CA1268520A - Methods of assay - Google Patents
Methods of assayInfo
- Publication number
- CA1268520A CA1268520A CA000481357A CA481357A CA1268520A CA 1268520 A CA1268520 A CA 1268520A CA 000481357 A CA000481357 A CA 000481357A CA 481357 A CA481357 A CA 481357A CA 1268520 A CA1268520 A CA 1268520A
- Authority
- CA
- Canada
- Prior art keywords
- electron
- ligand
- electrode
- specific binding
- electrons
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/806—Electrical property or magnetic property
Abstract
Abstract 'Methods of Assay' In its broadest aspect, the present invention provides a method of assaying a ligand in a sample using electrochemical apparatus containing an electrode and components comprising:
(a) the sample, (b) a specific binding partner to the ligand, (c) if desired, at least one further reagent selected from ligand analogues (as herein defined) and specific binding partners, and (d) an electron-source or electron-acceptor;
at least one of components (b) and, if present, (c) being labelled with an electron-transfer mediator capable of aiding the transfer of electrons from the electron-source to the electrode, or from the electrode to the electron-acceptor, which method includes the step of determining whether, and, if desired, the extent to which, the said transfer of electrons is perturbed by complex formation and/or by controlled external influences.
The electrochemical apparatus will typically comprise two or three electrodes, including one working electrode onto which one or more of the components may advantageously be immobilised.
An artificial generation or enhancement of perturbation of electron transfer is preferably performed by displacement of the complex formed relative to the unbound labelled component.
(a) the sample, (b) a specific binding partner to the ligand, (c) if desired, at least one further reagent selected from ligand analogues (as herein defined) and specific binding partners, and (d) an electron-source or electron-acceptor;
at least one of components (b) and, if present, (c) being labelled with an electron-transfer mediator capable of aiding the transfer of electrons from the electron-source to the electrode, or from the electrode to the electron-acceptor, which method includes the step of determining whether, and, if desired, the extent to which, the said transfer of electrons is perturbed by complex formation and/or by controlled external influences.
The electrochemical apparatus will typically comprise two or three electrodes, including one working electrode onto which one or more of the components may advantageously be immobilised.
An artificial generation or enhancement of perturbation of electron transfer is preferably performed by displacement of the complex formed relative to the unbound labelled component.
Description
6~3~2~
METHODS OF ASSAY
The present invention relates to methods of assay of one of a pair of specific binding partners, and to kits of reagents for carrying out these methods.
There is today a great need for rapid and accurate methods oE assaying biologically active substances (which may be at low concentration~, particularly in body fluids such as blood, saliva or urine. A wide variety of medical conditions, such as pregnancy, drug overdose, metabolic birth defects, hormonal disorders and diabetes can be diagnosed using such assay techniques.
Many assay methods rely on the formation of a complex between the species under assay ~hereinafter called "ligand") and another species to which it will bind specifically (hereinafter called "specific binding partner"). The extent of complex formation is a function of the amount of the ligand present.
The assay of ligand is determined by monitoring the extent of complex formation, or example by the use of chemical or biochemical labels. Several methods of labelling have been employed, for example radioisotopic or enzyme labelling, spin-labelling or labelling employing fluorescent or bioluminescent species.
The use of radioisotopic labels has been particularly widespread, due to the high degree of sensitivity and specificity obtainable. There are, however, serious disadvantages to the use of radioactive labels. Radioactive labels have limited shelf life due to spontaneous decay, necessita-ting frequent recalibration of the equipment, and their use requires adherence to strict safety precautions and is subject to legal regulatlon. These disadvantages inevitably lead to higher costs and necessity for 3
METHODS OF ASSAY
The present invention relates to methods of assay of one of a pair of specific binding partners, and to kits of reagents for carrying out these methods.
There is today a great need for rapid and accurate methods oE assaying biologically active substances (which may be at low concentration~, particularly in body fluids such as blood, saliva or urine. A wide variety of medical conditions, such as pregnancy, drug overdose, metabolic birth defects, hormonal disorders and diabetes can be diagnosed using such assay techniques.
Many assay methods rely on the formation of a complex between the species under assay ~hereinafter called "ligand") and another species to which it will bind specifically (hereinafter called "specific binding partner"). The extent of complex formation is a function of the amount of the ligand present.
The assay of ligand is determined by monitoring the extent of complex formation, or example by the use of chemical or biochemical labels. Several methods of labelling have been employed, for example radioisotopic or enzyme labelling, spin-labelling or labelling employing fluorescent or bioluminescent species.
The use of radioisotopic labels has been particularly widespread, due to the high degree of sensitivity and specificity obtainable. There are, however, serious disadvantages to the use of radioactive labels. Radioactive labels have limited shelf life due to spontaneous decay, necessita-ting frequent recalibration of the equipment, and their use requires adherence to strict safety precautions and is subject to legal regulatlon. These disadvantages inevitably lead to higher costs and necessity for 3
2~
high standards of sophistlcation of equipment, laboratory facili-ties and personnel.
We have now found that electron-transfer mediators which are capable of alding the transfer of electrons from an electron-source to an electrode (or the transfer of electrons from the electrode to an electron-acceptor) may be employed as labels to overcome the problems associated with known labels as discussed above and to provide a sensltive, specific and convenient assay method.
Thus, in its broadest aspect, the present invention provides a method of assaying a ligand in a sample using electro-chemical apparatus containing an electrode and components comprising:
(a) the sample, (b) a specific binding partner to the ligand, (c) if desired, at least one further reagent selected from ligand analogues (as herein defined) and specific binding partners to the ligand, and (d) an electron-source or electron-acceptor; at least one of components (b) and, if present, (c) being labelled with an electron-transfer mediator capable of aiding the transfe:r of electrons from -the electron-source to the electrode, or from the electrode to the electron-acceptor, which method includes the step o:E determing whether, and, if desired, the extent to which, the said transfer of electrons is perturbed by at least one of (i) complex formation and (ii) a controlled external influence which produces a perturbation of said transfer of e].ectrons as a function of said complex formation.
The method of the present invention can be used Eor eithe:r qualitative or quantitative assays, the assay being completed by comparing the determined perturbation with calibration data.
The term "ligand analogue" used herein refers to a species capable of complexing with the same specific binding partner as the ligand under assay, - 2a -~2~3521D
and includes inter alia within its scope a Icnown quantity of the ligand under assay.
Electron~sources or acceptors comprising component (d) may be single species or two or more co-operating species. Thus, for example, ascorbate I
H COH
H o ,"1, ,~
HO O
may function as an electron-source, apomorphine, substituted catechols (such as l-amino-2-(3,4-dihydrox-yphenyl)-ethane or 1-amino-2-(3,4,5-trihydroxyphenyl)-ethane) or aminophenols (such as p-aminophenol or l-amino-2-(2-amino-4,5-dihydroxyphenyl)-ethane) suitably being employed as labels for components (b) and, if present, (c), or dihydronicotinamide adenosine diphosphate (NADH) may function as an electron-source, with quinones (eg o-quinones such as the oxidised forms of dopamine (3-hydroxytyramine) and3, 4-dihydroxybenzylamine) as labels.
Alternatively, enzymes in co-operation with their substrates may be employed. Particular]y suitable enzymes include the so-called oxido-reductases, particularly, but not exclusively, flavo- and quino-protein enzymes, e.g. glucose oxidase, glucose dehydrogenase or methanol dehydrogenase. The term 2S "enzyme" used herein includes true enzymes, e.g.
2~D
of the types previously mentioned, and apoenzymes which may become activated in the presence of a cofactor. As an apoenzyme, for example, apoglucose oxidase may be used.
Other electron-sources and acceptors and suitable mediators therefor are known in the art.
The preferred electron-sources are oxidoreductase enzymes ~e.g. those mentioned above). Thus according to a preferred feature, the present invention provides a method of assaying a ligand in a sample as herein-before defined, wherein component (d) is an oxido-reductase enzyme in co-operation with a substrate therefor.
Suitable labels for components (b) and (c) for use in such a preferred method may, for example, be capable of accepting electrons from the enzyme and donating them to the electrode (during substrate oxidation), or may be capable of accepting electrons from the electrode and donating them to the enzyme (during substrate reduction). Such labels may, for example, be selected from the following:
(i) a polyviologen such as, for example, a compound of formula - CH2 CH2--N~N _ ¦L r~ r~3 ¦ n and derivatives thereof, e.g. side--chain alkyl derivatives, the preparation of which is described in Polymer Letters 9 pp 289-295 (1971), s~
~ii) a low molecular weight comDound selected from chloranils, fluoranils and bromanils (e~g. o-chloranil), (iii) ferrocene (bis-~5~cyclopentadienyl iron (II)) or a derivative thereof [including e.g. function-alised derivatives such as ferrocene monocarboxylic acid (FMCA), polymeric forms ('polyferrocenes') such as (ferrocene)4 or polyvinyl ferrocene and 'boron tetraferrocene' (B(ferrocene)4)~, tiv) compounds of biological ori~in possessing suitable enzyme compatability, e.g. Vitamin K, (v) N,N,N' ,N'-tetramethyl-4-phenylened;amine; and (vi) derivatives of phenazine methosulphate or phenazine ethosulphate.
~ ediators may interact with the enzyme at a site remote from or near to the active site for the substrate reaction.
Of the aforementioned electron-transfer mediators, the preferred are ferrocene and functionalised derivatives thereof. These compounds are desirable for this purpose because they are relatively cheap, stable, water soluble, non-toxic, and provide an easily electrochemically reversible system which in its reduced FeII state is not susceptible to oxidation by oxygen in the atmosphere.
Functionalisation may be required e.g. to permit attachment of the label to the reagent molecule.
The redox potential of ferrocene is +422 mV vs N~E. By introducing functional groups on to the ring system, this figure can be varied between +300 and +650 mV. Moreover, the water-solubility of carboxy-substituted ferrocenes is greater than that of the parent compound (see, e.g. Szentrimav R., l977, Amer. Chem. Soc. Symposium Series, 38, 154).
. . .
~6~35Z~
Thus, for example, in the case of ferrocene, it may be necessary to modify the ferrocene complex by providing one or both of the cyclopentadienyl groups with one or more side chains, e.g. of the formula -CHO
(CH2)nC 1 2 -(CH2)m~HR R
where n and m may be e.g. from 0 to 6 and R and R2, which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms (e.g. methyl). Additional functional groups may be incorporated into the side chain, typically those groups used in the chemical modification of proteins, for example mercuric chloride, precursors of nitrenes and carbenes, diazo or iodide groups.
Similar functionalisation may be desirable when ! mediators other than ferrocene are used.
The interaction between the mediator label and the enzyme may thus take the form of chemical bonding, or may take the form of non-chemical bonding or non-bonding interaction.
For a better understanding of the present inventionr reference is made to the accompanying drawings wherein:
Figure l(a) is a vertical cross-section of a suitable electrochemical apparatus for use in carrying out a method of assay according to the present invention;
Figure l(b) shows schematically an electrical circuit which may be used in conjunction with the apparatus illustrated in Figure lta) for cyclic voltammetry;
Figure 2 is a vertical cross-section of a working electrode suitable for use in a method of assay according to the invention, wherein component (b) is bound to a portion 26~
- 6a - 20208-1257 of the working electrode other than the working surface;
Flgure 3 is a cyclic voltammogram at a pyrolytic graphite electrode (voltage scan rate = 20 mvs lj of a conjugate of thyroxine (T4) and ferrocene monocarboxylic acid (FMCA) vs a standard calomel electrode (S.C.E.~, and Figures 4a and 4b are cyclic voltammograms at a pyrolytic graphite electrode of FMCA and T4 respectively vs S.C.E.
The working electrode from which the electrochemical readings will be taken will preferably be solid and have a electrically conductive working surface of e.g. carbon (preferably graphite, e.g. pyrolytic graphite), or metal, e.g. silver, gold or platinum.
If the electrode is of carbon, it may be present as a pre-formed rod or as an electrode shape made up of a paste of carbon particles. The nature of the surface of the electrode is usually important.
If metal, the surface can be roughened or chemically modified; if solid carbon, the surface can be previously heat-treated in an oven with oxygen excess or oxidized electrochemically. Thus, for example, when ascorbate is used as an electron-source, a carbon paste electrode of polished 'glassy carbon' sheets may advantageously be employed.
~2~
~ 7 ~ 20208-1257 In addition to the working electrode from which the electrochemical readings will be taken, the apparatus may comprise an auxiliary (counter) electrode and optionally a reference electrode, the electrodes being used in conjunction with a potentiostat and a sensitive current meter. The apparatus preferably contains an aqueous assay medium comprising inter alia p~ buffer. Means may be provided for incubating the assay medium at any desired temperature. As hereinbefore indicated, a suitable electrochemical apparatus is illustrated in vertical cross-section in Figure l~a) of the accompanying drawings. The working electrode l is composed of an elongate core 2 of steel tipped with a working surface 3 of pyrolytic graphite and having a coating 4 of epoxy resin. The auxiliary (counter) electrode 5 is of platinum. A calomel reference electrode 6 is shown, connected to the cell via a luggin capillary 7~ The cell and reference electrode are enclosed in a water jacket 8.
A variety of electrochemical methods exploiting any two of the three parameters potential (E), current (i) and time (t) may be used to measure the electrochemical characteristics of the components.
For example, electrochemical measurements can be made using differental pulse voltammetry, cyclic voltammetry or square-ware voltammetry. When cyclic voltammetry is used, a circuit such as, for example, that shown schematically in Figure l(b) of the accompanying drawings may be employed. In this Figure, C represents the auxiliary tcounter) electrode, W the working electrode and R the reference electrode.
This circuit may conveniently be used in conjunction with apparatus of the type shown in Figure l(a), the electochemical current i being measured using a potent;ostat.
