Method and reagent composition for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent composition includes epoxide groups capable of covalently binding to the target molecule. Optionally, the composition can contain photoreactive groups for use in attaching the reagent composition to the surface. The reagent composition can be used to provide activated slides for use in preparing microarrays of nucleic acids.

The present invention relates to reagents and support surfaces for immobilization of biomolecules, such as nucleic acids and proteins.
The immobilization of deoxyribonucleic acid (DNA) onto support surfaces has become an important aspect in the development of DNA-based assay systems, including the development of microfabricated arrays for DNA analysis. See, for instance, xe2x80x9cThe Development of Microfabricated Arrays of DNA Sequencing and Analysisxe2x80x9d, O""Donnell-Maloney et al., TIBTECH 14:401-407 (1996). Generally, such procedures are carried out on the surface of microwell plates, tubes, beads, microscope slides, silicon wafers or membranes.
A commonly used method for immobilizing cDNA""s or PCR products into arrays is to first coat glass slides with polylysine, then apply the DNA and illuminate with UV light to photocrosslink the DNA onto the polylysine (for example, see Schena M, Shalon D, Heller R, Chai A, Brown P O, Davis R W, xe2x80x9cParallel Human Genome Analysis: Microarray-based Expression Monitoring of 1000 Genesxe2x80x9d, Proc. Natl. Acad. Sci. USA 93(20):10614-9 (1996)). One disadvantage of this approach is that the UV crosslinking causes undesirable damage to the DNA that is not all useful for the immobilization. Another disadvantages of this approach is that UV crosslinking tends to be limited to longer nucleic acids (e.g., over about 100-mers), as provided by cDNA""s and PCR products (and in contrast to the shorter nucleic acids typically formed by synthesis and referred to as xe2x80x9coligonucleotidesxe2x80x9d). It appears that the potential damage induced by UV radiation (e.g., the formation of thymine dimers) is simply too great, and/or the extent of immobilization is insufficient, to permit shorter nucleic acids to be used. A population of longer nucleic acids, however, even when crosslinked by UV, will typically provide ample undamaged regions sufficient to permit accurate hybridization.
Only relatively few approaches to immobilizing DNA, to date, have found their way into commercial products. One such product for immobilizing oligonucleotides onto microwell plates is known as NUCLEOLINK(trademark), and is available from Nalge Nunc International (see, e.g., Nunc Tech Note Vol. 3, No. 17). In this product, the DNA is reacted with a carbodiimide to activate 5xe2x80x2-phosphate groups, which then react with functional groups on the surface. Disadvantages of this approach are that it requires the extra step of adding the carbodiimide reagent as well as a five hour reaction time for immobilization of DNA, and it is limited to a single type of substrate material.
As another example, Pierce has introduced a proprietary DNA immobilization product known as REACTI-BIND(trademark) DNA Coating Solutions (see Instructionsxe2x80x94REACTI-BIND(trademark) DNA Coating Solution Jan. 1, 1997). This product is a solution that is mixed with DNA and applied to surfaces such as polystyrene or polypropylene. After overnight incubation, the solution is removed, the surface washed with buffer and dried, after which it is ready for hybridization. Although the product literature describes it as being useful for all common plastic surfaces used in the laboratory, it does have some limitations. For example, Applicants were not able to demonstrate useful immobilization of DNA onto polypropylene using the manufacturer""s instructions. Furthermore, this product requires large amounts of DNA. The instructions indicate that the DNA should be used at a concentration between 0.5 and 5 xcexcg/ml.
Corning sells a product called xe2x80x9cDNA-BIND(trademark)xe2x80x9d for use in attaching DNA to the surface of a well in a microwell plate (see, e.g., the DNA-BIND(trademark) xe2x80x9cApplication Guidexe2x80x9d). The surface of the DNA-BIND(trademark) plate is coated with an uncharged, nonpolymeric, low molecular weight, heterobifunctional reagent containing an N-oxysuccinimide (NOS) reactive group. This group reacts with nucleophiles such as primary amines. The heterobifunctional coating reagent also contains a photochemical group and spacer arm which covalently links the reactive group to the surface of the polystyrene plate. Thereafter, amine-modified DNA can be covalently coupled to the NOS surface. The DNA is modified by adding a primary amine either during the synthesis process to the nascent oligomer or enzymatically to the preformed sequence. Since the DNA-BIND(trademark) product is polystyrene based, it is of limited use for those applications that require elevated temperatures such as thermal cycling. Corning also sells an aminosilane-coated glass slide, under the tradename CMT-GAPS(trademark) coated slides, which uses the same protocol as polylysine-coated slides for immobilizing DNA into microarrays.
TeleChem International, Inc. sells slides coated with an aldehyde silane as well as an aminosilane-coated slide. The aldehyde silane slides have very high backgrounds when fluorescence is used for detection. They also require an additional reduction step for stable immobilization.
Finally, SurModics, Inc., the assignee of the present invention, has recently introduced a coated glass slide, under the tradename 3D-Link(trademark) that consists of a hydrophilic polymer containing amine-reactive ester groups immobilized onto the surface. For best results, this product also requires amine modification of the DNA to be immobilized. As expected, however, the reactive ester groups tend to be hydrolytically unstable, which limits the amount of time arrays can be printed without some loss of performance to approximately eight hours.
The role of epoxide groups, in the course of binding or immobilizing nucleic acids, has been described in various ways as well. For instance, Shi et al., U.S. Pat. No. 5,919,626 describe the attachment of unmodified nucleic acids to silanized solid phase surfaces. The method involves the use of conventional nonpolymeric reagents such as mercapto-silanes and epoxy-silanes which bond to the surface by forming siloxane bonds with OH groups on the glass surface.
See also, U.S. Pat. No. 5,925,552 (Keogh, et al., xe2x80x9cMethod for Attachment of Biomolecules to Medical Devices Surfacesxe2x80x9d), which provides a method for forming a coating of an immobilized biomolecule on a surface of a medical device to impart improved biocompatibility for contacting tissue and bodily fluids. One such method includes converting a biomolecule comprising an unsubstituted amide moiety into an amine-functional material, combining the amine-functional material with a medical device biomaterial surface comprising a chemical moiety (such as, for example, an aldehyde moiety, an epoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety) which is capable of forming a chemical bond with the amine-functional material, to bond the two materials together to form an immobilized biomolecule on a medical device biomaterial surface. Included within the long list of biomolecules described as being useful in this patent were xe2x80x9ca DNA segment, a RNA segment, a nucleic acidxe2x80x9d and others.
Also on the subject of epoxides, Nagasawa et al., (J. Appl. Biochem. 7:430-437, 1985) describe the use of Sepharoses activated with epichlorohydrin or bisoxirane (both of which provide epoxide groups) for immobilizing DNA as immunosorbents for DNA antibodies. See also, Wheatley, et al., J. Chromatog. A 726:77-90 (1996) and Potuzak, et al., Nucl. Acids Res. 5:297-303 (1978).
To date, however, there appears to be no description in the art, let alone commercial products, that provide an optimal combination of such properties as hydrolytic stability, ease of use, minimized DNA damage (due to exposure to crosslinking radiation), and the ability to immobilize underivatized nucleic acids and/or shorter nucleic acid segments. In turn, there appear to be no products presently available, nor descriptions in the art, that provide or suggest the ability to use polymer-pendent epoxide groups adapted to immobilize either short or long nucleic acids, let alone in both derivatized and underivatized forms, and suitable for immobilization onto surfaces.
Finally, Surmodics, Inc., the assignee of the present invention, has previously described a variety of applications for the use of photochemistry, and in particular, photoreactive groups, e.g., for attaching polymers and other molecules to support surfaces. See, for instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329, 5,563,056, 5,637,460, 5,714,360, 5,741,551, 5,744,515, 5,783,502, 5,858,653, and 5,942,555.
