A sensing electrode for use in methods of electrochemical analysis comprising an electrically conducting electrode coated with an electroconductive polymer membrane having immobilised therein or absorbed thereto adaptor molecules avidin, streptavidin, anti-fitc antibodies through which the sensing electrode can be made specific for an analyte under test by the binding of receptors specific for the analyte.

The invention relates to methods of electrochemical detection of analytes and to sensing electrodes for use in methods of electrochemical detection.
Electrochemical analysis of analytes, such as various antigens, antibodies, DNA molecules etc, in biological fluids using biosensors is one of the most promising and attractive methods of instrument analysis. The sustained interest and large number of publications in this field are explained by a number of basic advantages of the method, namely high sensitivity, simplicity and the use of relatively simple and inexpensive equipment.
It is known in the art to construct biosensor devices based on the use of electroconductive polymer films, such as polypyrrole or polythiophene, which transduce a chemical signal associated with the presence of an analyte into a measurable electrical signal (see [1] and [2]).
EP-A-0 193 154 describes an electrode for use in electrochemical detection, the electrode being coated with a polypyrrole or polythiophene film. Bioreceptors complementary to the analyte to be tested are adsorbed onto the surface of the electroconductive polymer film after polymerisation. WO 89/11649 describes an alternative process for the production of polymeric electrodes for use in electrochemical assays. In this process bioreceptor molecules having the desired binding specificity are incorporated into a film of electroconductive polymer during polymerisation. Using the processes described in EP-A-0 193 154 and WO 89/11649 for each given assay it is necessary to synthesise a different sensing electrode having immobilized bioreceptors capable of specifically binding to the analyte for which one wants to test.
The applicants"" published application PCT/GB98/00548 describes a potentiometric method of electrochemical analysis using an electrochemical sensing electrode comprising a metallic potentiometric electrode again coated with a layer of electroconductive polymer containing immobilised bioreceptor molecules which bind specifically to the analyte under test. The presence of analyte is indicated by a change in surface charge of the sensing electrode upon binding of analyte to the immobilised bioreceptors. The analyte detection procedure is carried out by first assembling an electrochemical cell comprising the sensing electrode and a reference electrode connected together by means of a measuring device immersed in a working buffer solution of fixed pH. A base value of potential difference between the sensing electrode and the reference electrode is recorded, the sensing and reference electrodes are then brought into contact with a solution of higher ionic strength suspected of containing the analyte but with pH the same as the working buffer and potential difference is again recorded. The sensing and reference electrodes are finally transferred to clean working buffer and potential is again recorded. The change in potential difference between the sensing electrode and the reference electrode resulting from a change in ionic strength of the buffer at constant pH in the presence of analyte is proportional to the concentration of analyte.
As shown in references [13, 14, 15, 16] and in [3], the response (amount and rate of potential change) of the sensing electrode with polymer film containing bioreceptors to a step-change in ionic strength of the ambient solution (so-called xe2x80x9cion-stepxe2x80x9d procedure) is to a large extent determined by the charge on the polymer film. Apart from the material from which it is made, the polymer film charge is determined by the charge of the receptor molecules bound in it. If the receptor charge changes as a result of an affinity reaction with a specific analyte, the response of the sensing electrode will also change as a result of the ion-step procedure carried out after contact between the sensing electrode and the test fluid. It should be noted that, because of the amphoteric nature of the majority of analytes, the receptor charge depends on the pH of the solution and it is therefore very important to maintain a constant pH of the solution during the ion-step procedure.
Thus, in the previously described methods, based on measurement of the change in response of the sensing electrode to the ion-step procedure carried out before and after contact between the sensing electrode and test fluid, it is possible to make a determination as to the presence in the test fluid of analyte specific to the receptors bound on the sensing electrode. In the ideal case, the variation in the charge of the receptors in the membrane and, hence, the change in the sensing electrode response is directly proportional to the concentration in the test fluid of analyte specific to the receptors bound on the sensing electrode. However, in real conditions, the charge of the same analyte can vary considerably, which produces inconsistent quantitative results. Moreover, the affinity reactions are not always accompanied by a change in receptor charge. This normally occurs when testing small or non-charged antigens [14].
In summary, the shortcomings of the prior art methods of electrochemical detection based on the use of electroconductive polymer electrodes include complexity and limited amenability to industrialisation of the sensing electrode manufacturing process, inconsistency of the characteristics of the sensing electrodes obtained, limited ability to store the sensing electrodes without loss of performance. In addition, the previously described protocols for electrochemical detection, particularly the method described in PCT/GB98/00548, are of limited use for the detection of small and non charged molecules or molecules whose isoelectric point is close to the isoelectric point of the receptors immobilised on the surface of the sensing electrode.
The present invention provides a method of electrochemical analysis of an analyte in a sample which is to a large extent free of the shortcomings inherent in the methods described above in that it widens the scope of application by virtue of the ability to analyse small and non-charged molecules, provides strictly quantitative results, makes the electrode manufacturing process more amenable to industrial production methods, increases the productivity of the analysis, improves reproducibility and therefore enhances reliability of the results obtained.
Thus, in a first aspect the invention provides a sensing electrode for use in methods of electrochemical detection of an analyte, the sensing electrode comprising an electrically conductive electrode coated with an electroconductive polymer with adaptor molecules selected from the group consisting of avidin, streptavidin, anti-FITC antibodies and a molecule capable of binding to at least one class of receptor molecules immobilised therein or adsorbed thereto.
One of the principal problems inherent in electrochemical analysis methods using sensing electrodes is the problem of retention over time of the native properties of the receptors fixed on the sensing electrodes. Relative progress has been achieved in this field only for a limited number of enzyme sensing electrodes [7]. For the majority of electrochemical sensing electrodes using refined receptors known in the literature [8, 9, 10], their useful storage life is simply not stated. The retention of the native properties of immobilised receptors is particularly critical where antibodies are used as the receptors, which is attributable to their inherent high degree of conformational variability.
In contrast, it is known that antibodies and other biomolecules retain their useful properties over very long periods of time when stored in the form of concentrated solutions; therefore the problem of prolonged storage of the sensing electrodes without loss of working characteristics may be overcome by rapid immobilisation of receptors before use or even during the electrochemical detection procedure.
This problem is solved in the declared invention by use of so-called adaptor molecules which are immobilized in or adsorbed to the electroconductive polymer. The purpose of the adaptor molecules is to link receptor molecules specific to the analyte under test to the surface of the sensing electrode. As will be discussed below, with the use of adaptor molecules it is possible to temporally separate steps in the production of the sensing electrodes. Thus, it is feasible to manufacture the electrodes with immobilised/adsorbed adaptor molecules, store them for an extended period of time and then fix the specific receptors onto the electrode either before or during the electrochemical analysis. With the selection of appropriate adaptor molecules it is also possible to manufacture xe2x80x98universalxe2x80x99 sensing electrodes containing adaptor molecules capable of binding to a whole range of different receptor molecules. Specificity for the analyte under test is conferred on the xe2x80x98universalxe2x80x99 sensing electrode simply by binding to the adaptor molecules receptors of the appropriate specificity. It is therefore no longer necessary to incorporate receptors of the desired specificity during the electrodeposition process.
The proteins avidin and streptavidin are preferred for use as adaptor molecules. Avidin, a protein obtained from raw eggs, consists of four identical peptide sub-units, each of which has one site capable of bonding with a molecule of the co-factor biotin. Biotin (vitamin H) is an enzyme co-factor present in very minute amounts in every living cell and is found mainly bound to proteins or polypeptides. The ability of biotin molecules to enter into a binding reaction with molecules of avidin or streptavidin (a form of avidin isolated from certain bacterial cultures, for example Streptomyces aviation) and to form virtually non-dissociating xe2x80x9cbiotin-avidinxe2x80x9d complexes during this reaction (dissociation constant xcx9c10xe2x88x9215 Mol/l) is well known [11, 12].
Investigations carried out by the authors of the declared invention have shown that avidin and streptavidin immobilised in an electroconductive polymer film, retain their native properties for an extended period of time (at least one year and possibly longer) and can be used throughout this period to link with biotin conjugated receptors. Techniques which allow the conjugation of biotin to a wide range of different molecules are well known in the art. Thus sensing electrodes with immobilised avidin or streptavidin can easily made specific for a given analyte merely by binding of the appropriate biotinylated receptors.