In homogeneous assay systems, the formation of the complex between the ligand and the specific s~
binding partner or, in the case of competitlve assays, between the ligand analogue and the specific bindin~ partner, may cause a change ~e.g. a decrease) in the ability of electrons to flow e.g~ from the enzyme to the electrode and vice versa via the mediator. This may, for example, result from:
1. the blockage of access between the mediator and the enzyme by the formation of the complex, thus impairing electron transfer;
10 2. the blockage of access between the mediator and the electrode by the formation of the complex, thus impairing electron transfer;
high standards of sophistlcation of equipment, laboratory facili-ties and personnel.
We have now found that electron-transfer mediators which are capable of alding the transfer of electrons from an electron-source to an electrode (or the transfer of electrons from the electrode to an electron-acceptor) may be employed as labels to overcome the problems associated with known labels as discussed above and to provide a sensltive, specific and convenient assay method.
Thus, in its broadest aspect, the present invention provides a method of assaying a ligand in a sample using electro-chemical apparatus containing an electrode and components comprising:
(a) the sample, (b) a specific binding partner to the ligand, (c) if desired, at least one further reagent selected from ligand analogues (as herein defined) and specific binding partners to the ligand, and (d) an electron-source or electron-acceptor; at least one of components (b) and, if present, (c) being labelled with an electron-transfer mediator capable of aiding the transfe:r of electrons from -the electron-source to the electrode, or from the electrode to the electron-acceptor, which method includes the step o:E determing whether, and, if desired, the extent to which, the said transfer of electrons is perturbed by at least one of (i) complex formation and (ii) a controlled external influence which produces a perturbation of said transfer of e].ectrons as a function of said complex formation.
The method of the present invention can be used Eor eithe:r qualitative or quantitative assays, the assay being completed by comparing the determined perturbation with calibration data.
The term "ligand analogue" used herein refers to a species capable of complexing with the same specific binding partner as the ligand under assay, - 2a -~2~3521D
and includes inter alia within its scope a Icnown quantity of the ligand under assay.
Electron~sources or acceptors comprising component (d) may be single species or two or more co-operating species. Thus, for example, ascorbate I
H COH
H o ,"1, ,~
HO O
may function as an electron-source, apomorphine, substituted catechols (such as l-amino-2-(3,4-dihydrox-yphenyl)-ethane or 1-amino-2-(3,4,5-trihydroxyphenyl)-ethane) or aminophenols (such as p-aminophenol or l-amino-2-(2-amino-4,5-dihydroxyphenyl)-ethane) suitably being employed as labels for components (b) and, if present, (c), or dihydronicotinamide adenosine diphosphate (NADH) may function as an electron-source, with quinones (eg o-quinones such as the oxidised forms of dopamine (3-hydroxytyramine) and3, 4-dihydroxybenzylamine) as labels.
Alternatively, enzymes in co-operation with their substrates may be employed. Particular]y suitable enzymes include the so-called oxido-reductases, particularly, but not exclusively, flavo- and quino-protein enzymes, e.g. glucose oxidase, glucose dehydrogenase or methanol dehydrogenase. The term 2S "enzyme" used herein includes true enzymes, e.g.
2~D
of the types previously mentioned, and apoenzymes which may become activated in the presence of a cofactor. As an apoenzyme, for example, apoglucose oxidase may be used.
Other electron-sources and acceptors and suitable mediators therefor are known in the art.
The preferred electron-sources are oxidoreductase enzymes ~e.g. those mentioned above). Thus according to a preferred feature, the present invention provides a method of assaying a ligand in a sample as herein-before defined, wherein component (d) is an oxido-reductase enzyme in co-operation with a substrate therefor.
Suitable labels for components (b) and (c) for use in such a preferred method may, for example, be capable of accepting electrons from the enzyme and donating them to the electrode (during substrate oxidation), or may be capable of accepting electrons from the electrode and donating them to the enzyme (during substrate reduction). Such labels may, for example, be selected from the following:
(i) a polyviologen such as, for example, a compound of formula - CH2 CH2--N~N _ ¦L r~ r~3 ¦ n and derivatives thereof, e.g. side--chain alkyl derivatives, the preparation of which is described in Polymer Letters 9 pp 289-295 (1971), s~
~ii) a low molecular weight comDound selected from chloranils, fluoranils and bromanils (e~g. o-chloranil), (iii) ferrocene (bis-~5~cyclopentadienyl iron (II)) or a derivative thereof [including e.g. function-alised derivatives such as ferrocene monocarboxylic acid (FMCA), polymeric forms ('polyferrocenes') such as (ferrocene)4 or polyvinyl ferrocene and 'boron tetraferrocene' (B(ferrocene)4)~, tiv) compounds of biological ori~in possessing suitable enzyme compatability, e.g. Vitamin K, (v) N,N,N' ,N'-tetramethyl-4-phenylened;amine; and (vi) derivatives of phenazine methosulphate or phenazine ethosulphate.
~ ediators may interact with the enzyme at a site remote from or near to the active site for the substrate reaction.
Of the aforementioned electron-transfer mediators, the preferred are ferrocene and functionalised derivatives thereof. These compounds are desirable for this purpose because they are relatively cheap, stable, water soluble, non-toxic, and provide an easily electrochemically reversible system which in its reduced FeII state is not susceptible to oxidation by oxygen in the atmosphere.
Functionalisation may be required e.g. to permit attachment of the label to the reagent molecule.
The redox potential of ferrocene is +422 mV vs N~E. By introducing functional groups on to the ring system, this figure can be varied between +300 and +650 mV. Moreover, the water-solubility of carboxy-substituted ferrocenes is greater than that of the parent compound (see, e.g. Szentrimav R., l977, Amer. Chem. Soc. Symposium Series, 38, 154).
. . .
~6~35Z~
Thus, for example, in the case of ferrocene, it may be necessary to modify the ferrocene complex by providing one or both of the cyclopentadienyl groups with one or more side chains, e.g. of the formula -CHO
(CH2)nC 1 2 -(CH2)m~HR R
where n and m may be e.g. from 0 to 6 and R and R2, which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms (e.g. methyl). Additional functional groups may be incorporated into the side chain, typically those groups used in the chemical modification of proteins, for example mercuric chloride, precursors of nitrenes and carbenes, diazo or iodide groups.
Similar functionalisation may be desirable when ! mediators other than ferrocene are used.
The interaction between the mediator label and the enzyme may thus take the form of chemical bonding, or may take the form of non-chemical bonding or non-bonding interaction.
For a better understanding of the present inventionr reference is made to the accompanying drawings wherein:
Figure l(a) is a vertical cross-section of a suitable electrochemical apparatus for use in carrying out a method of assay according to the present invention;
Figure l(b) shows schematically an electrical circuit which may be used in conjunction with the apparatus illustrated in Figure lta) for cyclic voltammetry;
Figure 2 is a vertical cross-section of a working electrode suitable for use in a method of assay according to the invention, wherein component (b) is bound to a portion 26~
- 6a - 20208-1257 of the working electrode other than the working surface;
Flgure 3 is a cyclic voltammogram at a pyrolytic graphite electrode (voltage scan rate = 20 mvs lj of a conjugate of thyroxine (T4) and ferrocene monocarboxylic acid (FMCA) vs a standard calomel electrode (S.C.E.~, and Figures 4a and 4b are cyclic voltammograms at a pyrolytic graphite electrode of FMCA and T4 respectively vs S.C.E.
The working electrode from which the electrochemical readings will be taken will preferably be solid and have a electrically conductive working surface of e.g. carbon (preferably graphite, e.g. pyrolytic graphite), or metal, e.g. silver, gold or platinum.
If the electrode is of carbon, it may be present as a pre-formed rod or as an electrode shape made up of a paste of carbon particles. The nature of the surface of the electrode is usually important.
If metal, the surface can be roughened or chemically modified; if solid carbon, the surface can be previously heat-treated in an oven with oxygen excess or oxidized electrochemically. Thus, for example, when ascorbate is used as an electron-source, a carbon paste electrode of polished 'glassy carbon' sheets may advantageously be employed.
~2~
~ 7 ~ 20208-1257 In addition to the working electrode from which the electrochemical readings will be taken, the apparatus may comprise an auxiliary (counter) electrode and optionally a reference electrode, the electrodes being used in conjunction with a potentiostat and a sensitive current meter. The apparatus preferably contains an aqueous assay medium comprising inter alia p~ buffer. Means may be provided for incubating the assay medium at any desired temperature. As hereinbefore indicated, a suitable electrochemical apparatus is illustrated in vertical cross-section in Figure l~a) of the accompanying drawings. The working electrode l is composed of an elongate core 2 of steel tipped with a working surface 3 of pyrolytic graphite and having a coating 4 of epoxy resin. The auxiliary (counter) electrode 5 is of platinum. A calomel reference electrode 6 is shown, connected to the cell via a luggin capillary 7~ The cell and reference electrode are enclosed in a water jacket 8.
A variety of electrochemical methods exploiting any two of the three parameters potential (E), current (i) and time (t) may be used to measure the electrochemical characteristics of the components.
For example, electrochemical measurements can be made using differental pulse voltammetry, cyclic voltammetry or square-ware voltammetry. When cyclic voltammetry is used, a circuit such as, for example, that shown schematically in Figure l(b) of the accompanying drawings may be employed. In this Figure, C represents the auxiliary tcounter) electrode, W the working electrode and R the reference electrode.
This circuit may conveniently be used in conjunction with apparatus of the type shown in Figure l(a), the electochemical current i being measured using a potent;ostat.
In homogeneous assay systems, the formation of the complex between the ligand and the specific s~
binding partner or, in the case of competitlve assays, between the ligand analogue and the specific bindin~ partner, may cause a change ~e.g. a decrease) in the ability of electrons to flow e.g~ from the enzyme to the electrode and vice versa via the mediator. This may, for example, result from:
1. the blockage of access between the mediator and the enzyme by the formation of the complex, thus impairing electron transfer;
10 2. the blockage of access between the mediator and the electrode by the formation of the complex, thus impairing electron transfer;
3. alteration of the conformation of the mediator by the formation of the complex so that the free passsage of electrons between the enzyme and mediator is inhibited; or
4. alteration of the conformation of the mediator by the formation of the complex so that the free passage of electrons between the mediator and electrode is inhibited.
In a typical homogeneous assay, therefore, formation of the complex perturbs an electrochemical characterisitic of the components of the solution.
It is not necessary for a full voltammogram to be determined in measuring the electrochemical characteristic; it may be sufficient, for example, for an appropriate poised potential to be selected and readings of current taken at that point. The degree of perturbation can then be related to the amount of ligand present in the sample, from calibration data obtained with similar system using known amounts of ligand.
Although the order of introduction of the components (a~, (b) and, if present, (c) into the apparatus may not be critical, it is preferable that a complex is formed after introduction of the final one of components (a), (b) and, if present, (c), but not prior thereto. It is, however, also possible for there to be complex present before the final one oE these components is added, in which case the final component will become complexed by displacing one component of the complex. It may be necessary to incubate these components for a period of time to allow the complexing reaction to approach equilibrium before component (d) is added. Addition of component (d~ should not affect the complexing reaction, but these components must be present before measurements can be taken at the working electrode.
The method of the present invention is applicable to e.g. 'direct' assays (in which component (c) is absent) r 'displacement' assays or 'competitive' assays (in which a ligand analogue is present in component (c)). rrhe method of the invention may employ the so-called "sandwich" technique, using a solid phase binding partner. Depending on the order of the complexiny reacLions, the forward, fast forward, reverse and simultaneous variations are all possible according to the present invention.
The solid phase may comprise the electrode surface, or may take the form of particles, beads etc.
The solid phase binding partner may be prepared by any one of a number of conventional techniques for immobilising reagents onto solid supports.
In a method of the invention exploiting the time (t) parameter, the rate of perturbation of the electrochemical characteristic as a result of complex formation may be determined. Conveniently, the initial rate of perturbation will be measured.
Such a method is applicable, for example, to a competitive assay in which the ligand and labelled ligand analogue compete for complexing with the specific binding partner. Thus, the initial rate of perturbation is related to the concentration of ligand present and from a calibration plot the - 10 ~
initial rate of perturbation v. concentration of ligand present, the ligand assay can be readily determinedO
The method of assay involving a determination of the rate of perturbation is also applicable to non-competitive assays where the labelled ligand analogue is absent and sufficient labelled specific bindlng partner is employed to enable all the ligand introduced to be complexed.
Measurement of, for example, the absolute electrochemical current generated after a standard incubation period may enhance the ease and sensitivity of the assay.
In a typical heterogeneous assay, formation of the complex causes no (or only a slight) perturbation in an electrochemical characteristic of the components.
In that case, it will generally be necessary artifically to generate or enhance a perturbation by controlled external influences. Although the magnitude of the external influence may have some bearing on the change induced, and must therefore be consistent with any such influence employed in calibration experiments, it is thought that any change produced in the perturbation remains a function of the ligand/specific binding partner complex. [Artificial generation or enhancement of a perturbation may also be desirable in homogeneous assays]
The artificial generation or enhancement of the perturbation is preferably performed by displacement of the complex relative to the unbound labelled component, or example by providing component (b) in an insolubilised form coupled (e.g. in conventional manner1 to a solid support. Alternatively, the complex can be further complexed with a species which will bind specifically to the complex, coupled to a solid support, with subsequent displacement of the support and coupled molecules. In extreme cases, the displacement may constitute complete ~2~
~ 20208-1257 removal of the complex from the apparatus, but in genera] the complex will be ~isplaced within the apparatus.
The solid support may, for example, comprise S the electrode surface or may take the form of conventional solid phase particles, beads etc. The solid support may be magnetic or magnetisable to facilitate displacement or separation. Thus, for example, magnetic supports (e.g. in the form of particles or beads) may be composed of ferromagnetic or paramegnetic materials such as metals (e.g. iron, nickel or cobalt), metal alloys (e.g. magnetic alloys of aluminium, nickel, cobalt and copper), metal oxides (e.g. Fe3O4 ~-Fe3O3, CrO2, CoO, NiO or Mn2O3), magnetoplumbites or solid solutions (e.g. solid solutions of magnetite with ferric oxide). The preferred material for magnetic supports is magnetite (Fe3O4) or haematite (~-Fe2O3).
Particles may be non-colloidal or collo;dal.