The present invention provides a method and epoxide-based reagent composition for covalent attachment of target molecules onto the surface of a substrate, such as microscope slides, microwell plates, tubes, silicon wafers, beads or membranes. In a preferred embodiment, the method and composition are used to immobilize nucleic acid probes onto microscope slides, e.g., for use in printing DNA microarrays. The method and reagent of the present invention can be used to covalently immobilize either derivatized (e.g., amine-derivatized) or underivatized (i.e., not having a group added for the purpose of thermochemical reaction with an epoxide group) nucleic acids, and are particularly useful for underivatized nucleic acids. The immobilization method of this invention is very convenient to perform. In a preferred embodiment, the method involves the steps of coating a support with the reagent of this invention, printing the nucleic acid array, incubating the slide in a humid environment, blocking excess epoxide groups and washing the slide, after which it is ready for a hybridization assay.
Parent application U.S. Ser. No. 09/227,913, describes, inter alia, a comprehensive method and reagent composition for covalent attachment of target molecules onto the surface of a substrate, using a reagent that contains one or more thermochemically reactive groups (i.e., groups having a reaction rate dependent on temperature). Suitable groups are selected from the group consisting of activated esters such as N-oxysuccinimide (xe2x80x9cNOSxe2x80x9d), epoxide, azlactone, activated hydroxyl and maleimide groups.
The present application is particularly concerned with reagents having epoxide groups, in the manner described above, and provides further examples and advantages concerning the use of such epoxide-based reagents. Such advantages include, for instance, the ability to use the reagents to attach underivatized DNA, in addition to DNA derivatized to contain amine or other reactive groups, as described in parent application U.S. Ser. No. 09/227,913. Such advantages also include improved resistance to hydrolysis demonstrated by epoxides, as compared for instance, to NOS groups.
The present invention provides a method for immobilizing biomolecules, such as biopolymers selected from nucleic acids, proteins, and polysaccharides, the method comprising the steps of:
a) providing a solid support having a surface,
b) providing a reagent comprising one or more epoxide groups, and optionally also comprising one or more photogroups,
c) coating the reagent on the support surface (e.g., covalently attaching the polymeric reagent to the support surface by activation of the photogroups),
d) providing a biopolymer having a corresponding thermochemical reactive group,
e) attaching the biopolymer to the support by reacting its corresponding reactive group with the bound epoxide group,
f) optionally blocking the remaining epoxide groups (e.g., using an amine reagent), and
g) using the resultant coated support surface for its intended purpose, e.g., for the immobilization of biomolecules such as nucleic acids.
Applicants have found that polymers (and particularly hydrophilic polymers) containing epoxide groups, of the type described herein, have several advantages for DNA immobilization over previously used methods. These polymers, when coated onto silane-modified glass slides, for instance, provide an improved method for immobilizing underivatized DNA. Therefore, using these reagents, it is not necessary to modify the DNA with amines or other functional groups. Furthermore, the epoxide groups are significantly more stable to hydrolysis than are the amine-reactive ester groups. Compared with UV crosslinking of DNA onto polylysine or aminosilane, the coated surfaces of this invention are more convenient to use and tend to result in fewer undesirable side reactions, thereby resulting in less modification of the DNA.
A reagent composition of the invention preferably provides one or more epoxide (also known as xe2x80x9coxiranexe2x80x9d) groups pendent on a polymeric backbone, such as a hydrophilic polyacrylamide backbone. Optionally, and preferably, the reagent composition can also provide one or more pendent photoreactive groups. The photoreactive groups (alternatively referred to herein as xe2x80x9cphotogroupsxe2x80x9d) can be used, for instance, to attach reagent molecules to the surface of the support upon the application of a suitable energy source such as light. The epoxide groups, in turn, can be used to form covalent bonds with appropriate functional groups on the target molecule.
Optionally, the composition and method of this invention can be provided in the manner described in parent application U.S. Ser. No. 08/940,213. In such an embodiment, the reagent composition can be used for attaching a target molecule to the surface of a substrate, and comprises one or more groups for attracting the target molecule to the reagent, and one or more epoxide groups for forming covalent bonds with corresponding functional groups on the attracted target molecule. Optionally, such a composition further provides photogroups for use in attaching the composition to a surface. In one embodiment, for instance, a plurality of photogroups and a plurality of ionic groups (e.g., cationic groups) are attached to a hydrophilic polymer backbone. This polymer can then be coimmobilized with a second polymer backbone that provides the above-described epoxide groups for immobilization of target molecules. Suitable ionic groups include quaternary ammonium salts, protonated tertiary amines and other cationic groups such as phosphonium compounds. Also included are tertiary amine groups capable of being protonated when placed in an acid environment. Quaternary ammonium salts include alkyl quaternary ammonium compounds, such as [3-(methacryloylamino)propyl]trimethylammonium chloride (MAPTAC), as well as aromatic quaternary ammonium groups such as pyridinium compounds. Phosphonium compounds include polymers prepared from monomers such as tributyl(4-vinylbenzyl)phosphonium chloride, and are described in J. Appl. Polymer Sci. 53:1237 (1994), the disclosure of which is also incorporated by reference.
The invention further provides a method of attaching a target molecule, such as a DNA molecule, to a surface, by employing a reagent as described herein. In turn, the invention provides a surface having target molecules such as nucleic acids attached thereto by means of such a reagent, as well as a material (e.g., microscope slide) that provides such a surface.
Generally, the reagent molecules will first be attached to the surface by activation of the photogroups, thereafter the target molecule, (e.g., a nucleic acid) is contacted with the bound reagent under conditions suitable to permit it to come into binding proximity with the bound polymer. The target molecule is thermochemically coupled to the bound reagent by reaction between the reactive groups of the bound reagent and appropriate functional groups on the target molecule.
The invention further provides a method of attaching a target molecule, such as a nucleic acid, to a surface, by employing a reagent as described herein. As used herein, with regard to describing the present invention, the word xe2x80x9coligonucleotidexe2x80x9d (or xe2x80x9coligoxe2x80x9d) shall refer to a synthetic nucleic acid (as opposed to enzymaticaly prepared), and one that is typically shorter (e.g., on the order of 100-mer or less) than cDNA or PCR products formed enzymatically. The term xe2x80x9cnucleic acidxe2x80x9d, in turn, will refer to all such products collectively.
The invention further provides a surface having nucleic acids attached thereto by means of such a reagent, as well as a material (e.g., a slide or microwell plate) that provides such a surface. In yet another aspect, the invention provides a composition comprising a reagent of this invention in combination with a target molecule that contains one or more functional groups reactive with the thermochemically reactive group of the reagent.
A preferred reagent composition of the present invention comprises a hydrophilic polymer bearing one or more pendent epoxide groups adapted to form a covalent bond with corresponding reactive groups of a target molecule, and also bearing one or more pendent photoreactive groups adapted to be used for attaching the reagent to a surface, either before, during and/or after reaction between the reagent and the target molecules. Optionally, a composition can include other components, in addition to the reagent polymer component, such as polymers having pendent ionic groups, and the like.
In another embodiment of the invention, it is possible to immobilize the reagent composition, and in turn the target molecules, without the use of the photoreactive group. For instance, the surface of the material to be coated can be provided with thermochemically reactive groups, which can be used to immobilize hydrophilic polymers having epoxide groups as described above. For example, a surface may be treated with an ammonia plasma to introduce a limited number of reactive amines on the surface of the material. If this surface is then treated with a hydrophilic polymer having epoxide groups, then the polymer can be immobilized through reaction of the epoxide groups with amines on the surface. Preferably, the concentration of epoxide groups on the polymer is in sufficient excess, relative to the concentration of amines on the surface to insure that a sufficient number of reactive groups remain following the immobilization to allow coupling with the nucleic acid sequence.
