetabular assembly according to claim 1, wherein the liner is a metallic material which opposes at least part of a concave inner surface of the cup. 3. An acetabular assembly according to claim 2, wherein the liner is integral with the insert. 4. An acetabular assembly according to claim 3 wherein, the insert comprises a first layer of polyethylene fused to said metallic liner material. 5. An acetabular assembly according to claim 4, wherein the metallic liner is titanium alloy or chrome cobalt and engages at least part of the inner concave surface of the cup. 6. An acetabular assembly according to claim 6 wherein the circumferential wall of the cup is increased in length a predetermined circumferential distance beyond the equator. 7. An acetabular assembly according to claim 5 wherein the thickness of the liner at its apex influences the circumferential distance that the cup extends beyond its equator. 8. An acetabular assembly according to claim 7 wherein the liner is in tight fitting engagement with part of the inner concave surface of the cup. 9. An acetabular assembly according to claim 8 further comprising a gap between an outer surface of the liner and said inner surface of the cup to prevent unwanted engagement between the outer surface of the liner and said inner surface of the cup. 10. An acetabular assembly according to claim 9 wherein the circumferential wall of the cup is extended between [2 MM-5 MM] from the equator. 11. An acetabular assembly according to claim 10 wherein; the insert has a generally spherical outer surface opposing a generally spherical concave surface of the liner. 12. An acetabular assembly according to claim 11 wherein the outer surface of the metallic liner includes a straight wall region extending from near its equator in a direction of the apex and an arcuate portion in the region of its apex, the arcuate portion opposing the inner concave surface of the cup. 13. An acetabular assembly according to claim 12, wherein the cup has a substantially spherical outer surface. 14. An acetabular assembly according to claim 13 wherein the insert includes a profile part which keys into a back surface of the metallic liner. 15. An acetabular assembly according to claim 14 wherein, the cup includes a flattened region at its apex. 16. An acetabular assembly according to claim 15 wherein the axial distance from the apex of the acetabular cup to its equator is less that the radial distance from the circumferential wall at the equator to its axis. 17. An acetabular assembly according to claim 16 wherein, the straight wall region of the liner about is disposed at an angle within the range of 15-25 degrees from an axis of symmetry of the assembly. 18. An acetabular assembly according to claim 17 wherein, the straight wall region of the liner changes to said arcuate portion at a location less than half the circumferential distance from its equator to its apex. 19. An acetabular assembly according to claim 18 where the insert is made of polyethylene. 20. An acetabular assembly according to claim 19 wherein the liner is made of either chrome cobalt or titanium alloy. 21. An insert for an acetabular cup; the insert including a convex outer surface and a concave inner surface defining a body including a recess which receives a femoral head component; wherein the convex outer surface of the insert body receives thereon a liner which provides a wear inhibiting surface opposing at least part of an inner surface of the cup which receives and retains said insert. 22. An insert for an acetabular cup according to claim 21, wherein the liner is formed from a metallic material which opposes at least part of a concave inner surface of the cup. 23. An insert for an acetabular cup according to claim 22 wherein, the liner is integral with the insert. 24. An insert for an acetabular cup according to claim 23 wherein the insert body is polyethylene and the metallic liner is fused to said polyethylene. 25. An insert for an acetabular cup according to claim 24 wherein, the metallic liner is titanium alloy or chrome cobalt and when the insert is inserted by the cup, engages at least part of an inner surface of the acetabular cup. 26. An insert for an acetabular cup according to claim 25 wherein a circumferential wall of the cup is increased in length a predetermined circumferential distance beyond an equator of the cup. 27. An insert for an acetabular cup according to claim 26 wherein the thickness of the liner at its apex influences the circumferential distance that the wall of the cup is extended from the equator. 28. An insert for an acetabular cup according to claim 27 wherein the liner is in tight fitting engagement with part of the inner concave surface of the cup. 29. An insert for an acetabular cup according to claim 28 wherein, when the insert is inserted in the cup a gap is left between an outer surface of the liner and an opposing inner surface of the cup. 30. An insert for an acetabular cup according to claim 29 wherein the circumferential wall of the cup is extended in length between 2 mm-5 mm from the equator. 31. An insert for an acetabular cup according to claim 30 wherein; the insert has a generally spherical outer surface opposing a generally spherical concave surface of the liner. 32. An insert for an acetabular cup according to claim 31 wherein the outer surface of the metallic liner includes a straight wall region extending from near its equator in the direction of the apex and an arcuate portion in the region of its apex, the arcuate portion opposing the inner concave surface of the cup. 33. An insert for an acetabular cup according to claim 32 wherein the insert includes a profile part which keys into a back surface of the metallic liner. 34. An insert for an acetabular cup according to claim 33 wherein, the straight wall region of the liner is disposed at an angle within the range of 15-25 degrees from its axis of symmetry. 35. An insert for an acetabular cup according to claim 34 wherein, the straight wall region of the liner changes to said arcuate portion at a location less than half the circumferential distance from its equator to its apex. 36. An insert for an acetabular cup according to claim 35 wherein the insert is made of polyethylene. 37. An insert for an acetabular cup according to claim 36 wherein the liner is made of either chrome cobalt or titanium alloy. 38. An acetabular cup assembly for repair of an acetabulum; the assembly comprising an acetabular cup generally defining a hemisphere having an exterior convex surface and an interior at least partially concave surface, an apex and an equator; an insert for engagement with said cup and which receives a femoral head; a liner between said insert and said cup providing a wear inhibiting surface; wherein a wall of the acetabular cup extends a predetermined circumferential distance beyond its equator in a direction away from its apex. 39. An acetabular cup assembly according to claim 38 wherein the predetermined distance the wall extends beyond the equator is related to the thickness of the liner which is interposed between the insert and said interior surface of said cup. 40. An acetabular cup assembly according to claim 39 wherein the equatorial extension distance may be adjusted to accommodate liners of different thicknesses. 41. A method of construction of an acetabular cup assembly comprising the steps of; a) forming an acetabular cup having a generally convex outer surface for engaging acetabular bone and a generally concave inner surface together defining a cup wall which has an apex and an equator; b) taking an insert capable of insertion inside the acetabular cup; the insert including a convex outer surface and a concave recess which receives a femoral head c) applying a wear inhibiting liner to the convex outer surface of the insert so that when the insert is inserted in the acetabular cup the liner is disposed between the generally concave inner surface of the cup and the convex outer surface of the insert; 42. A method according to claim 41 comprising the further step of increasing in length the circumferential wall of the cup a predetermined circumferential distance beyond the equator to accommodate the liner. 43. A method according to claim 42 comprising the step of fusing the insert to the liner so that the liner is integral with the insert. 44. A method according to claim 43 comprising the additional step of inserting the insert into the acetabular cup so that at least part of an outer wall of the liner engages at least part of the inner wall of the cup and the remainder of the outer wall of the liner defines a gap or space between the liner and the cup. 45. A method according to claim 44 wherein the metallic liner is titanium alloy or chrome cobalt and the insert is polyethylene. 46. A method according to claim 45 wherein the circumferential wall of the cup is extended between 2 mm-5 mm from the equator. 47. A method according to claim 46 wherein the outer surface of the metallic liner includes a straight wall region extending from near its equator in the direction of the apex and an arcuate portion in the region of its apex, the arcuate portion opposing the inner concave surface of the cup. 48. A method according to claim 47 wherein the cup has a substantially spherical outer surface. 49. A method according to claim 48 wherein the acetabular cup is formed so that an axial distance from the apex of the outer surface of the acetabular cup to its equator is less that the radial distance from the outer surface of the circumferential wall at the equator to its axis of symmetry. 50. A method according to claim 49 including the step of disposing the straight wall region of the liner at an angle within the range of 15-25 degrees from its axis of symmetry 51. A method according to claim 41 wherein the insert is made of polyethylene. 52. A method according to claim 51 wherein the liner is made of either chrome cobalt or titanium alloy. 53. An liner for attachment with an insert for an acetabular cup; the insert including a convex outer surface and a concave inner surface defining a body including a recess which receives a femoral head component; wherein the convex outer surface of the insert body receives thereon the liner which provides a wear inhibiting surface opposing at least part of an inner surface of the cup which receives and retains said insert. 54. A liner for an acetabular cup according to claim 53, wherein the liner is formed from a metallic material which opposes at least part of a concave inner surface of the cup. 55. A liner for an acetabular cup according to claim 54 wherein, the liner is integral with the insert. 56. A liner for an acetabular cup according to claim 55 wherein the insert body is polyethylene and the metallic liner is fused to said polyethylene. 57. A liner for an acetabular cup according to claim 56 wherein, the metallic liner is titanium alloy or chrome cobalt and when the insert is inserted by the cup, engages at least part of an inner surface of the acetabular cup. 58. A liner for an acetabular cup according to claim 57 wherein a circumferential wall of the liner and cup is increased in length a predetermined circumferential distance beyond an equator of the cup. 59. A liner for an acetabular cup according to claim 58 wherein the thickness of the liner at its apex influences the circumferential distance that the wall of the cup is extended from the equator. |
<SOH> BACKGROUND <EOH>The present invention relates to acetabular components and more particularly relates to an acetabular prosthesis assembly for implantation within an acetabulum and which includes a cup for fixation to bone, an insert for insertion in the cup and a liner which provides a wear surface opposing a concave surface of the in the cup. |
Illuminated sign |
A sign for conveying information, having a plastic body, illuminating means such as light emitting diodes (LEDs) mounted within the body and a reflective coating applied on a surface of the body carrying information to reflect light emitted from the illumination means back into the body of the sign. The sing may have the LEDs mounted to face an inside surface of the sign capable of scattering light to a transparent surface to produce an illuminated area. Scattering the light form scattering surface will produce a substantially even intensity of light across the illuminated area of the sign after its transmission form the sign. |
1. A sign for conveying information, said sign comprising a body, at least one illuminating means mounted within the body, wherein the body is provided with light transmission means for transmitting light from the body, said at least one illuminating means being positioned such that light emitted from the illuminating means is directed substantially to a light scattering interior surface of the body, the light being scattered from said light scattering surface prior to its transmission from the sign via the light transmission means. 2. A sign according to claim 1, wherein the body is made from a plastic material. 3. A sign according to claim 1, wherein, the light transmission means is provided by a surface of the body which is a least in part, transparent. 4. A sign according to claim 1, wherein, a second light transmission means is provided by a space between surfaces of the body. 5. A sign according to claim 1, wherein, a third light transmission means is provided by an aperture in at least one of the surfaces. 6. A sign according to claim 1, wherein the light scattering interior surface and the light transmission means are arranged opposite one another in the body. 7. A sign according to claim 1, wherein the illuminating means is at least one light emitting diode (LED). 8. A sign according to claim 1, wherein the at least one light emitting diode is enclosed in a watertight housing. 9. A sign according to claim 8, wherein the watertight housing is made from molded plastic. 10. A sign for conveying information, said sign comprising a plastics body, illuminating means mounted within the body and a reflective coating applied on a surface of the body carrying information to reflect light emitted from the illumination means back into the body of the sign. 11. A sign according to claim 10, wherein the sign body is formed of acrylic. 12. A sign according to claim 10, wherein the sign is formed in a casting process. 13. A sign according to claim 10, wherein the illuminating means comprises at least one light emitting diode (LED). 14. A sign according to claim 10, wherein the illuminating means comprises a plurality of light emitting diodes. 15. A sign according to claim 14, wherein the light emitting diodes are distributed evenly throughout the body of the sign. 16. A sign according to claim 10, wherein the reflective coating is provided upon the entire surface of the sign body which carries the information. 17. A sign according to claim 10, wherein the reflective coating is provided only on selected areas of the surface of the sign body carrying the information. 18-19. (Canceled) |
Method for improving the operational reliabilty of dosing devices |
The dispensing of sample liquids is performed, particularly in case of high-throughput screening, by means of automatic dosing devices. Between individual dosing processes, the standstill of the dosing device may lead to an outgassing of the sample liquid existing in the dosing device, and to crystallization. In order to improve the operational safety, it is provided according to the method of the invention that a stand-by routine is initiated after termination of a dosing process. In the stand-by routine, liquid is dispensed via a dosing orifice of the dosing device. The dispensing step is repeated after a predetermined period of time. |
1. A method for improving the operational safety of dosing devices for chemical and/or biological liquids, wherein a stand-by routine is initiated upon termination of a dosing process, the stand-by routine comprising the following steps: dispensing liquid through a dosing orifice of the dosing device, and repeating the dispensing step after a predetermined period of time. 2. The method according to claim 1 wherein the liquid is dispensed in the form of droplets. 3. The method according to claim 1 or 2 wherein, in a liquid dispensing step, a plurality of droplets, particularly more than twenty, preferably more than fifty and most preferably more than eighty, is dispensed. 4. The method according to claim 1 wherein, in a liquid dispensing step, a predetermined quantity of liquid, particularly from 1 μl to 1 ml, is continuously dispensed for rinsing the dosing device. 5. The method according to any one of claims 1-4 wherein, during a stand-by routine comprising a plurality of liquid dispensing steps, part of the liquid dispensing steps include a droplet dispensing step and part of the liquid dispensing steps include a rinsing step. 6. The method according to claim 5 wherein, between two rinsing steps, a plurality of droplet dispensing steps, preferably more than ten and particularly more than twenty droplet dispensing steps, are performed. 7. The method according to any one of claims 1-6 wherein the time interval between two successive dispensing steps is from 20 to 80 seconds, particularly from 30 to 50 seconds. 8. The method according to any one of claims 1-7 wherein the time interval between two successive dispensing steps is reduced with increased length of the stand-by routine. 9. The method according to any one of claims 1-8 wherein, in the droplet dispensing step, sample liquid is dispensed. 10. The method according to any one of claims 1-9 wherein, in the liquid dispensing step, rinsing liquid is dispensed. 11. The method according to any one of claims 1-10 wherein the stand-by routine is automatically initiated upon lapse of a predetermined period of time of preferably from 30 to 60 seconds after termination of the dosing process. |
Methods and compositions for utilizing changes of hybridization signals during approach to equilibrium |
The present invention provides methods for utilizing the changes of hybridization levels in time during approach to equilibrium duplex formation for identifying specific hybridization to polynucleotide probes. In the invention, the changes of hybridization levels at one or more polynucleotide probes by a sample comprising a plurality of nucleic acid molecules having different sequences are monitored during their progress towards equilibrium and the continuing increase of hybridization signals beyond cross-hybridization is used as an indication of specific binding. The invention also provides methods of comparing specificities of different polynucleotides probes. The invention further provides methods for ranking and selecting polynucleotide probes that are specific to particular nucleic acids and methods for enhancing the detection of nucleic acids. The invention further provides methods for determining the orientation of nucleotide sequences. |
1. A method for determining whether specific hybridization to a polynucleotide probe by one or more nucleic acid molecules in a sample occurs, said sample comprising a plurality of nucleic acid molecules having different nucleotide sequences, said method comprising (1) contacting a plurality of molecules of said probe with said sample under conditions such that hybridization can occur; (2) determining change in hybridization levels of said probe measured at at least two different hybridization times, wherein each of said at least two different hybridization times corresponds to a different length of time said one or more nucleic acid molecules in said sample is allowed to hybridize with said probe; and (3) comparing said change with a threshold value, said threshold value indicating specific hybridization of one or more nucleic acid molecules in said sample to said probe, wherein specific hybridization is determined to have occurred when said change is above said threshold value. 2. The method of claim 1, wherein said at least two different hybridization times consists of a first hybridization time and a second hybridization time. 3. The method of claim 2, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time. 4. The method of claim 3, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 80% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 5. The method of claim 4, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 90% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 6. The method of claim 5, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 95% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 7. The method of claim 2, wherein said first hybridization time is 1 to 4 hours. 8. The method of claim 3, wherein said time scale of cross-hybridization equilibrium is determined from a measured hybridization curve representing progression of level of hybridization of said probe with a second sample, said second sample not containing nucleic acid molecules specifically hybridizable to said probe. 9. The method of claim 3, wherein said time scale of cross-hybridization equilibrium is determined from a measured hybridization curve representing progression of level of hybridization of a reference probe, wherein said reference probe has a sequence which is not specifically hybridizable to any known or predicted sequences in said plurality of nucleic acid molecules. 10. The method of claim 9, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 3% mismatched bases in said reference probe. 11. The method of claim 10, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 10% mismatched bases in said reference probe. 12. The method of claim 11, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 30% mismatched bases in said reference probe. 13. The method of claim 9, wherein said reference probe has a sequence which is a reverse complement of a sequence in said plurality of nucleic acid molecules. 14. The method of claim 9, wherein said reference probe has a sequence which is a reverse complement of said probe. 15. The method of any one of claims 2-14, wherein said second hybridization time is at least 2 times as long as said first hybridization time. 16. The method of claim 15, wherein said second hybridization time is at least 10 times as long as said first hybridization time. 17. The method of claim 15, wherein said second hybridization time is at least 16 times as long as said first hybridization time. 18. A method for determining whether specific hybridization to a polynucleotide probe by one or more nucleic acid molecules in a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs, said method comprising (1) contacting a polynucleotide array comprising said probe with said sample under conditions such that hybridization can occur, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining hybridization levels of said probe at at least two different hybridization times, wherein each of said at least two different hybridization times corresponds to a different length of time said one or more nucleic acid molecules in said sample is allowed to hybridize with said probe; (3) determining change of hybridization level by comparing hybridization levels measured at said at least two different hybridization times; and (4) comparing said change with a threshold value, said threshold value indicating specific hybridization of one or more nucleic acid molecules in said sample to said probe, wherein specific hybridization is determined to have occurred when said change is above said threshold. 19. The method of claim 18, wherein said at least two hybridization times consists of a first Hybridization time and a second hybridization time. 20. The method of claim 19, wherein said comparing comprises determining the ratio of said second hybridization level 12 and said first hybridization level I1. 21. The method of claim 19, wherein said comparing comprises determining a quantity as described by equation xdev = I 2 - I 1 err ( I 1 ) 2 + err ( I 2 ) 2 wherein I2 is said second hybridization level and I1 is said first hybridization level, and wherein said err(I1) and err(I2) are expected error in I1 and I2, respectively. 22. The method of claim 21, wherein said err(I1)2+err(I2)2 is defined by equation err(I1)2+err(I2)2=σ12+σ22+f2(I22+I12) wherein σ12 is the variance for I1, σ22 is the variance for I2 and f is the fractional multiplicative error level. 23. The method of claim 19, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time. 24. The method of claim 23, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 80% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 25. The method of claim 24, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 90% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 26. The method of claim 25, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 95% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 27. The method of claim 19, wherein said first hybridization time is 1 to 4 hours. 28. The method of any one of claims 19-27, wherein said second hybridization time is at least 2 times as long as said first hybridization time. 29. The method of any one of claims 19-27, wherein said second hybridization time is at least 10 times as long as said first hybridization time. 30. The method of any one of claims 19-27, wherein said second hybridization time is at least 16 times as long as said first hybridization time. 31. A method for determining whether specific hybridization to a polynucleotide probe by one or more nucleic acid molecules in a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs, said method comprising (1) contacting a polynucleotide array comprising said probe and at least one reference probe with said sample under conditions such that hybridization can occur, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining time scale of cross-hybridization equilibrium by measuring a hybridization curve representing progression of level of hybridization of said reference probe, wherein said reference probe has a sequence which is not complementary to any known or predicted sequences in said plurality of nucleic acid molecules; (3) determining hybridization level of said probe at at least two different hybridization times, wherein each of said at least two different hybridization times corresponds to a different length of time said one or more nucleic acid molecules in said sample is allowed to hybridize with said probe; (4) determining change of hybridization level by comparing hybridization levels measured at said at least two different hybridization times; and (5) comparing said change with a threshold value, said threshold value indicating specific hybridization of one or more nucleic acid molecules in said sample to said probe, wherein specific hybridization is determined to have occurred when said change is above said threshold value. 