Displacement of the solid support, may, or example, be effected by urging the support into the vicinity of the electrode. In the case of magnetic supports (e~g. particles), the methods described in our copending Canadian Patent Application No. 486,546 may suitably be employed. Thus, for example, a magnetic electrode (e.g. comprising a permanent magnet or an electromagent) may be used, or a non-magnetic electrode may be used in which case the particles will be urged into and retained in the vicinity of the electrode by the application of an external magnetic field.
The component (b) may be immobilised directly on to the magnetic support, or may be immobilised via one or more other 'spacer' molecules, including partners in specific binding interactions. Immobilisation of reagents may generally be achieved by conventional techniques such as, for example, adsorbtion, covalent bonding or cross-linking, or a combination of these techniques, e.g. adsorption of a chemical with one or more functional groups followed by covalent bonding or cross-linking of the reagent. Alternatively, substantially non-chemical means may be employed.
Suitable immobilisation techniques are known in the art~
Other methods for artifically generating or enhancing the perturbation include, for example, removing excess uncomplexed labelled reagent, e.g.
by draining from the apparatus or by coupling to a suitable solid support and removing the said solid support from the apparatus.
All of the variations described above for homogeneous assays (including direct, competitive, sandwich and displacement techniques and methods in which a rate of perturbation is measured rather than an absolute perturbation) are equally applicable to heterogeneous assays.
The methods of the present invention may generally be simpler than known methods, in that they may eliminate the need for separation of uncomplexed and complexed phases before the assaying step.
However, as indicated above, the invention also includes within its scope methoas in which reagents are employed immobilised on a solid surface, in which methods it may be necessary or desirable to separate the solid (complexed) and uncomplexed phases before the assaying step. Such separation may take the form of complete removal of the solid phase from the assay medium or may, for example, take the form of sedimentation or concentration of the solid phase in one region of the assay medium.
If electrode-immobilised components are employed, the need for separate addition of the component to the electrochemical apparatus may be eliminated.
Additionally, the direct interaction between the electrode and the electrode-immobilised species may lead to an improvement in the sensitivity of the perturbation measurements.
According to a further feature of the present invention, therefore, there are provided methods of assay of a ligand in a sample as hereinbefore defined wherein one or more of the components (b) and, if present, (c) is/are immobilised on the working electrode or a suitable solid surface.
The immobiLized component(s) may be bound to the working surface of the working electrode or to a portion of the working electrode other than the working surface.
The immobilised component is preferably that component which is labelled. The said component may be immobilised via the label as long as the ability of the label to mediate electron transfer is not impaired.
Thus, for example, a polyviologen label may be covalently bonded to a metal electrode. The large polyviologen molecule projects from the electrode surface and this is believed to facilitate interaction with the enzyme. Alternatively, chloranil and/or fluoranil may be disseminated throughout an electrode composed of particulate carbon.
In one embodiment, the system comprises an electrode, e.g. a carbon (for example pyrolytic graphite) electrode, or a suitable solid surface carrying an immobilised layer of ferrocene dicarboxylic acid, l,l'-dimethylferrocene lDMF) or polyvinylferrocene 30 (having an average molecular weight of about 16000), the molecules of which are also coupled to reagent lb) or, if present (c), as labels thereof.
2~
The carbon electrode core or suitable solid surface can be integral or a stiEf paste of particles.
Normally, any solid surface employed will present a porous surface for the ferrocene or ferrocene derivative, which may be adhered thereto ln a number of ways, for examples:
(a) for monomeric ferrocene or a monomeric ferrocene derivative, by deposition from a solution in a readily evaporatable liquid e.g. an organic solvent such as toluene;
(b) for a ferrocene polymeric derivative, e.g.
polyvinyl-ferrocene of average molecular weight about 16000 (for a method of synthesis see J. Polymer Sci. 1976, 14, 2433), deposition from a readily evaporatable organic solvent for the polymer such as chloroform;
(c) for a polymerisable ferrocene-type monomerr by electrochemically induced polymerisation in situ, elg. by dissolving vinylferrocene in an organic electrolyte containing tertiary butyl ammonium perchlorate in concentration about lM and depositing at a potential of -700 mV to induce deposition of vinylferrocene radicals as a polymer in situ; or 5 (d) by covalent modification of the solid surEace e.g. by carbodiimide cross-linking of the ferrocene or ferrocene derivative onto the surface (e.g. a carbon electrode).
Alternatively, the component may be immobilised directly on the solid surface by any of the conventional techniques used for coupling reagents to solid supports.
If desired, the electrode-immobilised component may be bound to a portion of the electrode other than the working surface. The electrode may in these circumstances be constructed so as to ensure that the immobilised component remains sufficiently close to the working surface to enable the assay 2~
to be carried out effectively. Such an electrode is illustrated in vertical cross-section in Figure 2 of the accompanying drawings, this being particularly suitable for "sandwich" immunoassays in which the immobilised component is an unlabelled specific binding partner (e.g. a capture antibody). The electrode of Figure 2 comprises an upwardly facing graphite working surface 1 in the base of a cell, the wall of which is formed by a polystyrene projection 2 from the body of the electrode. It is on this wall that a suitable specific binding partner may be immobilised (e.g. by adsorption). The electrical connection is provided by an insulated wire 3 secured to the bottom of the working surface by silver-loaded epoxy resin 4, the arrangement being encasedin epoxy resin 5 and sealed with polypropylene 6.
It will be appreciated that, when component (b) is electrode-immobilised, it is not possible artifically to generate or enhance a perturbation by displacement of the resulting complex. However, a perturbation may still be artifically generated or enhanced, for example by complexing any uncomplexed labelled component remaining in solution with a species which will complex specifically with that component, coupled to a solid support, with subsequent displacement of the support and coupled molecules.
In a further aspect, the present invention provides kits o~ reagents and/or apparatus for carrying out the assays of the invention. Suitable kits may comprise an electrochemical apparatus containing a working electrode, an auxiliary electrode and optionally a reference electrode, and an aqueous assay medium with suitable components present (either in solution or immobilised). Other components (e.g. further reagents etc) and the sample to be assayed may conveniently be introduced through an entry port provided in the apparatus.
The apparatus may be automated so that the components are added in a predetermined sequence, 2~
and the incubation temperature may be controlled.
Advantageously the apparatus may be pre-calibrated and provided with a scale whereby the perturb~tion in the electrochemical characteristic oE the components may be read off directly as an amount of ligand in the sample.
Examples of ligands which may be assayed by the method of the invention are given in Table I below, together with an indication of a suitable specific binding partner in each instance.
Table I
-Ligand Specific Binding Partner antigen specific antibody antibody antigen hormone hormone receptor hormone receptor hormone polynucleotide complementary polynucleotide strand strand avidin biotin biot.in avidin protein A immunoglobulin immunoglobulin protein A
enzyme enzyme cofactor (subst.rate) enzyme co~actor enzyme (substrate) lectins specific carbohydrate specific carbohydrate lectins of lectins The method of the invention has very broad applicability, but in particu.lar may be used to assay: hormones, including peptide hormones (e.g.
thyroid stimulating hormone (TSII), human chorionic gonadotrophin (HCG), lutenising hormone (LH), follicle 8~
stimulating hormone (FSH), insulin and prolactin) or non-peptide horrnones (e.g. steroid hormones such as cortisol, estradiol, progesterone and testosterone and thyroid hormones such as thyroxine (T4) and triiodothyronine), proteins (e.g. carcinoembryonic antigen (CEA) and alphafetoprotein (AFP)), drugs (e.g. digoxin), sugars, toxins or vitamins.
The invention will be particularly described hereinafter with reference to an antibody or an ]0 antigen as the ligand. However, the invention is not to be taken as being limited to assays of antibodies or antigens.
It will be understood that the term "antibody"
used herein includes within its scope a) any of the various classes or sub-classes of immunoglobulin, e.g. IgG, IgM, derived from any of the animals conventionally used, e.g. sheep, rabbits, goats or mice, b) monoclonal antibodies, c) intact molecules or "fragments" of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody, i.e. fragments devoid of the Fc portion (e.g., Fab, Fab', F(ab')2) or the so-called "half-molecule" fragments obtained by reductive cleavage of the disulphide bonds connecting the heavy chain components in the intact antibody.
The method of preparation of fragments of antibodies is well known in the art and will not be described herein.
The term "antigen" as used herein will be understood to include both permanently antigenic species (for example, proteins, bacteria, bacteria fragments, cells, cell fragments and viruses) and haptens which may be rendered antigenic under suitable conditions.
%~
Incorporation of, Eor example, a ferrocene label into the molecular structure of an antibody may for example be achieved by any of the following methods:
(i) providing the label with one or rnore functional groups capable of bonding interactions with the molecular structure of the antibody;
(ii) using cross-linking groups;
(iii) using the avidin-biotin binding system, (i.e.
avidin-labelled antibody binding with biotin-labelled ferrocene molecules or biotin-labelled antibody binding with avidin-labelled ferrocene).
Similar methods may be applied as desired for labelling an antigen molecule. Suitable methods are known in the art and will not be discussed in detail here. For example, the incorporation of ferrocene into certain steroids is described in Journal of Organometallic Chemistry, 160 (1978) pp. 223-230.
2~ Methods of purifying the labelled antibody or antigen are also known and include, for example, dialysis, density-gradient ultracentrifugation, gel filtration and ion-exchange chromatography.
The attachment of the label to the antibody or antigen can be via any portion of the molecular structure of the antibody or antigen, so long as immunological activity thereof is retained.
Immobilisation of an antibody or antiyen molecule onto the electrode or other suitable solid surface may be effected by various methods. The attachment of the antibody or antigen to the electrode or solid surface can be via any portion of the molecular structure so long as specific immunological activity is retained at the antibody or antigen binding site.
Thus, for example, electrode-immobilisation of unlabelled antibody or antigen reagent may be achieved by bonding interactions between functional ~%~ i2~
qroups on the antibody or anti~en molecule and the electrode, or by cross-linking or adsorption onto the surface of the electrode. Binding of reagents to the electrode may be accompli~hed by methods analogous to known methods for binding such reagents to solid supports ~e.g. particles and beads), for example those described in published European Patent Application No. 0 105 714.
Electrode-immobilisation of labelled reagent, may, for example, be achieved by any of the ollowing methods:
(i) incorporating a label molecule into the molecular structure of free reagent and subse~uently immobilising the reagent onto an electrode at a site remote from the label in the same way as described above for unmodified reagents;
(ii) incorporating a label molecule into the molecular structure of a pre-immobilised reagent;
(iii) incorporating a bifunctional label into the molecular structure of free antibody or antigen so as to enable one function to interact with the electrode; or (iv) incorporating a bifunctional label onto the electrode, so as to enable one function to interact with the molecular structure of free antibody or antigen.
The preferred label for use w;th an enzyme/substrate electron-source or electron-acceptor is ferrocene monocarboxylic acid.
When ascorbate i5 used as an electron-~source, and a catechol or aminophenol as a label, electrode-immobi];sation of labelled reagent is preferably achieved by adsorption or chemical reaction via the label on a suitably modified carbon electrode.
A discussion of methods of attachment of such species to carbon electrodes is given in Analytical Chemistry, Vol. 55, 9 (19~3), p. 1576.
~ i2~
sy way of example only, the invention inclucles inter _lia the following embodiments:
~- = antibody O = antigen M = mediator label E = electron-source or acceptor (e.y. enzyme + substrate) ~ = electrode surface o - solid phase (non-electrode); ? indicates ligand under assay 1. Direct Antibody Assay a) Soluble E + ~M add ~? ~ ? ~O M + E
~M
b1 Immobilised on electrode ~M ~ ~ ~- add ~? ~M~:~?
In both these assays the formation of the immune complex decreases the efficacy of the mediator, the change in signal being a measure of antibody concentration.
2 Direct Antiqen Assay .
a) Soluble M -C add O-7 ?-o~
~ E ~ + E
M -~ ~ M
The immune reaction alters the ability of the mediator/antibody complex to shuttle electrons to or from the electrode. Therefore the signal changes.
b) Immobilised ~M~ add ~? ~?
~ ~ E --~ ~ ~ E
--M ~ `
3. Competitive Antigen Assay a) Soluble M -O M-O>--E ~ cldd ~?> E -- M~ d ~ ~ E + M~
M~ ~-? ?~
Competition between the mediator-labelled antigen and the antigen under assay for the available antibody results in some of the mediator being perturbed, the signal relating to the concentration of antigen under assay.
b) Immobilised The immobilised system can take two forrns:
(i) On electrode surface ~M -O ~ E ~ ~? ~ ~ + E ? o~ M ~ ~?
M ~ M ~ o? M -O E
After separation~ the signal measured depends upon the ratio of the antigen under assay to mediator-labelled antigen. The electrode supplies a means of easy separation.
(ii) On solid phase (e.g. magnetic particles) M~ M~> o M~ c~ M ~ odd > E ~ M
E + E +
M~ ?~ ?~>0 ? ~ ? ~> O
1 sepa~clte AssAy ~ E
The sedimenting of the immune complex reduces the amount of mediator in solution hence perturbing the signal.
~8 4. Displacement Antiqen Assay M--0~ 0>--E ~ acld o-? E t M
M
?~
Displacement of mediator/antigen complex from the antibody by the antigen under assay results in an increase in signal.
In a typical homogeneous assay, therefore, formation of the complex perturbs an electrochemical characterisitic of the components of the solution.
It is not necessary for a full voltammogram to be determined in measuring the electrochemical characteristic; it may be sufficient, for example, for an appropriate poised potential to be selected and readings of current taken at that point. The degree of perturbation can then be related to the amount of ligand present in the sample, from calibration data obtained with similar system using known amounts of ligand.
Although the order of introduction of the components (a~, (b) and, if present, (c) into the apparatus may not be critical, it is preferable that a complex is formed after introduction of the final one of components (a), (b) and, if present, (c), but not prior thereto. It is, however, also possible for there to be complex present before the final one oE these components is added, in which case the final component will become complexed by displacing one component of the complex. It may be necessary to incubate these components for a period of time to allow the complexing reaction to approach equilibrium before component (d) is added. Addition of component (d~ should not affect the complexing reaction, but these components must be present before measurements can be taken at the working electrode.