A polymeric backbone can be either synthetic or naturally occurring, and is preferably a synthetic copolymer of the epoxide monomer and diluent or other monomers resulting from addition or condensation polymerization. Naturally occurring polymers, such as polysaccharides or polypeptides can be used as well. Preferred diluent monomers are biologically inert, in that they do not provide a biological function that is inconsistent with, or detrimental to, their use in the manner described.
Suitable diluent monomers for use in preparing a reagent of this invention include acrylics such as hydroxyethyl acrylate, hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate; acrylamide derivatives, such as acrylamide, methacrylamide, and acryloyl morpholine. Other synthetic polymers can be synthesized to include pendent epoxide groups in the manner described herein, including vinyls such as polyvinyl pyrrolidone and polyvinyl alcohol; nylons such as polycaprolactam, polylauryl lactam, polyhexamethylene adipamide and polyhexamethylene dodecanediamide; polyurethanes and polyethylene oxide, as well as combinations and copolymers thereof.
A reagent of the present invention preferably includes a hydrophilic polymer bearing a desired average number of photogroups and epoxide groups per average unit length or molecular weight, the combination dependent upon the reagent selected. The epoxide monomer can also be selected to provide any desired effective spacer distance between the polymeric backbone and the epoxide groups. In this manner, the reagent can be bonded to a surface or to an adjacent reagent molecule, to provide the other groups with sufficient freedom of movement to demonstrate optimal activity. The diluent comonomers are preferably hydrophilic (e.g., water soluble), with acrylamide and vinylpyrrolidone being particularly preferred.
Epoxide-containing polymers of the present invention can be prepared from monomers, such as glycidyl acrylate, glycidyl methacrylate, allylglycidyl ether, and glycidyl vinyl ether. Useful monomers are available from a variety of sources, for instance glycidyl acrylate (Pfaltz and Bauer Chemicals, cat. # G03480), glycidyl methacrylate (Aldrich cat # 15,123-8), allylglycidyl ether (Aldrich, cat # A3,260-8), glycidyl vinyl ether (Aldrich, cat. # 45,865-1), and glycidyl vinylbenzyl ether (Aldrich cat. # 45,867-8).
Epoxide monomers can also be made, such as by reaction of 2-isocyanatoethylmethacrylate with glycidol. Epoxide polymers can also be prepared by reacting hydroxyl polymers (e.g., polyhydroxypropylacrylamide) with epichlorohydrin. Other epoxide monomers and polymers can be made by those skilled in the art having spacers of various lengths and with various polarities. Other examples of suitable monomers are described in U.S. Pat. No. 5763,629.
Epoxide-containing polymers can also be synthesized by reacting hydroxyl- or amine-containing polymers with diepoxides. Currently, an epoxide activated-Sepharose is available (Sigma) that is made by reacting Sepharose gel beads with 1,4-butanedioldiglycidyl ether. This, or other diepoxides, (e.g., ethylene glycol diglycidyl ether, diepoxyoctane or diepoxydecane) can be used for derivatizing either amine or hydroxyl polymers to make polyepoxides. For example, polyhydroxypropylacrylamide or a copolymer containing a photomonomer can be reacted with an excess of 1,4-butanedioldiglycidyl ether to make a polyepoxide that can then be immobilized onto a surface.
Useful epoxide monomers include those of the general formula:
Where R1 is either CH3 or H and X is a noninterfering radical, preferably selected from the group:
where m=2xe2x88x926 and n=1xe2x88x9210;
where n=1xe2x88x9210;
xe2x80x94(CH2)mxe2x80x94Oxe2x80x94(CH2)xe2x80x94
where m=0 or 1, and;
where m=1xe2x88x9220 and n=1xe2x88x9210.
Without intending to be bound by theory, it would appear that epoxide groups can be used to immobilize underivatized DNA in a covalent fashion, and presumably due to a mechanism of reacting with amine groups on the purine and pyrimidine rings, such as cytosine and adenine, or with terminal hydroxyl groups.
Reagents of the invention optionally carry one or more pendent latent reactive (preferably photoreactive) groups covalently bonded to the polymer backbone. Photoreactive groups are defined herein, and preferred groups are sufficiently stable to be stored under conditions in which they retain such properties. See, e.g., U.S. Pat. No. 5,002,582, the disclosure of which is incorporated herein by reference. Latent reactive groups can be chosen that are responsive to various portions of the electromagnetic spectrum, with those responsive to ultraviolet and visible portions of the spectrum (referred to herein as xe2x80x9cphotoreactivexe2x80x9d) being particularly preferred.
Photoreactive groups respond to specific applied external stimuli to undergo active specie generation with resultant covalent bonding to an adjacent chemical structure, e.g., as provided by the same or a different molecule. Photoreactive groups are those groups of atoms in a molecule that retain their covalent bonds unchanged under conditions of storage but that, upon activation by an external energy source, form covalent bonds with other molecules.
The photoreactive groups generate active species such as free radicals and particularly nitrenes, carbenes, and excited states of ketones upon absorption of electromagnetic energy. Photoreactive groups may be chosen to be responsive to various portions of the electromagnetic spectrum, and photoreactive groups that are responsive to e.g., ultraviolet and visible portions of the spectrum are preferred and may be referred to herein occasionally as xe2x80x9cphotochemical groupxe2x80x9d or xe2x80x9cphotogroupxe2x80x9d.
Photoreactive aryl ketones are preferred, such as acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles (i.e., heterocyclic analogs of anthrone such as those having N, O, or S in the 10-position), or their substituted (e.g., ring substituted) derivatives. The functional groups of such ketones are preferred since they are readily capable of undergoing the activation/inactivation/reactivation cycle described herein. Benzophenone is a particularly preferred photoreactive moiety, since it is capable of photochemical excitation with the initial formation of an excited singlet state that undergoes intersystem crossing to the triplet state. The excited triplet state can insert into carbon-hydrogen bonds by abstraction of a hydrogen atom (from a support surface, for example), thus creating a radical pair. Subsequent collapse of the radical pair leads to formation of a new carbon-carbon bond. If a reactive bond (e.g., carbon-hydrogen) is not available for bonding, the ultraviolet light-induced excitation of the benzophenone group is reversible and the molecule returns to ground state energy level upon removal of the energy source. Photoactivatible aryl ketones such as benzophenone and acetophenone are of particular importance inasmuch as these groups are subject to multiple reactivation in water and hence provide increased coating efficiency. Hence, photoreactive aryl ketones are particularly preferred.
The azides constitute a preferred class of photoreactive groups and include arylazides (C6R5N3) such as phenyl azide and particularly 4-fluoro-3-nitrophenyl azide, acyl azides (xe2x80x94COxe2x80x94N3) such as benzoyl azide and p-methylbenzoyl azide, azido formates (xe2x80x94Oxe2x80x94COxe2x80x94N3) such as ethyl azidoformate, phenyl azidoformate, sulfonyl azides (xe2x80x94SO2xe2x80x94N3) such as benzenesulfonyl azide, and phosphoryl azides (RO)2PON3 such as diphenyl phosphoryl azide and diethyl phosphoryl azide. Diazo compounds constitute another class of photoreactive groups and include diazoalkanes (xe2x80x94CHN2) such as diazomethane and diphenyldiazomethane, diazoketones (xe2x80x94COxe2x80x94CHN2) such as diazoacetophenone and 1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (xe2x80x94Oxe2x80x94COxe2x80x94CHN2) such as t-butyl diazoacetate and phenyl diazoacetate, and beta-keto-alpha-diazoacetates (xe2x80x94COxe2x80x94CN2xe2x80x94COxe2x80x94Oxe2x80x94) such as t-butyl alpha diazoacetoacetate. Other photoreactive groups include the diazirines (xe2x80x94CHN2) such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes (xe2x80x94CHxe2x95x90Cxe2x95x90O) such as ketene and diphenylketene. Photoactivatible aryl ketones such as benzophenone and acetophenone are of particular importance inasmuch as these groups are subject to multiple reactivation in water and hence provide increased coating efficiency.