32. The method of claim 31, wherein said at least two different hybridization times consists of a first hybridization time and a second hybridization time. 33. The method of claim 32, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time. 34. The method of claim 33, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 80% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 35. The method of claim 34, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 90% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 36. The method of claim 35, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 95% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 37. The method of claim 32, wherein said first hybridization time is 1 to 4 hours. 38. The method of claim 31, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 3% mismatched bases in said reference probe. 39. The method of claim 38, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 10% mismatched bases in said reference probe. 40. The method of claim 39, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 30% mismatched bases in said reference probe. 41. The method of claim 31, wherein said reference probe has a sequence which is a reverse complement of a sequence in said plurality of nucleic acid molecules. 42. The method of claim 31, wherein said reference probe has a sequence which is a reverse complement of said probe. 43. The method of any one of claims 32-42, wherein said second hybridization time is at least 2 times as long as said first hybridization time. 44. The method of claim 43, wherein said second hybridization time is at least 10 times as long as said first hybridization time. 45. The method of claim 44, wherein said second hybridization time is at least 16 times as long as said first hybridization time. 46. The method of claim 32, wherein said comparing comprises determining the ratio of said second hybridization level and said first hybridization level. 47. The method of claim 32, wherein said comparing comprises determining a quantity as described by equation xdev = I 2 - I 1 err ( I 1 ) 2 + err ( I 2 ) 2 wherein I2 is said second hybridization level and I1 is said first hybridization level, and wherein said err(I1) and err(I2) are expected error in I1, and I2, respectively. 48. The method of claim 47, wherein said err(I1)2+err(I2)2 is defined by equation err(I1)2+err(I2)2=σ12+σ22+f2(I22+I12) wherein σ12 is the variance for I1, σ22 is the variance for I2 and f is the fractional multiplicative error level. 49. A method for determining the relative abundance of a nucleotide sequence in a plurality of samples, each of said plurality of samples comprising a plurality of nucleic acid molecules having different nucleotide sequences, said method comprising (1) determining for each sample a difference in hybridization levels measured at a first hybridization time and a second, different hybridization time to a probe that is specific to said nucleotide sequence; and (2) comparing said difference among said plurality of samples, thereby determining the relative abundance of said nucleotide sequence; wherein each of said first hybridization time and second hybridization time corresponds to a different length of time said sample is allowed to hybridize with said probe. 50. The method of claim 49, wherein said first hybridization time is close to time scale for reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time. 51. A method for determining the relative abundance of a nucleotide sequence in a plurality of samples, each of said plurality of samples comprising a plurality of nucleic acid molecules having different nucleotide sequences, said method comprising (1) contacting one or more polynucleotide arrays comprising said probe with one or more of said plurality of samples under conditions such that hybridization can occur, said polynucleotide arrays comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining for each of said plurality of samples a first hybridization level of said probe at a first hybridization time; (3) determining for each of said plurality of samples a second hybridization level of said probe at a second hybridization time, said second hybridization time is different from said first hybridization time; (4) determining for each of said plurality of samples a difference in said first and second hybridization levels; and (5) comparing said difference among said plurality of samples, thereby determining the relative abundance of said nucleotide sequence; wherein each of said first hybridization time and second hybridization time corresponds to a different length of time said sample is allowed to hybridize with said probe. 52. The method of claim 51, wherein each of said plurality of samples is labeled with a distinguishable dye, and wherein said plurality of samples are contacted with a single polynucleotide array simultaneously. 53. The method of claim 51 or 52, wherein said plurality of samples consists of at least 3 samples. 54. The method of claim 53, wherein said plurality of samples consists of at least 5 samples. 55. The method of claim 54, wherein said plurality of samples consists of at least 10 samples. 56. A method for comparing hybridization specificity of a first probe and a second probe, said method comprising comparing (a) a first hybridization curve representing progression of level of hybridization of said first probe and (b) a second hybridization curve representing progression of level of hybridization of said second probe, wherein each said hybridization curve comprises hybridization levels measured at a plurality of different hybridization time, wherein each of said plurality of hybridization times corresponds to a different length of time said probe is allowed to hybridize with a sample. 57. The method of claim 56, wherein each of said plurality of hybridization curves is measured in real time. 58. The method of claim 56, wherein each of said plurality of hybridization curves is measured in a plurality of different experiments. 59. A method for comparing hybridization specificity of a first probe and a second probe, said method comprising (1) determining a first hybridization curve representing progression of level of hybridization of said first probe; (2) determining a second hybridization curve representing progression of level of hybridization of said second probe; and (3) comparing said first hybridization curve and said second hybridization curve, hereby comparing hybridization specificity of said first probe and said second probe. 60. The method of claim 59, wherein said comparing comprises determining the value of a metric representing the difference between said first hybridization curve and said second hybridization curve. 61. The method of claim 60, wherein said metric is the difference in areas underneath said first hybridization curve and said second hybridization curve. 62. A method for comparing hybridization specificity of a first probe and a second probe, said method comprising (1) contacting a polynucleotide array comprising said first probe and second probe with a sample comprising a plurality of nucleic acid molecules under conditions such that hybridization can occur, wherein said plurality comprises at least one nucleic acid molecule comprising a nucleotide sequence complementary to said first probe and at least one nucleic acid molecule comprising a nucleotide sequence complementary to said second probe, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining a first hybridization curve I1(t) representing progression of level of hybridization of said sample to said first probe; (3) determining a second hybridization curve I2(t) representing progression of level of hybridization of said sample to said second probe; and (4) comparing said first curve and said second curve, thereby comparing hybridization specificity of said first probe and said second probe. 63. The method of claim 62, wherein said comparing comprises determining a curve representing the ratio of said first hybridization curve and said second hybridization curve. 64. The method of claim 62, wherein said comparing comprises determining a curve as described by equation xdev = I 2 ( t ) - I 1 ( t ) err ( I 1 ( t ) ) 2 + err ( I 2 ( t ) ) 2 wherein said err(I1(t)) and err(I2(t)) are expected error in I1 and I2, respectively. 65. The method of claim 64, wherein said err(I1(t))2+err(I2(t))2 is defined by equation err(I1(t))2+err(I2(t))2=σ12+σ22+f2(I2(t)2+I1(t)2) wherein σ12 is the variance for I1(t), σ22 is the variance for I2(t) and f is the fractional multiplicative error level. 66. The method of claim 62, wherein said comparing comprises determining the value of a metric representing the difference between said first hybridization curve and said second hybridization curve. 67. The method of claim 66, wherein said metric is the difference in areas underneath said first hybridization curve and said second hybridization curve. 68. A method for determining whether specific hybridization to a polynucleotide probe by a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs, said method comprising comparing-(a) a first hybridization curve representing progression of level of hybridization of said probe and (b) a second hybridization curve representing progression of level of hybridization of a reference probe, wherein said reference probe has a sequence which is not complementary to any known or predicted sequences in said sample. 69. The method of claim 68, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 3% mismatched bases in said reference probe. 70. The method of claim 69, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 10% mismatched bases in said reference probe. 71. The method of claim 70, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 30% mismatched bases in said reference probe. 72. The method of claim 68, wherein said reference probe has a sequence which is a reverse complement of a sequence in said plurality of nucleic acid molecules. 73. The method of claim 68, wherein said reference probe has a sequence which is a reverse complement of said probe. 74. The method of claim 68, wherein said comparing comprises determining the value of a metric representing the difference between said first hybridization curve and said second hybridization curve. 75. The method of claim 74, wherein said metric is the difference in areas underneath said first hybridization curve and said second hybridization curve. 76. A method for determining whether specific hybridization to a polynucleotide probe by one or more nucleic acid molecules in a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs, said method comprising (1) determining a first hybridization curve representing progression of level of hybridization of said probe; (2) determining a second hybridization curve representing progression of level of hybridization of a reference probe, wherein said reference probe has a sequence which is not complementary to any known or predicted sequences in said sample; and (3) comparing said first hybridization curve and said second hybridization curve, thereby determining whether specific hybridization to said polynucleotide probe by one or more nucleic acid molecules in said sample occurs. 77. The method of claim 76, wherein said comparing comprises determining the value of a metric representing the difference between said first hybridization curve and said second hybridization curve. 78. The method of claim 77, wherein said metric is the difference in areas underneath said first hybridization curve and said second hybridization curve. 79. A method for determining whether specific hybridization to a polynucleotide probe by one or more nucleic acid molecules in a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs, said method comprising (1) contacting a polynucleotide array comprising said probe and at least one reference probe with said sample under conditions such that hybridization can occur, said reference probe having a sequence which is not complementary to any known or predicted sequences in said sample, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining a first hybridization curve representing progression of level of hybridization of said sample to said probe; (3) determining a second hybridization curve representing progression of level of hybridization of said sample to said reference probe; and (4) comparing said first hybridization curve and said second hybridization curve, thereby determining whether specific hybridization to said polynucleotide probe by said one or more nucleic acid molecules in said sample occurs. 80. The method of claim 78, wherein said comparing comprises determining a curve representing the ratio of said first hybridization curve and said second hybridization curve. 81. The method of claim 78, wherein said comparing comprises determining a curve as described by equation xdev = I 2 ( t ) - I 1 ( t ) err ( I 1 ( t ) ) 2 + err ( I 2 ( t ) ) 2 wherein I2 is said second hybridization level and I1 is said first hybridization level, and wherein said err(I1) and err(I2) are expected error in I1 and I2, respectively. 82. The method of claim 81, wherein said err(I1(t))2+err(I2(t))2 is defined by equation err(I1(t))2+err(I2(t))2=σ12+σ22+f2(I2(t)2+I1(t)2 wherein σ12 is the variance for I1(t), σ22 is the variance for I2(t) and f is the fractional multiplicative error level. 83. The method of claim 78, wherein said comparing comprises determining the value of a metric representing the difference between said first hybridization curve and said second hybridization curve. 84. The method of claim 83, wherein said metric is the difference in areas underneath said first hybridization curve and said second hybridization curve. 85. A method for determining the difference in time scale of reaching hybridization equilibrium between specific and non-specific hybridization to a polynucleotide probe by a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences, said method comprising (1) determining time scale of reaching hybridization equilibrium from a first hybridization curve representing progression of level of hybridization of said probe, wherein said probe has a sequence which is specifically hybridizable to one or more sequences in said sample; (2) determining time scale of reaching hybridization equilibrium from a second hybridization curve representing progression of level of hybridization of a reference probe, wherein said reference probe has a sequence which is not complementary to any known or predicted sequences in said sample; and (3) determining the difference in time scales of reaching hybridization equilibrium at said probe and said reference probe. 86. The method of claim 85, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 3% mismatched bases in said reference probe. 87. The method of claim 86, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 10% mismatched bases in said reference probe. 88. The method of claim 87, wherein said reference probe hybridizes to any known or predicted sequences in said plurality of nucleic acid molecules with at least 30% mismatched bases in said reference probe. 89. The method of claim 85, wherein said reference probe has a sequence which is a reverse complement of a sequence in said sample and which is different from any known or predicted sequence in said sample. 90. The method of claim 85, wherein said reference probe has a sequence which is a reverse complement of said probe and which is different from any other known or predicted sequences in said sample. 91. A method for ranking a plurality of probes according to their binding specificities to their respective complementary sequence, said method comprising comparing hybridization curves representing progression of level of hybridizations of said probes. 92. A method for ranking a plurality of probes according to their binding specificities to heir respective complementary sequences, said method comprising (1) determining a plurality of hybridization curves, each representing progression of level of hybridization of one of said plurality of probes; and (2) comparing pair wise said plurality of curves, thereby ranking said plurality of probes according to their binding specificities. 93. The method of claim 92, wherein said comparing pair wise comprises determining the value of a metric representing the difference between said pair of hybridization curves. 94. The method of claim 93, wherein said metric is the difference in areas underneath said pair of hybridization curves. 95. A method for ranking a plurality of probes according to their binding specificities to their respective complementary sequence, said method comprising (1) contacting a polynucleotide array comprising said plurality of probes with a sample comprising a plurality of nucleotide sequences under conditions such that hybridization can occur, wherein said plurality of nucleotide sequences comprises nucleotide sequences that are complementary to said plurality of probes, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining a plurality of hybridization curves, each representing progression of level of hybridization of one of said plurality of probes; and (3) comparing pair wise said plurality of curves, thereby ranking said plurality of probes according to their binding specificities. 96. The method of claim 95, wherein each of said plurality of nucleotide sequences that are complementary to said plurality of probes has known abundance in said sample. 97. The method of claim 95, wherein each of said plurality of nucleotide sequences that are complementary to said plurality of probes has equal abundance in said sample. 98. The method of claim 95, wherein said plurality of nucleotide sequences further comprises nucleotide sequences that are not complementary to any of said plurality of probes. 99. The method of claim 95, wherein said comparing pair wise comprises determining the value of a metric representing the difference between said pair of hybridization curves. 100. The method of claim 99, wherein said metric is the difference in areas underneath said pair of hybridization curves. 101. A method for ranking a plurality of probes according to their binding specificities to their respective complementary sequences, said method comprising (1) determining a plurality of hybridization curves, each representing progression of level of hybridization of one of said plurality of probes; (2) determining a hybridization curve representing progression of level of hybridization of a reference probe; (3) comparing each of said plurality of hybridization curves of said plurality of probes with said hybridization curve of said reference probe; (4) ranking said plurality of probes according their relative specificities to said reference probe, thereby ranking said plurality of probes according to their binding specificities. 102. The method of claim 101, wherein said comparing comprises determining the value of a metric representing the difference between said hybridization curve in said plurality of hybridization curves and said hybridization curve of said reference probe. 103. The method of claim 102, wherein said metric is the difference in areas underneath said hybridization curve in said plurality of hybridization curves and said hybridization curve of said reference probe. 104. The method of claim 101, wherein said reference curve represents cross-hybridization. 105. The method of claim 101, wherein said reference curve represents specific hybridization with known specificity. 106. A method for ranking a plurality of probes according to their binding specificities to their respective complementary sequence, said method comprising (1) contacting a polynucleotide array comprising said plurality of probes and at least one reference probe with a sample comprising a plurality of nucleotide sequences under conditions such that hybridization can occur, wherein said plurality of nucleotide sequences in said sample comprises nucleotide sequences that are complementary to said plurality of probes, and wherein said reference probe has a sequence which is not complementary to any known or predicted sequences in said sample, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining a plurality of hybridization curves, each representing progression of level of hybridization of one of said plurality of probes, and a reference hybridization curve representing progression of level of hybridization of said reference probe; (3) comparing each of said plurality of curves representing progression of level of hybridization of said plurality of probes and said reference hybridization curve representing progression of level of hybridization of said reference probe; and (4) ranking said plurality of probes according to their respective relative specificity with said reference probe, thereby ranking said plurality of probes according to their binding specificities. 107. The method of claim 106, wherein each of said plurality of nucleotide sequences that are complementary to said plurality of probes has known abundance in said sample. 108. The method of claim 106, wherein each said plurality of nucleotide sequences that are complementary to said plurality of probes has equal abundance in said sample. 109. The method of claim 106, wherein said plurality of nucleotide sequences further comprises nucleotide sequences that are not complementary to any of said plurality of probes. 110. The method of claim 106, wherein said comparing comprises determining the value of a metric representing the difference between each of said hybridization curves and said reference hybridization curve. 111. The method of claim 110, wherein said metric is the difference in areas underneath said pair of hybridization curves. 112. The method of claim 106, wherein said reference probe has a sequence which is not specifically hybridizable to any known or predicted sequences in said sample. 113. The method of claim 106, wherein said reference probe has a sequence which is specifically hybridizable to a sequence in said sample with known specificity. 114. A method for selecting a plurality of probes having similar binding specificities to their respective complementary sequence, said method comprising (1) contacting a polynucleotide array comprising said plurality of probes and at least 2 one reference probe with a sample comprising a plurality of nucleotide sequences under conditions such that hybridization can occur, wherein said plurality of nucleotide sequences comprises nucleotide sequences that are complementary to said plurality of probes, and wherein said reference probe has a sequence which is specifically hybridizable to a sequence in said sample with a known specificity, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining a plurality of hybridization curves, each representing progression of level of hybridization of one of said plurality of probes, and a reference hybridization curve representing progression of level of hybridization of said reference probe; (3) comparing each of said plurality of curves representing progression of level of hybridization of said plurality of probes and said reference hybridization curve representing progression of level of hybridization of said reference probe; and (4) selecting probes that have similar specificities as compared to said reference probe, thereby selecting probes having similar binding specificities. 115. The method of claim 114, wherein said comparing comprises determining the value of a metric representing the difference between each of said hybridization curves and said reference hybridization curve. 116. The method of claim 115, wherein said metric is the difference in areas underneath said pair of hybridization curves. 117. A method for determining the presence or absence of each of one or more nucleotide sequences in a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences, said method comprising (1) contacting a polynucleotide array comprising a plurality of probes specifically hybridizable to said one or more sequences with said sample under conditions such that hybridization can occur, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining for each of said probes hybridization level at at least two different hybridization times, wherein each of said at least two different hybridization times corresponds to a different length of time said sample is allowed to hybridize with said probe; (3) determining for each of said probes change of hybridization level by comparing hybridization levels measured at said at least two different hybridization times; and (5) comparing each said change with a threshold value, said threshold value indicating presence of said nucleotide sequences in said sample. 118. The method of claim 117, wherein said at least two different hybridization times consists of a first hybridization time and a second hybridization time. 119. The method of claim 118, wherein said comparing comprises determining for each of said plurality of probes the ratio of said second hybridization level 12 and said first hybridization level I1. 120. The method of claim 118, wherein said comparing comprises determining for each of said plurality of probes a quantity as described by equation xdev = I 2 - I 1 err ( I 1 ) 2 + err ( I 2 ) 2 wherein I2 is said second hybridization level and I1 is said first hybridization level and wherein said err(I1) and err(I2) are expected error in I1 and I2, respectively. 121. The method of claim 120, wherein said err(I1)2+err(I2)2 is defined by equation err(I1)2+err(I2)2=σ12+σ22+f2 (I22+I12) wherein σ12 is the variance for I1, σ22 is the variance for I2 and f is the fractional multiplicative error level. 122. The method of claim 118, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time. 123. The method of claim 122, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 80% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 124. The method of claim 123, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 90% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 125. The method of claim 124, wherein said first hybridization time is long enough for hybridization level of said probe to reach at least 95% of cross-hybridization equilibrium level and said second hybridization time is longer than said first hybridization time. 126. The method of claim 118, wherein said first hybridization time is 1 to 4 hours. 