The method of the present invention is applicable to e.g. 'direct' assays (in which component (c) is absent) r 'displacement' assays or 'competitive' assays (in which a ligand analogue is present in component (c)). rrhe method of the invention may employ the so-called "sandwich" technique, using a solid phase binding partner. Depending on the order of the complexiny reacLions, the forward, fast forward, reverse and simultaneous variations are all possible according to the present invention.
The solid phase may comprise the electrode surface, or may take the form of particles, beads etc.
The solid phase binding partner may be prepared by any one of a number of conventional techniques for immobilising reagents onto solid supports.
In a method of the invention exploiting the time (t) parameter, the rate of perturbation of the electrochemical characteristic as a result of complex formation may be determined. Conveniently, the initial rate of perturbation will be measured.
Such a method is applicable, for example, to a competitive assay in which the ligand and labelled ligand analogue compete for complexing with the specific binding partner. Thus, the initial rate of perturbation is related to the concentration of ligand present and from a calibration plot the - 10 ~
initial rate of perturbation v. concentration of ligand present, the ligand assay can be readily determinedO
The method of assay involving a determination of the rate of perturbation is also applicable to non-competitive assays where the labelled ligand analogue is absent and sufficient labelled specific bindlng partner is employed to enable all the ligand introduced to be complexed.
Measurement of, for example, the absolute electrochemical current generated after a standard incubation period may enhance the ease and sensitivity of the assay.
In a typical heterogeneous assay, formation of the complex causes no (or only a slight) perturbation in an electrochemical characteristic of the components.
In that case, it will generally be necessary artifically to generate or enhance a perturbation by controlled external influences. Although the magnitude of the external influence may have some bearing on the change induced, and must therefore be consistent with any such influence employed in calibration experiments, it is thought that any change produced in the perturbation remains a function of the ligand/specific binding partner complex. [Artificial generation or enhancement of a perturbation may also be desirable in homogeneous assays]
The artificial generation or enhancement of the perturbation is preferably performed by displacement of the complex relative to the unbound labelled component, or example by providing component (b) in an insolubilised form coupled (e.g. in conventional manner1 to a solid support. Alternatively, the complex can be further complexed with a species which will bind specifically to the complex, coupled to a solid support, with subsequent displacement of the support and coupled molecules. In extreme cases, the displacement may constitute complete ~2~
~ 20208-1257 removal of the complex from the apparatus, but in genera] the complex will be ~isplaced within the apparatus.
The solid support may, for example, comprise S the electrode surface or may take the form of conventional solid phase particles, beads etc. The solid support may be magnetic or magnetisable to facilitate displacement or separation. Thus, for example, magnetic supports (e.g. in the form of particles or beads) may be composed of ferromagnetic or paramegnetic materials such as metals (e.g. iron, nickel or cobalt), metal alloys (e.g. magnetic alloys of aluminium, nickel, cobalt and copper), metal oxides (e.g. Fe3O4 ~-Fe3O3, CrO2, CoO, NiO or Mn2O3), magnetoplumbites or solid solutions (e.g. solid solutions of magnetite with ferric oxide). The preferred material for magnetic supports is magnetite (Fe3O4) or haematite (~-Fe2O3).
Particles may be non-colloidal or collo;dal.
Displacement of the solid support, may, or example, be effected by urging the support into the vicinity of the electrode. In the case of magnetic supports (e~g. particles), the methods described in our copending Canadian Patent Application No. 486,546 may suitably be employed. Thus, for example, a magnetic electrode (e.g. comprising a permanent magnet or an electromagent) may be used, or a non-magnetic electrode may be used in which case the particles will be urged into and retained in the vicinity of the electrode by the application of an external magnetic field.
The component (b) may be immobilised directly on to the magnetic support, or may be immobilised via one or more other 'spacer' molecules, including partners in specific binding interactions. Immobilisation of reagents may generally be achieved by conventional techniques such as, for example, adsorbtion, covalent bonding or cross-linking, or a combination of these techniques, e.g. adsorption of a chemical with one or more functional groups followed by covalent bonding or cross-linking of the reagent. Alternatively, substantially non-chemical means may be employed.
Suitable immobilisation techniques are known in the art~
Other methods for artifically generating or enhancing the perturbation include, for example, removing excess uncomplexed labelled reagent, e.g.
by draining from the apparatus or by coupling to a suitable solid support and removing the said solid support from the apparatus.
All of the variations described above for homogeneous assays (including direct, competitive, sandwich and displacement techniques and methods in which a rate of perturbation is measured rather than an absolute perturbation) are equally applicable to heterogeneous assays.
The methods of the present invention may generally be simpler than known methods, in that they may eliminate the need for separation of uncomplexed and complexed phases before the assaying step.
However, as indicated above, the invention also includes within its scope methoas in which reagents are employed immobilised on a solid surface, in which methods it may be necessary or desirable to separate the solid (complexed) and uncomplexed phases before the assaying step. Such separation may take the form of complete removal of the solid phase from the assay medium or may, for example, take the form of sedimentation or concentration of the solid phase in one region of the assay medium.
If electrode-immobilised components are employed, the need for separate addition of the component to the electrochemical apparatus may be eliminated.
Additionally, the direct interaction between the electrode and the electrode-immobilised species may lead to an improvement in the sensitivity of the perturbation measurements.
According to a further feature of the present invention, therefore, there are provided methods of assay of a ligand in a sample as hereinbefore defined wherein one or more of the components (b) and, if present, (c) is/are immobilised on the working electrode or a suitable solid surface.
The immobiLized component(s) may be bound to the working surface of the working electrode or to a portion of the working electrode other than the working surface.
The immobilised component is preferably that component which is labelled. The said component may be immobilised via the label as long as the ability of the label to mediate electron transfer is not impaired.
Thus, for example, a polyviologen label may be covalently bonded to a metal electrode. The large polyviologen molecule projects from the electrode surface and this is believed to facilitate interaction with the enzyme. Alternatively, chloranil and/or fluoranil may be disseminated throughout an electrode composed of particulate carbon.
In one embodiment, the system comprises an electrode, e.g. a carbon (for example pyrolytic graphite) electrode, or a suitable solid surface carrying an immobilised layer of ferrocene dicarboxylic acid, l,l'-dimethylferrocene lDMF) or polyvinylferrocene 30 (having an average molecular weight of about 16000), the molecules of which are also coupled to reagent lb) or, if present (c), as labels thereof.
2~
The carbon electrode core or suitable solid surface can be integral or a stiEf paste of particles.
Normally, any solid surface employed will present a porous surface for the ferrocene or ferrocene derivative, which may be adhered thereto ln a number of ways, for examples:
(a) for monomeric ferrocene or a monomeric ferrocene derivative, by deposition from a solution in a readily evaporatable liquid e.g. an organic solvent such as toluene;
(b) for a ferrocene polymeric derivative, e.g.
polyvinyl-ferrocene of average molecular weight about 16000 (for a method of synthesis see J. Polymer Sci. 1976, 14, 2433), deposition from a readily evaporatable organic solvent for the polymer such as chloroform;
(c) for a polymerisable ferrocene-type monomerr by electrochemically induced polymerisation in situ, elg. by dissolving vinylferrocene in an organic electrolyte containing tertiary butyl ammonium perchlorate in concentration about lM and depositing at a potential of -700 mV to induce deposition of vinylferrocene radicals as a polymer in situ; or 5 (d) by covalent modification of the solid surEace e.g. by carbodiimide cross-linking of the ferrocene or ferrocene derivative onto the surface (e.g. a carbon electrode).
Alternatively, the component may be immobilised directly on the solid surface by any of the conventional techniques used for coupling reagents to solid supports.
If desired, the electrode-immobilised component may be bound to a portion of the electrode other than the working surface. The electrode may in these circumstances be constructed so as to ensure that the immobilised component remains sufficiently close to the working surface to enable the assay 2~
to be carried out effectively. Such an electrode is illustrated in vertical cross-section in Figure 2 of the accompanying drawings, this being particularly suitable for "sandwich" immunoassays in which the immobilised component is an unlabelled specific binding partner (e.g. a capture antibody). The electrode of Figure 2 comprises an upwardly facing graphite working surface 1 in the base of a cell, the wall of which is formed by a polystyrene projection 2 from the body of the electrode. It is on this wall that a suitable specific binding partner may be immobilised (e.g. by adsorption). The electrical connection is provided by an insulated wire 3 secured to the bottom of the working surface by silver-loaded epoxy resin 4, the arrangement being encasedin epoxy resin 5 and sealed with polypropylene 6.
It will be appreciated that, when component (b) is electrode-immobilised, it is not possible artifically to generate or enhance a perturbation by displacement of the resulting complex. However, a perturbation may still be artifically generated or enhanced, for example by complexing any uncomplexed labelled component remaining in solution with a species which will complex specifically with that component, coupled to a solid support, with subsequent displacement of the support and coupled molecules.
In a further aspect, the present invention provides kits o~ reagents and/or apparatus for carrying out the assays of the invention. Suitable kits may comprise an electrochemical apparatus containing a working electrode, an auxiliary electrode and optionally a reference electrode, and an aqueous assay medium with suitable components present (either in solution or immobilised). Other components (e.g. further reagents etc) and the sample to be assayed may conveniently be introduced through an entry port provided in the apparatus.
The apparatus may be automated so that the components are added in a predetermined sequence, 2~
and the incubation temperature may be controlled.
Advantageously the apparatus may be pre-calibrated and provided with a scale whereby the perturb~tion in the electrochemical characteristic oE the components may be read off directly as an amount of ligand in the sample.
Examples of ligands which may be assayed by the method of the invention are given in Table I below, together with an indication of a suitable specific binding partner in each instance.
Table I
-Ligand Specific Binding Partner antigen specific antibody antibody antigen hormone hormone receptor hormone receptor hormone polynucleotide complementary polynucleotide strand strand avidin biotin biot.in avidin protein A immunoglobulin immunoglobulin protein A
enzyme enzyme cofactor (subst.rate) enzyme co~actor enzyme (substrate) lectins specific carbohydrate specific carbohydrate lectins of lectins The method of the invention has very broad applicability, but in particu.lar may be used to assay: hormones, including peptide hormones (e.g.
thyroid stimulating hormone (TSII), human chorionic gonadotrophin (HCG), lutenising hormone (LH), follicle 8~
stimulating hormone (FSH), insulin and prolactin) or non-peptide horrnones (e.g. steroid hormones such as cortisol, estradiol, progesterone and testosterone and thyroid hormones such as thyroxine (T4) and triiodothyronine), proteins (e.g. carcinoembryonic antigen (CEA) and alphafetoprotein (AFP)), drugs (e.g. digoxin), sugars, toxins or vitamins.
The invention will be particularly described hereinafter with reference to an antibody or an ]0 antigen as the ligand. However, the invention is not to be taken as being limited to assays of antibodies or antigens.
It will be understood that the term "antibody"
used herein includes within its scope a) any of the various classes or sub-classes of immunoglobulin, e.g. IgG, IgM, derived from any of the animals conventionally used, e.g. sheep, rabbits, goats or mice, b) monoclonal antibodies, c) intact molecules or "fragments" of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody, i.e. fragments devoid of the Fc portion (e.g., Fab, Fab', F(ab')2) or the so-called "half-molecule" fragments obtained by reductive cleavage of the disulphide bonds connecting the heavy chain components in the intact antibody.
The method of preparation of fragments of antibodies is well known in the art and will not be described herein.
The term "antigen" as used herein will be understood to include both permanently antigenic species (for example, proteins, bacteria, bacteria fragments, cells, cell fragments and viruses) and haptens which may be rendered antigenic under suitable conditions.
%~
Incorporation of, Eor example, a ferrocene label into the molecular structure of an antibody may for example be achieved by any of the following methods:
(i) providing the label with one or rnore functional groups capable of bonding interactions with the molecular structure of the antibody;
(ii) using cross-linking groups;
(iii) using the avidin-biotin binding system, (i.e.
avidin-labelled antibody binding with biotin-labelled ferrocene molecules or biotin-labelled antibody binding with avidin-labelled ferrocene).
Similar methods may be applied as desired for labelling an antigen molecule. Suitable methods are known in the art and will not be discussed in detail here. For example, the incorporation of ferrocene into certain steroids is described in Journal of Organometallic Chemistry, 160 (1978) pp. 223-230.
2~ Methods of purifying the labelled antibody or antigen are also known and include, for example, dialysis, density-gradient ultracentrifugation, gel filtration and ion-exchange chromatography.
The attachment of the label to the antibody or antigen can be via any portion of the molecular structure of the antibody or antigen, so long as immunological activity thereof is retained.
Immobilisation of an antibody or antiyen molecule onto the electrode or other suitable solid surface may be effected by various methods. The attachment of the antibody or antigen to the electrode or solid surface can be via any portion of the molecular structure so long as specific immunological activity is retained at the antibody or antigen binding site.
Thus, for example, electrode-immobilisation of unlabelled antibody or antigen reagent may be achieved by bonding interactions between functional ~%~ i2~
qroups on the antibody or anti~en molecule and the electrode, or by cross-linking or adsorption onto the surface of the electrode. Binding of reagents to the electrode may be accompli~hed by methods analogous to known methods for binding such reagents to solid supports ~e.g. particles and beads), for example those described in published European Patent Application No. 0 105 714.
Electrode-immobilisation of labelled reagent, may, for example, be achieved by any of the ollowing methods:
(i) incorporating a label molecule into the molecular structure of free reagent and subse~uently immobilising the reagent onto an electrode at a site remote from the label in the same way as described above for unmodified reagents;
(ii) incorporating a label molecule into the molecular structure of a pre-immobilised reagent;
(iii) incorporating a bifunctional label into the molecular structure of free antibody or antigen so as to enable one function to interact with the electrode; or (iv) incorporating a bifunctional label onto the electrode, so as to enable one function to interact with the molecular structure of free antibody or antigen.