Although avidin and streptavidin are the preferred adaptor molecules it is within the scope of the invention to use alternative adaptor molecules, in particular molecules capable of specifically binding to at least one class of receptor molecules. Included within this group of alternative adaptor molecules are protein A, protein G and lectins. These molecules all share the ability to bind to at least one class of receptor molecules, by which is meant that they are able to specifically bind to a common binding site motif which is present in each member of a group of receptor molecules, the dissociation constant for the binding interaction being less than 10xe2x88x928 Mol/l. By way of example, protein A (a 42 kD polypeptide isolated from Staphyloccus aureus or obtained by recombinant DNA technology) binds to immunoglobulins, particularly IgG, from a wide range of mammalian species at the Fc region; and protein G (IgG Fc receptor Type III, see Bjorck, L. and Kronvall, G., J. Immunol., 133, 969 (1984)) also binds to the Fc region of IgG molecules from a wide range of mammalian species. Lectins are proteins which bind to sugar moieties which may be present on glycoproteins or carbohydrates. Each type of lectin has specificity for a given sugar moiety and thus will be able to bind a range of glycoproteins or complex carbohydrates carrying the correct sugar moiety.
In a still further embodiment anti-FITC antibodies can be used as the adaptor molecules. In this embodiment, the specificity of the sensing electrode for analyte can be conferred by binding to the anti-FITC antibodies FITC labelled receptors of the appropriate specificity.
The use of adaptor molecules in/on the electroconductive polymer film also considerably improves the reliability of the results obtained during electrochemical analysis by reducing non-specific interactions of the components of the test solution during contact with the sensing electrode, which is linked to the blocking of the free surface of the electroconductive polymer by adaptor molecules. The use of adaptor molecules also increases the technical efficiency of the sensing electrode manufacturing process, for example by eliminating the need for an additional surface blocking procedure.
The potentiometric sensing electrodes of the invention are inexpensive to manufacture and so for convenience can be produced in a disposable format, intended to be used for a single electrochemical detection experiment or a series of detection experiments and then thrown away. The invention further provides an electrode assembly including both a sensing electrode and a reference electrode required for electrochemical detection. As will be discussed below, suitable reference electrodes include silver/silver chloride and calomel electrodes. Conveniently, the electrode assembly could be provided as a disposable unit comprising a housing or holder manufactured from an inexpensive material equipped with electrical contacts for connection of the sensing electrode and reference electrode.
The sensing electrodes of the invention can be used in a wide range of electrochemical analysis procedures, including (but not limited to) double antibody sandwich assays for antigens, double antigen sandwich assays for antibodies, competitive assays for antigens, competitive assays for antibodies, serological assays for the determination of human antibodies (e.g. Rubella IgG antibodies using labelled antihuman antibodies) and IgM assays (e.g. IgM-Rubella antibodies).
In a second aspect the invention provides a method of producing a sensing electrode for use in methods of electrochemical detection of an analyte, the sensing electrode comprising an electrically conductive electrode coated with an electroconductive polymer with adaptor molecules selected from the group consisting of avidin, streptavidin and a molecule capable of binding to at least one class of receptor molecules immobilized therein, the method comprising the steps of:
(a) preparing an electrochemical polymerisation solution comprising monomeric units of the electroconductive polymer and adaptor molecules,
(b) immersing the electrode to be coated in the electrochemical polymerisation solution, and
(c) applying a cyclic electric potential between the electrode and the electrochemical polymerisation solution to coat the electrode by electrochemical synthesis of the polymer from the solution, said cyclic electric potential being applied for at least one full cycle.
The invention further provides a method of producing a sensing electrode for use in methods of electrochemical detection of an analyte in a sample, the electrode comprising an electrically conductive electrode coated with an electroconductive polymer with adaptor molecules selected from the group consisting of avidin, streptavidin and a molecule capable of binding to at least one class of receptor molecules adsorbed thereto, the method comprising steps of:
(a) preparing an electrochemical polymerisation solution comprising monomeric units of the electroconductive polymer,
(b) immersing the electrode to be coated in the electrochemical polymerisation solution,
(c) applying a cyclic electric potential between the electrode and the electrochemical polymerisation solution to coat the electrode by electrochemical synthesis of the polymer from the solution, said cyclic electric potential being applied for at least one full cycle; and
(d) contacting the coated electrode with a solution comprising adaptor molecules such that the adaptor molecules are adsorbed onto the electroconductive polymer coating of the electrode.
According to the methods of the invention a film of electroconductive polymer is deposited onto the surface of an electrically conductive electrode by electrochemical synthesis from a monomer solution. The electrically conductive electrode is preferably a standard potentiometric electrode possessing metallic or quasi-metallic conductivity which is stable in aqueous media. As will be illustrated in the examples included herein, electrodeposition of the electroconductive polymer film is carried out using a solution containing monomers, a polar solvent and a background electrolyte. Pyrrole, thiophene, furan or aniline are the preferred monomers. Deionised water is preferably used as the polar solvent.
As is well krown to persons skilled in the art, electroccnductive molymers are often doped at the electrochemical synthesis stage in order to modify the structure and/or conduction Properties of the polymer. A typical dopant anion is sulphate (SO42xe2x88x92) which is incorporated during the polymerisation process, neutralising the positive charge on the polymer backbone. Sulphate is not readily released by ion exchange and thus helps to maintain the structure of the polymer. In the present invention it is preferred to use dopant anions having maximum capability for ion exchange with the solution surrounding the polymer in order to increase the sensitivity of the electrodes. This is accomplished by using a salt whose anions have a large ionic radius as the background electrolyte when preparing the electroehemical oolymerisation solution. Suitable salts whose anions knave large ionic radius include sodium dodecyl sulphate and dextran sulphate. The concentration of these salts in the electrochemical polymerisation solution is varied according to the type of test within the range 0.005-0.05 M.
As reported in a number of papers [4, 5], the ease with which ion exchange takes place and the rapidity with which ion equilibrium is attained for electroconductive polymers immersed in a solution are essentially dependent on the size of the dopant anion introduced at the electrodeposition stage: the larger the ionic radius of the dopant anion, the more readily ion-exchange reactions take place and the more rapidly a state of equilibrium is reached. This is directly linked to the valuse and rate of change of the potential of the xe2x80x9cmetal electrode-electroconductive polymerxe2x80x9d system in response to variation in the ion composition of the solution [6].
The electroconductive polymer membrane performs a dual function, serving both to bind the receptor to the surface of the sensing electrode, and to render the sensing electrode sensitive to variations in the composition of the buffer solution. In particular, changes in the composition of the buffer solution which affect the redox composition of the electroconductive polymer result in a corresponding change in the steady state potential of the sensing electrode.
Adaptor molecules may either be immobilized in the electroconductive polymer film at the electrochemical synthesis stage by adding adaptor molecules to the electrochemical polymerisation solution or may be adsorbed onto the surface of the electroconductive polymer film after electrochemical polymerisation. In the former case, a solution of adaptor molecules may be added to the electrodeposition solution immediately before the deposition process. The deposition process works optimally if the storage time of the finished solution does not exceed 30 minutes. Depending on the particular type of test, the concentration of adaptor molecules in the solution may be varied in the range 5.00-100.00 xcexc/ml. Procedures for electrodeposition of the electroconductive polymer from the solution containing adaptor molecules are described in the examples included herein. On completion of electrodeposition process, the sensing electrode obtained may be rinsed successively with deionised water and 0.01 M phosphate-saline buffer solution and, depending on the type of test, may then be placed in a special storage buffer solution containing microbial growth inhibitors or bactericidal agents (e.g gentamicin), or dried in dust-free air at room temperature.
Where the adaptor molecules are to be adsorbed after completion of the electrodeposition process the following protocol may be used (although it is hereby stated that the invention is in no way limited to the use of this particular method), the sensing electrode is first rinsed with deionised water and placed in freshly prepared 0.02M carbonate buffer solution, where it is held for 15-60 minutes. The sensing electrode is then placed in contact with freshly-prepared 0.02M carbonate buffer solution containing adaptor molecules at a concentration of 1.00-50.00 xcexcg/ml, by immersing the sensing electrode in a vessel filled with solution, or by placing a drop of the solution onto the surface of the sensing electrode. The sensing electrode is incubated with the solution of adaptor molecules, typically for 1-24 hours at +4xc2x0 C. After incubation, the sensing electrode is rinsed with deionised water and placed for 1-4 hours in a 0.1M phosphate-saline buffer solution. Depending on the type of test, the sensing electrode may then be placed either in a special storage buffer solution containing microbial growth inhibitors or bactericidal agents, or dried in dust-free air at room temperature.