127. The method of any one of claims 118-126, wherein said second hybridization time is at least 2 times as long as said first hybridization time. 128. The method of any one of claims 118-126, wherein said second hybridization time is at least 10 times as long as said first hybridization time. 129. The method of any one of claims 118-126, wherein said second hybridization time is at least 16 times as long as said first hybridization time. 130. A method for determining the orientation of a nucleotide sequence in a sample, said method comprising (1) contacting a polynucleotide array comprising a forward polynucleotide probe comprising said sequence in forward direction and a reverse polynucleotide probe comprising said sequence in reverse direction with said sample under conditions such that hybridization can occur, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining hybridization levels of said forward polynucleotide probe at a first plurality of hybridization times, wherein each of said first plurality of hybridization times corresponds to a different length of time said sample is allowed to hybridize with said forward polynucleotide probe; (3) determining hybridization levels of said reverse polynucleotide probe at a second plurality of hybridization times, wherein each of said second plurality of hybridization times corresponds to a different length of time said sample is allowed to hybridize with said reverse polynucleotide probe; (4) determining change of hybridization level of said forward polynucleotide probe by a method comprising comparing hybridization levels measured at said first plurality of hybridization times; (5) determining change of hybridization level of said reverse polynucleotide probe by a method comprising comparing hybridization levels measured at said second plurality of hybridization times; and (6) determining the orientation of said nucleotide sequence by a method comprising comparing said change of hybridization level of said forward polynucleotide probe with said change of hybridization level of said reverse polynucleotide probe. 131. The method of claim 130, wherein said first plurality of hybridization times consists of a first hybridization time and a second hybridization time and wherein said second plurality of hybridization times consists of a third hybridization time and a fourth hybridization time. 132. The method of claim 131, wherein said first and said third hybridization times are 1 to 4 hours, respectively. 133. The method of claim 132, wherein said second hybridization time is at least 2 times as long as said first hybridization time, and wherein said fourth hybridization time is at least 2 times as long as said third hybridization time. 134. The method of claim 133, wherein said second hybridization time is at least 16 times as long as said first hybridization time, and wherein said fourth hybridization time is at least 16 times as long as said third hybridization time. 135. The method of claim 134, wherein said second hybridization time is at least 48 times as long as said first hybridization time, and wherein said fourth hybridization time is at least 48 times as long as said third hybridization time. 136. The method of claim 135, wherein said second hybridization time is at least 72 times as long as said first hybridization time, and wherein said fourth hybridization time is at least 72 times as long as said third hybridization time. 137. The method of claim 131, wherein said comparing in said step (4) comprises determining the ratios of said second hybridization level and said first hybridization level, and wherein said comparing in said step (5) comprises determining the ratios of said fourth hybridization level and said third hybridization level. 138. The method of claim 131, wherein said comparing in said step (6) comprises determining (i) for said forward polynucleotide probe a quantity xdevf as described by equation xdev f = I f 2 - I f 1 err ( I f 1 ) 2 + err ( I f 2 ) 2 and (ii) for said reverse polynucleotide probe a quantity xdev, as described by equation xdev r = I r4 - I r3 err ( I r3 ) 2 + err ( I r4 ) 2 wherein said If1 and If2 are hybridization levels of said forward polynucleotide probe at said first and second hybridization times, respectively, wherein said Ir3 and Ir4 are hybridization levels of said reverse polynucleotide probe at said third and fourth hybridization times, respectively, and said err(If1), err(f2), err(Ir3) and err(Ir4) are expected errors in said hybridization levels If1, If2, Ir3 and Ir4, respectively. 139. The method of claim 138, wherein said nucleotide sequence is determined as forward when xdevf>th1 xdevf−xdevr>th2 or as reverse when xdevr>th1 xdevr−xdevf>th2 wherein th1 and th2 are predetermined threshold values. 140. The method of any one of claims 131-135, wherein said first hybridization time and said third hybridization time are the same, and wherein said second hybridization time and said fourth hybridization time are the same. 141. The method of claim 140, wherein the orientation of said nucleotide sequence is determined by calculating a quantity t according to equation t = I f2 - I r4 σ I f2 - I r4 wherein said If2 is hybridization level of said forward polynucleotide probe at said second hybridization time and said Ir4 is hybridization level of said reverse polynucleotide probe at said fourth hybridization time, wherein said σtf2−Ir4 is error of the difference between If2 and Ir4, and wherein said nucleotide sequence is determined as forward if t>th, and reverse if t<−th, th being a predetermined threshold value. 142. The method of any one of claims 136-139, wherein said first hybridization time and said third hybridization time are the same, and wherein said second hybridization time and said fourth hybridization time are the same. 143. The method of claim 141, wherein hybridization levels of said forward and reverse polynucleotide probes are measured concurrently at said second and fourth hybridization times. 144. The method of claim 142, wherein hybridization levels of said forward and reverse polynucleotide probes are measured concurrently at said first and third hybridization times and at said second and fourth hybridization times. 145. A method of determining the orientation of a nucleotide sequence in the genome of an organism, comprising (i) repeating the method of any one of claims 130-139 with a plurality of samples of said organism, each said sample being subject to a different condition, and (ii) determining said orientation of said nucleotide sequence by combining results from said plurality of samples. 146. The method of any one of claims 130-139, wherein said sample comprising nucleic acid molecules pooled from a plurality of samples of an organism, each said sample being subject to a different condition. 147. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein each probe on said array comprises a different nucleotide sequence consists of 5 to 1,000 nucleotides. 148. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein each probe on said array comprises a different nucleotide sequence consists of 10 to 600 nucleotides. 149. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein each probe on said array comprises a different nucleotide sequence consists of 10 to 200 nucleotides. 150. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein each probe on said array comprises a different nucleotide sequence consists of 10 to 1100 nucleotides. 151. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein each probe on said array comprises a different nucleotide sequence consists of 110 to 30 nucleotides. 152. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein each probe on said array comprises a different nucleotide sequence consists of 40 to 80 nucleotides. 153. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 1117, wherein each probe on said array comprises a different nucleotide sequence consists of 60 nucleotides. 154. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein said nucleic acid molecules in said sample are labeled. 155. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 117, wherein said nucleic acid molecules in said sample are labeled with dye molecules. 156. The method of any one of claims 18, 31, 51, 62, 79, 95, 106, 114, or 1117, wherein said nucleic acid molecules in said sample are labeled with radioactive molecules. 157. A computer system for identifying specific hybridization to a polynucleotide probe, said computer system comprising a processor, and a memory coupled to said processor and encoding one or more programs, wherein the one or more programs cause the processor to perform a method comprising: (1) comparing hybridization levels of said probe at a first hybridization time and a second hybridization time, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time; and (2) determining the difference of hybridization levels from said comparing, said difference representing a metric for identifying specific hybridization. 158. A computer system for comparing hybridization specificity of a first probe and a second probe, said computer system comprising a processor, and a memory coupled to said processor and encoding one or more programs, wherein the one or more programs cause the processor to perform a method comprising: (1) comparing a first hybridization curve representing progression of level of hybridization of said first probe and a second hybridization curve representing progression of level of hybridization of said second probe; and (2) determining the value of a metric from said comparing, said metric representing the difference between first hybridization curve and said second hybridization curve. 159. A computer system for ranking a plurality of probes according to their binding specificities, said computer system comprising a processor, and a memory coupled to said processor and encoding one or more programs, wherein the one or more programs cause the processor to perform a method comprising: (1) comparing each of two or more hybridization curves, each of said two or more hybridization curves representing progression of level of hybridization of one of said two or more probes, to a reference hybridization curve representing progression of level of hybridization of a reference probe; (2) determining the value of a metric for each of the two or more probes from each of said comparings, the value of said metric for each of the two or more probes representing the difference between each of the two or more hybridization curves and the reference hybridization curve; and (3) ranking the two or more probes according to the value of the metric for each of said two or more probes. 160. A computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the processor to execute the steps of: (1) comparing hybridization levels of said probe at a first hybridization time and a second hybridization time, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time; and (2) determining the difference of hybridization levels from said comparing, said difference representing a metric for identifying specific hybridization. 161. A computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the processor to execute the steps of: (1) comparing a first hybridization curve representing progression of level of hybridization of said first probe and a second hybridization curve representing progression of level of hybridization of said second probe; and (2) determining the value of a metric from said comparing, said metric representing the difference between first hybridization curve and said second hybridization curve. 162. A computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the processor to execute the steps of: (1) comparing each of two or more hybridization curves, each of said two or more hybridization curves representing progression of level of hybridization of one of said two or more probes, to a reference hybridization curve representing progression of level of hybridization of a reference probe; (2) determining the value of a metric for each of the two or more probes from each of said comparings, the value of said metric for each of the two or more probes representing the difference between each of the two or more hybridization curves and the reference hybridization curve; and (3) ranking the two or more probes according to the value of the metric for each of said two or more probes. |
<SOH> 2. BACKGROUND OF THE INVENTION <EOH>Rapid and accurate determination of the identities and abundances of nucleic acid species in a sample containing many different nucleic acid sequences is of great interest in biological and medical fields, e.g., in gene discovery and expression profiling. Presently, methods based on DNA arrays are widely used for the detection and measurement of particular sequences in complex samples. In such methods the identity and abundance of a nucleic acid sequence in a sample is determined by measuring the level of hybridization of the nucleic acid sequence to probes that comprise complementary sequences. Although various formats of DNA arrays are currently used, all DNA array technologies employ nucleic acid “probes,” (i.e., nucleic acid molecules having defined sequences) to selectively hybridize to, and thereby identifying and measuring the abundances of, complementary nucleic acid sequences in a sample. In these technologies, a set of nucleic acid probes, each of which has a defined sequence, is immobilized on a solid support in such a manner that each different probe is immobilized to a predetermined region. The set of immobilized probes or the array of immobilized probes is contacted with a sample containing labeled nucleic acid species so that nucleic acids having sequences complementary to an immobilized probe hybridize or bind to the probe. After separation of, e.g., by washing off, any unbound material, the bound, labeled sequences are detected and measured. The amount of labeled sequence hybridized to each probe in the array is used as a measure of the abundance of the sequence species in the cells (see, e.g., Schena et al., 1995, Science 270:467-470; Lockhart et al., 1996 , Nature Biotechnology 14:1675-1680; Blanchard et al., 1996 , Nature Biotechnology 14:1649; Ashby et al., U.S. Pat. No. 5,569,588). Using DNA array expression assays, complex mixtures of labeled nucleic acids, e.g., mRNAs or nucleic acids derived from mRNAs from a cell or a population of cells, can be analyzed. DNA array technologies have made it possible, inter alia, to monitor the expression levels of a large number of genetic transcripts at any one time (see, e.g., Schena et al., 1995 , Science 270:467-470; Lockhart etal., 1996 , Nature Biotechnology 14:1675-1680; Blanchard et al, 1996 , Nature Biotechnology 14:1649; Ashby et al., U.S. Pat. No. 5,569,588, issued Oct. 29, 1996; Shoemaker et al., U.S. patent application Ser. No. 09/724,538, filed on Nov. 28, 2000). DNA array technologies have also found applications in gene discovery, e.g., in identification of exon structures of genes (see, e.g., Shoemaker et al., U.S. patent application Ser. No. 09/724,538, filed on Nov. 28, 2000). Of the two main formats of DNA arrays, spotted DNA arrays are prepared by depositing DNA fragments with sizes ranging from about a few tens of bases to a few kilobases onto a suitable surface (see, e.g., DeRisi et al., 1996 , Nature Genetics 14:457460; Shalon et al., 1996 , Genome Res. 6:689-645; Schena et al., 1995 , Proc. Natl. Acad. Sci. U.S.A. 93:10539-11286; and Duggan et al., Nature Genetics Supplement 21:10-14). For example, in blotting assays, such as dot or Southern Blotting, nucleic acid molecules may be first separated, e.g., according to size by gel electrophoresis, transferred and immobilized to a membrane filter such as a nitrocellulose or nylon membrane, and allowed to hybridize to a single labeled sequence (see, e.g., Nicoloso, M. et al., 1989 , Biochemical and Biophysical Research Communications 159:1233-1241; Vernier, P. et al., 1996 , Analytical Biochemistry 235:11-19). Spotted cDNA arrays are prepared by depositing PCR products of cDNA fragments with sizes ranging from about 0.6 to 2.4 kb, from full length cDNAs, ESTs, etc., onto a suitable surface (see, e.g., DeRisi et al, 1996 , Nature Genetics 14:457-460; Shalon et al., 1996 , Genome Res. 6:689-645; Schena et al., 1995 , Proc. Natl. Acad Sci U.S.A. 93:10539-11286; and Duggan et al., Nature Genetics Supplement 21:10-14). Alternatively, high-density oligonucleotide arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface are synthesized in situ on the surface by, for example, photolithographic techniques (see, e.g., Fodor et al., 1991 , Science 251:767-773; Pease et al, 1994 , Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026; Lockhart et al, 1996 , Nature Biotechnology 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; 5,510,270; 5,445,934; 5,744,305; and 6,040,138). Methods for generating arrays using inkjet technology for in situ oligonucleotide synthesis are also known in the art (see, e.g., Blanchard, International Patent Publication WO 98/41531, published Sep. 24, 1998; Blanchard et al., 1996 , Biosensors and Bioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arrays in Genetic Engineering , Vol. 20, J. K. Setlow, Ed., Plenum Press, New York at pages 111-123). However, as is well known in the art, although hybridization is selective for complementary sequences, other sequences which are not perfectly complementary may also hybridize to a given probe at some level. Binding affinity of target nucleic acids to surface immobilized probe sequences during hybridization depends on both the sequence similarity of different target sequences in a sample and the hybridization stringency condition, e.g., the hybridization temperature and the salt concentrations. Binding kinetics also depends on the relative concentrations of different nucleic acids in a sample. Therefore, when measured at a given time under a given hybridization stringency condition, different target sequences with different degrees of similarity may hybridize to a given probe at different degrees. For polynucleotide probes targeted at, i.e., complementary to, low-abundance species, or target at nucleic acid species of closely resembled (i.e., homologous) sequences, such “cross-hybridization” can significantly contaminate and confuse the results of hybridization measurements. For example, cross-hybridization is a particularly significant concern in the detection of single nucleotide polymorphisms (SNP's) since the sequence to be detected (i.e., the particular SNP) must be distinguished from other sequences that differ by only a single nucleotide. Several approaches have been devised to reduce cross-hybridization. Cross-hybridization can be minimized by regulating either the hybridization stringency condition, e.g., the temperature and salt concentrations, during hybridization and/or during post-hybridization washings. For example, “highly stringent” wash conditions may be employed so as to destabilize the majority of but the most stable duplexes such that measured hybridization signals represent the abundances of sequences that hybridize most specifically, and are therefore the most complementary, to a given probe. Exemplary highly stringent conditions include, e.g., hybridization to filter-bound DNA in 5×SSC, 1% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989 , Current Protocols in Molecular Biology , Vol., Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, N.Y., at p. 2.10.3). Highly stringent conditions allow detection of allelic variants of a nucleotide sequence, e.g., about 1 mismatches per 10-30 nucleotides. Alternatively, “moderate-” or “low-stringency” wash conditions may be used to allow identification of sequences which are similar, but not identical, to the perfectly complementary sequence to a given probe, such as sequences from different members of a multi-gene family, or homologous genes in different organisms. Moderate- or low-stringency conditions are also well known in the art (see, e.g., Sambrook et al., supra; Ausubel, F. M. et al., supra). Exemplary moderately stringent wash conditions include, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra). Exemplary low-stringency washing conditions include, e.g., washing in 5×SSC or in 0.2×SSC/0.1% SDS at room temperature (Ausubel et a!, 1989, supra). A ‘high’ stringency condition for one sequence could be a ‘moderate’ or even ‘low’ stringency condition for another sequence. The effect of cross-hybridization on measured hybridization levels can also be reduced by selecting and using polynucleotide probes that are most specific for a particular target nucleic acid molecule of interest. For example, sensitivity- and specificity-based probe design and selection methods are developed (see, e.g., PCT publication WO 01/05935). Multiple different oligonucleotide probes which are complementary to different, distinct sequences of a target nucleic acid are also used (see, e.g., Lockhart et al. (1996) Nature Biotechnology 14:1675-1680; Graves et al. (1999) Trends in Biotechnology 17:127-134). Contributions of cross-hybridization to measured hybridization levels can also be removed by subtracting signals from suitable reference probes which serve to measure the levels of cross-hybridization. In one example, polynucleotide probes having intentional mismatches are used as the reference probes. The hybridization to (or dissociation from) the target nucleic acid molecule is compared to that of the perfect match oligonucleotide probe so that a cross-hybridization component may be subtracted from the total hybridization signal (see, e.g., Graves et al., supra; Fodor et al., 1991 , Science 251:767-773; Pease et al, 1994 , Proc. Natl. Acad. Sci. USA. 91:5022-5026; Lockhart et al., 1996 , Nature Biotechnology 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; 5,510,270; 5,445,934; 5,744,305; and 6,040,138). In another example, polynucleotide probes of reverse complementary sequences are used as the reference probes (see, Shoemaker et al., U.S. patent application Ser. No. 09/781,814, filed on Feb. 12, 2001; and Shoemaker et al., U.S. patent application Ser. No. 09/724,538, filed on Nov. 28, 2000). In another type of approaches, differences in equilibrium binding and wash dissociation kinetics between perfect and non-perfect match duplexes are utilized to distinguish and remove cross-hybridization from hybridization data (see, e.g., Friend et al., U.S. Pat. No. 6,171,794, issued on Jan. 9, 2001; and Burchard et al., U.S. Patent application Ser. No. 09/408,582, filed on Sep. 29, 1999). These methods are premised on the discovery that non-perfect duplexes tend to wash off more quickly, or at a lower stringency, than the perfect duplexes. Therefore, perfect and non-perfect match duplexes can be distinguished using wash dissociation histories. In U.S. Pat. No. 6,171,794, multiple cross-hybridization components are distinguished by comparison of wash dissociation curve with template dissociation histories. In U.S. patent application Ser. No. 09/408,582, a robust way of estimating the total contribution due to non-perfect duplexes using wash dissociation histories is described. Various techniques have also been developed to study the hybridization kinetics of polynucleotides immobilized in solution or agarose or polyacrylamide gels (see, e.g., Mazumder et al., 1998 , Nucleic Acids Research 26:1996-2000; Ikuta S. et al., 1987 , Nucleic Acids Research 15:797-811; Kunitsyn, A. et al., 1996 , Journal of Biomolecular Structure and Dynamics 14:239-244; Day, 1. N. M. et al., 1995 , Nucleic Acids Research 23:2404-2412), as well as hybridization to polynucleotide probes immobilized on glass plates (Beattie, W. G. et al., 1995 , Molecular Biotechnology 4:213-225) including oligonucleotide microarrays (Stimpson, D. I. et al., 1995 , Proc. Natl. Acad. Sci. U.S.A. 92:6379-6383). For example, the nucleotide sequence similarity of a pair of nucleic acid molecules can be distinguished by allowing the nucleic acid molecules to hybridize, and following the kinetic and equilibrium properties of duplex formation (see, e.g., Sambrook, J. et al., eds., 1989 , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., at pp. 9.47-9.51 and 11.55-11.61; Ausubel et al., eds., 1989 , Current Protocols in Molecular Biology , Vol I, Green Publishing Associates, Inc., John Wiley & Sons, Inc., New York, at pp. 2.10.1-2.10.16; Wetmur, J. G., 1991 , Critical Reviews in Biochemistry and Molecular Biology 26:227-259; Persson, B. et al., 1997 , Analytical Biochemistry 246:34-44; Albretsen, C. et al., 1988 , Analytical Biochemistry 170:193-202; Kajimura, Y. et al., 1990, GATA 7:71-79; Young, S. and Wagner, R. W., 1991 , Nucleic Acids Research 19:2463-2470; Guo, Z. et al., 1997 , Nature Biotechnology 15:331-335; Wang, S. et al., 1995 , Biochemistry 34:9774-9784; Niemeyer, C. M. et al., 1998 , Bioconjugate Chemistry 9:168-175). The exact hybridization or wash conditions that are optimal for any given assay will depend on the exact nucleic acid sequence or sequences of interest, and, in general, must be empirically determined. There is no single hybridization or washing condition which is optimal for all different nucleic acid sequences. In fact, even the most optimized conditions allow only partial discrimination of similar sequences, especially when such sequences have a high degree of similarity, or when some of the similar sequences are present in excess amounts or at high concentrations. Therefore, there is a need to develop methods for determination of specific hybridization and removal of contributions from cross-hybridized species in hybridization measurements. There is also a need to develop methods for experimentally selecting and ranking probes comprising sequences that most specifically hybridize to target sequences of interest. Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. |