The preferred label for use w;th an enzyme/substrate electron-source or electron-acceptor is ferrocene monocarboxylic acid.
When ascorbate i5 used as an electron-~source, and a catechol or aminophenol as a label, electrode-immobi];sation of labelled reagent is preferably achieved by adsorption or chemical reaction via the label on a suitably modified carbon electrode.
A discussion of methods of attachment of such species to carbon electrodes is given in Analytical Chemistry, Vol. 55, 9 (19~3), p. 1576.
~ i2~
sy way of example only, the invention inclucles inter _lia the following embodiments:
~- = antibody O = antigen M = mediator label E = electron-source or acceptor (e.y. enzyme + substrate) ~ = electrode surface o - solid phase (non-electrode); ? indicates ligand under assay 1. Direct Antibody Assay a) Soluble E + ~M add ~? ~ ? ~O M + E
~M
b1 Immobilised on electrode ~M ~ ~ ~- add ~? ~M~:~?
In both these assays the formation of the immune complex decreases the efficacy of the mediator, the change in signal being a measure of antibody concentration.
2 Direct Antiqen Assay .
a) Soluble M -C add O-7 ?-o~
~ E ~ + E
M -~ ~ M
The immune reaction alters the ability of the mediator/antibody complex to shuttle electrons to or from the electrode. Therefore the signal changes.
b) Immobilised ~M~ add ~? ~?
~ ~ E --~ ~ ~ E
--M ~ `
3. Competitive Antigen Assay a) Soluble M -O M-O>--E ~ cldd ~?> E -- M~ d ~ ~ E + M~
M~ ~-? ?~
Competition between the mediator-labelled antigen and the antigen under assay for the available antibody results in some of the mediator being perturbed, the signal relating to the concentration of antigen under assay.
b) Immobilised The immobilised system can take two forrns:
(i) On electrode surface ~M -O ~ E ~ ~? ~ ~ + E ? o~ M ~ ~?
M ~ M ~ o? M -O E
After separation~ the signal measured depends upon the ratio of the antigen under assay to mediator-labelled antigen. The electrode supplies a means of easy separation.
(ii) On solid phase (e.g. magnetic particles) M~ M~> o M~ c~ M ~ odd > E ~ M
E + E +
M~ ?~ ?~>0 ? ~ ? ~> O
1 sepa~clte AssAy ~ E
The sedimenting of the immune complex reduces the amount of mediator in solution hence perturbing the signal.
~8 4. Displacement Antiqen Assay M--0~ 0>--E ~ acld o-? E t M
M
?~
Displacement of mediator/antigen complex from the antibody by the antigen under assay results in an increase in signal.
5. Sandwich Antit~en AssaY
a) On electrode M~ tx~ ~? ~ - M sc~tate M ~ ~ , ~ ~> ~ odd,E ASSAY
~ ~< ~M ~
The binding of mediator-labelled antibody to the electrode via the antigen gives a measure of antigen concentration.
b) On solid phase ~ O ~1~-M
'~M t~dO~ sepatate,~d ~- M
~ - M ~ retain Supetnatant ~- M
2 5 ¦Odd E
ASSAY
This will only work if the free mediator is assayed, but still gives a measure of antigen concentration.
- 2~ -
a) On electrode M~ tx~ ~? ~ - M sc~tate M ~ ~ , ~ ~> ~ odd,E ASSAY
~ ~< ~M ~
The binding of mediator-labelled antibody to the electrode via the antigen gives a measure of antigen concentration.
b) On solid phase ~ O ~1~-M
'~M t~dO~ sepatate,~d ~- M
~ - M ~ retain Supetnatant ~- M
2 5 ¦Odd E
ASSAY
This will only work if the free mediator is assayed, but still gives a measure of antigen concentration.
- 2~ -
6. Competitive Sandwich AntibodY Assay a) On electrode ~ -M ~ sepQ~Qte ~ --r ASSAY
Competition between mediator-labelled antibody and the antibody under assay produces a signal which relates to the concentration of the antibody under assay.
b) On solid phase add <O>--M
--< 2=I~ --<0~--? r~aia ~ rr~ / Qdd E~ ASSAY
The mechanism of action is the same as the electrode immobilised system but the free mediator is used to give a signal.
The following Examples are intended to illustrate the invention more fully:
Example 1:
Evaluation of a conjuqate o~ thyroxine ~T4) and ferrocene monocarboxylic acid (FMCA) (T4-FMCA) as a mediator for glucose oxidase.
Preparation of starting materials (i) Ferrocene-modified thyroxine (T4) hormone (hapten) To stirred cooled (-lO~C) dimethylformamide (3 mls), was added ferrocene monocarboxylic acid (FMCA) (1 mmole) and then triethylamine (1.0 mmole). The mixture was then further cooled to -15C and stirred for 30 minutes and then allowed to warm up to room temperature and stirred for a further hour. The resulting carboxycarbonic anhydride of ferrocene was added dropwise to a solution of T4 in sodium hydroxide / ethanol and stirred for 24 hours, after which the sample was filtered and then concentrated by rotary evaporation. The resulting precipitate was washed in carbon tetrachloride, then ethyl acetate several times, the insoluble ferrocene monocarboxylic acid-T4 conjugate (T4-FMCA) then being filtered out of the ethyl acetate solution.
(ii) Purification of Ferrocene-modified thyroxine con~ugate (T4-FMCA) The T4-FMCA conjugate was purified by high performance liquid chromatography using a C18 reverse phase column~ After puriEication, the T4 and ferrocene concentrations in the conjugate were measured by radioimmunoassay and electrochemistry respectively.
(iil) Characteristics of the T4-FMCA coniuqate Incubation of T4-FMCA with excess anti-T4 ~L2~
antibody showed that 7~.4~ o~ the T4-E~MCA
was bound to the antibody.
The electrochemistry o~ the conjugate was also compared with that of the T4 and FMCA.
The conjugate shows two peaks on the forward wave of the cyclic voltammogram at a pyrolytic graphite electrode (Voltage Scan Rate = 20mVs-l), the first being at +310 mV vs a standard calomel electrode (S.C.E.~ (the ferrocene peak), the second at +410 mV V5 S.C.~. (T4) Figure 3. On the return wave only one peak at a potential of +255 mV vs S.C.E. was observed.
Cyclic voltammetry at pyrolytic graphite electrodes (Voltage Scan Rate = 5mVs-l) of the unmodified components of the conjugate showed that FMC~ [0.2 mM FMCA in Tris ~50 mM; pH 7.4] has peaks at -~320 mV and +2G0 mV vs S.C.E. for the forward and return waves respectively (Figure 4a) whilst T4 exhibits a single peak at -~420 mV vs S.C.E. on the forward wave (Figure 4b).
(iv) Anti-T4 Antibody Anti-T4 antibody was a conventional polyclonal antiserum obtained by immunising sheep with T4 conjugated to a high molecular weight protein (keyhole limpet haemocyanin).
(v) Standard Solutions of T4 T4 (sodium salt) was obtained from Sigma London Chemical Company, England. Standard solutions were made by disolving the T4 in sodium hydroxide (0.1 M) and then diluting with Tris-HCl buffer ~50 mM p~l 7.4? to the desired concentration.
(vi) Ap~aratus used to measure the electrochemistry of T~-F _ Cyclic voltammetry was performed using a three electrode electrochemical cell with a pyrolytic graphite working electrode as shown in figure l(a).
Evaluation of the performance of the T4-FMCA conjuqate as an electron transfer mediator for qlucose oxidase 40 ul of a solution of T4-FMCA (3 x 10 7 molar, in sodium proprionate buffer, 200 mM/l, pH 6.0~
was added to the electrochemical cell along with 40 ul of glucose (a molar solution containing lOOmM/l of magnesium chloride) and 320 ul of Tris/HCl buffer (10 mM/l, pH 7.4). After measurement of the electro-chemical current, the above experiment was repeatedwith 20 ul of a solution of glucose oxidase (1 mg of enzyme per ml in water) added, but only 300 ,ul of buffer. Again the electrochemical current was measured.
In a third series of experiments, 20 ~1 of the anti-T4 antiserum was added to 40 ul of the T4-FMCA conjugate and 280 ,ul of the buffer. ~fter an incubation period of 20 minutes, 20 ~1 of glucose oxidase solution and 40 ul of g]ucose solution were added and the electrochemical current remeasured.
In a fourth series of experiments, 20 ,ul of a solution of T4 tO.5 mM per litre in Tris/HLC buffer, lOmM/l, p~ 7~4), 20 ul of the anti-T4 antiserum, 40 ~1 of the T~-FMCA conjugate and 260 ~1 of Tris/HCl buffer were mixed and incubated for 20 minutes.
After addition of 20 ~1 of the solution of glucose oxidase and 40 ~1 of the glucose solution, the electrochemical current was measured.
In all cases, the electrochemical current was measured at a temperature of 37 + 0.5C and at a voltage scan rate of 5 mV/S. The results are presented in Table ].
The results show that the T4-FMCA will act as an electron transfer mediator for glucose oxidase, its ability to mediate being perturbed upon binding with anti-T4 antibody.
ELECTROCHEMICAL
EXPERIMENTCURRENT (~A) T4-FMCA -~ glucose + buffer Ø58 T4-FMCA + glucose + buffer +
glucose oxidase 1.58+ 0~04 T4-FMCA + glucose + buffer +
glucose oxidase + anti-T4-antibody 1.35+ 0.01 T4-FMCA -~ glucose + buffer -~
glucose oxidase ~ anti-T4-antibody 1.53+ 0.05 + T4 standard Example 2: Ferrocene-modified IqG antibod~
To a stirred cooled (-8C) solution of ferrocene monocarboxylic acid (1 mmole) in tetrahydrofuran (dried), isobutyl chloroformate (1 mmole) and triethyl-amine (1 mmole) were added with stirring. The mixture was stirred for thirty minutes and then allowed to warm up to room temperature and stirred for a further hour. The resulting carboxycarbonic anhydride of ferrocene was added dropwise to a cooled (2C) solution of IgG (500 mg) in 50 ml of O.lM sodium bicarbonate solution. The reaction mixture was stirred at 4C for 24 hours and then dialysed exhaustively against borate buffer pH 8.5.
It was spun and gel filtered on S-200 gel.
The iron content was determined by atomic absorption.
Competition between mediator-labelled antibody and the antibody under assay produces a signal which relates to the concentration of the antibody under assay.
b) On solid phase add <O>--M
--< 2=I~ --<0~--? r~aia ~ rr~ / Qdd E~ ASSAY
The mechanism of action is the same as the electrode immobilised system but the free mediator is used to give a signal.
The following Examples are intended to illustrate the invention more fully:
Example 1:
Evaluation of a conjuqate o~ thyroxine ~T4) and ferrocene monocarboxylic acid (FMCA) (T4-FMCA) as a mediator for glucose oxidase.
Preparation of starting materials (i) Ferrocene-modified thyroxine (T4) hormone (hapten) To stirred cooled (-lO~C) dimethylformamide (3 mls), was added ferrocene monocarboxylic acid (FMCA) (1 mmole) and then triethylamine (1.0 mmole). The mixture was then further cooled to -15C and stirred for 30 minutes and then allowed to warm up to room temperature and stirred for a further hour. The resulting carboxycarbonic anhydride of ferrocene was added dropwise to a solution of T4 in sodium hydroxide / ethanol and stirred for 24 hours, after which the sample was filtered and then concentrated by rotary evaporation. The resulting precipitate was washed in carbon tetrachloride, then ethyl acetate several times, the insoluble ferrocene monocarboxylic acid-T4 conjugate (T4-FMCA) then being filtered out of the ethyl acetate solution.
(ii) Purification of Ferrocene-modified thyroxine con~ugate (T4-FMCA) The T4-FMCA conjugate was purified by high performance liquid chromatography using a C18 reverse phase column~ After puriEication, the T4 and ferrocene concentrations in the conjugate were measured by radioimmunoassay and electrochemistry respectively.
(iil) Characteristics of the T4-FMCA coniuqate Incubation of T4-FMCA with excess anti-T4 ~L2~
antibody showed that 7~.4~ o~ the T4-E~MCA
was bound to the antibody.
The electrochemistry o~ the conjugate was also compared with that of the T4 and FMCA.
The conjugate shows two peaks on the forward wave of the cyclic voltammogram at a pyrolytic graphite electrode (Voltage Scan Rate = 20mVs-l), the first being at +310 mV vs a standard calomel electrode (S.C.E.~ (the ferrocene peak), the second at +410 mV V5 S.C.~. (T4) Figure 3. On the return wave only one peak at a potential of +255 mV vs S.C.E. was observed.
Cyclic voltammetry at pyrolytic graphite electrodes (Voltage Scan Rate = 5mVs-l) of the unmodified components of the conjugate showed that FMC~ [0.2 mM FMCA in Tris ~50 mM; pH 7.4] has peaks at -~320 mV and +2G0 mV vs S.C.E. for the forward and return waves respectively (Figure 4a) whilst T4 exhibits a single peak at -~420 mV vs S.C.E. on the forward wave (Figure 4b).
(iv) Anti-T4 Antibody Anti-T4 antibody was a conventional polyclonal antiserum obtained by immunising sheep with T4 conjugated to a high molecular weight protein (keyhole limpet haemocyanin).
(v) Standard Solutions of T4 T4 (sodium salt) was obtained from Sigma London Chemical Company, England. Standard solutions were made by disolving the T4 in sodium hydroxide (0.1 M) and then diluting with Tris-HCl buffer ~50 mM p~l 7.4? to the desired concentration.
(vi) Ap~aratus used to measure the electrochemistry of T~-F _ Cyclic voltammetry was performed using a three electrode electrochemical cell with a pyrolytic graphite working electrode as shown in figure l(a).
Evaluation of the performance of the T4-FMCA conjuqate as an electron transfer mediator for qlucose oxidase 40 ul of a solution of T4-FMCA (3 x 10 7 molar, in sodium proprionate buffer, 200 mM/l, pH 6.0~
was added to the electrochemical cell along with 40 ul of glucose (a molar solution containing lOOmM/l of magnesium chloride) and 320 ul of Tris/HCl buffer (10 mM/l, pH 7.4). After measurement of the electro-chemical current, the above experiment was repeatedwith 20 ul of a solution of glucose oxidase (1 mg of enzyme per ml in water) added, but only 300 ,ul of buffer. Again the electrochemical current was measured.