Upon activation of the photoreactive groups, the reagent molecules are covalently bound to each other and/or to the material surface by covalent bonds through residues of the photoreactive groups. Exemplary photoreactive groups, and their residues upon activation, are shown as follows.
Copolymers can be prepared using epoxide-containing monomers as described above, using techniques known to those skilled in the art. Preferably, the epoxide monomers and comonomers undergo free radical polymerization of vinyl groups using azo initiators such as 2,2xe2x80x2-azobisisobutyronitrile (AIBN) or peroxides such as benzoyl peroxide. The comonomers selected for the polymerization are chosen based on the nature of the final polymer product. For example, a photoreactive polymer containing epoxide groups can be prepared by the use of a monomer mixture that includes one or more monomers containing a photoreactive group and one or more second monomers containing an epoxide group.
An epoxide-functionalized polymeric reagent of this invention can be prepared by appropriate derivatization of a preformed polymer or, more preferably, by polymerization of a set of comonomers to give the desired substitution pattern. The latter approach is preferred because of the ease of changing the ratio of the various comonomers and by the ability to control the level of incorporation into the polymer.
The composition of the final polymer can be controlled by mole ratio of the monomers charged to the polymerization reaction. Typically the epoxide monomers are used at relatively low mole percentages of the total monomer content of the polymerization reaction, with the remainder of the composition consisting of a relatively low mole percent of photomonomers, and the remainder of monomers which are neither photoreactive nor thermochemically reactive toward the nucleic acid sequence. Examples of such monomers include, but are not limited to, acrylamide, acrylic acid and N-vinylpyrrolidone. Based on the relative reactivities of the monomers used, the distribution of the monomers along the backbone is largely random.
In a preferred embodiment, for instance, a reagent composition is formed having between about 5 mole % and about 25 mole % epoxide monomer (more preferably between about 10 mole % and about 20 mole %); between about 0.1 mole % and about 5 mole % photomonomer (more preferably between about 0.5 mole % and about 2 mole %), and the remainder (to 100 mole %) other monomers.
The present invention provides a method and reagent composition for covalent attachment of target molecules onto the surface of a substrate, such as slides formed of organosilane-pretreated glass, organosilane-pretreated silicon, silicon hydride, or plastic. In one embodiment, the method and composition are used to immobilize nucleic acid probes onto plastic materials such as microwell plates, e.g., for use in hybridization assays. In a preferred embodiment the method and composition are adapted for use with substantially flat or molded surfaces, such as those provided by organosilane-pretreated glass, organosilane-pretreated silicon, silicon hydride, or plastic (e.g., polymethylmethacrylate, polystyrene, polycarbonate, polyethylene, or polypropylene). The reagent composition can then be used to covalently attach a target molecule such as a biomolecule (e.g., a nucleic acid) which in turn can be used for specific binding reactions (e.g., to hybridize a nucleic acid to its complementary strand).
Support surfaces can be prepared from a variety of materials, including but not limited to plastic materials selected from the group consisting of crystalline thermoplastics (e.g., high and low density polyethylenes, polypropylenes, acetal resins, nylons and thermoplastic polyesters) and amorphous thermoplastics (e.g., polycarbonates and poly(methyl methacrylates). Suitable plastic or glass materials provide a desired combination of such properties as rigidity, surface uniformity, resistance to long term deformation, and resistance to thermal degradation.
The present invention provides a method for immobilizing biomolecules such as biopolymers, and particularly those selected from nucleic acids, proteins, polysaccharides, the method comprising the steps of:
a) providing a solid support having a surface,
b) providing a reagent comprising one or more epoxide groups, and optionally also comprising one or more photogroups,
c) coating the reagent on the support surface (e.g., by dipping, spraying, roll-coating or knife-coating), and covalently attaching the polymeric reagent to the support surface, e.g., by activation of the photogroups,
d) providing a biopolymer (e.g., nucleic acid, protein, polysaccharide) having one or more corresponding thermochemical reactive groups (e.g., amine, hydroxyl, sulfhydryl),
e) attaching the biopolymer to the support by reacting its corresponding reactive group with the bound epoxide group,
f) optionally blocking the remaining unreacted epoxide groups (e.g., by the use of an amine reagent), and
g) using the resultant coated support surface for its intended purpose, e.g., immobilizing biomolecules, such as nucleic acids for use in hybridization (e.g., on slides for arrays, or in microplate wells),
In a preferred embodiment, the present invention provides a method of attaching a target molecule to the surface of a substrate, the method comprising:
a) providing a reagent composition comprising a polymeric backbone having one or more pendent epoxide groups adapted to form covalent bonds with corresponding functional groups on the target molecule,
b) coating and immobilizing the reagent composition on the substrate surface,
c) providing a solution comprising a target molecule having one or more functional groups thermochemically reactive with corresponding epoxide groups provided by the reagent composition,
d) applying an amount (e.g., in the form of discrete small sample volume spots) of the solution on the surface of the substrate surface, and
e) incubating the combination under conditions suitable to permit the epoxide groups provided by the reagent composition to form covalent bonds with corresponding functional groups provided by the target molecule in order to attach the target molecule to the surface.
Preferably, for use in preparing and using coated slides, the reagent is adapted to be coated and immobilized on a surface in a manner that permits:
i) a small sample volume of a solution containing the target molecule to be applied in the form of a discrete spot on the reagent-coated surface,
ii) target molecule present in the sample volume to become covalently attached to the bound reagent by reaction between its functional groups and the epoxide groups, and
iii) substantially all unattached target molecule to be washed from the spot without undue detectable amounts of target molecule in the area surrounding the spot.
In a preferred embodiment, the target molecules are preferably applied to the epoxide polymer-coated surface in a solution at relatively low ionic strength and slightly alkaline pH (e.g., in 150 mM phosphate buffer, pH 8.5). Optimal coupling can be achieved by incubating the surface at high humidity (e.g., 75% relative humidity), which can be achieved by placing the surface within an enclosed storage box containing saturated NaCl at room temperature) for several hours, or most preferably, overnight. The excess uncoupled epoxide groups are then blocked with a solution containing 50 mM ethanolamine and 0.1% sodium dodecyl sulfate (SDS) in 0.1 M Tris buffer, pH 9 for 30 minutes at 50xc2x0 C. The surface is then rinsed with deionized water and washed with 4xc3x97standard saline citrate (xe2x80x9cSSCxe2x80x9d)(0.6 M NaCl+0.06 M sodium citrate) containing 0.1% SDS at 50xc2x0 C. for about 15 to about 60 minutes. The surface is then rinsed with deionized water and spun dry in a centrifuge.
When used for preparing microarrays, e.g., to attach capture probes (e.g., oligonucleotides or cDNA) to the microarray surface, such capture probes are generally delivered to the surface in a volume of less than about 1 nanoliter per spot, using printing pins adapted to form the spots into arrays having center to center spacing of about 200 xcexcm to about 500 xcexcm. Unlike the coupling of DNA from solution and onto the surface of coated microplate wells, nucleic acids printed in arrays of extremely small spot sizes tend to dry quickly, thereby altering the parameters affecting the manner in which the nucleic acids contact and couple with the support. In addition to the design and handling of the printing pins, other factors can also affect the spot size and/or the ultimate hybridization signals, including: salt concentrations, type of salts and wetting agents in the printing buffer; hydrophobic/hydrophilic properties of the surfaces; the size and/or concentration of the nucleic acids; and the drying environments.
In a preferred embodiment, the reagent composition can be used to prepare activated slides having the reagent composition photochemically immobilized thereon. The slides can be stably stored and used at a later date to prepare microarrays by immobilizing underivatized or amine-derivatized DNA. The coupling of the capture DNA to the surface takes place at pH 8-9 in a humid environment following printing the DNA solution in the form of small spots.