<SOH> 3. SUMMARY OF THE INVENTION <EOH>The present invention provides methods for utilizing the changes of hybridization levels during approach to equilibrium duplex formation in hybridization measurements. In the invention, changes of hybridization levels of polynucleotide probes are monitored at a plurality of hybridization times, e.g., during their progress towards equilibrium, and a continuing increase of hybridization levels beyond the time scale of cross-hybridization equilibrium is used as an indication of specific binding. The invention is based, at least in part, on the discovery that specificity of binding of nucleotide sequences to probes (i.e., the ratio of specific to non-specific duplexes) increases with time. The invention provides methods for determining whether specific hybridization to a polynucleotide probe by a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs. The methods determine change of hybridization level of the probe measured at a plurality of different hybridization times. The presence of specific hybridization at the probe is identified when the value of such change of hybridization level is above a predetermined threshold level. In preferred embodiments, hybridization levels measured at a first hybridization time and a second, different hybridization time is compared. Preferably, the first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium. More preferably, the first hybridization time is long enough for hybridization level at the probe to reach at least 80%, 90% or 95% of cross-hybridization equilibrium level. In a preferred embodiment, the first hybridization time is in the range of 14 hours. Preferably, the second hybridization time is longer than the first hybridization time. More preferably, the second hybridization time is at least 2, 4, 6, 10, 12, 16, 18, 48 or 72 times as long as the first hybridization time. In a preferred embodiment, the second hybridization time is in the range of 48-72 hours. In one embodiment, the time scale of cross-hybridization equilibrium is determined from a measured hybridization curve representing progression of hybridization level of the probe(s) with a sample which does not contain nucleic acid molecules specifically hybridizable to said probe(s). In another embodiment, the time scale of cross-hybridization equilibrium is determined from a measured hybridization curve representing progression of hybridization level of a reference probe, which has a sequence that is not specifically hybridizable to any known or predicted sequences in the sample. In one embodiment, the reference probe is a synthetic probe. In preferred embodiments, multiple synthetic probes are used so that the hybridization curve can be more reliably determined statistically. As examples, and not intended to be limiting, the reference probe hybridizes to any known or predicted sequences in a sample with at least 3%, 5%, 10%, 20% or 30% mismatched bases in said reference probe. In other embodiments, the reference probe has a sequence that is a reverse complement of a sequence or has a sequence that has reverse nucleotide order to a sequence in said plurality of nucleic acid molecules or is a reverse complement or has a reverse nucleotide order of the probe. In preferred embodiments, the invention provides methods for determining whether specific hybridization to polynucleotide probe occurs using polynucleotide probe arrays. In the embodiments, hybridization levels of probes are measured by contacting a polynucleotide array comprising the probes with a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences. In specific embodiments, the sample comprises more than 1,000, 5,000, 10,000, 50,000, or 100,000 nucleic acid molecules of different nucleotide sequences. In one embodiment, whether specific hybridization to a polynucleotide probe by a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs is determined by a method comprising (1) contacting a polynucleotide array comprising said probe with said sample under conditions such that hybridization can occur; (2) determining hybridization levels of said probe at a plurality of different hybridization times; (3) determining change of hybridization level by comparing hybridization levels measured at said plurality of different hybridization times; and (4) representing specific hybridization using said change, thereby determining whether specific hybridization of said probe occurs. Alternatively, whether specific hybridization to a polynucleotide probe by a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences occurs is determined by a method comprising (1) contacting a plurality of polynucleotide arrays, each comprising said probe, with said sample under conditions such that hybridization can occur; (2) determining hybridization levels of said probe at each said polynucleotide array at a plurality of different hybridization times; (3) determining change of hybridization level by comparing hybridization levels measured at said plurality of different hybridization times; and (4) representing specific hybridization using said change, thereby determined whether specific hybridization of said probe occurs. Preferably, specific hybridization at the probe is identified when the value of such change of hybridization level is above a predetermined threshold level. In a preferred embodiment, hybridization levels measured at a first hybridization time and a second hybridization time is compared and specific hybridization is identified if the change in hybridization levels is above a predetermined threshold. Preferably, the first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium. More preferably, the first hybridization time is long enough for hybridization level at the probe to reach at least 80%, 90% or 95% of cross-hybridization equilibrium level. Preferably, the second hybridization time is longer than the first hybridization time. More preferably, the second hybridization time is at least 2, 4, 6, 10, 12, 16, 18, 48 or 72 times as long as the first hybridization time. In a preferred embodiment, the ratio of said second hybridization level and said first hybridization level is determined and used as a measure of specific hybridization of the probe. In another preferred embodiment, a quantity xdev as described by equations (7) or (8), infra, is determined and used as a measure of specific hybridization of the probe. Preferably, each different probe on the polynucleotide array comprises a different nucleotide sequence consists of 5 to 1000, 10 to 600, 10 to 200, 10 to 100, 10 to 30, 40-80 nucleotides. More preferably, each different probe on the polynucleotide array comprises a different nucleotide sequence consists of 60 nucleotides. The sample is preferably labeled. In one embodiment, the sample is labeled with fluorescent dye molecules. In another embodiment, the sample is labeled with radioactive molecules. The present invention also provides methods for determining the relative abundance of one or more nucleotide sequences in a plurality of samples, each of said plurality of samples comprising a plurality of nucleic acid molecules having different nucleotide sequences. In one embodiment, the method comprises (1) determining for each sample difference in hybridization levels measured at a first hybridization time and a second, different hybridization time to a probe that is specific to said nucleotide sequence; and (2) comparing the differences among the plurality of samples. Preferably, the first hybridization time is close to time scale for reaching cross-hybridization equilibrium at the probe and the second hybridization time is longer than the first hybridization time. In a preferred embodiment, hybridization levels of probes are measured by contacting a polynucleotide array comprising the probes with a sample comprising a plurality of nucleic acid molecules having different nucleotide sequences under conditions such that hybridization can occur. In one embodiment, hybridization levels of probes are measured by (1) contacting one or more polynucleotide arrays comprising said probe with one or more of said plurality of samples under conditions such that hybridization can occur; (2) determining for each of said plurality of samples a first hybridization level of said probe at a first hybridization time; (3) determining for each of said plurality of samples a second hybridization level of said probe at a second, different hybridization time; (4) determining for each of said plurality of samples difference in said first and second hybridization levels; and (5) comparing said difference among said plurality of samples. Preferably, each different probe on the polynucleotide array comprises a different nucleotide sequence consists of 5 to 1000, 10 to 600, 10 to 200, 10 to 100, 10 to 30, 40-80 nucleotides. More preferably, each different probe on the polynucleotide array comprises a different nucleotide sequence consists of 60 nucleotides. The samples are preferably labeled. In one embodiment, a sample labeled with a fluorescence dye is measured. In some embodiments, more than one samples are measured using the same array, each sample is labeled with a different fluorescent dye having a distinguishable emission spectra such that different samples are labeled with different and distinguishable dyes. The differently labeled samples are contacted with a single polynucleotide array simultaneously. In preferred embodiments, at least 3, 5 or 10 samples, distinctively labeled, are measured. In other embodiments, the sample is labeled with radioactive molecules. The present invention also provides methods for comparing hybridization specificity among different probes. In the methods, hybridization specificities of different probes are compared by comparing the hybridization curves representing progressions of hybridization levels of the probes. Such hybridization curves representing progression of hybridization level can be measured in real time. Alternatively, progression of hybridization signal can be obtained by measuring hybridization levels in different experiments, in each of which a particular hybridization time is used (time correlated measurement). Hybridization curves are preferably compared by determining the value of a metric that represents the difference between the hybridization curves. In one embodiment, the metric is the difference in areas underneath the different hybridization curves. Hybridization curves can also be compared by determining a curve that represents the difference between the hybridization curves. In one embodiment, a ratio curve is determined. In another embodiment, a curve of xdev as defined infra is determined. In some embodiments, the hybridization curve of a probe is compared with the hybridization curve of a reference probe which has a sequence that is not specifically hybridizable to any known or predicted sequences in the sample using any of the method described above. Such embodiment offers a method for identifying specific hybridization of the probe. As examples, and not intended to be limiting, the reference probe can be a probe that is not specifically hybridizable to any known or predicted sequences in the sample, e.g., a probe that hybridizes to any known or predicted sequences in the sample with at least 3%, 5%, 10%, 20% or 30% mismatched bases in the probe. In other embodiments, the reference probe has a sequence that is a reverse complement of a sequence or has a sequence that has reverse nucleotide order to a sequence in said plurality of nucleic acid molecules or is a reverse complement or has a reverse nucleotide order of the probe. The invention also provides methods for determining the difference in time scale of reaching hybridization equilibrium between specific and non-specific hybridization to a polynucleotide probe. In one embodiment, the time scales of equilibrium specific and non-specific hybridization are determined from measured hybridization curve of the probe and a reference probe. As examples, and not intended to be limiting, the reference probe can be a probe that is not specifically hybridizable to any known or predicted sequences in the sample, e.g., a probe that hybridizes to any known or predicted sequences in the sample with at least 3%, 5%, 10%, 20% or 30% mismatched bases in the probe. In other embodiments, the reference probe has a sequence that is a reverse complement of a sequence or has a sequence that has reverse nucleotide order to a sequence in said plurality of nucleic acid molecules or is a reverse complement or has a reverse nucleotide order of the probe. The invention further provides methods for ranking a plurality of probes according to their binding specificities to their respective complementary sequences. In one embodiment, hybridization specificities of different probes are compared pair wise by comparing pair of the hybridization curves representing progressions of hybridization levels of the probes. The hybridization curves can be measured in real time, or alternatively, in time correlated measurement. Each pair of hybridization curves is preferably compared by determining the value of a metric that represents the difference between the pair of hybridization curves. In one embodiment, the metric is the difference in areas underneath the different hybridization curves. Hybridization curves can also be compared by determining a curve that represents the difference between the hybridization curves. In one embodiment, a ratio curve is determined. In another embodiment, a curve of xdev as defined infra is determined. Probes are then ranked according to their relative specificities. In another embodiment, hybridization curve of each of the plurality of probes is compared with the hybridization curve of one or more reference probes. In one embodiment, the one or more reference probes each having a sequence that is not specifically hybridizable to any known or predicted nucleotide sequences in the sample. As examples, and not intended to be limiting, the one or more reference probes in this embodiment can be probes that are not specifically hybridizable to any known or predicted sequences in the sample, e.g., a probe that hybridizes to any known or predicted sequences in the sample with at least 3%, 5%, 10%, 20% or 30% mismatched bases in the probe. In other embodiments, the reference probe has a sequence that is a reverse complement of a sequence or has a sequence that has reverse nucleotide order to a sequence in said plurality of nucleic acid molecules or is a reverse complement or has a reverse nucleotide order of the probe. In still other embodiments, the reference probe has a sequence that is a complement of a sequence or has a sequence that is complementary to a sequence in said plurality of nucleic acid molecules. The probes are then ranked according to their relative specificities with the reference probe(s), e.g., in order of lower to higher specificities starting from the one with a specificity most close to the reference. In another embodiment, the one or more reference probes each having a sequence that is specifically hybridizable to a nucleotide sequence in the sample, i.e., having a sequence that is complementary to a sequence in the sample, with a known specificity. In such an embodiment, the specificities of probes are ranked in according to specificity as compared to the known specificity of the reference probe. In still another embodiment, hybridization curve of each of the plurality of probes is compared with the hybridization curve of a reference probe having known specificity to a sequence in the sample and probes having similar specificities as the reference probe are selected. Preferably, hybridization curves of probes of interest and/or reference probes are measured using polynucleotide probe arrays. In such embodiments, hybridization levels of probes are measured by contacting a polynucleotide array comprising the probes of interest and/or reference probes with a sample comprising a plurality of nucleic acid molecules having nucleotide sequences that are complementary to probes of interest and/or reference probes. Preferably, each different probe on the polynucleotide array comprises a different nucleotide sequence consists of 5 to 1000, 10 to 600, 10 to 200, 10 to 100, 10 to 30, 40-80 nucleotides. More preferably, each different probe on the polynucleotide array comprises a different nucleotide sequence consists of 60 nucleotides. The sample is preferably labeled. In one embodiment, the sample is labeled with fluorescent dye molecules. In another embodiment, the sample is labeled with radioactive molecules. In one embodiment, each of the nucleotide sequences that are known to be complementary to the probes of interest and/or references probes has known abundance in said sample. In another embodiment, each of the nucleotide sequences that are known to be complementary to the probes of interest and/or references probes has equal abundance in said sample. Preferably, the sample also comprises nucleotide sequences that are not specifically hybridizable to any of probes of interest and/or references probes. The invention also provides methods for detecting the presence or absence of nucleotide sequences in a sample comprising a plurality of different nucleotide sequences. In the method the presence of a nucleotide is identified by the presence of specific hybridizations to polynucleotide probes having predetermined sequences. The presence of specific hybridization to a probe is determined by methods described in supra. In a preferred embodiment, the presence or absence of one or more nucleotide sequences in a sample is determined using one or more microarrays comprising probes specifically hybridizable to such nucleotide sequences. In the embodiment, one or more polynucleotide arrays comprising a plurality of probes specifically hybridizable to predetermined sequences are contacted with the sample and a first hybridization level I 1 of at a first hybridization and a second hybridization level I 2 of at a second hybridization time are determined for each of the probes. Change of hybridization level from I 1 to I 2 is then measured using a suitable metric, e.g., ratio of I 2 to I 1 , difference of I 2 to I 1 or the quantity xdev of I 2 to I 1 , for each probe is then determined. The presence of a nucleotide sequence is then identified if the value of the metric is greater than a predetermined threshold level, whereas the absence of a nucleotide sequence is identified if the value of the metric is less than a predetermined threshold level. The threshold level depends on the metric used and the sequences of interest as well as experimental conditions, e.g., stringency condition, and may be determined by those skilled in the art. In a preferred embodiment, a threshold level of 2, 4 or 10 is used for xdev. The invention also provides methods for determining the orientation of a nucleotide sequence in a sample by comparing specific hybridization to a forward probe comprising the sequence in forward direction and a reverse probe comprising the sequence in reverse direction. In the methods, the presence or absence of specific hybridization to one or the other probe in a pair of forward and reverse probes are determined and specific hybridization to one but not the other probe in the pair is used to identify the orientation of the sequence. In preferred embodiments, specific hybridizations to the forward and/or reverse probes are determined by the methods utilizing changes of hybridization levels during approach to hybridization equilibrium. In more preferred embodiments, kinetic methods are used to determine specific hybridizations to both the forward and reverse probes. When kinetic methods are used, hybridization levels of the forward and reverse probes are both measured at a plurality of hybridization times so that specific hybridization to the forward or the reverse probe can be determined. The hybridization levels at the forward and reverse probes can be measured concurrently or separately. In a preferred embodiment, the method for determining the orientation of a nucleotide sequence comprises: (1) contacting a polynucleotide array comprising a forward polynucleotide probe comprising said sequence in forward direction and a reverse polynucleotide probe comprising said sequence in reverse direction with said sample under conditions such that hybridization can occur, said polynucleotide array comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different predetermined nucleotide sequences; (2) determining hybridization levels of said forward polynucleotide probe at a first plurality of hybridization times, wherein each of said first plurality of hybridization times corresponds to a different length of time said sample is allowed to hybridize with said forward polynucleotide probe; (3) determining hybridization levels of said reverse polynucleotide probe at a second plurality of hybridization times, wherein each of said second plurality of hybridization times corresponds to a different length of time said sample is allowed to hybridize with said reverse polynucleotide probe; (4) determining change of hybridization level of said forward polynucleotide probe by a method comprising comparing hybridization levels measured at said first plurality of hybridization times; (5) determining change of hybridization level of said reverse polynucleotide probe by a method comprising comparing hybridization levels measured at said second plurality of hybridization times; and (6) determining the orientation of said nucleotide sequence by a method comprising comparing said change of hybridization level of said forward polynucleotide probe with said change of hybridization level of said reverse polynucleotide probe. In preferred embodiments, the first plurality of hybridization times consists of a first hybridization time and a second hybridization times, whereas the second plurality of times consists of a third hybridization time and a fourth hybridization times. In a preferred embodiment, the first and third hybridization times are 1 to 4 hours. In another preferred embodiment, the second and the fourth hybridization times are at least 2, 4, 12, 16, 48 or 72 times as long as said first and third hybridization times, respectively. In more preferred embodiments, the first and the third hybridization times are the same, and the second and the fourth hybridization times are the same. In preferred embodiments, the orientation of the nucleotide sequence is determined by comparing the xdev's for the forward probe and the reverse probe. In another embodiment, the orientation of the nucleotide sequences is determined by comparing the hybridization levels of the forward probe and the reverse probe measured at the second hybridization times. The invention also provides computer systems which can be used to practice the methods of the invention. In one embodiment, the invention provides a computer system for identifying specific hybridization to a polynucleotide probe, said computer system comprising a processor, and a memory coupled to said processor and encoding one or more programs, wherein the one or more programs cause the processor to perform a method comprising: (1) comparing hybridization levels of said probe at a first hybridization time and a second hybridization time, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time; and (2) determining the difference of hybridization levels from said comparing, said difference representing a metric for identifying specific hybridization. In another embodiment, the invention provides a computer system for comparing hybridization specificity of a first probe and a second probe, said computer system comprising a processor, and a memory coupled to said processor and encoding one or more programs, wherein the one or more programs cause the processor to perform a method comprising: (1) comparing a first hybridization curve representing progression of hybridization level of said first probe and a second hybridization curve representing progression of hybridization level of said second probe; and (2) determining the value of a metric from said comparing, said metric representing the difference between first hybridization curve and said second hybridization curve. In still another embodiment, the invention provides a computer system for ranking a plurality of probes according to their binding specificities, said computer system comprising a processor, and a memory coupled to said processor and encoding one or more programs, wherein the one or more programs cause the processor to perform a method comprising: (1) comparing each of two or more hybridization curves, each of said two or more hybridization curves representing progression of hybridization level of one of said two or more probes, to a reference hybridization curve representing progression of hybridization level of a reference probe; (2) determining the value of a metric for each of the two or more probes from each of said comparings, the value of said metric for each of the two or more probes representing the difference between each of the two or more hybridization curves and the reference hybridization curve; and (3) ranking the two or more probes according to the value of the metric for each of said two or more probes. The invention also provide computer program which can be used to practice the methods of the invention. In one embodiment, the invention provides computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the processor to execute the steps of: (1) comparing hybridization levels of said probe at a first hybridization time and a second hybridization time, wherein said first hybridization time is close to the time scale for substantially reaching cross-hybridization equilibrium and said second hybridization time is longer than said first hybridization time; and (2) determining the difference of hybridization levels from said comparing, said difference representing a metric for identifying specific hybridization. In another embodiment, the invention provides computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the processor to execute the steps of: (1) comparing a first hybridization curve representing progression of hybridization level of said first probe and a second hybridization curve representing progression of hybridization level of said second probe; and (2) determining the value of a metric from said comparing, said metric representing the difference between first hybridization curve and said second hybridization curve. In still another embodiment, the invention provides computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the processor to execute the steps of: (1) comparing each of two or more hybridization curves, each of said two or more hybridization curves representing progression of hybridization level of one of said two or more probes, to a reference hybridization curve representing progression of hybridization level of a reference probe; (2) determining the value of a metric for each of the two or more probes from each of said comparings, the value of said metric for each of the two or more probes representing the difference between each of the two or more hybridization curves and the reference hybridization curve; and (3) ranking the two or more probes according to the value of the metric for each of said two or more probes. |
Method and device for producing an aqueous acrylamide solutions using a biocatalysts |
The invention relates to a method and a device for producing an aqueous acrylamide solution by the hydration of acrylnitrile in an aqueous solution in the presence of a biocatalyst. |