In a third series of experiments, 20 ~1 of the anti-T4 antiserum was added to 40 ul of the T4-FMCA conjugate and 280 ,ul of the buffer. ~fter an incubation period of 20 minutes, 20 ~1 of glucose oxidase solution and 40 ul of g]ucose solution were added and the electrochemical current remeasured.
In a fourth series of experiments, 20 ,ul of a solution of T4 tO.5 mM per litre in Tris/HLC buffer, lOmM/l, p~ 7~4), 20 ul of the anti-T4 antiserum, 40 ~1 of the T~-FMCA conjugate and 260 ~1 of Tris/HCl buffer were mixed and incubated for 20 minutes.
After addition of 20 ~1 of the solution of glucose oxidase and 40 ~1 of the glucose solution, the electrochemical current was measured.
In all cases, the electrochemical current was measured at a temperature of 37 + 0.5C and at a voltage scan rate of 5 mV/S. The results are presented in Table ].
The results show that the T4-FMCA will act as an electron transfer mediator for glucose oxidase, its ability to mediate being perturbed upon binding with anti-T4 antibody.
ELECTROCHEMICAL
EXPERIMENTCURRENT (~A) T4-FMCA -~ glucose + buffer Ø58 T4-FMCA + glucose + buffer +
glucose oxidase 1.58+ 0~04 T4-FMCA + glucose + buffer +
glucose oxidase + anti-T4-antibody 1.35+ 0.01 T4-FMCA -~ glucose + buffer -~
glucose oxidase ~ anti-T4-antibody 1.53+ 0.05 + T4 standard Example 2: Ferrocene-modified IqG antibod~
To a stirred cooled (-8C) solution of ferrocene monocarboxylic acid (1 mmole) in tetrahydrofuran (dried), isobutyl chloroformate (1 mmole) and triethyl-amine (1 mmole) were added with stirring. The mixture was stirred for thirty minutes and then allowed to warm up to room temperature and stirred for a further hour. The resulting carboxycarbonic anhydride of ferrocene was added dropwise to a cooled (2C) solution of IgG (500 mg) in 50 ml of O.lM sodium bicarbonate solution. The reaction mixture was stirred at 4C for 24 hours and then dialysed exhaustively against borate buffer pH 8.5.
It was spun and gel filtered on S-200 gel.
The iron content was determined by atomic absorption.
Claims (11)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of assaying a ligand in a sample using electrochemical apparatus containing an electrode and components comprising:
(a) the sample, (b) a specific binding partner to the ligand, (c) if desired, at least one further reagent selected from ligand analogues and specific binding partners to the ligand, and (d) an electron-source or electron-acceptor;
at least one of components (b) and, if present, (c)being labelled with an electron-transfer mediator capable of aiding the transfer of electrons from the electron-source to the electrode, or from the electrode to the electron-acceptor, which method includes the step of determining whether, and, if desired, the extent to which, the said transfer of electrons is perturbed by at least one of (i) complex formation and (ii) a controlled external influence which produces a perturbation of said transfer of electrons as a function of said complex formation.
(a) the sample, (b) a specific binding partner to the ligand, (c) if desired, at least one further reagent selected from ligand analogues and specific binding partners to the ligand, and (d) an electron-source or electron-acceptor;
at least one of components (b) and, if present, (c)being labelled with an electron-transfer mediator capable of aiding the transfer of electrons from the electron-source to the electrode, or from the electrode to the electron-acceptor, which method includes the step of determining whether, and, if desired, the extent to which, the said transfer of electrons is perturbed by at least one of (i) complex formation and (ii) a controlled external influence which produces a perturbation of said transfer of electrons as a function of said complex formation.
2. A method as claimed in claim 1 wherein component (d) is an oxidoreductase enzyme in co-operation with a substrate therefor.
3. A method as claimed in claim 1 wherein the controlled external influence, when present, comprises displacement of the complex formed relative to the unbound labelled component.
4. A method as claimed in claim 1 wherein one or more of the components (b) and, if present, (c) is/are immobilised on the working electrode or a suitable solid surface.
5. A method as claimed in claim 1 wherein the electron-transfer mediator comprises ferrocene or a derivative thereof.
6. A method as claimed in claim 5 wherein the electron-transfer mediator comprises a derivative of ferrocene containing one or more side chains of the formula -CHO, -(CH2)n COOH or -(CH2)m NR1R2 (where n and m are each from 0 to 6 and R1 and R2, which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms.)
7. A method as claimed in claim 1 wherein the perturbation on the transfer of electrons is determined from a perturbation in the peak current observed under the application of a preselected potential across the components.
8. A method as claimed in claim 1 wherein the ligand is an antigen or an antibody.
9. A kit for carrying out a method of assay as claimed in claim 1 comprising in separate containers:
(i) a specific binding partner to the ligand or a specific binding partner to the ligand and at least on further reagent selected from ligand analogues and specific binding partners, component (i) or at least one of the components (i) being labelled with an electron transfer mediator, (ii) an oxidoreductase enzyme capable of donating electrons to said electron transfer mediator or accepting electrons from said electron transfer mediator; and (iii) a substrate for said enzyme.
(i) a specific binding partner to the ligand or a specific binding partner to the ligand and at least on further reagent selected from ligand analogues and specific binding partners, component (i) or at least one of the components (i) being labelled with an electron transfer mediator, (ii) an oxidoreductase enzyme capable of donating electrons to said electron transfer mediator or accepting electrons from said electron transfer mediator; and (iii) a substrate for said enzyme.
10. A kit for carrying out a method of assay as claimed in claim 1 comprising in one or more containers:
(i) a specific binding partner to the ligand or a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners, component (i) or at least one of the components (i) being labelled with an electron transfer mediator; and (ii) a non-enzyme electron source capable of donating electrons to said electron transfer mediator or a non-enzyme electron acceptor capable of accepting electrons from said electron transfer mediator.
(i) a specific binding partner to the ligand or a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners, component (i) or at least one of the components (i) being labelled with an electron transfer mediator; and (ii) a non-enzyme electron source capable of donating electrons to said electron transfer mediator or a non-enzyme electron acceptor capable of accepting electrons from said electron transfer mediator.
11. A kit as claimed in claim 9 or claim 10 which further comprises an electrochemical apparatus containing a working electrode.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8417301 | 1984-07-06 | ||
GB848417301A GB8417301D0 (en) | 1984-07-06 | 1984-07-06 | Assay |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1268520A true CA1268520A (en) | 1990-05-01 |
Family
ID=10563527
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000481357A Expired - Lifetime CA1268520A (en) | 1984-07-06 | 1985-05-13 | Methods of assay |
Country Status (8)
Country | Link |
---|---|
US (1) | US4945045A (en) |
EP (1) | EP0167248B1 (en) |
AT (1) | ATE100203T1 (en) |
AU (1) | AU597562B2 (en) |
CA (1) | CA1268520A (en) |
DE (1) | DE3587720T2 (en) |
GB (1) | GB8417301D0 (en) |
IL (1) | IL75174A (en) |
Families Citing this family (207)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3479522D1 (en) * | 1983-12-16 | 1989-09-28 | Medisense Inc | Assay for nucleic acids |
GB8417301D0 (en) | 1984-07-06 | 1984-08-08 | Serono Diagnostics Ltd | Assay |
US5185256A (en) * | 1985-06-21 | 1993-02-09 | Matsushita Electric Industrial Co., Ltd. | Method for making a biosensor |
GB8523631D0 (en) * | 1985-09-25 | 1985-10-30 | Pena Ltd Paul De | Bioelectrochemical cell |
US5770459A (en) * | 1986-04-30 | 1998-06-23 | Igen International, Inc. | Methods and apparatus for improved luminescence assays using particle concentration, electrochemical generation of chemiluminescence detection |
FR2599844B1 (en) * | 1986-06-09 | 1990-05-11 | Centre Nat Rech Scient | SOLID CONDUCTIVE PHASE FOR IMMUNOENZYMATIC ASSAY, PREPARATION METHOD THEREOF AND USE IN IMMUNOENZYMATIC ASSAY PROCESSES |
US5935779A (en) * | 1988-11-03 | 1999-08-10 | Igen International Inc. | Methods for improved particle electrochemiluminescence assay |
US6881589B1 (en) | 1987-04-30 | 2005-04-19 | Bioveris Corporation | Electrochemiluminescent localizable complexes for assay compositions |
US5962218A (en) * | 1988-11-03 | 1999-10-05 | Igen International Inc. | Methods and apparatus for improved luminescence assays |
US5779976A (en) * | 1988-11-03 | 1998-07-14 | Igen International, Inc. | Apparatus for improved luminescence assays |
US5705402A (en) * | 1988-11-03 | 1998-01-06 | Igen International, Inc. | Method and apparatus for magnetic microparticulate based luminescence assay including plurality of magnets |
US5746974A (en) * | 1988-11-03 | 1998-05-05 | Igen International, Inc. | Apparatus for improved luminescence assays using particle concentration, electrochemical generation of chemiluminescence and chemiluminescence detection |
SE8902043L (en) * | 1988-11-10 | 1990-05-11 | Pharmacia Ab | PROCEDURE CHARACTERIZES MACROMOLECULES |
US5063081A (en) * | 1988-11-14 | 1991-11-05 | I-Stat Corporation | Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor |
US4927502A (en) * | 1989-01-31 | 1990-05-22 | Board Of Regents, The University Of Texas | Methods and apparatus using galvanic immunoelectrodes |
US5198367A (en) * | 1989-06-09 | 1993-03-30 | Masuo Aizawa | Homogeneous amperometric immunoassay |
CA2027694C (en) * | 1989-10-20 | 2002-03-05 | Tadashi Matsunaga | Process and apparatus for detecting sensitized leukocyte or antigen |
JPH0795170B2 (en) * | 1990-08-21 | 1995-10-11 | ダイソー株式会社 | Polyviologen modified electrode and its application |
US5824477A (en) * | 1990-09-12 | 1998-10-20 | Scientific Generics Limited | Electrochemical denaturation of double-stranded nucleic acid |
US6197508B1 (en) | 1990-09-12 | 2001-03-06 | Affymetrix, Inc. | Electrochemical denaturation and annealing of nucleic acid |
US5527670A (en) * | 1990-09-12 | 1996-06-18 | Scientific Generics Limited | Electrochemical denaturation of double-stranded nucleic acid |
ZA92803B (en) | 1991-02-06 | 1992-11-25 | Igen Inc | Method and apparatus for magnetic microparticulate based luminescene asay including plurality of magnets |
GB2255637B (en) * | 1991-03-20 | 1995-11-15 | Marconi Gec Ltd | Separation method |
GB9201481D0 (en) * | 1992-01-23 | 1992-03-11 | Scient Generics Ltd | Treatment of nucleic acid material |
JPH05236997A (en) * | 1992-02-28 | 1993-09-17 | Hitachi Ltd | Chip for catching polynucleotide |
FR2692357A1 (en) * | 1992-06-12 | 1993-12-17 | Centre Nat Rech Scient | Redox-labeled antigen, immunoassay method with electrochemical detection and assay kit. |
GB9225354D0 (en) * | 1992-12-04 | 1993-01-27 | Univ Manchester | Immunoassay |
US5494831A (en) * | 1993-08-30 | 1996-02-27 | Hughes Aircraft Company | Electrochemical immunosensor system and methods |
US6071699A (en) | 1996-06-07 | 2000-06-06 | California Institute Of Technology | Nucleic acid mediated electron transfer |
US5824473A (en) * | 1993-12-10 | 1998-10-20 | California Institute Of Technology | Nucleic acid mediated electron transfer |
IL108726A (en) * | 1994-02-22 | 1999-12-31 | Yissum Res Dev Co | Electrobiochemical method and system for the determination of an analyte which is a member of a recognition pair in a liquid medium and electrodes therefor |
ATE175723T1 (en) | 1994-03-15 | 1999-01-15 | Scient Generics Ltd | ELECTROCHEMICAL DENATUSATION OF DOUBLE STRANDED NUCLEIC ACID |
US5620850A (en) | 1994-09-26 | 1997-04-15 | President And Fellows Of Harvard College | Molecular recognition at surfaces derivatized with self-assembled monolayers |
US5866434A (en) * | 1994-12-08 | 1999-02-02 | Meso Scale Technology | Graphitic nanotubes in luminescence assays |
AU5920696A (en) * | 1995-05-18 | 1996-11-29 | Igen, Inc. | Method for derivitizing electrodes and assay methods using s uch derivitized electrodes |
US6127127A (en) * | 1995-06-27 | 2000-10-03 | The University Of North Carolina At Chapel Hill | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
US6132971A (en) * | 1995-06-27 | 2000-10-17 | The University Of North Carolina At Chapel Hill | Microelectronic device |
US6346387B1 (en) * | 1995-06-27 | 2002-02-12 | Xanthon, Inc. | Detection of binding reactions using labels detected by mediated catalytic electrochemistry |
US6387625B1 (en) | 1995-06-27 | 2002-05-14 | The University Of North Carolina At Chapel Hill | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
