Electrobiochemical method and system for the determination of an analyte which is a member of a recognition pair in a liquid medium, and electrodes thereof

• US 5,942,388 A
• Filed: 02/21/1995
• Issued: 08/24/1999
• Est. Priority Date: 02/22/1994
• Status: Expired due to Fees

• Current Assignee: Biosensor Applications Sweden AB
• Original Assignee: Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem)
• Inventors: Cohen, Yael, Rubin, Shai, Riklin, Azalia, Dagan, Arie, Willner, Itamar, Blonder, Ron
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/390,978
• Time in Patent Office: 1,645 Days
• Field of Search: 204/193, 204/402, 204/403, 204/416, 204/420, 204/422, 204/280, 422/82.01, 422/82.03, 435/7.1, 435/6, 435/7.8, 435/7.95, 435/962, 436/518, 436/806, 436/825, 436/524, 436/525
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: G01N 33/553 Metal or metal coated
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample

Abstract

An elcctrobiochemical system for the determination of the presence and optionally concentration of an analyte in a liquid medium, the system comprising an electrode having immobilized thereon a member of a recognition pair, the other member of said pair being said analyte, the presence of said analyte in the medium resulting in formation of a pair complex, being a complex between said immobilized member and said analyte; the system further comprising redox molecules capable of changing their redox state by accepting electrons from or donating electrons to the electrode; the formation of the pair complex on the electrode bringing a change in the electrical response of the system, whereby the presence and optionally the concentration of said analyte in the medium can be determined.

Claims

1. An electrobiochemical system for determining the presence or concentration of an analyte in a liquid medium comprising:

an electrode, said electrode being made or coated with a metal selected from the group consisting of gold, platinum, silver and copper;

or said electrode being made from conductive glass;

redox molecules dissolved in the liquid medium for exchanging electrons with said electrode;

means for generating and measuring an electrical response of said electrode; and

,

molecules immobilized on said electrode that bind to said analyte so as to form a recognition pair complex comprising an immobilized molecule and a molecule of said analyte, wherein said recognition pair complex impairs the electron exchange between said redox molecules and said electrode thereby decreasing said electrical response and allowing the presence or concentration of said analyte to be determined;

the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associate with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attaching to, or chemisorption onto said conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im.

2. The system according to claim 1, wherein said recognition pair complex is selected from the group consisting of antigen-antibody, sugar-lectin, ligand-receptor, biotin-avidin, enzyme-substrate, oligonucleotide-DNA, oligonucleotide-protein, and oligonucleotide-cell.

3. The system according to claim 1, wherein said redox molecule is freely tumbling in solution.

4. The system according to claim 1, wherein the immobilized member of the recognition pair comprises a group reactive to exposure to light energy, said group having a first and a second state and converts from the first state to the second state by exposure to irradiation of light of a first wavelength and from the second to the first state by exposure to irradiation of light of a second wavelength, the exposure inducing a change in affinity of the immobilized member for binding to said analyte, whereby in the first state said immobilized member has a high affinity of binding to said analyte such that the analyte remains bound more tenaciously to the immobilized member than in said second state which incurs lower affinity state between said recognition pair.

5. A method for the determination of the presence and optionally the concentration of an analyte in a liquid medium, comprising the steps of:

(a) providing an electrode, said electrode being made or coated with a metal selected from the group consisting of gold platinum, silver and copper;

or said electrode being made from conductive glass;

(b) providing redox molecules dissolved in the liquid medium for exchanging electrons to or from said electrode;

means for generating and measuring an electrical response of said electrode;

(d) providing molecules immobilized on said electrode that bind to said analyte so as to form a recognition pair complex comprising an immobilized molecule and a molecule of said analyte, wherein said recognition pair complex impairs the electron exchange between said redox molecules and said electrode thereby decreasing said electrical response and allowing the presence or concentration of said analyte to be determined;

the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associating with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attach to, or chemisorption onto said conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im;

(e) contacting the electrode with the liquid medium;

(f) measuring the decrease in electrical response so as to determine the presence or concentration of the analyte in the medium.

6. The method according to claim 5, wherein said immobilized molecule bound to said analyte comprises a recognition pair that is selected from the group consisting of antigen-antibody, sugar-lectin, ligand-receptor, biotin-avidin, enzyme-substrate, oligonucleotide-DNA, oligonucleotide-protein, and oligonucleotide-cell.