When the adaptor molecules are avidin or streptavidin, the above-described methods of the invention for producing a sensing electrode may optionally comprise a further step of contacting the coated electrode with a solution comprising specific receptors conjugated with biotin such that said biotinylated receptors bind to molecules of avidin or streptavidin immobilised in or adsorbed to the electroconductive polymer coating of the electrode via a biotin/avidin or biotin/streptavidin binding interaction.
Research carried out both by the authors of the declared invention, and by others [12], has shown that the biotinylation of receptors under optimal conditions does not alter their properties (affinity, storage qualities, etc.) compared with their non-biotinylated equivalents.
Conjugation of biotin with the corresponding receptors, a process known to those skilled in the w art as biotinylation, can be carried out using one of the known procedures, for example as described in [12]. In addition, a number of ready-made preparations of biotinylated antibodies of different specificity are commercially available, e.g. Anti-Human IgG or Anti-Human IgA goat biotin-labelled antibodies made by Calbiochem-Novabiochem, USA.
One of the significant advantages of using biotinylated receptors is the ability to vary the specificity of the sensing electrode, by producing a reaction between the avidin or streptavidin bound on the sensing electrode and the corresponding biotinylated receptors. As discussed previously, the sensing electrode with bound avidin/streptavidin is in effect a xe2x80x98universal sensing electrodexe2x80x99 and specificity to the desired molecules under test is conferred by the binding of the appropriate biotinylated receptors. To make the sensing electrode with bound avidin or streptavidin specific to the analyte under test, a reaction is carried out between the avidin or streptavidin bound on the sensing electrode with biotinylated receptors, for which purpose the sensing electrode is brought into contact with a solution of the latter at room temperature, either by immersing the sensing electrode in a vessel filled with solution, or by placing drop of solution on the sensing electrode surface (concentration of biotinylated receptors in the solution is generally 0.1-100 xcexcg/ml; contact time 3-15 minutes).
The receptor molecules can be any molecule capable of specifically binding to another molecule (an analyte). Suitable types of receptors include monoclonal and polyclonal antibodies, chimaeric antibodies, fragments of antibodies which retain the ability to recognise antigen (e.g. Fab and Fab2 fragments), recombinant proteins and fragments thereof, synthetic peptides, antigens, single-strand DNA, RNA or PNA molecules, hormones, hormone receptors, enzymes, chemical compounds etc.
As discussed above, the electrochemical detection methods known in the art using potentiometric sensing electrodes are of limited use in the detection of small and non-charged antigens. In order to overcome this problem, and to obtain strictly quantitative results, use may be made of secondary receptors or competing molecules conjugated with a charge label.
Accordingly, in a further aspect the invention provides a method of electrochemical detection of an analyte in a sample, which method comprises the steps of:
(a) providing a sensing electrode having an electroconductive polymer coating, the coating having immobilised therein or adsorbed thereto receptors which specifically bind to the desired analyte to be detected in the sample;
(b) treating the sensing electrode by immersion in a test solution comprising the sample so that said desired analyte binds to said immobilised or adsorbed receptors;
(c) contacting the sensing electrode with a solution comprising secondary receptors capable of binding to said analyte at a site spatially distinct from the site of binding to the immobilised or adsorbed receptors, said secondary receptors being conjugated with a charge label;
(d) monitoring the electric potential difference between the treated sensing electrode and a reference electrode when both are immersed in an electrolyte; and
(e) monitoring the electric potential difference between the sensing electrode and a reference electrode following a change in the ionic strength of the electrolyte at constant pH.
The affinity reaction steps of the above-described method are equivalent to a standard sandwich assay well known to those skilled in the art. The sandwich format of analysis is particularly useful for the detection of polyvalent antigens in which case the receptors and labelled secondary receptors used in the test are antibodies which bind to different, spatially distinct epitopes on the antigen. The sandwich format can also be used where the antigen carries two or more identical epitopes which are spatially separated. In this latter case, the receptors and labelled secondary receptors used in the test may be antibodies of identical specificity.
It is also within the scope of the invention to perform the electrochemical analysis in a competitive assay format. Therefore, the invention also provides a method of electrochemical detection of an analyte in a sample comprising the steps of:
(a) providing a sensing electrode having an electroconductive polymer coating, the coating having immobilised therein or adsorbed thereto receptors which are capable of binding to the desired analyte to be detected in the sample;
(b) treating the sensing electrode by immersion in a test solution comprising the sample so that said desired analyte binds to said immobilised or adsorbed receptors;
(c) contacting the sensing electrode with a solution comprising competing molecules capable of binding to said immobilised or adsorbed receptors, said competing molecules being conjugated with a charge label;
(d) monitoring the electric potential difference between the treated sensing electrode and a reference electrode when immersed in an electrolyte; and
(e) monitoring the electric potential difference between the sensing electrode and a reference electrode following a change in the ionic strength of the electrolyte at constant pH.
In this competitive electrochemical assay the competing molecules could be labelled analyte or labelled structural analogs of the analyte which are capable of binding to the same analyte binding site on the immcbilized/adsorbed receptors (see FIG. 5B and FIG. 7B). The use of labelled analyte as the competing molecule is particularly preferred for the detection of small analyte molecules (e.g. digoxin, as described in the examples included herein). Alternatively, the competing molecule may bind to a different site on the immobilized/adsorbed receptor. For example, if the immobilized receptor is an antibody, the competing molecule could be an anti-immunoglobulin antibody (preferably Fab-specific) or even an anti-idiotype antibody of the appropriate specificity (see FIG. 5A and FIG. 7A).
As would be readily understood by persons skilled in the art, with reference to FIGS. 5A, 5B, 7A and 7B of the present application, the competitive detection methods are usually dependent on their being an excess of receptor sites on the surface of the sensing electrode. Those receptors which do not bind analyte will be available for binding to the competing molecule. Assuming that the total number of receptor sites remains constant, the amount of bound competing molecule will be inversely proportional to the amount of analyte present.
In order to transduce the chemical signal associated with the concentration of,the analyte into a measurable electrical signal the charge label which is conjugated to the secondary receptors or competing molecules can be any charge label having the following properties:
(i) carries a net charge (positive or negative) at the pH of the electrolyte of part (d); and
(ii) the magnitude of this charge changes in response to a change in the ionic strength of the electrolyte at constant pH.
Preferably the charge label is highly charged, i.e. has a net charge at the pH of the electrolyte of part (d) of greater than one electrostatic unit (e). Suitable charge labels include gold, ferrocene and latex microspheres. The magnitude of the charge on the charge label affects the redox composition of the electroconductive polymer coating on the sensing electrode such that the step change in ionic strength between steps (d) and (e) results in a detectable change in potential difference between the sensing electrode and the reference electrode. The charge label is only brought into close proximity of the electroconductive polymer on formation of receptor/analyte/secondary receptor complexes (sandwich assay) or receptor/competing molecule complexes (competitive assay). The use of a charge label thus makes it possible to obtain correct qualitative results and considerably extends the spectrum of testable analytes by virtue of the ability to test small and non-charged analytes.
Latex microspheres are the preferred type of charge label conjugated with the secondary receptors or competing molecules. Conjugation with latex microspheres may be carried out using one of the known techniques, for example as described in [17] or [18], or using special commercially available kits for the conjugation of antibodies with latex imicrospheres, for example xe2x80x9cCarbodiimide Kit for Carboxylated Beadsxe2x80x9d made by Polysciences Inc., USA, following the protocol supplied by the manufacturer. Certain ready-made latex conjugates are commercially available from specialist manufacturers, e.g. Polysciences Inc., USA.
As an alternative to the use of a charge label, it is also possible to perform electrochemical detection procedures equivalent to those described above using secondary receptors or competing molecules conjugated with an enzyme labels in order to transduce the chemical signal associated with the concentration of the analyte into a measurable electrical signal.
Accordingly, the invention further provides a method of electrochemical detection of an analyte in a sample, which method comprises the steps of:
(a) providing a sensing electrode having an electroconductive polymer coating, the coating having immobilized therein or adsorbed thereto receptors which are capable of binding to the desired analyte to be detected in the sample;