Activated slides of the present invention are particularly well suited to replace conventional (e.g., amino-silylated) glass slides in the preparation of microarrays using manufacturing and processing protocols, reagents and equipment such as micro-spotting robots (e.g., as available from Cartesian), and a micro-spotting device (e.g., as available from TeleChem International). Suitable spotting equipment and protocols are commercially available, such as the xe2x80x9cArrayltxe2x80x9d(trademark) ChipMaker 3 spotting device. This product is said to represent an advanced version of earlier micro-spotting technology, employing 48 printing pins to deliver as many as 62,000 samples per solid substrate (e.g., 1 inch by 3 inch standard slide).
The use of such an instrument, in combination with conventional (e.g., poly-L-lysine coated) slides, is well known in the art. See, for instance, U.S. Pat. No. 5,087,522 (Brown et al.) xe2x80x9cMethods for Fabricating Microarrays of Biological Samplesxe2x80x9d, and the references cited therein, the disclosures of each of which are incorporated herein by reference.
For instance, the method and system of the present invention can be used to provide a substrate, such as a coated glass slide, with a surface having one or more microarrays. Each microarray preferably provides at least about 10/cm2 (and preferably at least about 100/cm2 distinct target molecules (e.g., polynucleotide or polypeptide biopolymers). Each distinct target molecule 1) is disposed at a separate, defined position in the array, 2) has a length of at least 10 subunits, 3) is present in a defined amount between about 50 attomoles and about 10 nanomoles, and 4) is deposited in selected volume in the volume range of about 0.01 nanoliters to about 100 nanoliters. These regions (e.g., discrete spots) within the array can be generally circular in shape, with a typical diameter of between about 75 microns and about 1000 microns (and preferably between about 100 and about 200 microns). The regions are also preferably separated from other regions in the array by about the same distance (e.g., center to center spacing of about 100 microns to about 1000 microns). A plurality of analyte-specific regions can be provided, such that each region includes a single, and preferably different, analyte specific reagent (xe2x80x9ctarget moleculexe2x80x9d).
Those skilled in the art, given the present description, will be able to identify and select suitable reagents depending on the type of target molecule of interest. Target molecules include, but are not limited to, plasmid DNA, cosmid DNA, expressed sequence tags (ESTs), bacteriophage DNA, genomic DNA (includes, but not limited to yeast, viral, bacterial, mammalian, insect), RNA, complementary DNA (cDNA), peptide nucleic acid (PNA), and oligonucleotides.
The invention will be further described with reference to the following non-limiting Examples. It will be apparent to those skilled in the art that many changes can be made in the embodiments described without departing from the scope of the present invention. Thus the scope of the present invention should not be limited to the embodiments described in this application, but only by embodiments described by the language of the claims and the equivalents of those embodiments. Unless otherwise indicated, all percentages are by weight. Structures of the various xe2x80x9cCompoundsxe2x80x9d identified throughout these Examples can be found in Table 1 included below.


Preparation of an electrolyte film for lithium/polymer electrolyte batteries by extruding a powder mixture containing a polymer, a lithium salt and an additive consisting of an ultra fine powder of a metal oxide, such as silica, aluminum, or titanium oxide with a particle size between about 7 and 40 nm. The role of the additive is to prevent the formation of adhesive solid blocks in the powder mixture and to allow the introduction of this mixture in the extruder. Electrolyte compositions, electrolytes as well as electrochemical generators obtained according to the invention are also described.

a) Field of the Invention
The present invention concerns additives for obtaining non-adhesive powders for the extrusion of electrolytes of polymer electrolyte batteries, hereinafter ACEP. More specifically the invention relates to a process for the preparation of an electrolyte film for lithium/polymer electrolyte batteries. The invention also concerns compositions for the preparation of an electrolyte film for the batteries mentioned above, the electrolytes contained in these compositions as well as the electrochemical generators including these electrolytes.
b) Description of Prior Art
Lithium/polymer electrolyte ACEP batteries are manufactured by superposing three main types of films: a film of positive electrode containing a material which is electrochemically active such as vanadium oxide, a film of electrolyte comprising a polymer and a lithium salt, and a lithium film. Each of these films has a thickness between 15 and 50 .mu.m, for a total thickness of elementary film of the battery of 100 to 150 .mu.m. About thirty meters of elementary film 15 cm wide are typically required to give a battery of 100 wh.
The film of electrolyte is mainly manufactured by solvent coating, however melt extrusion is also considered because of its numerous advantages. The amorphous copolymers used in ACEP batteries can be easily extruded to provide an electrolyte without salt. Positive electrodes containing an excess of salt must then be prepared by solvent coating, but these are more difficult to obtain with the desired properties of uniformity and adhesion on the collector than positive electrodes containing a regular amount of salt.
When trying to extrude copolymers with a salt, the following problem occurs: when the copolymer powder is mixed with a lithium salt powder, large adhesive solid blocks of polymer-salt complex are obtained, which prevent the introduction of the mixture in the extruding screw, and which cannot be reduced in the form of pellets. The same problem appears with mixtures of salt and crystalline polymer of the polyethylene oxide type.
A partial solution consists in introducing the polymer and salt separately into the extruder, however, the measurement of the relative quantities of each of the powders is not very accurate and, moreover, since the powders have no time to mix perfectly, the films obtained are not very homogeneous.
According to the invention, an approach is proposed which consists in utilizing additives preventing the formation of solid adhesive blocks and enabling to obtain mixtures of polymer and salt in the form of fine powders which are easy to introduce into an extruder.
It is an object of the present invention to propose additives, based on ultra fine powders, whose granulometry is between about 7 and 40 nm, for example silica or other mineral compounds, for example oxides, which have no effects on the electrochemical performance of ACEP batteries.
The addition of silica or aluminum powders in ACEP electrolytes has been proposed by Wieczorek et al. (2nd Int. Symp. Polym. Elect., 1990, pp. 339-347) but only for the more conventional purpose of improving the mechanical and thermal properties of the electrolytes prepared by solvent coating, and with much larger particles (2 to 30 .mu.m), which do not allow the above desired effects to be obtained when extruding the polymers and the lithium salts.
It is also an object of the invention to provide additives enabling to obtain non-adhesive powders of polymeric electrolyte-salt mixtures which may be introduced into an extruder to prepare said electrolytes by melt process.
Another object of the invention is the utilization of additives, for example based on pyrogenated silica powders of the AEROSIL.TM. type, consisting of ultra fine particles (7 to 40 nm), preferably chemically treated at their surfaces to eliminate siloxane or silanol polar groups.
The invention relates to a process for preparing an electrolyte film for lithium/polymer electrolyte batteries, by extrusion of a polymer and a lithium salt which can be used in said electrolyte. The process is characterized in that the extrusion of said polymer with said lithium salt is carried out in the presence of ultra fine powders of a metal oxide whose granulometry is between about 7 and 40 nm.
Preferably the metal oxides are selected from silicon, aluminum, and titanium oxides and pyrogenated silica is particularly suitable. The silica preferably is has one having a BET surface of 50 to 400 m.sup.2 /g and contain more than 99.8% of silica.
According to another preferred embodiment of the invention, a polymer powder is first mixed separately with some pyrogenated silica powder, a lithium salt powder is mixed separately with some pyrogenated silica powder, the two mixtures are combined and a last amount of pyrogenated silica powder is added, then, the final mixture is mixed and extruded.
The invention also concerns a composition for the preparation of an electrolyte film for lithium/polymer electrolyte batteries characterized in that it comprises a polymer and a lithium salt which can be used in these electrolytes and an ultra fine powder of metal oxides with particle sizes between about 7 and 40 nm.
The invention also concerns electrolytes and electrochemical generators manufactured from compositions according to the invention.
The families of polymers which can be used in ACEP electrolytes described in Armand U.S. Pat. No. 4,303,748, as well as the amorphous, crosslinkable or non-crosslinkable copolymers and terpolymers, described more in detail in U.S. Pat. Nos. 4,578,326, 4,357,901 and Canadian Pat. No. 1,269,702 are preferably used for the preparation of electrolytes according to the invention.