1. Method for producing an aqueous acrylamide solution by the hydration of acrylonitrile in an aqueous solution in the presence of a biocatalyst, characterised in that the biocatalyst is separated from the aqueous acrylamide solution within ≦2 hours, preferably within ≦1 hour of the end of the reaction. 2. Method according to claim 1, characterised in that the biocatalyst is separated with a tubular centrifuge. 3. Method according to claim 1, characterised in that the biocatalyst is separated with an at least partially continuously operating, self-draining centrifuge. 4. Method according to claim 3, characterised in that the centrifuge is an annular gap centrifuge. 5. Method according to any one of claims 2 to 4, characterised in that the clear discharge from the centrifuge is preferably monitored using an optical means, particularly preferably a light barrier. 6. Method according to claim 5, characterised in that the monitoring is used to control the centrifuges. 7. Method according any one of claims 1 to 6, characterised in that the biocatalyst is flocculated before the separation. 8. Method according to claim 7, characterised in that aluminium sulphate is used as the flocculation agent. 9. Method according to claim 7, characterised in that an anionic polymer is used as the flocculation agent. 10. Method according to any one of claims 7 to 9, characterised in that the flocculation is performed at a pH value of 6.8 to 8.0, preferably 7.0 to 7.5. 11. Method according to any one of claims 1 to 10, characterised in that the aqueous acrylamide solution freed of biocatalyst is set to a pH value of 4.5 to 7.0, preferably 5.5 to 6.5. 12. Method according to any one of claims 1 to 11, characterised in that the separated biocatalyst is freed of acrylamide by at least a single, preferably multiple washing and separation. 13. Method according to claim 12, characterised in that the washing is performed with deionised water. 14. Method according to claim 12 or 13, characterised in that the acrylamide concentration in the biocatalyst is <10 ppm, preferably <5 ppm. 15. Method according to any one of claims 12 to 14, characterised in that the washing water is recycled in the process. 16. Method according to any one of claims 12 to 15, characterised in that the biocatalyst is sterilised after the washing. 17. Method according to any one of claims 1 to 16, characterised in that the biocatalyst is Rhodococcus rhodochrous filed under the deposition number 14230 with DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maschroder Weg 1b, D-38124 Braunschweig, Germany. 18. Device for the production of an aqueous acrylamide solution by the hydration of acrylonitrile in an aqueous solution in the presence of a biocatalyst with a reactor and a tubular centrifuge for separating the biocatalyst from the aqueous acrylamide solution. 19. Device for the production of an aqueous acrylamide solution by the hydration of acrylonitrile in an aqueous solution in the presence of a biocatalyst with a reactor and a self-draining, at least partially continuously operating centrifuge, to separate the biocatalyst from the aqueous acrylamide solution. 20. Device according to claim 19, characterised in that the centrifuge is an annular gap centrifuge. 21. Device according to any one of claims 18 to 20, characterised in that the clear discharge from the centrifuge is monitored using an optical means. 22. Device according to claim 21, characterised in that a signal is used to control the centrifuge. |
Buffer solution for electroporation and a method comprising the use of the same |
The invention relates to a buffer solution for suspending animal or human cells and for dissolving biologically active molecules in order to introduce said biologically active molecules into the cells using an electric current and to a method for introducing biologically active molecules into animal or human cells using an electric current and a buffer solution. The inventive buffer solution has a buffering capacity of at least 20 mmol*I−1*pH−1 and an ionic strength of at least 200 mmol*I−1 during a change to the pH value from pH 7 to pH 8 and at a temperature of 25° C. The use of a buffer solution of this type in the corresponding method allows biologically active molecules to be introduced into animal and human cells with a high degree of transfection efficiency and at the same time a low cell mortality. Different cell types, in particular dormant and actively dividing cells of low activity, can be successfully transfected in said buffer solution. |
1. A buffer solution for suspending animal or human cells and/or for dissolving biologically active molecules in order to introduce said biologically active molecules into the cells using electric current, the buffer solution (1) having a buffer capacity of at least 20 mmol×1−1pH−1 and an ionic strength of at least 200 mmol×1−1 at a change in the pH from about pH 7 to about pH 8 and at a temperature of 25° C., and (2) comprising a lower concentration of potassium ions (K+) and a higher concentration of sodium ions (Na+), if compared with the cytoplasm of the cells. 2. The buffer solution of claim 1 having a buffer capacity between 22 and 80 mmol×1−1×pH−1. 3. The buffer solution of claim 1 having a buffer capacity between 40 and 70 mmol×1−1×pH−1. 4. The buffer solution according to of claim 1, 2 or 3 having an ionic strength between 200 and 500 mmol×1−1. 5. The buffer solution of claim 1 having an ionic strength between 250 and 400 mmol×1−1. 6. The buffer solution of claim 1 further comprising a concentration of magnesium ions (Mg2+) of at least 10 mmol×1−1 magnesium ions(M2+). 7. The buffer solution of claim 6, wherein the concentration of magnesium ions is about 15 to 20 mmol×1−1 magnesium ions. 8. The buffer solution of claim 1 further comprising magnesium chloride (MgCl2) and/or magnesium sulphate (MgSO4). 9. The buffer solution of claim 1 comprising a concentration of potassium ions (K+) between 2 to 6 mmol×1−1 K+, and a concentration of sodium ions (Na+) between 100 to 150 mmol×1−1 Na+. 10. The buffer solution of claim 1 comprising HEPES and/or Na2HPO4/NaH2PO4 and/or Tris/HCl and/or K2HPO4/KH2PO4. 11. The buffer solution of claim 1 further comprising a compound selected from the group consisting of sodium chloride, sodium succinate, mannitol, glucose, sodium lactobionate and/or peptides. 12. The buffer solution of claim 1 comprising 4-6 mM KCl, 10-20 mM MgCl2 and 120-160 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2). 13. The buffer solution of claim 1 comprising 4-6 mM KCl, 10-20 mM MgCl2, 5-25 mM HEPES and 120-160 mM Na2HPO4NaH2PO4 (at a pH of about at least 7.2). 14. The buffer solution of claims 1 comprising 4-6 mM KCl, 10-20 mM MgCl2, 50-160 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2) and 5-100 mM sodium lactobionate or 5-100 mM mannitol or 5-100 mM sodium succinate or 5-100 mM sodium chloride. 15. The buffer solution of claim 1 comprising 4-6 mM KCl, 10-20 mM MgCl2, 5-25 mM HEPES, 50-160 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2) and 5-100 mM sodium lactobionate or 5-100 mM mannitol or 5-100 mM sodium succinate or 5-100 mM sodium chloride. 16. The buffer solution of claim 1 comprising 4-6 mM KCl, 10-20 mM MgCl2, 80-100 mM NaCl, 8-12 mM glucose, 0.3-0.5 mM Ca(NO3)2, 20-25 mM HEPES and 50-100 mM tris/HCl or 30-50 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2). 17. The buffer solution of claim 1 comprising 0.1-3.0 mM MgCl2, 50-200 mM K2HPO4/KH2PO4 (at a pH of about at least 6.5) and/or 1-50 mM mannitol and/or 1-50 mM sodium succinate. 18. A method for introducing biologically active molecules into animal or human cells by means of electric current, comprising: i. suspending the cells and dissolving the biologically active molecules in a buffer solution which has a buffer capacity of at least 20 mmol×1−1×pH−1 and an ionic strength of at least 200 mmol×1−1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C. to form a suspension, and ii. applying an electric voltage to the suspension so as to introduce said biologically active molecules into animal or human cells. 19. The method of claim 18, wherein the buffer solution has a buffer capacity between 22 and 80 mmol×1−1×pH−1. 20. The method of claim 18, wherein the buffer solution has a buffer capacity between 40 and 70 mmol×1−1×pH−1. 21. The method of claim 18, wherein the buffer solution has an ionic strength between 200 and 500 mmol×1−1. 22. The method of claim 18, wherein the buffer solution has an ionic strength between 250 and 400 mmol×1−1. 23. The method of claim 18, wherein the buffer solution comprises at least 10 mmol×1−1 magnesium ions (Mg2+). 24. The method of claim 23, wherein the buffer solution comprises 15 to 20 mmol×1−1 magnesium ions. 25. The method of claim 18, wherein the buffer solution comprises magnesium chloride (MgCl2) and/or magnesium sulphate (MgSO4). 26. The method of claims 18, wherein the buffer solution comprises a lower concentration of potassium ions (K+), preferably 2 to 6 mmol×1−1 K+, and a higher concentration of sodium ions (Na+), preferably 100 to 150 mmol×1−1 Na+, if compared with the cytoplasm of the cells. 27. The method of claim 1, wherein the buffer solution contains comprises HEPES and/or Na2HPO4/NaH2PO4 and/or Tris/HCl and/or K2HPO KH2PO4. 28. The method of claim 18, wherein the buffer solution comprises a compound selected from the group consisting of sodium chloride, sodium succinate, mannitol, glucose, sodium lactobionate and/or peptides. 29. The method of claim 18, wherein the buffer solution comprises 4-6 mM KCl, 10-20 mM MgCl2 and 120-160 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2). 30. The method of claim 18, wherein the buffer solution comprises 4-6 mM KCl, 10-20 mM MgCl2, 5-25 mM HEPES and 120-160 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2). 31. The method of claim 18, wherein the buffer solution comprises 4-6 mM KCl, 10-20 mM MgCl2, 50-160 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2) and 5-100 mM sodium lactobionate or 5-100 mM mannitol or 5-100 mM sodium succinate or 5-100 mM sodium chloride. 32. The method of claim 18, wherein the buffer solution comprises 4-6 mM KCl, 10-20 mM MgCl2, 5-25 mM HEPES, 50-160 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2) and 5-100 mM sodium lactobionate or 5-100 mM mannitol or 5-100 mM sodium succinate or 5-100 mM sodium chloride. 33. The method according to any one of claims 18 to 28, wherein the buffer solution contains comprises 4-6 mM KCl, 10-20 mM MgCl2, 80-100 mM NaCl, 8-12 mM glucose, 0.3-0.5 mM Ca(NO3)2, 20-25 mM HEPES and 50-100 mM tris/HCl or 30-50 mM Na2HPO4/NaH2PO4 (at a pH of about at least 7.2). 34. The method of claim 18, wherein the buffer solution comprises 0.1-3.0 mM MgCl2, 50-200 mM K2HPO4/KH2PO4 (at a pH of about at least 6.5) and/or 1-50 mM mannitol and/or 1-50 mM sodium succinate. 35. The method of claim 18, wherein the introduction of the biologically active molecules into the cells is achieved by a voltage pulse having a field strength of up to 2 to 10 kV×cm−1 and a duration of 10 to 200 μs and a current density of at least 2 A×cm−2. 36. The method of claim 35, wherein the introduction of the biologically active molecules into the cells is achieved by a current flow following said high-voltage pulse without interruption, having a 2-2 current density of 2 to 14 A×cm−2, preferably up to 5 A×cm−2, and a duration of 1 to 100 ms, preferably up to 50 ms. 37. The method of claim 35, wherein the biologically active molecules are transfected into the cell nucleus of animal or human cells by means of electric current. 38. The method of claim 18, wherein nucleic acids, proteins or peptides are introduced into quiescent or dividing animal or human cells. 39. The method of claim 18, wherein nucleic acids, proteins or peptides are introduced into primary animal or human cells. 40. The method of claim 38, wherein the nucleic acids are present in complexes or in compounds with peptides, proteins or other biologically active molecules. 41. The method of claim 18, wherein the cells are selected from the group consisting of primary human blood cells, pluripotent precursor cells of human blood, primary human fibroblasts and endothelial cells, muscle cells and melanocytes. 42-43. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>The introduction of biologically active molecules, such as for example, DNA, RNA or proteins, into living cells is an important instrument for studying biological functions of these molecules. A preferred method for introducing foreign molecules into the cells is electroporation which, unlike other methods, only causes slight permanent changes to the biological structure of the target cell by the transfer reagents. During electroporation the foreign molecules are introduced into the cells from an aqueous solution by a brief current flow wherein the cell membrane is made permeable for the foreign molecules by the action of short electrical pulses. As a result of the “pores” briefly formed in the cell membrane, the biologically active molecules initially enter the cytoplasm in which they can already exert their function to be studied if necessary. In cases where DNA is introduced into eukaryotic cells, this must enter the cell nucleus however, so that it is possible for the genetic information to be expressed. In the case of dividing cells, this can take place during the cell division wherein the DNA passively enters the cell nucleus after the temporary dissolution of the nuclear membrane. In studies of quiescent or weakly dividing cells, for example, primary animal cells, however, the DNA does not enter into the cell nucleus in this way so that corresponding methods cannot be used here or at least are very tedious. Moreover, especially when DNA is introduced into animal cells, so-called transfection, particular problems frequently occur as a result of the lability of the cells, since the survival rate of the cells influences the efficiency of the transfection as an important parameter. |
Apparatus for performing assess-press-pull massaging |
The invention concerns an apparatus for massaging the human body, for use in particular in performing an assess/press/pull type massage, comprising a working head (1) open in its lower part and containing means for forming a skin fold. The invention is characterised in that the means forming a skin fold comprise two pinching skin-pinching jaws (11, 12) pivoting about an axis (13) parallel to the plane of the skin and at the ends of which are fixed massaging fingers (11b, 12b), said apparatus comprising means for pivoting the two jaws (11, 12) towards each other and inversely. Additionally, the means forming a skin fold advantageously comprise a suction cup (19) mounted mobile between the two jaws (11, 12), connected to a vacuum source by a flexible conduit (27) and means (22, 24, 25) for moving the suction cup (19) vertically. |
1. Massage equipment for the human body, which can be used in particular for performing an assess press-pull type massage, comprising a working head (1) open in its lower part, capable of being moved on the skin, and containing means for forming a fold of skin, characterised in that the means for forming a fold of skin comprise two jaws for gripping the skin (11, 12) pivoting about an axis (13) parallel to the plane of the skin, carrying at their ends massage fingers (11b,12b) and means to make the two jaws (11, 12) pivot towards one another and inversely, in order to respectively grip and release the skin between them to form a fold of skin. 2. The equipment as claimed in claim 1 characterised in that a suction cup (19) mounted mobile between the two jaws (11,12), connected to the depression source by a flexible tube (27) and means (22, 24, 25) for moving the suction cup (19) into a lower end position in which it is located substantially in the plan of lower opening of the working head (1) and in contact with the skin, and an upper end position so as to be able to draw the zone of the skin upwards which is located between the massage fingers (11b, 12b) of the two jaws (11,12) 3. The equipment as claimed in claim 2 characterised in that the suction cup 19 is attracted to its lower end position by a spring (24) and to its upper end position by a separation traction cable (25). 4. The equipment as claimed in claim 3 characterised in that the suction cup (19) is mounted to pivot on a first end, located to one side of the pivot axis 13 of the two jaws (11, 12), a rocker bar (22), in turn swivel-mounted, at its second end, on a part of one of the jaws (12), about an axis (23) parallel to the pivot axis (13) and located on the other side of this axis (13). 5. The equipment as claimed in claim 3 characterised in that the separation traction cable (25) is housed in a sheath on which is interposed a visual display (26) showing the cable to indicate the axial position of the cable in its sheath and consequently the effective position of the suction cup (19). 6. The equipment as claimed in claim 1 characterised in that the two jaws (11, 12) are attached to locking traction cables (17) and release cables (18) respectively controlling the approach and spread of the jaws (11, 12) and their massage fingers. 7. The equipment as claimed in claim 1 characterised in that each of the jaws (11,12) comprises a lower arm (11a,12a) on which is fixed, by means of a button (14), a massage finger, which is interchangeable, and whereof the shape is adapted to the zone to be treated. 8. The equipment as claimed in claim 7 characterised in that each massage finger (11b,12b) comprises an external covering material (16) selected so as to adapt the adherence and slide of each massage finger to the quality of the skin and to the treatment to be carried out, with the aim of obtaining optimal comfort and efficacy of massage. 9. The equipment as claimed in claim 1 characterised in that it comprises an independent control unit (2) comprising coilers (7, 28, 29) mechanical cables (6, 17, 18, 21), a power source (32) to drive the coilers (7, 28, 29), a vacuum pump (31) and an electric control circuit (35), a supple sheath (3) connecting the control unit (2) to the working head (1) and containing the mechanical cables (6, 17, 18, 21), the tube (25) connecting the vacuum pump (31) to the suction cup (19) and electric conductors, a first cable (21) connected to a first coiler (29) being connected to the suction cup (19) to cause the movement of the suction cup (19) upwards, a second mechanical cable (17) and a third mechanical cable (18) being connected to the two jaws (11,12) to control the pivot movement of these jaws and to a second coiler (28). 10. The equipment as claimed in claim 9 characterised in that it comprises a bracket (4) attached to its upper end, located above the work table, at one end of the supple sheath (3), the bracket (4) being constituted by a tube through which extend the tube (27), the mechanical cables (17,18,21) and the electric conductors. 11. The equipment as claimed in claim 10 characterised in that the control unit (2) contains a third coiler (7) for a mechanical lifting cable (6) passing through the bracket (4) and on a return element (5), such as a pulley, located at the upper end of the bracket (4), said lifting cable (6) being attached to the upper part of the working head (1) in a point (8). 12. The equipment as claimed in claim 9 characterised in that the working head (1) carries control and adjustment buttons (33) and control indicators (36) which are connected by electrical conductors passing through the supple sheath (3), to components of the control unit (2). 13. The equipment as claimed in claim 1 characterised in that it comprises force sensors for measuring the force exerted between the jaws (11) and (12). 14. The equipment as claimed in claim 9 characterised in that the control unit 2 or the working head 1 comprises an automated component for coordinating les movements of the jaws 11 and 12 and of the suction cup 19. |
Hydrogen generation apparatus and method for using same |
A compact hydrogen generator for use with fuel cells and other applications includes a hydrogen membrane reactor having a combustion chamber and a reaction chamber. The two chambers are have a fluid connection and a heat exchange relationship with one another. The hydrogen generation apparatus also includes a fuel supply, a fuel supply line for transporting fuel from the fuel supply to the reaction chamber, an oxygen supply, an oxygen supply line for transporting oxygen form the oxygen supply to the combustion chamber, as well as a tail gas supply line for transporting tail gas supply line for transporting tail gases form the reaction chamber, a combustion by-product line for transporting combustion by-products for the combustion chamber, and a reaction product line for transporting hydrogen from the reaction chamber. |
1. A hydrogen generator comprising: a hydrogen membrane reactor having a combustion chamber in a fluid connection with and a heat exchange relationship with a reaction chamber, a fuel supply; a fuel supply line for transporting fuel from the fuel supply to the reaction chamber; an oxygen supply; an oxygen supply line for transporting oxygen from the oxygen supply to the combustion chamber; a tail gas supply line for transporting tail gases from the reaction chamber; a combustion by-product line for transporting combustion by-products from the combustion chamber; and a reaction product line for transporting hydrogen from the reaction chamber. 2. A hydrogen generator in accordance with claim 1 wherein the hydrogen membrane reactor comprises a top plate, a bottom plate, and a separation plate having first and second opposing surfaces, the top plate and the first surface of the separation plate together defining the reaction chamber, the bottom plate and the second surface of the separation plate together defining the combustion chamber, a hydrogen separation membrane having first and second opposing surfaces disposed between the top plate and the separation plate, the top plate and the first surface of the hydrogen separation membrane together defining a hydrogen exhaust zone and the separation plate and the second surface of the hydrogen separation membrane together defining a reaction zone and wherein the fuel supply line is for transporting fuel to the reaction zone, the tail gas supply line is for transporting tail gas from the reaction zone, and the reaction product line is for transporting hydrogen from the hydrogen exhaust zone. 3. A hydrogen generator in accordance with claim 2 wherein the combustion chamber comprises a plurality of combustion channels extending radially from the surface of the separation plate, the combustion channels creating a fluid path through the combustion chamber; the hydrogen exhaust zone comprises a plurality of hydrogen exhaust channels extending radially from the first surface of the hydrogen membrane, creating a fluid path through the hydrogen exhaust zone; and the reaction zone comprises a plurality of reaction channels extending radially from the second surface of the hydrogen membrane, creating a fluid path through the reaction zone. 4. A hydrogen generator in accordance with claim 3 wherein the height and width of each of the combustion channels, the hydrogen exhaust channels, and the reaction channels is between 0.01 mm and 10 mm. 5. A hydrogen generator in accordance with claim 3 wherein the height and width of each of the combustion channels, the hydrogen exhaust channels, and the reaction channels is between 0.5 mm and 5 mm. 6. A hydrogen generator in accordance with claim 2 wherein the tail gas supply line makes a fluid connection between the reaction zone and the combustion chamber. 7. A hydrogen generator in accordance with claim 2 wherein the tail gas supply line makes a fluid connection between the reaction zone and the oxygen supply line. 8. A hydrogen generator in accordance with claim 1 further comprising a fuel heat exchanger operably connected to the fuel supply line and one of the combustion by-product line or the reaction product line. 9. A hydrogen generator in accordance with claim 8 wherein the fuel heat exchanger is a counterflow-type heat exchanger. 10. A hydrogen generator in accordance with claim 9 wherein the fuel heat exchanger is a stacked-plate-type heat exchanger having channels with a height and a width between about 0.01 mm and 10 mm running between the stacked plates. 11. A hydrogen generator in accordance with claim 1 further comprising an oxygen heat exchanger operably connected to the oxygen supply line and one of the combustion byproduct line or the reaction product supply line. 12. A hydrogen generator in accordance with claim 11 wherein the oxygen heat exchanger is a counterflow-type heat exchanger. 13. A hydrogen generator in accordance with claim 12 wherein the oxygen heat exchanger is a stacked-plate-type heat exchanger having channels with a height and a width between about 0.01 mm and 10 mm running between the stacked plates. 14. A hydrogen generator in accordance with claim 1 further comprising a fuel heat exchanger operably connected to the fuel supply line and the combustion by-product line and an oxygen heat exchanger operably connected to the oxygen supply line and the reaction product supply line. 15. A hydrogen generator in accordance with claim 1 further comprising a hydrogen reservoir in fluid connection with the reaction product supply line. 16. A hydrogen generator in accordance with claim 1 further comprising a hydrogen fuel cell in fluid connection with the reaction product supply line. 17. A hydrogen generator in accordance with claim 1 wherein the fuel supply is an ammonia supply. 18. A hydrogen generator in accordance with claim 1 further comprising a combustion catalyst in the combustion chamber. 19. A hydrogen generator in accordance with claim 2 further comprising a combustion catalyst packed in or coated on the internal surfaces of the combustion channels. 20. A hydrogen generator in accordance with claim 1 further comprising a reaction catalyst in the reaction chamber. 21. A hydrogen generator in accordance with claim 20 further comprising a reaction catalyst packed in or coated on the internal surfaces of the reaction channels. 22. A hydrogen generator in accordance with claim 17 further comprising an ammonia adsorbent supply in fluid communication with the reaction product line. 23. A hydrogen generator in accordance with claim 1 wherein the fuel supply is a hydrocarbon supply. 24. A hydrogen generator in accordance with claim 23 wherein the hydrocarbon supply is methanol, propane, butane, or kerosene supply. 25. A hydrogen membrane reactor comprising a top plate, a bottom plate, and a separation plate having first and second opposing surfaces, the top plate and the first surface of the separation plate together defining the reaction chamber, the bottom plate and the second surface of the separation plate together defining the combustion chamber, the combustion chamber having a plurality of combustion channels extending radially from the surface of the separation plate, the combustion channels creating a fluid path through the combustion chamber, a hydrogen separation membrane having first and second opposing surfaces disposed between the top plate and the separation plate, the top plate and the first surface of the hydrogen separation membrane together defining a hydrogen exhaust zone, the hydrogen exhaust zone having a plurality of hydrogen exhaust channels extending radially from the first surface of the hydrogen membrane, the hydrogen exhaust channels creating a fluid path through the hydrogen exhaust zone, and the separation plate and the second surface of the hydrogen separation membrane together defining a reaction zone; the reaction zone having a plurality of reaction channels extending radially from the second surface of the hydrogen membrane, the reaction channels creating a fluid path through the reaction zone; a fuel inlet into the reaction zone; an oxygen inlet into the combustion chamber, a tail gas outlet out of the reaction zone; a hydrogen outlet out of the hydrogen exhaust zone; and a by-product outlet out of the combustion chamber. 