US6361951B1 (en) * | 1995-06-27 | 2002-03-26 | The University Of North Carolina At Chapel Hill | Electrochemical detection of nucleic acid hybridization |
US5968745A (en) * | 1995-06-27 | 1999-10-19 | The University Of North Carolina At Chapel Hill | Polymer-electrodes for detecting nucleic acid hybridization and method of use thereof |
US6180346B1 (en) | 1995-06-27 | 2001-01-30 | The Universtiy Of North Carolina At Chapel Hill | Electropolymerizable film, and method of making and use thereof |
US6165335A (en) | 1996-04-25 | 2000-12-26 | Pence And Mcgill University | Biosensor device and method |
US6130037A (en) * | 1996-04-25 | 2000-10-10 | Pence And Mcgill University | Biosensor device and method |
US7381525B1 (en) | 1997-03-07 | 2008-06-03 | Clinical Micro Sensors, Inc. | AC/DC voltage apparatus for detection of nucleic acids |
US7014992B1 (en) * | 1996-11-05 | 2006-03-21 | Clinical Micro Sensors, Inc. | Conductive oligomers attached to electrodes and nucleoside analogs |
US6096273A (en) * | 1996-11-05 | 2000-08-01 | Clinical Micro Sensors | Electrodes linked via conductive oligomers to nucleic acids |
US7045285B1 (en) * | 1996-11-05 | 2006-05-16 | Clinical Micro Sensors, Inc. | Electronic transfer moieties attached to peptide nucleic acids |
JP3394262B2 (en) | 1997-02-06 | 2003-04-07 | セラセンス、インク. | Small volume in vitro analyte sensor |
US6013459A (en) | 1997-06-12 | 2000-01-11 | Clinical Micro Sensors, Inc. | Detection of analytes using reorganization energy |
US6036924A (en) | 1997-12-04 | 2000-03-14 | Hewlett-Packard Company | Cassette of lancet cartridges for sampling blood |
US6686150B1 (en) * | 1998-01-27 | 2004-02-03 | Clinical Micro Sensors, Inc. | Amplification of nucleic acids with electronic detection |
AU764926B2 (en) * | 1998-01-27 | 2003-09-04 | Clinical Micro Sensors, Inc. | Amplification of nucleic acids with electronic detection |
US6063573A (en) * | 1998-01-27 | 2000-05-16 | Clinical Micro Sensors, Inc. | Cycling probe technology using electron transfer detection |
US6391005B1 (en) | 1998-03-30 | 2002-05-21 | Agilent Technologies, Inc. | Apparatus and method for penetration with shaft having a sensor for sensing penetration depth |
US6175752B1 (en) | 1998-04-30 | 2001-01-16 | Therasense, Inc. | Analyte monitoring device and methods of use |
US9066695B2 (en) | 1998-04-30 | 2015-06-30 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
US8688188B2 (en) | 1998-04-30 | 2014-04-01 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
US8465425B2 (en) | 1998-04-30 | 2013-06-18 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
US6949816B2 (en) | 2003-04-21 | 2005-09-27 | Motorola, Inc. | Semiconductor component having first surface area for electrically coupling to a semiconductor chip and second surface area for electrically coupling to a substrate, and method of manufacturing same |
US8346337B2 (en) | 1998-04-30 | 2013-01-01 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
US8974386B2 (en) | 1998-04-30 | 2015-03-10 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
US8480580B2 (en) | 1998-04-30 | 2013-07-09 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
AU4215999A (en) | 1998-06-01 | 1999-12-20 | Roche Diagnostics Corporation | Redox reversible imidazole-osmium complex conjugates |
WO1999064847A1 (en) * | 1998-06-08 | 1999-12-16 | Xanthon, Inc. | Electrochemical probes for detection of molecular interactions and drug discovery |
US7087148B1 (en) | 1998-06-23 | 2006-08-08 | Clinical Micro Sensors, Inc. | Binding acceleration techniques for the detection of analytes |
US20050244954A1 (en) * | 1998-06-23 | 2005-11-03 | Blackburn Gary F | Binding acceleration techniques for the detection of analytes |
US6761816B1 (en) | 1998-06-23 | 2004-07-13 | Clinical Micro Systems, Inc. | Printed circuit boards with monolayers and capture ligands |
US6281006B1 (en) | 1998-08-24 | 2001-08-28 | Therasense, Inc. | Electrochemical affinity assay |
US6251260B1 (en) | 1998-08-24 | 2001-06-26 | Therasense, Inc. | Potentiometric sensors for analytic determination |
US6740518B1 (en) | 1998-09-17 | 2004-05-25 | Clinical Micro Sensors, Inc. | Signal detection techniques for the detection of analytes |
US6591125B1 (en) | 2000-06-27 | 2003-07-08 | Therasense, Inc. | Small volume in vitro analyte sensor with diffusible or non-leachable redox mediator |
US6338790B1 (en) | 1998-10-08 | 2002-01-15 | Therasense, Inc. | Small volume in vitro analyte sensor with diffusible or non-leachable redox mediator |
US6541617B1 (en) | 1998-10-27 | 2003-04-01 | Clinical Micro Sensors, Inc. | Detection of target analytes using particles and electrodes |
US6833267B1 (en) * | 1998-12-30 | 2004-12-21 | Clinical Micro Sensors, Inc. | Tissue collection devices containing biosensors |
CA2360927C (en) * | 1999-01-23 | 2018-03-06 | Minerva Biotechnologies Corporation | Interaction of colloid-immobilized species with species on non-colloidal structures |
US20050148101A1 (en) * | 1999-01-23 | 2005-07-07 | Bamdad Cynthia C. | Interaction of colloid-immobilized species with species on non-colloidal structures |
WO2000043791A2 (en) * | 1999-01-25 | 2000-07-27 | Minerva Biotechnologies Corporation | Rapid and sensitive detection of aberrant protein aggregation in neurodegenerative diseases |
AU2898500A (en) | 1999-03-02 | 2000-09-21 | Helix Biopharma Corporation | Biosensor device and method |
US20020177135A1 (en) * | 1999-07-27 | 2002-11-28 | Doung Hau H. | Devices and methods for biochip multiplexing |
US7312087B2 (en) | 2000-01-11 | 2007-12-25 | Clinical Micro Sensors, Inc. | Devices and methods for biochip multiplexing |
EP2322645A1 (en) | 1999-06-18 | 2011-05-18 | Abbott Diabetes Care Inc. | Mass transport limited in vivo analyte sensor |
US7935481B1 (en) | 1999-07-26 | 2011-05-03 | Osmetech Technology Inc. | Sequence determination of nucleic acids using electronic detection |
US6616819B1 (en) | 1999-11-04 | 2003-09-09 | Therasense, Inc. | Small volume in vitro analyte sensor and methods |
US6824669B1 (en) * | 2000-02-17 | 2004-11-30 | Motorola, Inc. | Protein and peptide sensors using electrical detection methods |
US6753143B2 (en) | 2000-05-01 | 2004-06-22 | Clinical Micro Sensors, Inc. | Target analyte detection using asymmetrical self-assembled monolayers |
JP4382265B2 (en) * | 2000-07-12 | 2009-12-09 | 日本電気株式会社 | Method and apparatus for forming silicon oxide film |
EP1325332B1 (en) * | 2000-10-03 | 2006-12-20 | Minerva Biotechnologies Corporation | Magnetic in situ dilution |
US7456028B2 (en) * | 2000-10-16 | 2008-11-25 | Board Of Trustees Of The University Of Arkansas, N.A. | Electrochemical method for detecting water born pathogens |
EP1348034B1 (en) * | 2000-11-15 | 2016-07-20 | Minerva Biotechnologies Corporation | Oligonucleotide identifiers |
US8641644B2 (en) | 2000-11-21 | 2014-02-04 | Sanofi-Aventis Deutschland Gmbh | Blood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means |
US6560471B1 (en) | 2001-01-02 | 2003-05-06 | Therasense, Inc. | Analyte monitoring device and methods of use |
US7435384B2 (en) * | 2001-01-08 | 2008-10-14 | Leonard Fish | Diagnostic instrument with movable electrode mounting member and methods for detecting analytes |
WO2002078512A2 (en) | 2001-04-02 | 2002-10-10 | Therasense, Inc. | Blood glucose tracking apparatus and methods |
DE60238119D1 (en) | 2001-06-12 | 2010-12-09 | Pelikan Technologies Inc | ELECTRIC ACTUATOR ELEMENT FOR A LANZETTE |
US7749174B2 (en) | 2001-06-12 | 2010-07-06 | Pelikan Technologies, Inc. | Method and apparatus for lancet launching device intergrated onto a blood-sampling cartridge |
US7316700B2 (en) | 2001-06-12 | 2008-01-08 | Pelikan Technologies, Inc. | Self optimizing lancing device with adaptation means to temporal variations in cutaneous properties |
ES2357485T3 (en) | 2001-06-12 | 2011-04-26 | Pelikan Technologies Inc. | INTEGRATED BLOOD SAMPLE ANALYSIS SYSTEM WITH MULTIPLE USES SAMPLE TAKING MODULE. |
US9226699B2 (en) | 2002-04-19 | 2016-01-05 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling module with a continuous compression tissue interface surface |
US9795747B2 (en) | 2010-06-02 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
US8337419B2 (en) | 2002-04-19 | 2012-12-25 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US7699791B2 (en) | 2001-06-12 | 2010-04-20 | Pelikan Technologies, Inc. | Method and apparatus for improving success rate of blood yield from a fingerstick |
EP1404232B1 (en) | 2001-06-12 | 2009-12-02 | Pelikan Technologies Inc. | Blood sampling apparatus and method |
US7981056B2 (en) | 2002-04-19 | 2011-07-19 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
US7025774B2 (en) | 2001-06-12 | 2006-04-11 | Pelikan Technologies, Inc. | Tissue penetration device |
US7344894B2 (en) | 2001-10-16 | 2008-03-18 | Agilent Technologies, Inc. | Thermal regulation of fluidic samples within a diagnostic cartridge |
US20030175947A1 (en) * | 2001-11-05 | 2003-09-18 | Liu Robin Hui | Enhanced mixing in microfluidic devices |
US8154093B2 (en) | 2002-01-16 | 2012-04-10 | Nanomix, Inc. | Nano-electronic sensors for chemical and biological analytes, including capacitance and bio-membrane devices |
GB0205455D0 (en) | 2002-03-07 | 2002-04-24 | Molecular Sensing Plc | Nucleic acid probes, their synthesis and use |
US7648468B2 (en) | 2002-04-19 | 2010-01-19 | Pelikon Technologies, Inc. | Method and apparatus for penetrating tissue |
US7175642B2 (en) | 2002-04-19 | 2007-02-13 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
US7291117B2 (en) | 2002-04-19 | 2007-11-06 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8579831B2 (en) | 2002-04-19 | 2013-11-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US7491178B2 (en) | 2002-04-19 | 2009-02-17 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7371247B2 (en) | 2002-04-19 | 2008-05-13 | Pelikan Technologies, Inc | Method and apparatus for penetrating tissue |
US7713214B2 (en) | 2002-04-19 | 2010-05-11 | Pelikan Technologies, Inc. | Method and apparatus for a multi-use body fluid sampling device with optical analyte sensing |
US7297122B2 (en) | 2002-04-19 | 2007-11-20 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7547287B2 (en) | 2002-04-19 | 2009-06-16 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7674232B2 (en) | 2002-04-19 | 2010-03-09 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7563232B2 (en) | 2002-04-19 | 2009-07-21 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7374544B2 (en) | 2002-04-19 | 2008-05-20 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7524293B2 (en) | 2002-04-19 | 2009-04-28 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8702624B2 (en) | 2006-09-29 | 2014-04-22 | Sanofi-Aventis Deutschland Gmbh | Analyte measurement device with a single shot actuator |
US8267870B2 (en) | 2002-04-19 | 2012-09-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling with hybrid actuation |
US7232451B2 (en) | 2002-04-19 | 2007-06-19 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8372016B2 (en) | 2002-04-19 | 2013-02-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling and analyte sensing |
US7717863B2 (en) | 2002-04-19 | 2010-05-18 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7909778B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7901362B2 (en) | 2002-04-19 | 2011-03-08 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7331931B2 (en) | 2002-04-19 | 2008-02-19 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US9314194B2 (en) | 2002-04-19 | 2016-04-19 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US7976476B2 (en) | 2002-04-19 | 2011-07-12 | Pelikan Technologies, Inc. | Device and method for variable speed lancet |
US9795334B2 (en) | 2002-04-19 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US7229458B2 (en) | 2002-04-19 | 2007-06-12 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
WO2003088824A2 (en) | 2002-04-19 | 2003-10-30 | Pelikan Technologies, Inc. | Device and method for variable speed lancet |
US8360992B2 (en) | 2002-04-19 | 2013-01-29 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US7892183B2 (en) | 2002-04-19 | 2011-02-22 | Pelikan Technologies, Inc. | Method and apparatus for body fluid sampling and analyte sensing |
US7582099B2 (en) | 2002-04-19 | 2009-09-01 | Pelikan Technologies, Inc | Method and apparatus for penetrating tissue |
US7141058B2 (en) | 2002-04-19 | 2006-11-28 | Pelikan Technologies, Inc. | Method and apparatus for a body fluid sampling device using illumination |
US7244265B2 (en) | 2002-04-19 | 2007-07-17 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7485128B2 (en) | 2002-04-19 | 2009-02-03 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8784335B2 (en) | 2002-04-19 | 2014-07-22 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling device with a capacitive sensor |
US8221334B2 (en) | 2002-04-19 | 2012-07-17 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US7410468B2 (en) | 2002-04-19 | 2008-08-12 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7948041B2 (en) | 2005-05-19 | 2011-05-24 | Nanomix, Inc. | Sensor having a thin-film inhibition layer |
US8574895B2 (en) | 2002-12-30 | 2013-11-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus using optical techniques to measure analyte levels |