(b) treating the sensing electrode by immersion in a test solution comprising the sample so that the said analyte binds to said immobilized or adsorbed receptors;
(c) contacting the sensing electrode with a solution comprising secondary receptors capable of binding to said analyte at a site spatially distinct from the site of binding to immobilized or adsorbed receptors, said secondary receptors being conjugated with an enzyme;
(d) monitoring the electric potential difference between the treated sensing electrode and a reference electrode when both are immersed in an electrolyte; and
(e) monitoring the electric potential difference between the sensing electrode and a reference electrode following exposure to an electrolyte comprising the substrate for said enzyme.
Also within the scope of the invention is the corresponding competitive method of electrochemical detection comprising the steps of:
(a) providing a sensing electrode having an electroconductive polymer coating, the coating having immobilized therein or adsorbed thereto receptors which are capable of binding to the desired analyte to be detected in the sample;
(b) treating the sensing electrode by immersion in a test solution comprising the sample so that the said desired analyte binds to said immobilized or adsorbed receptors;
(c) contacting the sensing electrode with a solution comprising competing molecules capable of binding to said irmobilized or adsorbed receptors, said competing molecules being conjugated with an enzyme;
d) monitoring the electric potential difference between the treated sensing electrode and a reference electrode when both are immersed in an electrolyte; and
e) monitoring the electric potential difference between the sensing electrode and a reference electrode following exposure to an electrolyte comprising the substrate for said enzyme.
In one embodiment of the above-described methods the enzyme conjugated to the secondary receptors or competing molecules is capable of converting a substrate which directly affects the redox composition of the electroconductive polymer coating of the sensing electrode into a product which has no detectable effect on the redox composition of the electroconductive polymer.
In an alternative embodiment, the enzyme conjugated to the secondary receptors or competing molecules is capable of converting a substrate which has no detectable effect on the redox composition of the electrochemical polymer coating of the sensing electrode to a product capable of directly or indirectly affecting the redox composition of the electroconductive polymer. An example of such an enzyme is horseradish peroxidase. One way in which the product of the enzymic reaction may indirectly affect the redox composition of the electroconductive polymer is by causing a change in the pH of the electrolyte (for this embodiment the pH of the electrolyte is not buffered). An example of an, enzyme which generates such a product is urease.
In a still further embodiment, the enzyme conjugated to the secondary receptors or the competing molecules is capable of converting a product which has no detectable effect on the redox composition of the electroconductive polymer coating of the sensing electrode into a product which is a substrate for a second enzyme, the action of the second enzyme generating a second product which directly or indirectly affects the redox composition of the electroconductive polymer.
In all embodiments the conjugated enzyme is brought into close proximity of the electroconductive polymer by formation of receptor/analyte/secondary receptor complexes (sandwich format) or by formation of receptor/competing molecule complexes (competitive format).
The conjugation of secondary receptors or competing molecules with enzyme labels may be performed by any of the techniques known in the art (see, for example [19]). Use can also be made of widely available commercial preparations of conjugates of receptors of different specificity with different enzyme labels.
All of the above methods of electrochemical detection can be performed using any type of receptor capable of specifically binding to another molecule (an analyte). Suitable types of receptors include monoclonal and polyclonal antibodies, chimaeric antibodies, fragments of antibodies which retain the ability to recognise antigen (e.g. Fab and Fab2 fragments), recombinant proteins and fragments thereof, synthetic peptides, antigens, single-strand DNA, RNA or PNA molecules, hormones, hormone receptors, enzymes, chemical compounds etc.
Regardless of whether the secondary receptors or competing molecules are conjugated with a charge or enzyme label, the maximum degree of specificity and sensitivity for all of the detection methods of the invention is achieved by performing the affinity reactions (i.e. steps (a) to (c)) in a xe2x80x98sequentialxe2x80x99 format. This is particularly so when the analyte under test is a polyvalent antigen (i.e. the sandwich assay). In the sequential format the sensing electrode with bound receptors is first brought into contact with a test solution comprising the sample to be tested for the presence of the analyte. As used herein the term xe2x80x98samplexe2x80x99 includes within its scope any material which it is desired to test for the presence of analyte, including biological fluids such as whole blood, serum, plasma, urine, lymph, cerebrospinal fluid or semen, environmental fluids, materials used or produced in the food and drink industry or a dilution or extract of any of the above. The sample may also comprise a solution or extract of a solid material. The container used for the test solution may be the well of a microtiter plate, a micro-centrifuge tube or any other vessel of suitable size. The volume of test solution will generally be 5-200 xcexcl depending on the geometrical dimensions of the sensing electrode. The contact time between the sensing electrode and test solution is typically 3-30 minutes at 15-40xc2x0 C. with or without continuous mixing.
Following contact with the test solution the sensing electrode is transferred to a vessel containing solution of labelled secondary receptors. The vessel and volume of solution used are similar to those used for contact between the sensing electrode and test solution. The concentration of labelled secondary receptors or labelled competing molecules in the solution is typically 1-100 xcexcg/ml depending on the required sensitivity of the test. Contact is made for 3-30 minutes at 15-40xc2x0 C. with or without continuous mixing.
As an alternative to the xe2x80x98sequentialxe2x80x99 format it is possible to substantially reduce the total test time and simplify the test procedure by performing steps (b) and (c) simultaneously by contacting the sensing electrode with test solution to which has been added the appropriate labelled secondary receptors or labelled competing molecules for a contact time of about 5-60 minutes. The concentration of labelled secondary receptors or is labelled competing molecules added to the test solution is typically 1-100 xcexcg/ml depending on their type, specific features and required sensitivity of the test.
To eliminate possible non-specific interactions between the components of biological fluids under test and the surface of the sensing electrode, and also non-specific adsorption of labelled secondary receptors or labelled competing molecules onto the surface of the sensing electrode, which will distort the results obtained, various blocking agents may be added to the solution of labelled secondary receptors or labelled competing molecules. Suitable blocking agents include bovine serum albumin (0.5%-5%), human serum albumin (0.5-5 wt. %), dilute normal human or animal serum (5-10 vol. %), gelatin (10-50 vol. %), etc. In so doing, interaction of the labelled secondary receptors or labelled competing molecules with the sensing electrode is accompanied by simultaneous blocking of any free surface of the sensing electrode.
In all of the detection methods of the invention use can be made of sensing electrodes containing immobilized/adsorbed adaptor molecules. In particular, the receptor molecules may be attached to the surface of the sensing electrode via biotin/avidin, biotin/streptavidin, protein A/antibody, protein G/antibody, FITC/anti-FITC or lectin/sugar binding interactions.
The use of xe2x80x98universalxe2x80x99 sensing electrodes containing adaptor molecules allows the detection methods to be performed in a xe2x80x98one-potxe2x80x99 format. In this embodiment, the affinity reactions are performed in a homogeneous solution, providing maximum contact between the interacting molecules and ensuring maximum sensitivity and minimum duration of the test. In this case, the solution of receptors and the solution of labelled secondary receptors or labelled competing molecules are added simultaneously or sequentially to the test solution comprising the sample suspected of containing the analyte in a single reaction vessel. The concentrations of receptors and labelled secondary receptors or labelled competing molecules in the test solution are typically 0.1-100 xcexcg/ml and 1-100 xcexcg/ml respectively. The test solution is then incubated at 15-40xc2x0 C. for 5-60 minutes with or without continuous mixing to allow the binding reactions to take place. The sensing electrode containing the appropriate adaptor molecules is then brought into contact with the test solution, either by immersion into the vessel containing the test solution, or by placing a drop of test solution on the surface of the sensing electrode. The contact time between the sensing electrode and test solution is typically 3-30 minutes at 15-40xc2x0 C. Measurement of the amount of analyte bound on the sensing electrode is then performed using the xe2x80x9cion-stepxe2x80x9d procedure or by adding the appropriate enzyme substrate depending on whether the secondary receptors or competing molecules are labelled with a charge or enzyme label.
Once all the affinity reaction steps are completed, an electrochemical measuring cell is assembled by bringing the sensing electrode and a reference electrode, connected by a measuring instrument, into contact with an electrolyte solution (also referred to herein as a working solution) and the measuring device is used to record the sensing electrode potential relative to the reference electrode over a fixed time period. Commercially available reference electrodes of suitable size, or electrodes purpose-designed for implementation of the declared invention, may be used as the reference. The measuring instrument is a standard potentiometric measuring instrument or potentiostat. PC-compatible electronic measuring instruments purpose designed for implementation of the declared invention and controlled by custom software can also be used