26. A hydrogen membrane reactor in accordance with claim 25 wherein the height and width of each of the combustion channels, the hydrogen exhaust channels, and the reaction channels is between 0.01 mm and 10 mm. 27. A hydrogen membrane reactor in accordance with claim 26 wherein the height and width of each of the combustion channels, the hydrogen exhaust channels, and the reaction channels is between 0.5 mm and 5 mm. 28. A hydrogen membrane reactor in accordance with claim 25 further comprising a combustion catalyst packed in or coated on the internal surfaces of the combustion channels. 29. A hydrogen membrane reactor in accordance with claim 25 further comprising a reaction catalyst packed in or coated on the internal surfaces of the reaction channels. 30. A method for generating hydrogen comprising the steps of: a. flowing a hydrogen-producing fuel through a reaction zone and into a combustion chamber of a hydrogen membrane reactor, the combustion chamber in fluid connection with and in a heat exchange relationship with a reaction chamber, the reaction chamber including the reaction zone and containing a reaction catalyst initially at a temperature less than the reaction catalyst's light-off temperature and the reaction zone separated from a hydrogen exhaust zone by a hydrogen separation membrane; combusting the hydrogen-producing fuel to produce combustion by-products while raising the temperature of the reaction catalyst in the reaction zone and exhausting the by-products b. continuing step a for a period of time sufficient to raise the temperature of the reaction catalyst to above its light off temperature; c. flowing additional hydrogen-producing fuel into the reaction chamber, d. reacting the additional hydrogen-producing fuel to produce hydrogen and tail gases; and separating the hydrogen from the tail gases by selectively passing the hydrogen through the hydrogen membrane. 31. The method in accordance with claim 30 wherein the combustion chamber contains a combustion catalyst having a light-off temperature. 32. The method in accordance with claim 30 wherein the reaction catalyst has a light off temperature of less than 650° C. 33. The method in accordance with claim 31 wherein the combustion catalyst and the reaction catalyst each have a light off temperature of less than 650° C. 34. The method in accordance with claim 30 further comprising recirculating the tail gas from the reaction zone into the combustion chamber. 35. The method in accordance with claim 30 further comprising preheating the hydrogen-producing fuel prior to flowing the hydrogen producing fuel into the reaction zone. 36. The method in accordance with claim 30 further comprising flowing oxygen into the combustion chamber. 37. The method in accordance with claim 25 further comprising flowing preheated oxygen into the combustion chamber. 38. The method in accordance with claim 36 further comprising flowing preheated oxygen into the combustion chamber. 39. The method in accordance with claim 30 further comprising flowing the separated hydrogen into a hydrogen reservoir. 40. The method in accordance with claim 30 further comprising flowing the separated hydrogen into a hydrogen fuel cell. 41. The method in accordance with claim 30 wherein the hydrogen-producing fluid is ammonia. 42. The method in accordance with claim 41 further comprising flowing the separated hydrogen through an ammonia adsorbent. 43. The method in accordance with claim 42 wherein the reaction catalyst is a ruthenium catalyst, a nickel catalyst, an iron oxide catalyst, a rhodium catalyst, an iridium catalyst or a rhenium catalyst. 44. The method in accordance with claim 31 wherein the combustion catalyst is a platinum-rhodium catalyst. 45. The method in accordance with claim 30 wherein the hydrogen-producing fluid is a hydrocarbon. 46. The method in accordance with claim 30 wherein the hydrocarbon is methanol, propane, butane or kerosene. 47. A hydrogen generator comprising: a hydrogen membrane reactor including a combustion chamber having an inlet and an outlet in a heat exchange relationship with a reaction chamber having an inlet and an outlet; a fuel supply; a fuel supply line fluidly connecting the fuel supply to the reaction chamber inlet, an oxygen supply; an oxygen supply line fluidly connecting the oxygen supply to the combustion chamber inlet; a tail gas supply line making a fluid connection with the reaction chamber outlet; a combustion by-product line making a fluid connection with for transporting combustion by-product from the combustion chamber; and a reaction product line for transporting hydrogen from the reaction chamber. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention generally relates to the chemical arts. More particularly, the present invention relates to an apparatus and method for generating hydrogen gas by decomposing or reforming a liquid fuel. 2. General Background and State of the Art The growing popularity of portable electronic devices has produced an increased demand for compact and correspondingly portable electrical power sources to energize these devices. Developments in robotics and other emerging technology applications are further increasing the demand for small, independent power sources. At present, storage or rechargeable batteries are typically used to provide independent electrical power sources for portable devices. However, the amount of energy that can be stored in storage or rechargeable batteries is insufficient to meet the need of certain applications. Hydrogen/air fuel cells (H/AFCs) have enormous potential as a replacement for batteries. Because they can operate on very energy-dense fuels, fuel cell-based power supplies offer high energy-to-weight ratios compared with even state-of-the-art batteries. Fuel cells are of particular interest to the military, where significant efforts are being made to reduce the weight of power supplies that soldiers must carry to support high-tech, field-portable equipment. There is also considerable potential for utilizing fuel cell-based power supplies for commercial applications, particularly where small size and low weight are desirable. A common H/AFC is a polymer electrolyte membrane (PEM) fuel cell. PEM fuel cells are constructed of an anode and a cathode separated by a polymer electrolyte membrane. Functionally, fuel cells generate electricity by reacting hydrogen with oxygen to produce water. Since oxygen can typically be obtained from the ambient atmosphere, only a source of hydrogen must be provided to operate a fuel cell. Merely providing compressed hydrogen is not always a viable option, because of the substantial volume that even a highly compressed gas occupies. Liquid hydrogen, which occupies less volume, is a cryogenic liquid, and a significant amount of energy is required to maintain the extremely low temperatures required to maintain it as a liquid. Furthermore, there are safety issues involved with the handling and storage of hydrogen in the compressed gas form or in the liquid form. Several alternative approaches are available. These alternatives include ammonia decomposition and hydrocarbon reformation. Ammonia decomposition is relatively easy. Ammonia can be thermo-catalytically cracked at relatively low temperatures to produce a gas mixture that is 75% hydrogen by volume. Hydrocarbon fuels are somewhat more technically challenging, because hydrocarbon reformation requires relatively higher temperatures, and the simple cracking of hydrocarbons produces a solid residue which is undesirable in a fuel cell application. However, the reformation of hydrocarbon fuels offers the incentive of enabling a higher energy density fuel to be used, as compared with the use of ammonia as a fuel source, i.e., the production of a greater mass of hydrogen per unit mass of fuel. Consequently, there is a desideratum for an apparatus that has the flexibility to effectively and efficiently generate hydrogen from either ammonia or hydrocarbon fuel. The ammonia decomposition reaction can be represented as follows: in-line-formulae description="In-line Formulae" end="lead"? 2NH 3 →N 2 +2H 2 (1) in-line-formulae description="In-line Formulae" end="tail"? The simple hydrocarbon cracking reaction can be represented as follows: in-line-formulae description="In-line Formulae" end="lead"? C n H(2 n +2)→C n(solid) +(n+1)H 2 (2) in-line-formulae description="In-line Formulae" end="tail"? The formation of solid residues can be avoided through the use of oxidative cracking processes or by employing steam reforming. Oxidative cracking be represented as follows: in-line-formulae description="In-line Formulae" end="lead"? C n H(2 n +2)O 2 → n CO 2 +(n+1)H 2 (3) in-line-formulae description="In-line Formulae" end="tail"? Steam reforming can be represented as follows: in-line-formulae description="In-line Formulae" end="lead"? C n H(2 n +2)2 n H 2 O→ n CO 2 +(3 n +1)H 2 (4) in-line-formulae description="In-line Formulae" end="tail"? It is a drawback of ammonia decomposition that traces of un-reacted ammonia (typically <2000 ppm) remain in the product gas stream. One of the challenges of utilizing ammonia to produce hydrogen for a fuel cell is that H/AFCs do not tolerate ammonia in the hydrogen feed gas, so the trace amounts of ammonia in the hydrogen produced by an ammonia cracker must be removed before the remaining H 2 /N 2 mixture is supplied to a fuel cell. It is a drawback of hydrocarbon reformulation that the actual product is a mixed gas stream that contains substantial amounts of carbon monoxide (CO). Furthermore, the product is a gas stream that also contains partially oxidized hydrocarbons. Both carbon dioxide and partially oxidized hydrocarbons can poison the anode electro-catalysts used in PEM fuel cells. Thus, utilizing either ammonia decomposition, oxidative cracking or steam reforming requires additional steps to purify the hydrogen, or decompose the impurities. Such additional processes add size, cost, and complexity to a hydrogen generation system, making achieving a compact, low cost, and portable system more difficult. Therefore, it is also a desideratum to provide a hydrogen generation system that can be used to provide hydrogen to a fuel cell, which requires minimal or no additional processing to purify the hydrogen that is produced before such hydrogen can be used in a fuel cell. To compete with battery-based power supplies, such an H/AFC apparatus needs to be compact and reliable. It is a further desideratum to develop a portable hydrogen supply with a volume less than I liter and a mass less than 1 kg that can produces up to 50 watts of electrical power, with a total energy output of I kWh. Commercially available metal hydride storage cylinders are available in 920 gm cylinders that contain the equivalent of 100 W-h of hydrogen; thus, a total energy output of 1 kWh represents an order of magnitude increase in energy density over commercially available apparatuses. |
<SOH> SUMMARY OF THE INVENTION <EOH>Now in accordance with this invention there has been found a compact hydrogen generation apparatus for use with fuel cells and other applications. The hydrogen generator includes a hydrogen membrane reactor having a combustion chamber and a reaction chamber. The two chambers are have a fluid connection and a heat exchange relationship with one another. The hydrogen membrane reactor also includes a fuel inlet into the reaction zone, an oxygen inlet into the combustion chamber, a tail gas outlet out of the reaction zone, a hydrogen outlet out of the hydrogen exhaust zone, and a by-product outlet out of the combustion chamber. The hydrogen generation apparatus also includes a fuel supply, a fuel supply line for transporting fuel from the fuel supply to the reaction chamber, an oxygen supply, an oxygen supply line for transporting oxygen from the oxygen supply to the combustion chamber, as well as a tail gas supply line for transporting tail gases from the reaction chamber, a combustion byproduct line for transporting combustion by-products from the combustion chamber, arid a reaction product line for transporting hydrogen from the reaction chamber. In some embodiments, the hydrogen membrane reactor is formed of a top plate, a bottom plate, and a separation plate having first and second opposing surfaces. The top plate and the first surface of the separation plate together define the reaction chamber, while the bottom plate and the second surface of the separation plate together define the combustion chamber. A hydrogen separation membrane having first and second opposing surfaces is disposed between the top plate and the separation plate, so that the top plate and the first surface of the hydrogen separation membrane together define a hydrogen exhaust zone, while the separation plate and the second surface of the hydrogen separation membrane together defining a reaction zone. In these embodiments, the fuel supply line transports fuel to the reaction zone, the tail gas supply line transports tail gas from the reaction zone, and the reaction product line transports hydrogen from the hydrogen exhaust zone. And in some embodiments, the combustion chamber has a plurality of combustion channels extending radially from the surface of the separation plate and forming a fluid path through the combustion chamber, the hydrogen exhaust zone has a plurality of hydrogen exhaust channels extending radially from the first surface of the hydrogen membrane and forming a fluid path through the hydrogen exhaust zone, and the reaction zone has a plurality of reaction channels extending radially from the second surface of the hydrogen membrane and forming a fluid path through the reaction zone. The height and width of each of the combustion channels, the hydrogen exhaust channels, and the reaction channels is preferably between 0.01 mm and 10 mm and more preferably between 0.5 mm and 5 mm. In some embodiments, the tail gas supply line makes a direct fluid connection between the reaction zone and the combustion chamber. In other embodiments, the tail gas supply line makes an indirect fluid connection between the reaction zone and the combustion chamber via the oxygen supply line. Some embodiments additionally include a fuel heat exchanger operably connected to the fuel supply line and one of the combustion by-product line or the reaction product line. In preferred embodiments, the fuel heat exchanger is operably connected to the combustion byproduct line. Some embodiments additionally include an oxygen heat exchanger operably connected to the oxygen supply line and one of the combustion by-product line or the reaction product supply line. In preferred embodiments, the oxygen heat exchanger is operably connected to the reaction product line. In preferred embodiments, the fuel heat exchanger and/or the oxygen heat exchanger are counterflow-type heat exchangers. In more preferred embodiments, the fuel heat exchanger and/or the oxygen heat exchanger are stacked-plate-type heat exchangers having channels with a height and a width between about 0.01 mm and 10 mm running between the stacked plates. Some embodiments additionally include a hydrogen reservoir in fluid connection with the reaction product supply line. A hydrogen fuel cell in fluid connection with the reaction product supply line is included in some embodiments. In some embodiments, a combustion catalyst in included the combustion chamber. The combustion catalyst and the reaction catalyst can be packed in or coated on the internal surfaces of the combustion and/or reaction channels, respectively. In some embodiments, the fuel supply is an ammonia supply. These embodiments can additionally include an ammonia adsorbent supply in fluid communication with the reaction product line. In other embodiments, the fuel supply is a hydrocarbon supply Suitable hydrocarbon fuel supplies include methanol, propane, butane, and kerosene fuel supplies. Also in accordance with the invention there has been found a method for generating hydrogen. In a first step a hydrogen-producing fuel is flowed through the reaction zone and into the combustion chamber of the hydrogen membrane reactor. The reaction zone contains a reaction catalyst initially at a temperature less than the reaction catalyst's light-off temperature. In preferred embodiments, the light off temperature of the reaction catalyst is less than 6500 C. Suitable reaction catalysts include ruthenium catalysts, nickel catalysts, iron oxide catalysts, rhodium catalysts, iridium catalysts or rhenium catalysts. The hydrogen-producing fuel is then combusted to produce combustion by-products while raising the temperature of the reaction catalyst in the reaction zone and the combustion by-products are exhausted. Combustion of the hydrogen-producing fuel is continued for a period of time sufficient to raise the temperature of the reaction catalyst to above its light off temperature. Additional hydrogen-producing fuel is flowed into the reaction chamber and reacted to produce hydrogen and tail gases. The hydrogen is then separated from the tail gases by selectively passing the hydrogen through the hydrogen membrane. In some embodiments, the combustion chamber contains a combustion catalyst having a light-off temperature. In preferred embodiments, the combustion catalyst also has a light off temperature of less than 650° C. Suitable combustion catalysts include platinum-rhodium catalysts. In some embodiments, the tail gas is recirculated from the reaction zone into the combustion chamber. In some embodiments, the hydrogen-producing fuel is pre-heated prior to flowing the hydrogen producing fuel into the reaction zone. And some embodiments include flowing oxygen, preferably pre-heated oxygen into the combustion chamber. In some embodiments, the separated hydrogen is flowed into a hydrogen reservoir. In other embodiments the separated hydrogen is flowed into a hydrogen fuel cell. In some embodiments, the hydrogen-producing fluid is ammonia. And is some of these embodiments, the separated hydrogen is flowed through an ammonia adsorbent. In other embodiments, the hydrogen-producing fluid is a hydrocarbon. Preferred hydrocarbons include methanol, propane, butane, and kerosene. |
Method and a device for transferring a leader in a plant for drying of a web-formed material |
For the purpose of transferring a leader of a web-formed material (1) from a first treatment step to a subsequent second treatment step, the web-formed material (1) is divided by forming a strip (1a) in one edge portion of the web-formed material. The strip (1a) is passed in between two sharp edges (5a,6a) located close to each other, such that the two sharp edges (5a,6a) embrace the strip like a pair of shears. Thereafter, one of the sharp edges (6a) is moved towards the other (5a) so that said one edge (6a) passes said other edge (5a), hence cutting off the strip. With the same movement, the leader is given an impulse, in a direction essentially perpendicular to the strip (1a), towards a leader conveyor (7,8) which intercepts the leader and passes it into the second treatment step. |
1. A method for transferring a leader of a web-formed material, preferably a pulp web, from a first treatment step to a subsequent second treatment step, when threading the web-formed material, wherein the web-formed material is divided into a first, narrow part and a second, wide part by forming a strip by means of a longitudinal cut in one edge portion of the web-formed material and by forming a leader by means of a transverse cut across the strip, whereupon the formed leader is led or thrown against a leader conveyor which passes the leader into the second treatment step and pulls it there through, whereupon the width of the first part is successively increased until it corresponds to the whole width of the web-formed material, wherein the strip is passed in between two essentially straight, sharp edges, located close to each other, and/or that two essentially straight, sharp edges, located close to each other, are moved so as to embrace the strip like a fork or a pair of shears, one of the sharp edges is moved towards the other in a shears-like movement so that one edge passes the other, hence cutting of the strip and creating a leader, with the same movement, the leader is given an impulse, in a direction essentially perpendicular to the strip, towards a leader conveyor, and that the leader is intercepted, with the leader conveyor, and passed into the second treatment step. 2. A method according to claim 1, wherein one of the sharp edges is mounted on a first knife and the other sharp edge is mounted on a second knife, so arranged that the knives cross each other when the shears-like movement is started, preferably so as to make contact with each other when crossing each other. 3. A method according to claim 2, wherein the first knife is held fixed during the shears-like movement. 4. A method according to claim 2, wherein a translatory movement is imparted to the second knife, relative to the first knife, during the shears-like movement. 5. A method according to claim 2, wherein a rotary movement is imparted to the second knife, relative to the first knife, during the shears-like movement. 6. A method according to claim 1, wherein the direction of movement of the strip between the two knives is essentially vertical and downwardly-directed. 7. A method according to claim 4, wherein the second knife is moved above the first knife during the shears-like movement. 8. A method according to claims 5, wherein the second knife is rotated around an essentially vertical axis of rotation. 9. A method according to claim 1, wherein the formed leader is intercepted between two belts. 10. A method according to claim 9, wherein the leader is pulled through the second treatment step with one of the belts which, folded twice, clasps at least part of the strip. 11. A device when threading a web-formed material, preferably a pulp web, for the purpose of transferring a leader of the web-formed material from a first treatment step to a subsequent second treatment step, wherein the web-formed material is divided into a first, narrow part and a second, wide part by forming a strip by means of a longitudinal cut in one edge portion of the web-formed material and by forming a leader by means of a transverse cut across the strip, whereupon the formed leader is led or thrown against a leader conveyor which passes the leader into the second treatment step and pulls it therethrough, whereupon the width of the first part is successively increased until it corresponds to the whole width of the web-formed material, wherein a first knife with an essentially straight, sharp edge, and a second knife with an essentially straight, sharp edge, whereby the two knives are arranged close to each other, means for passing the strip in between the two knives and/or means for moving the two knives so that they embrace the strip like a pair of shears, means for moving one of the knives towards the other in a shears-like movement so that one of the sharp edges passes the other hence cutting off the strip and creating a leader, a leader conveyor adapted to intercept the leader and pass it into the second treatment step, and means to give the leader an impulse, synchronously with the movement of one of the knives in a direction essentially perpendicular to the strip towards the leader conveyor. 12. A device according to claim 11, wherein the sharp edge on the first knife and the sharp edge on the second knife are arranged so as to cross each other when the shears-like movement is started, preferably so as to make contact with each other when crossing each other. 13. A device according to claim 12, further comprising means for holding the first knife fixed during the shears-like movement. 14. A device according to claim 13, further comprising means for imparting a translatory movement to the second knife relative to the first knife during the shears-like movement. 15. A device according to claim 13, further comprising means for imparting a rotary movement to the second knife relative to the first knife during the shears-like movement. 16. A device according to claim 14, wherein the second knife is adapted to move essentially in a horizontal plane during the shears-like movement. 17. A device according to claim 16, wherein the second knife is placed above the first knife during the shears-like movement. 18. A device according to claim 14, further comprising two belts adapted to run while being in direct contact with each other. 19. A device according to claim 18, wherein the two belts run around respective turning rolls and are brought into contact with each other in a nip arranged close to the two knives. 20. A device according to claim 19, characterized in that wherein the nip is placed in essentially the same horizontal plane as the edge of the upper knife |