With respect to the lithium salts, the preferred families of salts are those used in ACEP electrolytes described in Armand U.S. Pat. No. 4,303,748, as well as the more elaborated salts based on terfluorosulfonimide of lithium (TFSI) or bisperhalogenoacyl or sulfonylimides of lithium (TFSM), crosslinkable or non-crosslinkable, described in U.S. Pat. Nos. 4,505,997, 4,818,694 and PCT WO92/02966 of Jul. 25, 1991.
The extrusion additives which are preferably used are the ultra fine powders ("fumes") of pyrogenated silica, commercially available from the Degussa Company under the trademark AEROSIL or from the Cabot Chemical Company under the trademark CABOSIL. The particles of non-treated silica have silanol and siloxane hydrophilic groups at their surfaces. The particles of silica which have been chemically treated with silane (AEROSIL type R) have hydrophobic CH.sub.3 or other groups at their surfaces. The particles which have been treated are particularly suitable for electrochemical applications because they do not react with lithium and lead to ACEP batteries with better electrochemical performances.
These particles normally have a granulometry of 7 to 40 nm, a BET surface of 50 to 400 m.sup.2 /g and contain more than 99.8% silica. Powders of the same type, except that they are made of aluminum, titanium or other metallic oxides are also suitable.
Although the preparation of powder mixtures presents no problem to one skilled in the art, it is preferred to proceed as follows. First, some polymer powder is mixed with a powder of, for example, AEROSIL to reduce the adhesive character of the polymer, some lithium salt powder is also mixed with AEROSIL powder to separate the grains of salt from one another. Then the two powder mixtures are mixed and some AEROSIL powder is added to complete the separation of the adhesive phases of polymer-salt complex. Of course, one could use any other metallic oxide powder.
The quantity of metal oxide powder, for example AEROSIL, which is required increases with the relative quantity of salt in the polymer and with the amorphous and adhesive character of the polymer-salt complex, as this will appear to one skilled in the art. Normally, 2% to 10% of AEROSIL by weight with respect to the polymer-salt complex enables to obtain the desired properties, with 5% as a typical value.

Additives for extruding polymer electrolytes


An improved golf driver club head wherein each driver club head has a tensioning device, which places the peripheral/outer structure of the club head in compression; thereby, resulting in a stronger, more rigid club head structure. In the preferred embodiment, the tensioning device comprises an elongated cylindrical member having respective end members. The top and bottom surfaces of the golf club head are each provided with a cylindrical depression or recess and an aperture or passage at the lower end of the depression. The passages are just large enough to accommodate the tensioning device, but are too small to pass the respective end members. During club head manufacture, the length of the device is selected to apply a compressive force between the head surfaces. Fine adjustment is accommodated by a threaded end member. The actual compression force may be selected to yield the best performance depending on the geometry, structure and material of the club head.

1. Field of the Invention
The present invention relates generally to the field of golf club heads and more specifically to a golf club wood-type head having a tensioning device for better energy transfer.
2. Background Art
Golf club wood heads that are made from metal (metal woods) and other more exotic high technology materials have distinct advantages over wooden club heads. Generally wooden golf club heads are less durable, vary in density and hardness and require great expertise through the manufacturing process. Metal and exotic materials woods on the other hand can be replicated consistently from the original design, use strong durable materials and are produced using proven manufacturing processes.
Metal and exotic material woods out-perform wooden clubs because they use harder, stronger materials. The use of these materials enables the weight to be transferred to the club head perimeter thereby stabilizing the club head as it comes into impact with the golf ball. Stabilizing the club head at impact reduces the characteristic oscillations that occur to the club head on the golfer""s down swing, resulting in a more efficient transfer of energy to the golf ball. The increased (more efficient) transfer of energy results in additional ball travel distance. Another benefit from a more stable club head is accuracy. If the club head is more stable at impact, less oscillation will occur during impact. This produces less spin to the golf ball and reduces deflection of the club head resulting in straighter shots.
Over the last decade golf club manufacturers have increased the size of metal wood driver club heads. This has happened for two basic reasons: One, golfers are always searching for ways to hit the golf ball longer and straighter. Larger club heads are more stable during impact and are easier to hit, resulting in consistently longer straighter hits. Two, manufacturing methods have improved, allowing the development of larger metal wood driver club heads utilizing extremely thin wall casting or forgings in the structure and in high performance materials such as titanium.
Regardless of manufacturer, most driver club head weights are within a few grams of each other. Typical driver club head weights will range from approximately 192 grams to 202 grams depending on club length, shaft type and manufacturer specifications. Generally, the club head will remain as a constant in order to provide proper balance, physical and mechanical properties which combine to produce a well performing golf club.
Metal wood (driver) club heads utilize thin wall castings or forgings to achieve their size, shape and weight. The larger the driver club head, the thinner the castings or forgings that are required to produce the intended resulting driver club head.
When a driver club head comes into contact with the golf ball, which is stationary, during the golf swing, the resultant collision, depending on the impact velocity and angle of attack, causes the golf ball to compress on the one hand and the driver club head to deform on the other.
The amount of deformation that occurs to the driver club head during impact with the golf ball depends on, but is not limited to the following criteria:
1. The size of the driver club head;
2. The material used to construct the driver club head;
3. The design and construction of the driver club head;
4. The wall thickness of the driver club head;
5. The velocity of the driver club head as it comes into contact with the golf ball;
6. The angle of attack at which the driver club head impacts the golf ball;
7. The distribution of mass within the club head;
8. The mass (weight) of the club head;
9. The stiffness of the club head structure;
10. The strength of the club head structure.
As driver club heads become larger, their ability to prevent deformation at impact with the golf ball becomes more difficult. Even with the use of high performance materials such as titanium or carbon fiber, the weight constraints common in most driver club heads make it imperative to design driver club heads using thin walls. As the driver club head comes into contact with the stationary golf ball during the downswing, the driver club head will oscillate and deform in an effort to find its way around the stationary golf ball. The golf ball compresses under the load during said impact with the club head initiating a lateral direction change of the golf ball away from the center of gravity of the club head resulting in the golf ball rolling in the direction of least resistance prior to the initiation of the rebound phase of the golf ball during impact with the club head. During this impact process, the stored energy that normally would be directed from the driver club head to the golf ball is momentarily redirected to the driver club head. The resulting redirection of energy transfer causes the driver club head structure to flex and deform. This occurrence constitutes a loss of energy that could be imparted from the driver club head to the golf ball. The resulting loss of energy from the driver club head to the golf ball during impact will result in a reduction of energy that can be transferred from the club head to the golf ball resulting in a loss of distance that the golf ball will travel.
The present invention comprises an improved golf driver club head wherein each driver club head has a tensioning device which places the peripheral/outer structure of the metal wood club head in compression thereby resulting in a stronger more rigid metal wood club head structure.
This is accomplished by affixing the tensioning device to the crown (top) surface of the metal wood club head and to the sole (bottom) surface of the metal wood club head. The device is affixed to the two surfaces of the metal wood club head and is pre-tensioned. The tensioning is accomplished by compressing the two metal wood club head surfaces together then affixing the device to the opposed surfaces so that the surfaces are held in state of relative compression. Another way this can be accomplished is to affix the device to the two metal wood club head surfaces either through a hole or boss and to adjust the device by compressing, rotating (screwing), gluing or other form of fastening.
In the preferred embodiment illustrated herein by way of example, the tensioning device comprises an elongated cylindrical member having respective end members. The top and bottom surfaces of the golf club head are each provided with a cylindrical depression or recess and an aperture or passage at the lower end of the depression. The passages are just large enough to accommodate the tensioning device, but are too small to pass the respective end members. During club head manufacture, the length of the device is selected to apply a compressive force between the head surfaces which may preferably be in the range of one pound to three hundred pounds. The actual compression force may be selected to yield the best performance depending on the actual geometry, structure and material of the club head.