For convenience the sensing electrode and reference electrode can be linked to the measuring instrument by means of a special holder equipped with electrical contacts for connection of the sensing electrode and reference electrode and connected to the measuring instrument by a cable or other means. A holder integral with the measuring instrument could also be used, making it possible to miniaturise the measuring system in terms of its overall dimensions.
Aqueous buffer solutions are used as the working solution: phosphate-saline, Tris-HCl, carbonate bicarbonate, acetate, borate, etc. The volume of working solution in the electrochemical cell is typically between 50 and 5000 xcexcl depending on the geometrical dimensions of the sensing electrode. The container for the buffer solution may be any suitably sized vessel in a material with minimal adsorption properties, e.g. the well of a standard microtiter plate. Another embodiment of the declared invention is a variant in which a low-volume ( less than 1 cm3) flow-through cell is used in conjunction with an integral holder for the sensing electrode and reference electrode, through which buffer solution can be pumped by means of a peristaltic pump or other means.
The potential of the sensing electrode relative to the reference electrode potential is recorded for a fixed time period using a chart recorder connected to a potentiometric measuring device or potentiostat, or by means of a special program where PC-compatible electronic instrumentation is used. In the latter case, the program measures the sensing electrode potential relative to the reference electrode potential at pre-determined time intervals (typically every 3-5 seconds for a total of 10-100 seconds) and displays the results in the form of points on the coordinates xe2x80x9csensing electrode signal-timexe2x80x9d. Recording of sensing electrode potential relative to the reference electrode potential is carried out to determine the background potential value V1 of the sensing electrode, and also to evaluate the background potential drift (xcex3) of the sensing electrode, which is calculated by linearisation of the curve xe2x80x9csensing electrode signal-timexe2x80x9d obtained using the least squares method.
If the secondary receptors or competing molecules are conjugated with a charge label, the amount of analyte bound to the sensing electrode is evaluated by changing the ionic strength of the electrolyte solution at constant pH, the so-called xe2x80x9cion-stepxe2x80x9d procedure.
In the ion-step procedure the ionic strength of the electrolyte solution may be modified (upwards or downwards) either by transferring the holder complete with sensing electrode and reference electrode from the initial working solution into a second working solution of the same composition but with a different ionic strength, or by adding a buffer solution of different (higher or lower) ionic strength directly to the working solution in which the sensing electrode and reference electrode are immersed. If a flow through cell is used, the ionic strength of the electrolyte solution can be modified by expelling the initial working solution from the cell using a buffer solution of different ionic strength.
Working solutions having different ionic strengths may be achieved by having different concentrations of salts, e.g. KCl, Na2SO4, etc., the use of which is based on the fact that they dissociate completely when added to the solution and do not bias the pH of the solution. The concentration of salts in the working solution ranges from 0.01 to 0.1 M.
If the secondary receptors or competing molecules are conjugated with an enzyme it is not necessary to perform the xe2x80x9cion-stepxe2x80x9d procedure. Instead, the composition of the working solution is modified by adding a suitable substrate for the enzyme. To this end, either the holder complete with sensing electrode and reference electrode can be transferred from the vessel containing the initial working solution to a vessel containing working solution plus substrate, or the substrate solution can be added directly to the original working solution in which the sensing electrode and reference electrode are immersed. If a flow-through cell is used, the composition of the working solution can be modified by expelling the initial working solution from the cell using a working solution containing substrate.
Substrates which may be used include ABTS ({2,2xe2x80x2-Azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid]}), TMB (3,3,5,5xe2x80x2-Tetraethylbenzidine), DAB (3,3xe2x80x2 Diaminobenzidine) (where the enzyme label is peroxidase), urea (where the enzyme label is urease), p-NPP (p-Nitrophenyl Phosphate), BCIP (5-bromo-4-chloro-3-indolylphosphate) (where the enzyme label is alkaline phosphatase).
The variation in sensing electrode potential relative to the reference electrode potential in response to a step change in ionic strength of the working solution or addition of an enzyme substrate is recorded for a fixed time period using a measuring instrument. Again, the recording is made either using a chart recorder connected to a potentiometric measuring device or potentiostat, or by means of a special program where PC-compatible electronic instrumentation is used. In the latter case, the program measures the sensing electrode potential relative to the reference electrode potential at pre-determined time intervals (typically every 3-5 seconds) and displays the results in the form of points on the coordinates xe2x80x9csensing electrode signalxe2x80x94timexe2x80x9d. Depending on the particular type of test, the time taken to record the variation in sensing electrode potential relative to reference electrode potential varies between 30 and 600 seconds.
In the case where the ionic strength of the buffer solution is changed, the curve obtained for the variation in sensing electrode potential relative to the reference electrode potential usually takes the form of a parabola, and represents the response of the sensing electrode to the change in ionic strength of the buffer solution, which is modulated by the total charge (isoelectric point) of the electroconductive polymer film.
If the analysis is performed as a sandwich assay, the variation in total charge of the polymer film is directly proportional to the quantity of the analyte under test. However, if the analysis is performed as a competitive assay, the variation in total charge of the polymer film is generally inversely proportional to the quantity of analyte under test.
On completion of this stage in the procedure, the final value V2 of sensing electrode potential relative to reference electrode potential is determined. The following quantitative characteristics of the change in sensing electrode potential relative to the reference electrode potential can then be calculated:
1. the area (integral) described by the curve obtained for the change in sensing electrode potential relative to reference electrode potential, S2:       S    2    =            ∫      T1      T2                          f        2                          xe2x80x83                          (        t        )                          xe2x80x83                          ⅆ        t
xe2x80x83where: T2xe2x88x92T1=total time period for recording of background potential drift or potential relative to the reference electrode; ƒ2xe2x80x94curve of xe2x80x9csensing electrode potential in millivolts versus timexe2x80x9d; txe2x80x94current recording time; and
2. difference in millivolts between the background and final potential of the sensing electrode:
xcex4=V2xe2x88x92V1
Based on the quantitative characteristics of the variation in sensing electrode potential in response to a change in ionic strength or composition of the working solution, a determination is made as to the quantitative content of target analyte in the test solution.
Using the values for xcex3, S2 and/or xcex4 obtained as described above it is possible to re-calculate the values to allow for the zero line drift xcex3, yielding the values S2xcex3 and/or xcex4xcex3, on the basis of which a determination is made of the quantity of target analyte in test solution. The corrected values S2xcex3 and xcex4xcex3 may be compared with a calibration curve of xe2x80x9canalytical result versus amount of target analytexe2x80x9d. As would be readily apparent to persons skilled in the art, data for construction of a calibration curve can be obtained in a manner similar to the procedure described above using a range of test solutions containing known amounts of the target analyte.
In a still further aspect the invention provides a method of electrochemical detection of an analyte in a sample, which method comprises the steps of:
(a) providing a sensing electrode comprising an electrically conductive electrode coated with a layer of electroconductive polymer with avidin or streptavidin immobilized therein or adsorbed thereto, said avidin or streptavidin molecules being attached to receptor molecules capable of binding the analyte to be detected attached via a biotin/avidin or biotin/streptavidin binding interaction;
(b) contacting the sensing electrode with a test solution comprising the sample so that said desired analyte binds to said immobilized or adsorbed receptor molecules;
(c) monitoring the potential of the sensing electrode relative to a reference electrode when both are immersed in an electrolyte; and
(d) monitoring the potential difference of the sensing electrode relative to the reference electrode following a change in the ionic strength or composition of the electrolyte at constant pH.
No This method of electrochemical detection is of use where the binding of the target analyte to the receptor is causes a change in charge on the surface of the sensing electrode which is sufficiently large to be measurable without the need for a separate charge or enzyme label. In particular, this method is useful in the electrochemical detection of nucleic acids. Hybridisation of target nucleic acids to nucleic acid probes (e.g. oligonucleotides) attached to the surface of the sensing electrode is accompanied by a change in charge sufficient large to be detectable by the xe2x80x9cion-stepxe2x80x9d procedure. There is thus no need to use secondary receptors or competing molecules conjugated with charge label. Materials suspected of containing specific nucleic acids (e.g. biological fluids) may commonly be subjected to an amplification step (e.g. PCR) before being subjected to the detection procedure. It is therefore within the scope of the invention to perform the electrochemical detection of specific nucleic acids on samples which have been subjected to an amplification procedure.