<SOH> BACKGROUND ART <EOH>In the manufacture of pulp, wet pulp is laid out on a transport wire. The pulp is drained and pressed, at a wet end, into a web-formed material with a dry content of 40-55%. Web-formed materials, such as pulp, are then usually dried, either in a contactless manner by blowing hot air against the web-formed material, or by contact with heated surfaces, primarily cylinders, to a dry content of 90-96%. Since it is difficult to transfer a wide web-formed material between two treatment steps, such as the wet end and the drying plant, a narrow strip, in this application called a leader, is cut at one edge portion of the web-formed material. (A commonly used synonym for “leader” is “tail”.) The leader is then applied to a leader conveyor which threads the web into the subsequent treatment step and pulls the leader therethrough. The leader is formed by two cuts, one longitudinal cut which creates the narrow strip, and one transverse cut, across the narrow strip, which thus creates the actual leader. The width of the web-formed material which is led through the subsequent treatment step is successively increased until finally the whole of the web-formed material is passed therethrough. The procedure must be repeated upon each transition between two treatment steps. The transfer of the leader from a wet end to the leader conveyor in a drying plant has often taken place by an operator manually tearing off an edge strip, created by a longitudinal cut. However, this results in a leader with a rough tip, which may be difficult to apply to a leader conveyor. Further, increased web speeds lead to increased difficulties when performing a manual transfer with the aid of an operator and hence the risks of personal injuries also increase. Thus, a possibility of manual treatment as well as safety reasons sometimes set a limit to the web speed and increase the production costs. From U.S. Pat. No. 4,671,151, a device for forming and deflecting a leader from a paper web is previously known. The paper web is divided by a longitudinal cut into a narrow part and a wide part. The narrow part is lifted by a guide plate towards and past a support. The guide plate and the support are v-shaped and adapted to each other to provide a cutting effect on the paper and form a leader when the guide plate passes the support. The speed of the guide plate must be high since the paper web may not move to a considerable extent during the actual forming of the leader. The proposed device must fulfil two contradictory requirements. On the one hand, the guide plate must pass the support with a very small clearance for a safe cutting, and, on the other hand, the guide plate must under all operating conditions and changes in temperature pass the support without knocking against it. It has been found that, when forming leaders of relatively thick webs with a low strength, such as pulp, it is difficult to attain a satisfactory forming of the leader with a good reproducibility. Other methods are suggested in U.S. Pat. No. 5,413,017, in which the transverse cut is created by two saw-toothed wheels rotating in opposite directions, and U.S. Pat. No. 4,904,344, in which mechanical tearing is utilized. These devices suffer from the same weaknesses as the device suggested in U.S. Pat. No. 4,671,151 when it comes to thick webs with a low strength. There is a great risk that the formed leader is folded or split up and cannot be intercepted by a leader conveyor. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to a method for the purpose of transferring a leader of a web-formed material from a first treatment step to a subsequent second treatment step, when threading the web-formed material, preferably a pulp web. The web-formed material is divided into a first, narrow part and a second, wide part by forming a strip by a longitudinal cut in one edge portion of the web-formed material. A leader is formed by a transverse cut across the strip. The leader is led or thrown against a leader conveyor which passes the leader into the second treatment step. The leader, and hence the first, narrow part of the web-formed material, is pulled through the second treatment step. The width of the first part is increased successively until it corresponds to the whole width of the web-formed material. In the method according to the invention, the strip is passed in between two sharp edges located close to each other, or, inversely, two sharp edges located close to each other are moved so as to embrace the strip like a fork. Thereafter, one of the sharp edges is moved towards the other in a shears-like movement such that one edge passes the other, hence cutting off the strip. With the same movement, the leader is given an impulse, in a direction essentially perpendicular to the strip, towards a leader conveyor which intercepts the leader and passes it into the second treatment step. The present invention also relates to a device for carrying out the above method. The device comprises a first knife with an essentially straight, sharp edge, and a second knife with an essentially straight, sharp edge. The two knives are arranged close to each other. Further, there are means for passing the strip in between the two knives and/or means for moving the two knives such that they embrace the strip like a pair of shears, and means for moving one knife towards the other in a shears-like movement such that one of the sharp edges passes the other, hence cutting off the strip and creating a leader. A leader conveyor is arranged to intercept the leader and pass it into the second treatment step, and a means is arranged to give the leader an impulse, synchronously with the movement of one of the knives, in a direction, essentially perpendicular to the strip, towards the leader conveyor. |
Ballet pointe shoe |
The invention relates to a custom-fitting, asymmetric ballet shoe with a shell (9) and liner (13) that enables the dancer to stand en pointe and perform the extreme movements required by ballet choreography with minimal discomfort, pain, and injury to the foot caused by the various aerial ballet maneuvers called for by both traditional and modern choreography. The shoe of the present invention is designed to redistribute the dancer's weight and the ground force reactions associated with dancing en pointe evenly across the toes while translating some of the force away from the distal aspect of the dancer's toes to the rest of the foot. The shoe has a removable cosmetic cover (18) to enable, inter alia, the color and design to be varied according to costume design and the cover's replacement when the fabric is worn, thereby extending the useful life of the remainder of the shoe. |
1. A ballet shoe for distributing the vertical forces exerted upon the foot of a dancer when the dancer is en pointe in an orthopaedically improved manner comprising: a shell comprising a shank and a toe box, said toe box having an inner surface and an outer surface; and a liner shaped to substantially fill the space between the toes of the dancer and said inner surface of said shell, said liner conforming to the three-dimensional topography of the dancer's toes. 2. The shoe of claim 1 wherein the liner further conforms to the portion of the forefoot of the dancer surmounting the lower portions of the metatarsal bones of the foot. 3. The shoe of claim 1 wherein said three-dimensional topography is attained when the foot is en pointe in an orthopaedically acceptable conformation. 4. The shoe of claim 1 wherein said liner is an orthopaedically acceptable foam. 5. The shoe of claim 1 wherein said liner is a dense closed cell foam. 6. The shoe of claim 5 wherein said foam is pelite. 7. The shoe of claim 1 wherein said shank is disposed to support at least a portion of the plantar surface of the foot. 8. The shoe of claim 7 wherein said shank supports the arch of the foot short of the heel. 9. The shoe of claim 1 wherein said toe box and said shank are integral. 10. The shoe of claim 1 wherein said toe box and said shank are molded. 11. The shoe of claim 1 wherein said toe box and said shank are molded from thermoplastic. 12. The shoe of claim 11 wherein said thermoplastic is polyethylene or polypropylene. 13. The shoe of claim 11 wherein said toe box and said shank form a unitary structure. 14. The shoe of claim 1 wherein said vertical forces are substantially uniformly transmitted to at least the lower portions of the metatarsal bones of the foot. 15. The shoe of claim 1 further comprising a cover for said toe box portion of said shell, said cover being adapted for enveloping the heel of the dancer. 16. A ballet shoe cover comprising: a resilient outsole; a portion adapted for enclosing a toe box of a shell, said shell comprising a toe box and a shank; and a portion enclosing the heel of a dancer. 17. The cover of claim 16 further comprising an inner layer and an outer layer. 18. The cover of claim 17 wherein said inner layer includes an opening for inserting said shank. 19. The cover of claim 16 wherein said toe box further comprises a toe platform, wherein said resilient outsole extends to cover said toe platform. 20. The cover of claim 16 wherein said resilient outsole is leather. 21. The cover of claim 16 further comprising a strap or ribbon. 22. The cover of claim 16 further comprising a lace or drawstring. 23. A method for making a ballet shoe comprising: providing a shell comprising a toe box and a shank, said toe box having an inner surface and an outer surface, said toe box being capable of holding the forefoot of a dancer including at least a portion of the foot surmounting the lower portions of the metatarsal bones thereof; and providing a liner to fit within said toe box of said shell, said liner being foam molded to substantially fill the space between the toes of the dancer and said inner surface of said shell, said liner being shaped to conform to the three-dimensional topography of the dancer's toes and to hold the toes of the dancer in an orthopaedically acceptable conformation. 24. The method of claim 23 wherein said molding is performed upon a negative cast taken of the lower forefoot of the dancer while in a balance sustaining environment effective to attain the three-dimensional topography of the toes when the foot is en pointe in an orthopaedically acceptable conformation. 25. The method of claim 24 wherein said balance sustaining environment is a particulate solid. 26. The method of claim 25 wherein said particulate solid is sand. 27. The method of claim 23 wherein said liner is a dense closed cell foam. 28. The method of claim 27 wherein said foam is pelite. 29. The method of claim 23 further comprising providing a cover for said toe box portion of said shell, wherein said cover is adapted for enveloping the heel of the dancer. 30. The method of claim 29 wherein said cover is replaceable. 31. The method of claim 29 further comprising providing a plurality of said covers. 32. The method of claim 31 wherein said covers vary aesthetically. 33. The method of claim 32 wherein said covers vary in color. 34. The method of claim 29 wherein said cover further comprises a resilient outsole. 35. The method of claim 23 wherein said toe box and said shank form a unitary structure. 36. The method of claim 23 wherein said toe box and said shank are molded from thermoplastic. 37. The method of claim 23 wherein said shell is selected from a standard set of shells. |
<SOH> BACKGROUND OF THE INVENTION <EOH>In ballet, movements include dance steps inspired by running, jumping, leaping and physical exertion by a soloist or an interaction between two or more individuals. The end result is a remarkably punishing regimen of movement being associated with virtually any ballet performance. Not surprisingly, the pursuit of perfection in ballet goes along with a remarkably high incidence of strain and injury. Indeed, the problem is so serious that few dancers are able to practice their profession into middle age. The demands of ballet create significant possibilities of strains and injury to a dancer's feet. While much of a dancer's training is devoted to exercises that strengthen the muscles and tendons of the dancer's feet, there is an ever-present discomfort, pain, and risk of injury inherent in the art of ballet. Almost half the injuries in ballet are to the foot and occur when ballerinas dance on the tip of their toes, that is “en-pointe.” In this position the leg and toes are approximately vertical to the dance surface (i.e., perpendicular to the floor). The dancer's technique or “line” in the pointe position is an imaginary vertical, longitudinal, straight line extending from the tip of the shoe through the center of the toe platform and up the leg. This “line” is considered ideal for balance during ballet dancing. Fatigue of muscles crossing the metatarsal-phalangeal joints is thought to be a causal factor of the foot problems experienced by ballet dancers. Tendonitis of flexor hallucis longus, acute intrinsic muscle spasm, and repetitive muscle strain injuries at midtarsal are common in ballerinas and occur when maneuvering to the pointe position. Fatigue also is thought to be a major factor in fractures to the phalanges, metatarsals, and sesamoid bones as acute fractures usually occur towards the end of a day when the feet of the dancer are very tired. In addition, although the pointe shoe is made with a firm toe box platform at the tip of the shoe providing a firm flat surface on which the dancer balances, serious problems are created by the fact that the toe line of the individual is seldom straight or regular or perpendicular to the ideal vertical line along the dancer's leg. The result is unequal weight distribution across the tips of the dancer's toes. Unequal weight distribution may result in extreme discomfort or pain to the dancer. In many cases permanent injury in the nature of toe contractures known as hammertoes, or other serious toe disfigurement, results from undue stress. The uneven weight distribution and excessive pressure on the weight bearing toes also results in a number of foot health problems such as stress-induced toe buckling or bending, blistering, skin irritation, painful corns, stress fractures, bunions (a bony enlargement of the big toe joint), and ingrown toenails to mention a few. In addition to the aforementioned health problems, the unequal weight distribution may cause the dancer's feet to tilt to one side or “sickle out” through lack of balance, resulting in poor technique or “line” in the pointe position. It is common for a ballet dancer to suffer constant pain, discomfort, and disfiguration of the toes in order to compensate for this natural imbalance of pressure on the toes in the pointe position in an effort to maintain acceptable technique or “line.” The pointe shoes themselves do offer some support but quickly breakdown and lose their beneficial characteristics, often ready to be discarded after one performance. The design and materials of the ballet slipper used by a dancer generally have been unchanged since the original conception of such “pointe shoes.” The traditional blocked ballet slipper is made by hand on a last, using layers of fabrics, cardboard, paper, or leather saturated with glue to form a reinforced toe box joined to a leather or cardboard shank. A reinforcing stiffener frequently is included in the shank. Usually the outer sole is made from leather. An outer fabric or “upper” is sewn to the sole and usually gathered in pleats under the toe. The connection between the toe and sole is not easy to manufacture and can come loose after prolonged use. This type of slipper is labor intensive and expensive to produce, although some improved casting methods have been developed to speed the laminating steps, for example as disclosed in U.S. Pat. No. 4,453,966 to Terlizzi. Moreover, the traditional ballet slipper requires extensive breaking-in before it is comfortable for use. Typically, a ballerina will break in the slipper by manually flexing it, applying force by way of slamming the slipper in a door or bashing it with a hammer, or soaking it in warm water or alcohol. It can take as much as three hours to prepare a single pair of slippers for a performance if they have been manufactured using an epoxy or other durable glue as a laminate. Once the slipper is broken in, it will have an extremely short useful life, usually no more than twenty to forty-five minutes during a performance. The short useful life is attributable to the deterioration of the toe box and or shank caused by the rapid breakdown of the glue used to form the laminates of the toe box. The breakdown can be accelerated by perspiration that occurs during energetic dancing. Once the shank and/or toe box have deteriorated, the slipper is useless because there will be no support for the dancer. A further problem encountered with the traditional ballet slipper is that the outer cover (typically a satin material) is slippery and can contribute to skids and falls when the ballerina is rising to the pointe position. The ballerina usually will darn the toes of the slipper and rub the tips in resin to minimize the chances of slipping. Nevertheless, falls do occur. Ballet shoes have not kept pace with the technical demands of ballet choreography. Ballet dancers' pointe shoes must fit very snugly in order to provide the support required for toe dancing. The stiff toe box that encases the toes must firmly hug the metatarsals to hold the foot in place when the dancer stands in the pointe position. If the shoe is too wide or too loose the foot will slide unrestrained down into the box, causing all the dancer's weight to be focused on the tips of her longest toes, resulting in pain as well as potentially contributing to problems such as arthritis, bunions, hammer toes, calluses, claw toe deformities, stress fractures, and bruised or lost toenails. However, the pointe shoe also must be wide enough at the metatarsal to allow the foot to spread out when landing from jumps and when passing through the position known as “demi-pointe” in transition from normal stance to en pointe. There exists a clear need for the development of footwear adapted to enable dancers to achieve all of the extremes in movement ballet choreography demands. The present invention satisfies such a need. The invention herein disclosed relates to a customized ballet shoe that enables the achievement of such dance steps while eliminating the pain, deformity, and injury associated with such movements by redistributing the dancer's weight and the forces of landing off of the distal aspect of the toes, as well as by absorbing the shock when a dancer is in pointe position, while at the same time improving the dancer's technique. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides ballet shoes comprising a shell and a liner. The shell includes a shank and a toe box. The toe box has an inner surface and an outer surface. The liner is shaped to conform to the three-dimensional topography of the dancer's toes and substantially fill the space between the toes of the dancer and the inner surface of the shell. In preferred embodiments, the shell is molded from thermoplastic and the liner is a dense closed cell foam. In certain embodiments, the ballet shoes of the invention include a cosmetic cover. The shoe of the present invention redistributes the dancer's weight and the ground force reactions associated with dancing en pointe evenly across the toes while translating some of the force away from the distal aspect of the dancer's toes to the rest of the foot. The present invention thus distributes the vertical forces exerted upon the foot of a dancer en pointe in an orthopaedically improved manner. Also provided are methods for manufacturing a ballet shoe of the invention. In one embodiment, these methods include production of a negative cast taken of the lower forefoot of the dancer en pointe while a balance sustaining environment surrounds the dancer's foot. The dancer's downward vertical force is distributed throughout the balance sustaining environment allowing the dancer to position herself en pointe without requiring her to modify her natural toe position. The negative cast is then used to create a liner for the ballet shoes of the invention. Toe disfiguration that the dancer experiences while en pointe with traditional pointe shoes will not be present in a mold created using a balance sustaining environment. The present invention also provides a removable cosmetic cover for the ballet shoe. Thus, a single shell can be used with an assortment of covers and the useful life of the shell of the ballet shoe is extended. The color and design of the cover can be varied according to costume design. |
Apparatus and method for multiple rich media formats video broadcasting |
Apparatus and method for concurrently converting multiple video and audio formats and sources into different formats. The main elements of the system are: a. A Media Format Matrix (MFM) module, having multiple input and output channels. b. A Communicator module, having various network interfaces. c. A Storage module. The implementation of the system is based on standard networking protocols, used to transfer the data between the internal MFM, Communicator and storage elements. The system platform is built around an internal switched network infrastructure. The management architecture provides a scalable infrastructure for controlling and monitoring single or multiple internal and external system modules, and in addition single, multiple or clusters of systems. |
1. A modular, storage-less, real-time scalable video/audio streaming and switching system comprising a media format matrix, said media format matrix comprising: a. at least one star topology packet switching fabric module, for managing network streaming inside said media format matrix; b. one or more controller modules connected with said at least one switching fabric module and with an external management unit, for configuring and controlling said media format matrix; and at least one of a group consisting of: c. one or more encoding modules connected with said switching fabric module, for receiving uncompressed video or audio and converting it into a predefined compressed format; d. one or more decoding modules connected with said switching fabric module, for receiving compressed video or audio and converting it into a predefined uncompressed format; e. one or more compressed stream interface modules connected with said switching fabric module, for transmitting compressed data between said switching fabric and an external media interface; f. one or more transcoding modules connected with said switching fabric module, for receiving compressed streams from said switching fabric, transcoding them into different compressed formats and sending said different compressed stream back to said switching fabric; g. one or more NIC modules connected with said switching fabric module, for connecting between said media format matrix and one or more distribution networks; and h. one or more pre-processing modules connected with said switching fabric module, for processing uncompressed streams. 2. The system of claim 1, additionally comprising a communicator device connected with said media format matrix, for inputting streams from local or wide area distribution networks into the matrix and for outputting streams from the matrix to local or wide area distribution networks. 3. The system of claim 1, wherein said external management unit is operable to communicate to at least one of said one or more controller modules a configuration of said matrix. 4. The system of claim 1, wherein said one or more switching fabric modules use a standard network protocol. 5. The system of claim 4, wherein said standard network protocol is IP over Ethernet. 6. The system of claim 1, wherein said media format matrix is connected with multiple input channels. 7. The system of claim 6, wherein said multiple input channels comprise compressed video/audio and uncompressed video/audio. 8. The system of either of claims 1 or 6, wherein said media format matrix is connected with multiple output channels. 9. The system of claim 8, wherein said multiple output channels comprise compressed video/audio and uncompressed video/audio. 10. The system of claim 1, wherein said media format matrix modules and said external management unit use a common management protocol. 11. The system of claim 10, wherein said common management protocol is SNMP. 12. The system of claim 1, wherein said one or more compressed stream interfaces additionally serve for scrambling and/or descrambling compressed signals. 13. The system of claim 1, comprising more than one switching fabric modules, and wherein each of said switching fabric modules is connected to at least another one of said switching fabric modules, and wherein said switching fabric modules create a cluster of connected switching fabric modules. 14. The system of claim 1, comprising more than one switching fabric modules, and wherein each of said switching fabric modules is connected to all of said media format matrix modules for enabling redundancy of said media format matrix modules. 15. The system of claim 1, wherein said one or more controller modules control external network elements. 16. The system of claim 15, wherein said external network elements comprise one of the group including router and video switcher. 17. The,system of claim 1, wherein said external management unit directly controls at least one of said media format matrix modules. 18. A method of encoding video/audio data streams, comprising the steps of: providing a streaming and switching system comprising a media format matrix, said media format matrix comprising: a. at least one star topology switching fabric module, for managing network streaming inside said media format matrix; b. one or more encoding modules connected with said switching fabric module, for receiving uncompressed video or audio and converting it into a predefined compressed format; c. one or more compressed stream interface modules connected with said switching fabric module, for transmitting compressed data between said switching fabric and an external media interface; d. one or more NIC modules connected with said switching fabric module, for connecting between said media format matrix and one or more distribution networks; and e. one or more controller modules connected with said switching fabric module and with an external management unit, for configuring and controlling said media format matrix; inputting an uncompressed data stream into the system; converting said data stream into a desired compressed format and/or bit rate; and transmitting said compressed data towards one of said switching fabric modules. 19. The method of claim 18, wherein said media format matrix additionally comprises one or more pre-processing modules connected with said switching fabric module; and additionally comprising, after said step of inputting an uncompressed data stream, a step of preprocessing said uncompressed data stream. 20. The method of claim 18, wherein said media format matrix additionally comprises one or more decoding modules; and additionally comprising, after said step of converting, a step of generating an additional copy of said compressed data and directing said additional copy to one of said decoding modules. 21. The method of claim 20, wherein said decoder module decodes said additional copy of said compressed data and wherein said decoded data is compared to said uncompressed data stream input into the system. 22. The method of claim 18, wherein said uncompressed data stream is input via an interface of one of said encoding modules; 23. The method of claim 19, wherein said uncompressed data stream is input via one of said pre-processing modules. 24. The method of claim 18, additionally comprising, after said step of transmitting, the steps of: directing said compressed data from said switching fabric module towards one of said NIC modules; and transmitting said compressed data to a distribution network. 25. The method of claim 18, additionally comprising, after said step of transmitting, the steps of: directing said compressed data from said switching fabric module towards one of said compressed stream interface modules; and transmitting said compressed data out of said media format matrix on a compressed stream interface. 26. A method of decoding video/audio data streams, comprising the steps of: providing a streaming and switching system comprising a media format matrix, said media format matrix comprising: a. at least one star topology switching fabric module, for managing network streaming inside said media format matrix; b. one or more decoding modules connected with said switching fabric module, for receiving compressed video or audio and converting it into a predefined uncompressed format; c. one or more interface means connected with said switching fabric module, for transmitting compressed data between said switching fabric and an external media interface; and d. one or more controller modules connected with said switching fabric module and with an external management unit, for configuring and controlling said media format matrix; inputting a compressed data stream into the system and transmitting it to one of said switching fabric modules; directing said compressed stream from said switching fabric module to one or more of said decoding modules; and decoding said stream to one or more uncompressed format. 