While the applicant is not the first to recognize the potential positive effect of a tensioned golf club head (see for example, prior art U.S. Pat. No. 5,501,453), applicant is believed to be the first to employ a tensioning device within the head cavity and the first to compress the top and bottom head surfaces toward one another to resist their further separation during ball impact.
It is therefore a principal object of the present invention to provide a metal wood golf club head having a tensioning device to resist expansion between the top and bottom surfaces of the head during impact with the golf ball.
It is another object of the invention to provide a metal wood golf club head having a tensioning device positioned inside the head for compressing the top and bottom head surfaces in the nominal condition of the club head.
It is yet another object of the invention to interconnect the interior top and bottom surfaces of a metal wood golf club head by a tensioning wire which resists separation of those surfaces during ball impact.
It is still another object of the invention to provide a method for compressing two opposing walls of a golf club metal wood head toward each other to resist their further separation at ball impact.
It is still another object of the invention to improve the performance of a golf club having a metal wood head by employing a tensioning device to compress two opposing walls of the head and thereby resist deformation of the head at ball impact.

Golf club head having a device for resisting expansion between opposing walls during ball impact


A tracheostomy dilator comprises a rod which is penetrated by an inner lumen. Both ends of the inner lumen are open, at the end facing the patient and at the end of the tracheostomy dilator facing away from the patient. The end facing the patient is provided with a thread. The tracheostomy dilator is used as screw dilator and can widen the tissue in one single dilation process such that a further tracheostomy cannula can be inserted into the trachea.

The invention concerns a tracheostomy dilator consisting of a rod having an inner lumen which is open at both ends and extends from the end of the rod facing the patient to the end of the rod facing away from the patient.
Tracheostomy dilators of this type have become known in connection with puncture tracheostomy according to Ciaglia et al., xe2x80x9cElective Percutaneous Dilatational Tracheostomyxe2x80x9d Chest 1985, volume 6, pages 715-719.
In the known puncture tracheostomy, the trachea is transcutanely punctured in a bronchoscopically controlled fashion, below the ring-like cartilage, preferably between the second and third tracheal ring, and successively extended via a Seldinger wire with stepped dilators, and after the desired extension, a tracheostomy cannula is inserted into the trachea. This tracheostomy method has been used in the meantime at least for a highly selective part of patients. The known method, however, also bears risks for the patient and consequently only very experienced anaesthetists are supposed to use this method. The known method is time-consuming and every dilation bears the danger of injuring the posterior tracheal wall.
A known further development of the percutaneous dilatation tracheostomy which has become known from Wolfgarten et al. xe2x80x9cPunktionstracheostomie mit einmaliger Trachealdilatationxe2x80x9d (puncture tracheostomy with single tracheal dilatation), Chirurg 2000, pages 63-65 and 723-724, consists e.g. in that the skin is cut between ring-like cartilage and incisura jugularis approximately at the height of the third tracheal ring. Subsequently, the skin tissue and cervical muscles are extended with a clamp until the trachea becomes visible. The trachea is punctured via the skin cut by means of a hollow needle under bronchoscopic control, a guiding wire is pushed under visual control into the trachea, and dilation by means of a conical dilator is effected once via the guiding wire to a size required for the tracheostomy cannula to be inserted.
It is a common feature of the known dilatation methods that the stoma channel and the anterior tracheal wall must be widened through a large feed force and long advancing distance. The tip of the dilator might come too close to the posterior tracheal wall and cause injuries.
For this reason, single bouginage seems to be less controllable than bouginage in several steps according to Ciaglia et al. since the feed forces for single bouginage are larger and the advancing distance can be better controlled via bouginage in several steps.
It is the object of the present invention to further develop the known tracheostomy dilators such that they can be used with improved control.
This object is achieved in accordance with the invention in that the end of the rod facing the patient comprises a first thread and the section of the rod comprising the first thread tapers towards the end facing the patient.
The inventive tracheostomy dilator therefore has the substantial advantage that during dilatation, also during single dilatation, the compression onto the anterior tracheal wall is reduced to a minimum. The inventive dilator is threaded onto a guiding wire which is inserted, in a fashion known per se, into the trachea, and then screwed into the tissue until it has penetrated the trachea in the desired manner. The user must not exert a feed impulse in the transverse direction onto the inventive dilator since the thread automatically produces a feeding motion of the dilator in correspondence with the selected thread pitch.
The inventive dilator penetrates the anterior tracheal wall in a smooth fashion compared to conventional dilatation which includes substantially large feed forces and protrudes the anterior wall of the inner lumen of the trachea such that the dilator tip possibly reaches a dangerous closeness to the posterior tracheal wall. The inventive dilator does not pose this danger since dilatation is carried out in a helical motion. Only dilatation is desired and not any compression of the tracheal wall which would be dangerous. Dilatation with screws minimizes compression onto the anterior tracheal wall. The feed forces for screw dilatation can be controlled to a large extent through turning of the dilator and are predetermined by the selected thread pitch. This ensures continuous dilatation in the radial direction without exerting too high an axial pressure onto the tissue to be dilated.
The rod in accordance with the invention tapers in the rod section comprising the thread towards the free end of the rod facing the patient. This is advantageous in that the tissue to be widened is smoothly stretched. Depending on the conical tapering of this section, dilatation is effected on a shorter or longer stretch.
Moreover, in a preferred embodiment of the inventive tracheostomy dilator, the first thread is formed as automatically cutting thread. This is advantageous in that the dilator can penetrate, e.g. be turned, into the tissue to be widened with little external force.
If the first thread of the tracheostomy dilator has different thread pitches across its length, it is possible to produce only slight expansion of the tissue in a first dilatation step by turning the dilator several times, and to produce larger dilatation with a smaller turning motion, accompanied by a larger axial advance, e.g. in a second dilatation step, wherein the thread tip of the inventive dilator is fixedly anchored in the tissue.
If the free end of the rod facing the patient is additionally chamfered, the dilator tip penetrates the tissue bordering the tip in a particularly smooth fashion.
In a preferred fashion, the diameter of the inner lumen is adjusted to the outer diameter of a guiding wire such that the inner volume is filled by the guiding wire without leaving any gaps. This is advantageous in that tissue cannot reach the inner lumen during dilatation.
The rod can also comprise a second thread connecting to the first thread which permits further controlled advance of the dilator. Additionally, the dilator is fixed in its position by the second thread. The second thread can extend to the end facing away from the patient, i.e. along the entire cylindrical shaft or only in the section of the cylindrical shaft facing the patient. The thread pitch can correspond to the pitch of the first thread or be different. It is also feasible that the thread pitch of the second thread varies along the shaft. The second pitch can be formed as a bar disposed onto the shaft or as depression in the bar wall.
In a further embodiment of the inventive tracheostomy dilator, the first and/or second thread has a surface showing little friction when contacting tissue. This has the advantage that, when turning the inventive dilator, tissue cannot be threaded onto the thread pitches. It is particularly advantageous to provide a hydrophilic layer on the outer surface and/or wet the outer surface with a sliding gel. Through provision of such friction-reducing measures, the thread section of the dilator can be turned into the tissue to be dilated with little force and in a controlled manner.
To trigger a controlled turning motion of the dilator, the end of the rod facing away from the patient is preferably provided with a handle to permit controlled turning of the inventive screw dilator by the user.
If the inventive tracheostomy dilator is produced from boil proof plastic or metal it can be sterilized a few times rather than disposed of after use.
In another embodiment of the invention, the tracheostomy dilator can be produced from two semi-shells which are connected to one another for the dilatation process either via a film hinge or in another fashion. When dilatation is finished, the screw dilator can be withdrawn after positioning of the tracheostomy cannula and be removed from the guiding wire outside of the patient. This embodiment has the advantage that the screw dilator must not be withdrawn across the entire length of the guiding wire.