Method of electrochemical analysis of an analyte


A chuck according to an embodiment of the present invention includes first and second support portions 10 each of which has a repetition structure of a convex portion 11 and a concave portion 13 on a base. A substrate is held by bringing each convex portion 11 into contact with the substrate and exhausting gas in each concave portion 13 such that the concave portion 13 has a negative pressure with respect to a space between the first support portion 10 and the second support portion 10. At least one support portion of the first support portion 10 and the second support portion 10 includes at least four convex portions 11 and three concave portions 13.

In a substrate-holding apparatus with which a lithography apparatus or another apparatus is equipped, good flatness of a substrate needs to be maintained to focus exposure light accurately.
PTL 1 discloses that the upper surface of a chuck is divided into rectangular sections by using a support portion that includes two convex portions and a concave portion therebetween. PTL 1 discloses that gas in the concave portion is exhausted to hold a substrate, and the amount of gas that is supplied to and exhausted from the rectangular sections is controlled in accordance with the result of measurement of the flatness of the substrate held, so that the flatness of the substrate is corrected.

Chuck, substrate-holding apparatus, pattern-forming apparatus, and method of manufacturing article


In the method for manufacturing a fluid dynamic bearing, the fluid dynamic bearing that lubricant is not injected into is prepared in a predetermined work space. The pressure in the work space is reduced. A delivery nozzle that delivers the lubricant is inserted into the storage region. The lubricant is delivered in the storage region so that the lubricant does not spill over from the storage region. The pressure in the work space is restored. Another way is that a lubricant is ejected after an oil repellent region for preventing the lubricant from leaking out is covered.

1. Field of the Invention
The present invention relates to a method for manufacturing a fluid dynamic bearing, a fluid dynamic bearing manufactured by the method, a motor that comprises the fluid dynamic bearing, and a disk drive device that comprises the motor. In particular, the present invention relates to technique to fill lubricant in a fluid dynamic bearing.
2. Description of the Related Art
A magnetic-disk drive device such as a hard disk drive and an optical-disk drive device such as a CD (Compact Disc) device and a DVD (Digital Versatile Disc) device are known as devices for recording and reading digital data. These devices comprise a motor for rotating a disk. Lately, the fluid dynamic bearing has been adopted in these motors in many situations.
Various methods for manufacturing the fluid dynamic bearing are proposed, such as a method disclosed in Japanese Patent Application Publication No. 2002-5170, a method disclosed in Japanese Patent Application No. 2005-98393, and a method disclosed in Japanese Patent Application Publication No. 2005-273908.

Method for manufacturing a fluid dynamic bearing, a fluid dynamic bearing, a motor, and a disk drive device

Samsung Electro-Mechanics Japan Advanced Technology Co., Ltd.


A method for treating patients with renal failure includes administering to them an effective amount of antibody or of a functional equivalent thereof to at least two of urea, creatinine, tumor necrosis factor alpha, interferon gamma, interleukin 6 and interleukin 1 beta. Soluble cytokine receptors also can be employed. The method can be used as a supplement to or as partial or complete replacement for dialysis. A pharmaceutical composition includes antibody or functional equivalent thereof to urea, creatinine, or both; antibody, functional equivalent or soluble cytokine receptor to tumor necrosis factor alpha, interferon gamma, interleukin 6, interleukin 1 beta or any combination thereof The composition can be included in a kit.

Chronic renal disease is a world-wide problem. In the United States, there were over 400,000 patients enrolled in Medicare-funded end-stage renal disease programs at the end of 2002. It is projected that by 2010, more than 650,000 patients will be enrolled in such programs, at a cost of over $28 billion a year. In spite of improvements in dialysis therapy, patients often experience a decline in quality of life. Mortality and morbidity continue to be significant.
Renal failure is associated with an inability of the kidneys to excrete large loads of electrolytes and other substances. As a result, patients often experience generalized edema, acidosis, high concentration of non-protein nitrogen compounds, especially urea, and high concentrations of other urinary retention products including creatinine. This condition is known as uremia.
Chronic kidney disease also is associated with the malnutrition, inflammation and atherosclerosis (MIA) syndrome. In fact, atherosclerotic cardiovascular disease is a leading cause of morbidity and mortality in end-stage renal disease (ESRD) patients. In many cases, ESRD patients also do not respond to erythrorpoietin therapy employed to manage their anemia.
It is well established that ESRD is a state of chronic systemic inflammation. The kidney is an organ that not only excretes cytokine metabolites but also contributes to cytokine production through its endocrinal function. It has been reported that the kidney may play a role in handling the clearance of pro-inflammatory cytokines. Even mild to moderate reduction in glomerular filtration rate appears to result in increased levels of circulating pro-inflammatory cytokines.
The hemodialysis procedure itself contributes to an inflammatory response and mortality in dialysis patients is higher than that in the general population across all age groups. Hemodialysis patients suffer from chronic inflammation for reasons such as potential blood contamination from endotoxin in the dialysis fluid, repeated contact of blood with artificial materials in the extracorporeal circuit, infections related to vascular access problems and so forth.
An approach for the non-specific elimination of circulating cytokines by continuous renal replacement therapies (CRRT) was proposed but reported controversial. In addition, it was found that treatment with oral pentoxyfilline could reduce T-cell expression of TNF-α and IFN-γ. The use of steroids, thalidomide, anti-thymocyte globulin, anti-lymphocyte globulin and of the anti-CD3 monoclonal antibody OKT3 has been suggested. Inhibitors of TNF-α have been approved by the U.S. FDA as therapies for patients with various autoimmune diseases including rheumatoid arthritis, ankylosing spondylitis, psoriasis and psoriatic arthritis and Crohn's disease; their use in systemic inflammatory response syndrome (SIRS) and for reversing the inflammatory process in erythropoietic resistance has been suggested. IL-1 receptor antagonist has been approved by U.S. FDA to treat rheumatoid arthritis and anti-IL-6 receptor antibody has been tested in patients with rheumatoid arthritis with good results. In addition, a humanized monoclonal antibody to IFN-γ is now in clinical testing for the treatment of Crohn's disease.

Method and composition for treatment of renal failure with antibodies and their equivalents as partial or complete replacement for dialysis


For recognizing shadowing events affecting a photovoltaic module, electric power produced by the module is recorded. For each position of the sun on a present day an expected value of the electric power is defined. Further, an ideal power course of the electric power over the present day is determined by fitting a curve that corresponds to cloudless sun without shadow casting obstacles to peak values of the electric power recorded for same positions of the sun during a plurality of previous days. For all positions of the sun at which the electric power produced on the present day falls short of the ideal power course a shadowing probability of not less than zero is defined whose magnitude depends on the level of accord of the electric power produced on the present day with the expected value at the position of the sun.

German utility model publication DE 201 02 619 U1 discloses a display for a solar power system. In the display, a day target curve of the power produced by the solar power system is calculated. This day target curve is then continuously fitted to the actually possible power of the particular solar power system by learning under error-free conditions. A precondition for this procedure is an additional measurement of the solarization intensity actually present. The first day target curve is calculated depending on the location of the solar power system (latitude and longitude) and the seasonal position of the sun which is adjusted daily or monthly. In this way, environmental limitations to the optimum orientation of the photovoltaic modules of the solar power system have an impact on the first daily curve. If, after the learning phase, the actual value of the power produced by the solar power system differs from the learned target curve by more than an adjustable degree, this is pointed out to the user. In the known display, repeated shadowing events affecting the photovoltaic modules of the solar power system have an influence on the learned target curve. However they are neither recognized as such nor assessed with regard to their importance.
A method of controlling a solar power system comprising several photovoltaic modules is known from DE 10 2006 008 178 A1. Here, comparative measurements between the individual photovoltaic modules are performed for a permanent power control. If deviations between the measurement values occur, these are interpreted as an indication that the photovoltaic system needs a revision. With regard to the deviations occurring, it shall be differentiated between whether it is an environmental disturbance like shadowing by clouds, trees or immovables, or an irreversible mechanical or electrical damage. Details enabling this differentiation, however, are not found in DE 10 2006 008 178 A1.
There still is a need for a method which enables recognizing shadowing of a photovoltaic module with high reliability and which thus provides a basis for suitably assessing the consequences of the respective shadowing.