US7811231B2 (en) | 2002-12-31 | 2010-10-12 | Abbott Diabetes Care Inc. | Continuous glucose monitoring system and methods of use |
US7587287B2 (en) | 2003-04-04 | 2009-09-08 | Abbott Diabetes Care Inc. | Method and system for transferring analyte test data |
GB0310270D0 (en) * | 2003-05-03 | 2003-06-11 | Univ Edinburgh | Biomolecular devices |
US8262614B2 (en) | 2003-05-30 | 2012-09-11 | Pelikan Technologies, Inc. | Method and apparatus for fluid injection |
US7850621B2 (en) | 2003-06-06 | 2010-12-14 | Pelikan Technologies, Inc. | Method and apparatus for body fluid sampling and analyte sensing |
US8066639B2 (en) | 2003-06-10 | 2011-11-29 | Abbott Diabetes Care Inc. | Glucose measuring device for use in personal area network |
WO2006001797A1 (en) | 2004-06-14 | 2006-01-05 | Pelikan Technologies, Inc. | Low pain penetrating |
EP1635700B1 (en) | 2003-06-13 | 2016-03-09 | Sanofi-Aventis Deutschland GmbH | Apparatus for a point of care device |
WO2005033659A2 (en) | 2003-09-29 | 2005-04-14 | Pelikan Technologies, Inc. | Method and apparatus for an improved sample capture device |
EP1680014A4 (en) | 2003-10-14 | 2009-01-21 | Pelikan Technologies Inc | Method and apparatus for a variable user interface |
US7822454B1 (en) | 2005-01-03 | 2010-10-26 | Pelikan Technologies, Inc. | Fluid sampling device with improved analyte detecting member configuration |
US8668656B2 (en) | 2003-12-31 | 2014-03-11 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for improving fluidic flow and sample capture |
WO2005089103A2 (en) | 2004-02-17 | 2005-09-29 | Therasense, Inc. | Method and system for providing data communication in continuous glucose monitoring and management system |
WO2006011062A2 (en) | 2004-05-20 | 2006-02-02 | Albatros Technologies Gmbh & Co. Kg | Printable hydrogel for biosensors |
US9775553B2 (en) | 2004-06-03 | 2017-10-03 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a fluid sampling device |
EP1765194A4 (en) | 2004-06-03 | 2010-09-29 | Pelikan Technologies Inc | Method and apparatus for a fluid sampling device |
US8652831B2 (en) | 2004-12-30 | 2014-02-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for analyte measurement test time |
US20090035876A1 (en) * | 2005-03-31 | 2009-02-05 | Inverness Medical Switzerland Gmbh | Electrochemical Assay |
US8112240B2 (en) | 2005-04-29 | 2012-02-07 | Abbott Diabetes Care Inc. | Method and apparatus for providing leak detection in data monitoring and management systems |
CA2609379C (en) | 2005-06-03 | 2016-11-29 | Allen J. Bard | Electrochemistry and electrogenerated chemiluminescence with a single faradaic electrode |
US7766829B2 (en) | 2005-11-04 | 2010-08-03 | Abbott Diabetes Care Inc. | Method and system for providing basal profile modification in analyte monitoring and management systems |
US7889347B2 (en) | 2005-11-21 | 2011-02-15 | Plexera Llc | Surface plasmon resonance spectrometer with an actuator driven angle scanning mechanism |
US7463358B2 (en) * | 2005-12-06 | 2008-12-09 | Lumera Corporation | Highly stable surface plasmon resonance plates, microarrays, and methods |
US7885698B2 (en) | 2006-02-28 | 2011-02-08 | Abbott Diabetes Care Inc. | Method and system for providing continuous calibration of implantable analyte sensors |
US8226891B2 (en) | 2006-03-31 | 2012-07-24 | Abbott Diabetes Care Inc. | Analyte monitoring devices and methods therefor |
US7620438B2 (en) | 2006-03-31 | 2009-11-17 | Abbott Diabetes Care Inc. | Method and system for powering an electronic device |
US20080071158A1 (en) | 2006-06-07 | 2008-03-20 | Abbott Diabetes Care, Inc. | Analyte monitoring system and method |
US8930203B2 (en) | 2007-02-18 | 2015-01-06 | Abbott Diabetes Care Inc. | Multi-function analyte test device and methods therefor |
US8732188B2 (en) | 2007-02-18 | 2014-05-20 | Abbott Diabetes Care Inc. | Method and system for providing contextual based medication dosage determination |
US8123686B2 (en) | 2007-03-01 | 2012-02-28 | Abbott Diabetes Care Inc. | Method and apparatus for providing rolling data in communication systems |
US8456301B2 (en) | 2007-05-08 | 2013-06-04 | Abbott Diabetes Care Inc. | Analyte monitoring system and methods |
US8461985B2 (en) | 2007-05-08 | 2013-06-11 | Abbott Diabetes Care Inc. | Analyte monitoring system and methods |
US8665091B2 (en) | 2007-05-08 | 2014-03-04 | Abbott Diabetes Care Inc. | Method and device for determining elapsed sensor life |
US7928850B2 (en) | 2007-05-08 | 2011-04-19 | Abbott Diabetes Care Inc. | Analyte monitoring system and methods |
CN101339154B (en) * | 2007-07-05 | 2013-03-27 | 五鼎生物技术股份有限公司 | Composite modified electrode test piece |
TWI336782B (en) * | 2007-07-05 | 2011-02-01 | Apex Biotechnology Corp | Composite modified electrode trip |
US8004669B1 (en) | 2007-12-18 | 2011-08-23 | Plexera Llc | SPR apparatus with a high performance fluid delivery system |
EP2265324B1 (en) | 2008-04-11 | 2015-01-28 | Sanofi-Aventis Deutschland GmbH | Integrated analyte measurement system |
US8103456B2 (en) | 2009-01-29 | 2012-01-24 | Abbott Diabetes Care Inc. | Method and device for early signal attenuation detection using blood glucose measurements |
US9375169B2 (en) | 2009-01-30 | 2016-06-28 | Sanofi-Aventis Deutschland Gmbh | Cam drive for managing disposable penetrating member actions with a single motor and motor and control system |
US20100213057A1 (en) | 2009-02-26 | 2010-08-26 | Benjamin Feldman | Self-Powered Analyte Sensor |
WO2010127050A1 (en) | 2009-04-28 | 2010-11-04 | Abbott Diabetes Care Inc. | Error detection in critical repeating data in a wireless sensor system |
WO2010138856A1 (en) | 2009-05-29 | 2010-12-02 | Abbott Diabetes Care Inc. | Medical device antenna systems having external antenna configurations |
JP2013501921A (en) * | 2009-08-07 | 2013-01-17 | ナノミックス・インコーポレーテッド | Biological detection based on magnetic carbon nanotubes |
US9314195B2 (en) | 2009-08-31 | 2016-04-19 | Abbott Diabetes Care Inc. | Analyte signal processing device and methods |
EP2473099A4 (en) | 2009-08-31 | 2015-01-14 | Abbott Diabetes Care Inc | Analyte monitoring system and methods for managing power and noise |
WO2011041469A1 (en) | 2009-09-29 | 2011-04-07 | Abbott Diabetes Care Inc. | Method and apparatus for providing notification function in analyte monitoring systems |
US8965476B2 (en) | 2010-04-16 | 2015-02-24 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
CN103118785B (en) | 2010-08-05 | 2015-11-25 | 雅培医护站股份有限公司 | The method of immunity using susceptible magnetic microballon to catch and device |
US9329175B2 (en) | 2010-08-05 | 2016-05-03 | Abbott Point Of Care Inc. | Oscillating immunoassay method and device |
US11402375B2 (en) | 2010-08-05 | 2022-08-02 | Abbott Point Of Care Inc. | Magnetic immunosensor with trench configuration and method of use |
EP2601526B1 (en) | 2010-08-05 | 2016-12-21 | Abbott Point Of Care, Inc. | Magnetic immunosensor and method of use |
US8956859B1 (en) | 2010-08-13 | 2015-02-17 | Aviex Technologies Llc | Compositions and methods for determining successful immunization by one or more vaccines |
WO2012075258A2 (en) | 2010-12-03 | 2012-06-07 | Abbott Point Of Care Inc. | Ratiometric immunoassay method and blood testing device |
US9050595B2 (en) | 2010-12-03 | 2015-06-09 | Abbott Point Of Care Inc. | Assay devices with integrated sample dilution and dilution verification and methods of using same |
US9061283B2 (en) | 2010-12-03 | 2015-06-23 | Abbott Point Of Care Inc. | Sample metering device and assay device with integrated sample dilution |
CN103282125B (en) | 2010-12-03 | 2015-05-20 | 雅培医护站股份有限公司 | Sample metering device and assay device with integrated sample dilution |
WO2013070794A2 (en) | 2011-11-07 | 2013-05-16 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods |
US9968306B2 (en) | 2012-09-17 | 2018-05-15 | Abbott Diabetes Care Inc. | Methods and apparatuses for providing adverse condition notification with enhanced wireless communication range in analyte monitoring systems |
EP2863332A1 (en) | 2013-10-15 | 2015-04-22 | One Drop Diagnostics Sàrl | System and method for controlling access to analytical results of a diagnostic test assay |
CN110635143B (en) * | 2019-10-23 | 2022-06-10 | 西北大学 | High-activity catalyst for electrocatalytic reaction and preparation method thereof |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3966556A (en) * | 1972-11-06 | 1976-06-29 | Syva Company | Compounds for enzyme amplification assay methadone analogs |
CA1086225A (en) * | 1976-03-18 | 1980-09-23 | Masuo Aizawa | Syphilis antibody diagnosis by antigen membrane and potentiometric method |
US4318980A (en) * | 1978-04-10 | 1982-03-09 | Miles Laboratories, Inc. | Heterogenous specific binding assay employing a cycling reactant as label |
US4600690A (en) * | 1978-08-07 | 1986-07-15 | Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University | Immunoassay |
US4340448A (en) * | 1978-08-28 | 1982-07-20 | University Of Pittsburgh | Potentiometric detection of hydrogen peroxide and apparatus therefor |
US4233144A (en) * | 1979-04-16 | 1980-11-11 | Technicon Instruments Corporation | Electrode for voltammetric immunoassay |
US4391904A (en) * | 1979-12-26 | 1983-07-05 | Syva Company | Test strip kits in immunoassays and compositions therein |
US4319980A (en) * | 1980-03-07 | 1982-03-16 | Rodman Jenkins | Method for treating coal to obtain a refined carbonaceous material |
EP0078636B2 (en) * | 1981-10-23 | 1997-04-02 | MediSense, Inc. | Sensor for components of a liquid mixture |
CA1223638A (en) * | 1983-05-05 | 1987-06-30 | Graham Davis | Assay systems utilising more than one enzyme |
CA1220818A (en) * | 1983-05-05 | 1987-04-21 | Hugh A.O. Hill | Assay techniques utilising specific binding agents |
CA1218704A (en) * | 1983-05-05 | 1987-03-03 | Graham Davis | Assay systems using more than one enzyme |
GB8328520D0 (en) * | 1983-10-25 | 1983-11-23 | Serono Diagnostics Ltd | Methods of assay |
DE3479522D1 (en) * | 1983-12-16 | 1989-09-28 | Medisense Inc | Assay for nucleic acids |
GB8417301D0 (en) * | 1984-07-06 | 1984-08-08 | Serono Diagnostics Ltd | Assay |
-
1984
- 1984-07-06 GB GB848417301A patent/GB8417301D0/en active Pending
-
1985
- 1985-05-13 CA CA000481357A patent/CA1268520A/en not_active Expired - Lifetime
- 1985-05-13 DE DE3587720T patent/DE3587720T2/en not_active Expired - Lifetime
- 1985-05-13 AU AU42411/85A patent/AU597562B2/en not_active Expired
- 1985-05-13 AT AT85303367T patent/ATE100203T1/en not_active IP Right Cessation
- 1985-05-13 IL IL75174A patent/IL75174A/en unknown
- 1985-05-13 EP EP85303367A patent/EP0167248B1/en not_active Expired - Lifetime
-
1988
- 1988-06-23 US US07/212,043 patent/US4945045A/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
ATE100203T1 (en) | 1994-01-15 |
GB8417301D0 (en) | 1984-08-08 |
EP0167248B1 (en) | 1994-01-12 |
DE3587720T2 (en) | 1994-06-01 |
IL75174A (en) | 1990-07-12 |
AU597562B2 (en) | 1990-06-07 |
AU4241185A (en) | 1986-01-09 |
DE3587720D1 (en) | 1994-02-24 |
EP0167248A3 (en) | 1986-12-30 |
EP0167248A2 (en) | 1986-01-08 |
IL75174A0 (en) | 1985-09-29 |
US4945045A (en) | 1990-07-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA1268520A (en) | Methods of assay | |
US5149630A (en) | Methods of assay | |
EP0142301B1 (en) | Methods of assay | |
EP0170446B1 (en) | Methods of assay | |
EP0241309A2 (en) | Measurement of electroactive species in solution | |
US6346387B1 (en) | Detection of binding reactions using labels detected by mediated catalytic electrochemistry | |
Sergeyeva et al. | Polyaniline label-based conductometric sensor for IgG detection | |
EP0125139A2 (en) | Assay techniques utilising specific binding agents | |
US6881536B1 (en) | Particle based electrochemiluminescent assays | |
JPH0721478B2 (en) | Working film for immunosensor | |
EP0096095A1 (en) | Semiconductor device, sensor and method for determining the concentration of an analyte in a medium | |
Aizawa | Immunosensors | |
Robinson et al. | Bioelectrochemical enzyme immunoassay of human choriogonadotropin with magnetic electrodes. | |
Mattiasson et al. | Novel approaches to enzyme-immunoassay | |
FI101830B (en) | Light-resistant, physical developer | |
JPH0285755A (en) | Immune sensor and detection of immune reaction | |
Fu | Magnetic-controlled non-competitive enzyme-linked voltammetric immunoassay for carcinoembryonic antigen | |
Robinson et al. | A homogeneous bioelectrochemical immunoassay for thyroxine | |
EP0155135B1 (en) | Methods of assay | |
Ikariyama et al. | [9] Bioaffinity sensors | |
Karube et al. | A catalytic immuno-reactor for the amperometric determination of human serum albumin | |
Suzawa et al. | Synthesis of electroactive protein hybrid, Fec-BSA-Dig, and its application to a novel homogeneous electrochemical immunoassay | |
Tang et al. | One‐Step Electrochemical Immunoassay for Carcinoembryonic Antigen in Human via Back‐Filling Immobilization of Gold Nanoparticles on DNA‐Modified Gold Electrodes | |
JP2745683B2 (en) | Immunoassay | |
de la Escosura-Muñiz et al. | Aurothiomalate as an electroactive label for the determination of immunoglobulin M using glassy carbon electrodes as immunoassay transducers |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MKEX | Expiry |