In a further inventive embodiment, a tracheostomy cannula can be disposed on the outer surface of the inventive screw dilator. This has the advantage that the tracheostomy cannula is advanced into the trachea during dilatation.
The inventive screw dilator reduces the still present risks of a percutaneous dilatation tracheostomy since dilatation is triggered through a radial motion component.
Further advantages can be extracted from the description and the enclosed drawing. The features mentioned above and below can be used in accordance with the invention either individually or collectively in any arbitrary combination. The embodiment described below is to be understood as exemplary representation of an inventive tracheostomy dilator. The embodiment shown in the figures is highly schematised and not to be taken to scale.


A method and system for retrieving large contiguous data files is provided. The method divides the data during the storage operation such that it spans two or more logical volumes. The data may be stored over the plurality of volumes in any convenient manner such as in the case of two logical volumes, storing the even address data on one device and the odd address data on the other device. During an access request (either a read or a write) a host controller receiving the access request will generate as many access requests as are associated with the storage of the data to associated storage controllers. The storage devices are coupled via separate busses to their respective storage controllers and thus several storage devices may communicate with their associated controllers simultaneously. The data may now be delivered to the host controller from the two or more storage devices simultaneously thus drastically improving data throughput. The host controller receives the data from the two or more storage controllers and re-assembles the data into a contiguous file and delivers it to the requesting device.

This invention relates generally to storage systems associated with computer systems and more particularly to providing a method for improving the data throughput associated with a storage system during long sequential input/output (I/O) transactions.
As it is known in the art, computer systems generally include a central processing unit, a memory subsystem and a storage subsystem. According to a networked or enterprise model of a computer system, the storage subsystem associated with or in addition to a local computer system, may include a large number of independent storage devices or disks housed in a single enclosure. This array of storage devices is typically connected to several computers over a network. Such a model allows for the centralization of data which is to be shared among many users and also allows a single point of maintenance for the storage functions associated with computer systems.
One type of storage subsystem known in the art is one which includes a number of redundant disk storage devices configured as an array. Such a system is typically known as a RAID storage system. One of the advantages of a RAID type storage system is that it provides a massive amount of storage (typically in the several gigabyte range) and depending upon the RAID configuration may provide several differing levels of fault tolerance. Fault tolerance is typically achieved by providing, in addition to the disk devices that are used for storing the data, a disk device which is used to store parity data. The parity data may be used in combination with the remaining data on the other disk devices to reconstruct data associated with a failed disk device.
A disk storage system such as the RAID system described above will typically include one or more front end (or host) adapters/controllers which are responsible for receiving and processing requests from the various host devices which may be connected to the storage system. Additionally, a RAID storage system as described above may also include several disk adapters/controllers which are used to control the transactions between the disk storage devices and the host controller/adapter described above. Some storage systems may also include a very large buffer (e.g. a cache memory) for buffering the data transfers between the disk adapters and the host adapters.
In addition, a requirement of typical present day storage systems is that they present (or emulate) a particular storage geometry to the host computer. The geometry includes the configuration of the storage devices (i.e. number of cylinders, heads, sectors per track, etc.). The geometry presented to the host may not be the actual physical configuration of the storage devices in the system. As a result, some level of translation must be carried out between the emulated storage parameters and the physical storage parameters.
The problem associated with front-end emulation and back-end control is to relegate the two responsibilities in two separate processors. The front-end processor manages all that concerns operation of the front-end (host) controller. That is, it maintains information about the file system such as list of logical volumes and logical subdivisions such as cylinders, tracks, etc. The back-end processor manages tasks which are transparent to the host such as data mirroring, data striping, RAID protection, concurrent copy and others. As such, the back-end processor is typically loaded with much more functionality than the front-end processor.
In a RAID storage system as described above, the storage system (front-end processor) may be connected to the host via a bus which has different physical characteristics (e.g., data throughput, contention time, etc.) than the bus which connects the physical devices to the storage controller (back-end processor). For example, the host adapter may be coupled to the network (i.e., host or requesting systems) via a so called wide SCSI bus operating at speeds which allow the transfer of data up to 20 megabits per second. The host adapter is then typically coupled to either a cache or directly to a disk adapter via a communication bus which allows for data transmission rates which are at least as fast as the wide SCSI bus. The disk adapters, however, may be connected to the associated disk storage devices by a so called narrow SCSI bus which runs at half the speed as a wide SCSI bus (i.e., data rates up to 10 megabits per second).
It will be appreciated by those of skill in the art that for the configuration described above, the transmission rate mismatch may result in a performance bottleneck during long sequential input/output activity. That is, during a read of large amounts of sequential data, the associated disk device will transmit its data to the disk controller at a rate, which is half the speed at which the host controller can transmit the data to the requesting device. Thus the host adapter, and, as a result, the host device spend an inordinate amount of time waiting for data and thus wasting processing time. Similarly, when a host device needs to write a large amount of sequential data to a particular disk storage device, the host device will be able to transmit the data to the host adapter at a rate which is twice as fast as the rate at which the disk adapter can transmit the data to storage device. As a result the host devices and its associated bus would be stalled while it waited for the disk adapter to transmit the associated data to the disk.
Two attempts at solving the above problem have included the use of pipelining techniques or so called prefetch mechanisms. During pipelining, for a read operation, the host adapter will be begin transferring the associated data to the requesting host device before the entire read has been satisfied from the disk adapter into the associated buffer or cache. The objective of this technique is to tune the data transfer such that data is transferred to the host device by the host adapter in blocks of data such that one block has been completely transferred to the host device just after the disk adapter has finished placing the next sequential block of data into the cache for transfer. A similar scheme is employed during writes of data with the transfer from data to the disk device from the host adapter.
The second approach which utilizes a prefetch scheme involves the use of caching algorithm which is designed to minimize the occurrences of cache misses. To do so, large amounts of data are prefetched from the disk storage device into the cache with the belief that the data prefetched will be that data which is requested by the host device and thus eliminate a second transaction from the disk adapter to the disk storage device to place the data in the cache for transfer to the host device. The drawback to the prefetching scheme is that its effectiveness depends crucially on the size of the cache and assumes that the disk adapter is not continuously busy fulfilling I/O requests.
It would be advantageous, therefore, to provide a system which allows long sequential I/O transactions between a disk storage device and a host device to occur without the usual bottlenecks associated with such a transfer.
In accordance with the present invention, a method of performing input/output operations between a requesting device and a responding device, where the responding device includes a plurality of storage devices, includes the following steps. In response to receipt of a write access request by a requesting device, where the write access is a write of a sequential block of data, portions of the block of sequential data are stored on each of the plurality of storage devices. In response to receipt of a read access request by the requesting device for the previously stored block of sequential data, an access request is generated to each of the storage devices on which the portions of data are stored. Each of the portions of the block of sequential data is retrieved from each of the plurality of storage devices and the retrieved portions are assembled into the original block of sequential data and the retrieved portions are transmitted to the requesting device as a block of sequential data. With such an arrangement, data may be delivered to a requesting device at a constant rate without stalling even though the communications path to any one of the storage devices is slower than the communication rate of the requesting device.
In accordance with another aspect of the present invention, a storage system is provided which includes a plurality of storage devices each configured to store blocks of data. The storage system further includes a plurality of storage controllers where each storage controller is coupled respectively to one of the storage devices. Each of the storage controllers is operable to transmit and receive data to and from its corresponding storage device. The storage system further includes a request controller which receives from a requesting device an access request for a sequential block of data. The request controller is coupled to each of the storage controller and is responsive to the access request for generating an access request to each of the plurality of storage controllers having the portions of data associated with the requested sequential block stored thereon. With such an arrangement, a storage system is provided which allows for increased performance for transactions involving large amounts of sequential data since the data may be stored and subsequently retrieved from several individual devices rather than retrieved as a stream from a single device.

Method and apparatus for performing input/output operations between a requesting device and responding device