Method of recognizing and assessing shadowing events


An apparatus and method for an articulating microsurgical instrument is disclosed herein. The articulating microsurgical instrument may be configured to be operable with a Doppler probe, bone grasper, soft tissue grasper/dissector, scissors, flexible forceps, or a suction/irrigation line configured to provide tools within a surgical location that can be adjusted to a desired angle of operation. A tip assembly may comprise an articulating portion at a distal tip and the articulating portion may be configured to deflect upon actuation of an articulation control. The articulation control may be a trigger assembly or a roller wheel. A bayonet-style handle may include a set of posts configured to interact with the one or more control wires during actuation of the articulation control. One or more control wires may be housed in a lumen and actuated using a articulation control of a handle assembly.

Articulating microsurgical instruments may be implemented in various types of surgeries, including neurosurgery, ear nose and throat (ENT) procedures, and cardiovascular procedures, for example. A surgeon, by holding the hand piece in their hand, can manipulate the working end to perform a desired task. Some conventional medical instruments utilize a flexible needle along with an ultrasound device external to the patient to measure spatial location of echos that contain a Doppler shift.


An apparatus diverts the flow of bulk solids into a bin from a discharge of a bulk solids conveyor. The apparatus contains a fitting that attaches to the discharge, a frame with a vertical conduit, at least two baffles that pivot on rotating shafts and are capable of variable positioning between a substantially vertical position that opens the vertical conduit of the frame and a substantially horizontal position that substantially closes the vertical conduit of the frame, and a drive motor to rotate the baffles.

Bulk solids, also known as bulk granular materials, are commodities that have relatively small particle sizes and are typically transported unpackaged in large quantities by ship, rail, or truck. Examples of bulk solids are grains, products from grains, and chemicals.
Bulk solids are handled (moved from container to container) using a variety of conveying equipment. Commonly used conveying equipment include conveyor belts, screw conveyors (also known as augers), drag flight conveyors, and pneumatic systems. Bulk solids are often loaded into bins by moving them to a central point above the bin and discharging them to pour down by gravity into the bin. The term "bin" is used herein to refer to containers of any type for bulk solids, whether resting on the ground or part of ships, rail cars, or trucks, and includes tanks, silos, hoppers, etc.
When bulk solids are poured onto a horizontal surface, a conical pile is formed. The internal angle between the surface of the pile and the horizontal surface is known as the angle of repose and is a function of the density, shape, surface area, and coefficient of friction of the bulk solids. Many bulk solids have angles of repose in the range of 30 to 45 degrees.
When bulk solids are poured into a bin from the discharge of a conveyor that is positioned in a central overhead location, the pouring must stop when the top of the conical pile reaches the top of the bin. Due to the angle of repose, unfilled space remains in the bin around the top of the perimeter. This wasted space often represents five or more percent of the total volume of the bin. To reduce the volume of wasted space, workmen sometimes manually shovel the solids into the voids. However, this is a slow and labor intensive process.
Baffles are used in various types of equipment to provide mixing and dispersion of solids. Pieces of equipment with stationary baffles are disclosed in McLemore, U.S. Pat. No. 2,515,725, Jul. 18, 1950; Girardi, U.S. Pat. No. 3,258,142, Jun. 28, 1966; and Connelly et al., U.S. Pat. No. 5,316,429, May 31, 1994. Dillman, U.S. Pat. No. 4,264,213, Apr. 28, 1981, discloses an asphalt mixing apparatus with two separate, manually movable baffles in a chute that improve mixing. None of these pieces of equipment contains a flow diverter having multiple baffles that move in coordination.
Accordingly, there is a demand for an improved apparatus for diverting the flow of bulk solids into a bin from a central overhead discharge.


A system and method for eliminating undercut when forming a C4 solder bump for BLM (Ball Limiting Metallurgy) and improving the C4 pitch. In the process, a barrier layer metal stack is deposited above a metal pad layer. A top layer of the barrier layer metals (e.g., Cu) is patterned by CMP with a bottom conductive layer of the barrier metal stack removed by etching. The diffusion barrier and C4 solder bump may be formed by electroless plating, in one embodiment, using a maskless technique, or by an electroplating techniques using a patterned mask. This allows the pitch of the C4 solder bumps to be reduced.

1. Field of the Invention
The present invention relates generally to the fabrication of semiconductor chips, and more particularly to a structure and novel methodology for fabricating a reliable, dimensionally accurate, high-integrity Ball Limiting Metallurgy (BLM) without undercut in Back-End-Of-Line (BEOL) semiconductor chip processing.
2. Description of the Prior Art
Controlled Collapse Chip Connection (C4) processes are well known in forming solder bumps in back-end-of line semiconductor fabrication, e.g., when chips are connected to their packaging. Typically, the formation of a C4 solder bump includes the conventional formation of a metallurgical system that includes the underlying final metal layer (pad), an under "bump" or Ball Limiting Metallurgy (BLM) and the solder ball. The BLM ideally should provide good adhesion to a wafer passivation and to the IC final metal pad, and, function as an effective solder diffusion barrier.
The drive to Lead-reduced and lead-free C4 processing together with reduced-pitch requirements limit the extendibility of current electroetch BLM technology, which routinely produces a final structure in which the baselayer BLM films (Ti, Cu) are dimensionally inset or "undercut" with respect to the outer dimension of the C4 solder ball. A typical process BLM structure for leaded C4 is comprised of an approximate ˜0.15 μm TiW base layer which underlies a film of CrCu of similar thickness over which resides a copper layer that is approximately ˜0.45 μm thick. A resist solder mask is used to electroplate the C4, and following a resist strip operation, the Cu/CrCu/TiW BLM is typically wet-etched using an electroetch process, with the C4 solder ball serving as the etch mask. This BLM technology can be used successfully with Pb-reduced (97/3 Pb/Sn) solder and C4 pitch that is no less than 4 on 8 mil (about 100 μm×200 μm). However, in current 65 nm technology, there is a requirement for C4 designs that are laid out on 3 on 6 mil pitch and which use both Pb-Reduced and Pb-Free C4 technology. The simple reduction in pitch from 4on8 to 3on6 minimizes contact area of the BLM to the final passivation film around the via such that undercut of the baselayer BLM films is no longer acceptable. The final via is set at ˜47 μm by electrical considerations, and there is a reliability constraint that the BLM must never be reduced in dimension sufficiently to expose the via sidewall. With an electroetch undercut that is typically 10-15 μm per edge, this constraint can no longer be met with 3on6 pitch. FIG. 1 illustrates the current BLM process and the wet etch undercut problem.
Particularly, FIGS. 1A-1D depict the BLM process steps according to the prior art.
As shown in FIG. 1A, there is depicted an underlying insulating layer 10, e.g., a fluorine-doped silicon oxide layer (FSG layer) having the final copper metallization layer 19 formed therein. Formed on top of the FSG layer 10 is the further insulator stack 11 comprising, for example, SiN, SiO2 and SiN layers. Formed thereabove and contacting the underlying Cu metallization 19 by a contact via is a metal bonding pad, e.g., Aluminum, and final passivation layer 12, e.g., polyimide. In FIG. 1A, there is depicted the process step of etching a final via 15 in the polyimide passivation layer 12. Then, as shown in FIG. 1B, current fabrication techniques implement Pb-free C4's using plating of the solder in a photoresist pattern, followed by wet etching of the BLM 20. In the prior art solder bump plating process, the BLM 20 includes the deposition of an adhesion layer 21, e.g., a titanium-tungsten alloy (TiW), followed by wetting layers 22 of, e.g., Cr—Cu (chromium-copper alloy) and copper (Cu). The wetting layers ensure the solder completely covers the patterned Ti—W adhesion layer (thereby ensuring a large contact area between the solder ball and the chip, and providing high mechanical strength. In the solder bump plating process, the wafer is cleaned to remove oxides or organic residue prior to metal deposition and to roughen the wafer passivation and bond pad surface to promote better adhesion of the BLM. The BLM barrier layer metals such as TiW, Cr—Cu, and Cu may then be sequentially sputtered or evaporated over the entire wafer so that the BLM 20 adheres to the wafer and passivation in addition to the bond pads. Next, a photoresist layer 30 is applied and then metal layers 40 (e.g., a Ni barrier layer followed by a C4 Sn-based solder) are electroplated over the bond pad to a height as determined by the patterned photoresist 30.
Then, as shown in FIG. 1C, after the solder bump is formed, the photoresist is stripped, leaving the BLM exposed on the wafer. The BLM 20 is subsequently etched from the wafer whereby the CrCu layer 22 is removed by electroetching, and the TiW layer 21 removed by using a wet etch process (e.g., an H2O2-based wet etch). FIG. 1C depicts the undercut 50 of approximately 10 μm-15 μm that results at each edge of the BLM due to the applied electroetch. Finally, as shown in FIG. 1D, the solder bump 40 is reflowed to form the Pb-free or Pb-reduced solder ball 40′.
For Pb-free and Pb-reduced technology, the BLM itself must normally include a top barrier layer to prevent consumption of the copper in the BLM layer by tin in the solder. Nickel is commonly used for this, in a thickness of 1-3 um. Pb-Free BLM technology is still being developed in the industry, but one Pb-Free BLM that is available comprises a stack of TiW—Cu—Ni, with the TiW layer about 0.15 μm in thickness, Cu layer from about 0.5 μm-2.0 μm in thickness, and the Ni layer about 1 μm in thickness. The TiW and Cu films are typically deposited by PVD, and the Ni is normally electroplated.
It is the case though, that problems associated with undercut of the BLM during wet electroetching of the Cu and TiW base layers exist. For example, the final structure such as shown in FIG. 1D, is at risk for reliability failure due to the minimal effective contact attach area of the remaining TiW base layer to the final passivation (polyimide) film 12, and/or with respect to the voids created by undercut 50 at each edge of the structure, which can act to entrap contaminants that act as stress nucleation sites for failure during thermal cycling.
While a partial prior solution to this problem that might include wet etch of the copper by electroetch, followed by TiW RIE to pattern the TiW base layer without undercut beneath the Cu layer is theoretically possible, in practice it does not work because of incomplete cleaning of the electroetched Cu layer in the field areas over the TiW film. The electroetch process invariably leaves minute residues which act to micromask the TiW film during the RIE, resulting in metallic residues between C4s that are of sufficient density to cause electrical shorting. This problem has proven to be unavoidable when Cu electroetch or wet-etching is used for BLM patterning.
It would be highly desirable to provide a C4 fabrication technique that results in an improved C4 pitch by eliminating the BLM undercut by a novel fabrication process that consequently increases the mechanical stability of the formed solder bump.