27. The method of claim 26, wherein said interface means comprises one or more compressed stream interface modules connected with said switching fabric module. 28. The method of claim 26, wherein said interface means comprises one or more NIC modules connected with said switching fabric module. 29. A method of transcoding video/audio data streams, comprising the steps of: providing a streaming and switching system comprising a media format matrix, said media format matrix comprising: a. at least one star topology switching fabric module, for managing network streaming inside said media format matrix; b. one or more interface means connected with said switching fabric module, for transmitting compressed data between said switching fabric and an external network; c. one or more transcoding modules connected with said switching fabric module, for receiving compressed streams from said switching fabric, transcoding them into different compressed formats and sending said differently compressed stream back to said switching fabric; and d. one or more controller modules connected with said switching fabric module and with an external management unit, for configuring and controlling said media format matrix; inputting a compressed data stream to the system; transmitting said compressed stream to one of said switching fabric modules; directing said compressed stream to one or more said transcoding modules; transcoding said compressed stream to a desired format or bit-rate or resolution; and transmitting said transcoded stream to one said switching fabric modules. 30. The method of claim 29, wherein said interface means comprises one or more compressed stream interface modules. 31. The method of claim 29, wherein said interface means comprises one or more NIC modules. 32. The method of claim 31, additionally comprising the steps of: transmitting said transcoded stream from said switching fabric module to one of said NIC modules; and transmitting said transcoded stream from said NIC module to a network. 33. The method of claim 30, additionally comprising the steps of: transmitting said transcoded stream from said switching fabric module to one of said compressed stream interface modules; and outputting said transcoded stream from said compressed stream interface module. 34. The method of claim 29, wherein said media format matrix additionally comprises one or more decoding modules; and additionally comprising the steps of: transmitting said transcoded stream from said switching fabric module to one of said decoding modules; decoding said transcoded stream; and monitoring said decoded stream. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The plurality of formats, interfaces, media types, access networks and physical connections involved in video/audio streaming makes the design, implementation and control of digital video/audio broadcast systems very complex. The following list describes exemplary common standards used today in broadcast systems: 1. Uncompressed Video/Audio: Analog Composite, Analog S-Video, Digital SDI 2. Video color systems: PAL, NTSC, SECAM 3. Digital Video/Audio compression formats: MPEG1, MPEG2, MPEG4, WMT (Microsoft Windows Media Technology), QT (Apple QuickTime), RN (Real Networks), H26L and other proprietary and non-proprietary formats. 4. Audio interfaces: Analog Balanced, Analog Unbalanced, Digital Balanced and Unbalanced. 5. Compressed Audio/Video transmission protocols (Media interfaces): DVB-ASI, DVB-S, DVB-C, DHEI, SDTI, DV etc. 6. Network interfaces: Ethernet, ATM, IP, SDH etc. 7. Network interface physical layer: Copper twisted pair, Optical cables, Coaxial cable, USB etc. 8. Network layer 4 protocols: UDP, RTP, TCP etc. 9. Storage interfaces: SCSI, IDE, IEEE1394-FireWire etc. 10. Management protocols: SNMP, Telnet, RS232, RTSP, XML etc. Existing video/audio processing systems require multiple and different apparatus, with individual and different control of every element. Some flexibility can be obtained by combining various building blocks (e.g. Encoders, Decoders, Network interfaces, Video interfaces), and connecting them by external wires to a common control unit. However, this practice requires coordination between many equipment vendors, consumes large office space, and hence lacks flexibility and scalability. There is a need for a modular, configurable system for seamless concurrent handling of various video and audio input/output requirements in a scalable, robust manner. |
<SOH> SUMMARY OF THE INVENTION <EOH>The apparatus and method of the present invention allow for multiple video and audio formats and sources to be converted into different formats in a concurrent mode of operation. The main elements of the system of the present invention are: a. A Media Format Matrix (MFM) module, having multiple input and output channels. b. A Communicator module, having various network interfaces. c. A Storage module. The implementation of the system is based on standard networking protocols, used to transfer the data between the internal MFM, Communicator and storage elements. The system platform is built around an internal switched network infrastructure. This internal infrastructure enables the introduction of multiple interfaces and processing modules to the system, thus providing extensibility for future elements that will be required as new technologies emerge. The use of standard network protocols for transmission of data and control inside the system provides flexibility, simplicity and scalability to the system. The internal network topology is designed to eliminate any single point of failure, thus increasing the system's reliability. The management architecture of the present invention provides a scalable infrastructure for controlling and monitoring single or multiple internal and external system modules, and in addition single, multiple or clusters of systems. The system of the present invention can be used for a large variety of video and audio streaming environments and applications, serving a large-scale number of receivers and transmitters. Using the concept of the present invention may serve as a basis for any video/audio streaming system architecture. |
Compositions containing plant seed oil |
The invention relates to palatable compositions comprising a plant seed oil selected from a Ribes fruit, evening primrose or borage plant, a hydrocolloid base, a sweetening agent and an aqueous phase wherein the hydrocolloid base consists of starch and at least one thickening agent or alternatively two or more thickening agents absent any starch. Compositions of the invention have improved sensory characteristics such as improved mouthfeel, taste and odour. |
1. A palatable composition comprising an oil phase, an aqueous phase, a hydrocolloid base and a sweetening agent; wherein the said oil phase contains a plant seed oil from a Ribes fruit, evening primrose or borage plant and the hydrocolloid base consists of starch and at least one thickening agent selected from the group consisting of xanhan gum, guar gum, carageenan, cellulose derivatives, alginic acid and salts thereof. 2. A palatable composition according to claim 1 wherein the Ribes fruit is Ribes nigrum, Ribes rubrum or Ribes ovacrispa. 3. A palatable composition according to claim 1 in the form of an oil-in-water emulsion comprising 0.1% to 30% w/w of the plant seed oil as the oil phase and 75% to 95% w/w of the aqueous phase. 4. A palatable composition according to claim 1 comprising 0.5% to 5% w/w of starch. 5. A palatable composition according to claim 1 comprising 0.5% to 6% w/w of sweetening agent. 6. A palatable composition according to claim 1 wherein the thickening agent is xanthan gum. 7. A palatable composition according to claim 1 comprising a hydrocolloid base consisting essentially of starch and two thickening agents. 8. A palatable composition according to claim 7 wherein a first thickening agent is xanthan gum. 9. A palatable composition according to claim 7 wherein a second thickening agent is selected from the group consisting of guar gum, carageenan, cellulose derivatives, and alginic acid and salts thereof. 10. A palatable composition comprising an oil phase, an aqueous phase, a hydrocolloid base and a sweetening agent; wherein said oil phase contains a plant seed oil from a Ribes fruit, evening primrose or borage plant, and the hydrocolloid base consists of two or more thickening agents. 11. A palatable composition according to claim 10 wherein the hydrocolloid base consists essentially of xanthan gum as a first thickening agent and a second thickening agent selected from guar gum, carageenan, cellulose derivatives, alginic acid and salts thereof. 12. A palatable composition according to claim 11 wherein the cellulose derivative is carboxymethyl cellulose. 13. A composition according to claim 10 in the form of an oil-in-water emulsion comprising 0.1% to 30% w/w of the plant seed oil as the oil phase and 75% to 95% w/w of the aqueous phase. 14. A palatable composition according to claim 10 comprising 0.5% to 5% w/w of starch. 15. A composition according to claim 10 comprising 0.5% to 6% w/w of sweetening agent. 16. A composition according to claim 1 having a viscosity of 6000 to 10000 cps. 17. A composition according to claim 1 further comprising trace elements, minerals and/or vitamins. 18. A composition according to claim 1 further comprising a preservative. 19. A composition according to claim 1 further comprising a taste modifier that is an alkali earth metal salt. 20. A composition according to claim 1 wherein the pH of the aqueous phase is equal to or less than 7. 21. A composition according to claim 20 wherein the pH is 3 to 5. 22. A composition according to claim 20 wherein the taste modifier is an amino acid. 23. A composition according to claim 22 wherein the amino acid is selected from the group consisting of leucine, lysine and aspartic acid. 24. A method of preparing a palatable composition according to claim 1 comprising the steps of: A. dissolving a sweetening agent and any other water soluble-ingredient(s) in water forming an aqueous phase; B. adjusting the pH of the aqueous phase if required; C. dissolving a hydrocolloid base in a plant seed oil to form an oil phase; D. admixing the aqueous phase of step (B) with the oil phase. |
Ceramic multilayer substrate manufacturing method and unfired composite multilayer body |
To produce a green composite laminate 11, a green multilayer collective substrate 13 containing low-temperature sinterable glass ceramic powder as a main ingredient is disposed between first and second shrinkage-restraining layers 14a and 14b containing alumina powder as a main ingredient. Grooves 16 are formed on one main surface 11a of the green composite laminate 11 such as to pass through the first shrinkage-restraining layer 14a and the green multilayer collective substrate 13 and reach the second shrinkage-restraining layer 14b, but not to reach the other main surface 11b of the green composite laminate 11. The green composite laminate 11 provided with the grooves 16 is sintered under conditions where the low-temperature sinterable glass ceramic powder is sintered and the green first and second shrinkage-restraining layers 14a and 14b are removed to prepare a plurality of ceramic multilayer substrates. This manufacturing method offers ceramic multilayer substrates with a high dimensional accuracy and substantially prevents defective divisions when a multilayer collective substrate is split into a plurality of ceramic multilayer substrates. |
1-5. (Cancelled). 6. A method for manufacturing a ceramic multilayer substrate, comprising: providing a green composite laminate comprising a green multilayer collective substrate comprising a laminate of a plurality of ceramic green layers containing ceramic powder as a main ingredient and having upper and lower main surfaces, and first and second shrinkage-restraining layers disposed respectively on the upper and lower main surfaces, the first and second shrinkage-restraining layers containing sintering-resistant powder that is not substantially sintered at a sintering temperature of the ceramic powder as a main ingredient; forming at least one groove extending through the first shrinkage-restraining layer and the green multilayer collective substrate and into but not completely through the second shrinkage-restraining layer; and sintering the green composite laminate with the groove under conditions where the ceramic powder is sintered and the sintering-resistant powder is not substantially sintered. 7. The method for manufacturing a ceramic multilayer substrate according to claim 6, wherein the ceramic green layers contain at least one of glass powder and crystallized glass powder. 8. The method for manufacturing a ceramic multilayer substrate according to claim 7, further comprising compressing the green composite laminate in the lamination direction before providing the groove. 9. The method for manufacturing a ceramic multilayer substrate according to claim 8, wherein the grove extends into about {fraction (1/10)} to {fraction (4/10)} of the thickness of the second shrinkage-restraining layer. 10. The method for manufacturing a ceramic multilayer substrate according to claim 9, wherein a plurality of said grooves are formed. 11. The method for manufacturing a ceramic multilayer substrate according to claim 10, wherein at least one groove is arranged in a longitudinal direction and at least one other groove is arranged in a lateral direction substantially perpendicular to the longitudinal direction. 12. The method for manufacturing a ceramic multilayer substrate according to claim 11, further comprising preparing a plurality of ceramic multilayer substrates by splitting the sintered laminate along the grooves of the sintered multilayer collective substrate. 13. The method for manufacturing a ceramic multilayer substrate according to claim 6, further comprising compressing the green composite laminate in the lamination direction before providing the groove. 14. The method for manufacturing a ceramic multilayer substrate according to claim 6, wherein the grove extends into about {fraction (1/10)} to {fraction (4/10)} of the thickness of the second shrinkage-restraining layer. 15. The method for manufacturing a ceramic multilayer substrate according to claim 6, wherein a plurality of grooves are formed. 16. The method for manufacturing a ceramic multilayer substrate according to claim 14, wherein at least one groove is arranged in a longitudinal direction and at least one other groove is arranged in a lateral direction substantially perpendicular to the longitudinal direction. 17. The method for manufacturing a ceramic multilayer substrate according to claim 6, further comprising forming at least two ceramic multilayer substrates by splitting the sintered multilayer collective substrate along a groove. 18. The method for manufacturing a ceramic multilayer substrate according to claim 6, further comprising manufacturing the green composite laminate. 19. The method for manufacturing a ceramic multilayer substrate according to claim 6, further comprising removing the substantially unsintered first and second shrinkage-restraining layers from the sintered multilayer collective substrate. 20. The method for manufacturing a ceramic multilayer substrate according to claim 19, wherein the ceramic green layers contain at least one of glass powder and crystallized glass powder, and the green composite laminate is compressed in the lamination direction before providing the groove, and wherein the grove is formed to extend into about {fraction (1/10)} to {fraction (4/10)} of the thickness of the second shrinkage-restraining layer. 21. The method for manufacturing a ceramic multilayer substrate according to claim 20, wherein a plurality of grooves are formed and the sintered multilayer collective substrate is split along the grooves into a plurality of ceramic multilayer susbtrate. 22. The method for manufacturing a ceramic multilayer substrate according to claim 21, wherein at least one groove is arranged in a longitudinal direction and at least one other groove is arranged in a lateral direction substantially perpendicular to the longitudinal direction. 23. A green composite laminate comprising: a green multilayer collective substrate having upper and lower main surfaces and comprising a laminate of a plurality of ceramic green layers containing ceramic powder as a main ingredient; first and second shrinkage-restraining layers disposed respectively on the upper and lower main surfaces of the green multilayer collective substrate, the first and second shrinkage-restraining layers containing sintering-resistant powder as a main ingredient, the sintering-resistant powder being not substantially sintered at the sintering temperature of the ceramic powder; and at least one groove extending through the first shrinkage-restraining layer and the green multilayer collective substrate and into but not through the second shrinkage-restraining layer. 24. The green composite laminate according to claim 23, wherein the grove extends into about {fraction (1/10)} to {fraction (4/10)} of the thickness of the second shrinkage-restraining layer. 25. The green laminate according to claim 24, having a plurality of said grooves. |
<SOH> BACKGROUND ART <EOH>In recent years, the performance of electronic components in the electronics field has been greatly enhanced, contributing to high-speed information processing, compact design, and multifunctional design of information processing apparatuses such as mainframes, mobile communicating terminals, and personal computers. A multichip module (MCM), having multiple semiconductor devices such as VLSIs and ULSIs on its ceramic substrate, is one of these electronic components. In such a module, a ceramic multilayer substrate having wiring conductors arranged three-dimensionally is often used in order to increase the packaging density of LSIs and achieve good electric connections between LSIs. A ceramic multilayer substrate is produced by firing a green multilayer collective substrate formed of a plurality of laminated ceramic green sheets. Unfortunately, green multilayer collective substrates, when fired by a conventional technique, shrink in the directions along the main surfaces and across the thickness, resulting in dimensional errors of about 0.4% to 0.6% particularly in the directions along the main surfaces. This may cause outer conductors to suffer from a degraded positional accuracy and inner conductors to be deformed, distorted, or broken. In view of the problems described above, Japanese Unexamined Patent Application Publication No. 4-243978 discloses a method for manufacturing ceramic multilayer substrates, which is described below. First, glass ceramic powder that is sinterable at temperatures of 1000° C. or less and alumina powder that is not sintered at the sintering temperature of this glass ceramic powder are prepared. Then, ceramic green sheets containing the glass ceramic powder are laminated on one another, and the resultant green multilayer substrate is then interposed between shrinkage-restraining layers containing the alumina powder. Thus, a green composite laminate is produced. The composite laminate is then fired under the sintering conditions used for the glass ceramic powder. At this time, the alumina powder contained in the shrinkage-restraining layers is not substantially sintered, and therefore shrinkage does not substantially occur in the shrinkage-restraining layers. The above-mentioned effect causes the shrinkage-restraining layers to restrain the green multilayer substrate, so that the multilayer substrate shrinks across the thickness only; shrinkage in the directions along the main surfaces is suppressed. Removing the shrinkage-restraining layers thereafter by appropriate means allows the ceramic multilayer substrate to be prepared. The ceramic multilayer substrate obtained by the non-shrinking process described above is highly reliable because it has high dimensional accuracy in the directions along the main surfaces, i.e., the lengthwise direction (X direction) and widthwise direction (Y direction) of the ceramic green sheets, and suffers from less camber and torsion. In order to efficiently produce such a ceramic multilayer substrate as described above, a multilayer collective substrate including a plurality of ceramic multilayer substrates is first produced and is then split along predetermined split lines into a plurality of ceramic multilayer substrates. In splitting the multilayer collective substrate as described above, grooves may be formed along respective predetermined split lines on a main surface of the multilayer collective substrate to facilitate the splitting of the multilayer collective substrate. These grooves are normally formed on a main surface of the green multilayer collective substrate using a cutter or a die. When the ceramic multilayer substrate is produced using shrinkage-restraining layers, as described above, grooves are formed as shown in FIG. 3 , which is disclosed in Japanese Unexamined Patent Application Publication No. 7-99263. FIG. 3 is a sectional view showing a part of a green composite laminate 101 which includes a green multilayer collective substrate 102 , a first shrinkage-restraining layer 103 , and a second shrinkage-restraining layer 104 , the green multilayer collective substrate 102 being interposed between the layers 103 and 104 . In FIG. 3 , wiring conductors provided on the multilayer collective substrate 102 are not shown and the dimension across the thickness is enlarged. In FIG. 3 , the green multilayer collective substrate 102 includes a plurality of ceramic green sheets 107 containing glass ceramic powder. The first shrinkage-restraining layer 103 and the second shrinkage-restraining layer 104 are formed of a predetermined number of laminated green sheets 108 containing sintering-resistant powder such as alumina powder which is not sintered at the sintering temperature used for the above-mentioned glass ceramic powder. This green composite laminate 101 is manufactured as follows. First, the ceramic green sheets containing glass ceramic powder are laminated and compressed in the lamination direction, and thereby the green multilayer collective substrate 102 containing a plurality of the ceramic green sheets 107 is produced. Next, grooves 106 are formed on one main surface of the green multilayer collective substrate 102 . Then, the green sheets 108 containing alumina powder are laminated so as to interpose the green multilayer collective substrate 102 , thus the first shrinkage-restraining layer 103 and the second shrinkage-restraining layer 104 are formed. As a result, the green composite laminate 101 is obtained. Subsequently, the green composite laminate 101 is compressed again in the lamination direction, and is then fired at the sintering temperature used for the glass ceramic powder contained in the ceramic green sheets 107 . Then, the first shrinkage-restraining layer 103 and the second shrinkage-restraining layer 104 , which are not substantially sintered in the firing step, are removed to produce the sintered multilayer collective substrate 102 with the grooves 106 . The sintered multilayer collective substrate 102 is split along the grooves 106 to prepare individual ceramic multilayer substrates. Before the grooves 106 are formed, as described above, on the green multilayer collective substrate 102 , a plurality of the laminated ceramic green sheets need to be pre-compressed sufficiently in order to prevent the individual ceramic green sheets 107 from shifting at the time the grooves are formed. However, when the green composite laminate 101 is compressed again, it is difficult to achieve a sufficiently tight bond between the compressed green multilayer collective substrate 102 and the uncompressed first shrinkage-restraining layer 103 and the second shrinkage-restraining layer 104 . As a result, the force by which the first shrinkage-restraining layer 103 and the second shrinkage-restraining layer 104 restrain the ceramic green sheets 107 becomes small, possibly failing to satisfactorily prevent shrinkage of the ceramic green sheets 107 during firing, i.e., shrinkage of the green multilayer collective substrate 102 in the surface directions. Another disadvantage is a low productivity arising from an unusual step of forming the grooves 106 intervening the step of laminating the ceramic green sheets 107 constituting the multilayer collective substrate and the green sheets 108 constituting the shrinkage-restraining layers. In view of the problems described above, an object of the present invention is to provide a method for manufacturing ceramic multilayer substrates with a high dimensional accuracy and a high reliability in a highly productive manner and to provide a green composite laminate obtained at a step in this method. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 illustrates an embodiment according to the present invention, and shows a sectional view of a part of a large-area composite laminate obtained at a step in manufacturing a ceramic multilayer substrate. FIG. 2 is a plan view of an array of split lines in the large-area composite laminate shown in FIG. 1 . FIG. 3 illustrates a known composite laminate, and shows a sectional view of a part of the large-area composite laminate obtained at a step in manufacturing a ceramic multilayer substrate. detailed-description description="Detailed Description" end="lead"? |
Method for distributed multicast routing in connection-oriented networks and network for applying this method |
A method for multicast routing for a telecommunications network comprising a plurality of communication nodes interconnected by multiple paths, for the establishment of a multicasting connection between a source node and a plurality of destination nodes, in which the destination nodes are informed that a multicast connection request is arriving from a particular source. Each destination node then sends a connection request to the source, and these requests are processed and relayed by each node within the network. The requests are propagated from node to node until they reach the source node, carrying the information on the cost accumulated along the path. Finally, the source node, having received the information on the costs of the possible paths, selects the path which has a satisfactory cost. |
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