Undercut-free BLM process for Pb-free and Pb-reduced C4

International Business Machines Corporation


A method for hardfacing a metal component surface (14, 16), especially a shroud surface of a turbine blade made of a TiAl alloy, with at least one metal material (18, 20), in particular a Co—Cr alloy. The hardfacing coating is produced separately from the component surface and is then joined to the component surface in a high-temperature soldering process. A turbine blade including such a hardfacing coating, primarily in a shroud region (2).

The blades of low-pressure turbines are frequently made of nickel-based alloys or superalloys, such as, for example, IN 713, MAR 227 and B 1900. In order to reduce abrasion, the Z-shaped contact faces of their shrouds are usually hardfaced with cobalt-chromium alloys (Co—Cr alloys or Stellites®). The height of the hardfacing is typically 2 mm in the finished state. Methods typically used for producing the hardfacing are tungsten inert gas welding, micro-plasma welding and laser-beam welding. However, if the turbine blades are made of titanium aluminide material (TiAl), they cannot be provided with a Stellite® hardfacing, because this may cause the titanium aluminide to mix with the Stellite®, as a result of which brittle phases and cracks may form in the hardfacing and the titanium aluminide base material of the shroud.


Disclosed are flashlights that optimize the light output and beam focus of both a flood beam and a spot beam. The disclosed flashlights include a housing member and a lens including a first focusing element adapted to shape a light beam into a spot beam and a second focusing element adapted to shape a light beam into a flood beam. A first LED is positioned to direct light through the first focusing element, and a second LED positioned to direct light through the second focusing element. The first LED has a smaller die than the second LED, and the distance between the first LED and the first focusing element is greater than the distance between the second LED and the second focusing element.

Flashlights have been produced that allow adjustment of the light beam in a continuous range of beam widths and/or focal distances ranging from a flood beam to a narrow beam, and vice versa. However, such systems typically compromise either the light output of the flood beam or the narrow focus (e.g., beam distance) of the spot beam. For instance, the brightness of the flood light may be compromised in order to achieve a long beam distance with the spot beam, or the beam distance of the spot beam may be compromised in order to achieve more light output in the flood mode, or both.


A fastening tab is provided having a substrate with a main portion, a user's end portion extending from the main portion, a primary mechanical fastening patch having a side edge coterminous with the side edge of the user's end portion of the substrate, and first and second auxiliary mechanical fastening patches on the main portion of the substrate. A method of making a fastening tab is also disclosed. The method includes attaching multiple discrete patches of mechanical fastener to a substrate web extending in the machine direction, cutting through the substrate web and the multiple patches of mechanical fastener in the machine direction with a continuous, meandering cut to provide two sub-webs, and optionally providing cross-web direction cuts through each sub-web to provide a plurality of fastening tabs. An absorbent article including the fastening tab and webs of fastening tabs are also included.

Mechanical fasteners, which are also called hook and loop fasteners, are useful for providing releasable attachment in numerous applications. For example, mechanical fasteners are widely used in wearable disposable absorbent articles to fasten such articles around the body of a person. In typical configurations, a hook strip or patch on a fastening tab attached to the rear waist portion of a diaper or incontinence garment, for example, can fasten to a landing zone of loop material on the front waist region, or the hook strip or patch can fasten to the backsheet (e.g., nonwoven backsheet) of the diaper or incontinence garment in the front waist region.
Fastening tabs for the left and right portions of a diaper or incontinence garment can sometimes be prepared from a common continuous web, where a cut or cuts having a repeating pattern can be used to form nested pairs of fastening tabs. Such processes typically minimize waste during the manufacturing of fastening tabs. See, for example, U.S. Pat. No. 5,312,387 (Rossini et al.); U.S. Pat. No. 5,399,219 (Roessler et al.); and U.S. Pat. No. 7,371,302 (Miyamoto et al.); U.S. Pat. No. 7,658,813 (Petersen); and European Patent No. 0233704 B1, published Jul. 15, 1992.
Some fastening tabs have more than one region of mechanical fastener (e.g., hook patches) on the tab, which has been proposed to decrease the likelihood of unintentional disengagement of the mechanical fastener members. See, for example, U.S. Pat. No. 5,957,908 (Kline et al.) and U.S. Pat. No. 5,851,205 (Hisada et al.)

Fastening tab and method of making the same


To provide an ion milling apparatus adapted to suppress the contamination of a beam forming electrode. The ion milling apparatus includes: an ion gun containing therein a beam forming electrode for forming an ion beam; a specimen holder for fixing a specimen to be processed by irradiation of an ion beam; a mask for shielding a part of the specimen from the ion beam; and an ion gun controller for controlling the ion gun.

The ion milling apparatus is a device for preparing a specimen for examination under a scanning electron microscope, transmission electron microscope or the like. The ion milling apparatus is a process unit which thins the specimen by utilizing a sputtering phenomenon where the specimen is bombarded with accelerated ions such that the ions sputter atoms and molecules. The specimen is processed as follows. A mask as a shielding plate against an ion beam is placed on a top side of the specimen to be processed. A protruded region from an edge face of the mask is sputtered whereby a flat and smooth cross-section along the mask edge face can be obtained. A process object of the ion milling apparatus includes metals, glass, ceramics, electronic components, composite materials and the like. In the case of the electronic components, for example, the ion milling technique is used for various purposes such as acquisition of internal structure and cross-sectional shape, film thickness evaluation, crystalline condition evaluation, failure analysis and cross-sectional analysis of foreign substances. The scanning electron microscopes employ the ion milling apparatus for acquisition of form images, compositional images of specimens and channeling images, X-ray analysis, crystal orientation analysis and the like.
Heretofore, there is known an ion milling apparatus having a configuration which includes: an ion beam source generating an ion beam to be applied to a specimen; a specimen chamber in which the specimen is placed and processed by the ion beam; an exhaust device for exhausting the specimen chamber so as to keep the chamber in vacuum; and a gas injection mechanism for injecting a gas for ion generation. The apparatus is provided with an acceleration electrode which accelerates ions of the ion beam source and also functions as a secondary electron suppressor. Thus, the apparatus has a machine configuration that eliminates the increase in distance between anion gun and the specimen so that a milling processing time can be shortened. Regarding the ion milling apparatus including the ion beam source for radiation of ion beam and a specimen holder for fixing the specimen, Patent Literature 1 discloses an ion milling apparatus featuring a good milling efficiency. This apparatus further includes a mask for covering a part of the specimen and has a configuration where a non-axisymmetric lens is disposed between the ion beam source and the mask.