7. The method according to claim 5, wherein the immobilized molecule has or is linked to a group reactive to exposure to light energy;

said group having a first and a second state and is capable of being converted from the first state to the second state by exposure to irradiation of light of a first wavelength and from the second to the first state by exposure to irradiation of light of a second wavelength;

the exposure inducing a change in affinity of the immobilized molecule for binding to said analyte, whereby in the first state said immobilized molecule has a high affinity of binding to said analyte such that the analyte remains bound more tenaciously to the immobilized molecule than in said second state which incurs lower affinity between the immobilized molecule and the analyte;

the method comprising the following additional steps;

(g) irradiating said electrode by a light having said first wavelength;

(h) rinsing the electrode so as to remove analyte molecules from the medium surrounding the electrode; and

(i) irradiating said electrode by light having a said second wavelength, whereby the electrode is ready for re-use.

8. The method according to claim 5, wherein the immobilized molecule comprises a photoreactive group, said group having a first and a second state and is capable of being converted from the first state to the second state by a mild thermal treatment;

the exposure inducing a change in affinity of the immobilized molecule for binding to said analyte, whereby in the first state said immobilized molecule has a high affinity of binding to said analyte such that the analyte remains bound more tenaciously to the immobilized molecule than in said second state which incurs low affinity state between the immobilized molecule and the analyte, the method comprising the following additional steps;

(g) irradiating said electrode by a light having said first wavelength;

(h) rinsing the electrode so as to remove analyte molecules from the medium surrounding the electrode; and

(i) subjecting the electrode to a mild thermal treatment, whereby the electrode is ready for re-use.

9. An electrode for use in the determination of the presence or concentration of an analyte, the analyte being a member of a recognition pair other than an enzyme-substrate pair, in a liquid medium, said electrode being made or coated with a metal selected from the group consisting of gold, platinum, silver and copper;

or said electrode being made from conductive glass;

so as to allow transport of electrons to or from a redox molecule and having immobilized thereon a layer of one member of said recognition pair other than the analyte, the immobilized member being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein

Z represents a sulfur containing moiety capable of chemical association with, attachment to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methozysilane or alkoxysilane residues which are capable of chemical association with, attachment to, or chemisorption onto said conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized member represented by Im, such that, binding of analyte to the immobilized member, modifies electron exchange between a redox molecule and said electrode whereby the presence or concentration of said analyte in a medium surrounding the electrode is determined.

10. A reusable electrode for use in an electro-chemical system for the determination of the presence or concentration of an analyte in a liquid medium, the electrode comprising:

an electrode material capable of electrical communication with a redox molecule;

there being immobilized on the electrode material a layer of a member of a recognition pair the other member of the pair being said analyte;

the electrical communication between the redox molecule and the electrode material being modified by binding of said member to said analyte;

the immobilized member has or is linked to a group reactive to exposure to light energy, said group having a first and a second state and is capable of being converted from the first state to the second state by exposure to irradiation of light of a first wavelength and from the second to the first state by exposure to irradiation of light of a second wavelength;

the exposure inducing a change in affinity of the immobilized member for binding to said analyte, whereby in the first state said immobilized member has a high affinity of binding to said agent such that the bound analyte is not readily dissociated and in said second state said immobilized member has a low affinity of binding to said analyte, such that the bound agent is readily dissociated and can be removed from the system and the electrode can then be exposed to light of a second wavelength inducing a change to said first state, whereby the electrode will be ready for re-use, whereby the presence or concentration of said analyte in a medium surrounding the electrode can be determined.

11. A method for the determination of the presence or concentration of an analyte in a liquid medium comprising:

providing an electrode, said electrode being made or coated with a metal selected from the group consisting of gold, platinum, silver and copper;

or said electrode being made from conductive glass;

placing an electrode in said liquid medium containing redox molecules for exchanging electrons with said electrode;

providing means for generating and measuring an electrical response of said electrode;

providing immobilized molecules on said electrode, said molecules binding to at least one molecule of said analyte so as to form a recognition pair complex comprising said immobilized molecule and at least one molecule of said analyte, wherein said recognition pair complex reduces the electron exchange between the redox molecule and said electrode thereby changing said electrical response and allowing the presence or concentration of said analyte to be determined;

the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associating with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attach to, or chemisorption onto said conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im; and

measuring the change in electrical response and based thereon determining the presence or concentration of said analyte.

12. A method for the determination of the presence or concentration of an analyte in a liquid medium comprising:

providing an electrode, said electrode being made or coated with a metal selected from the group consisting of gold, platinum, silver and copper;

or said electrode being made from conductive glass;

placing an electrode into said liquid medium containing analyte, said analyte molecules containing at least one redox moiety for exchanging electrons with said electrode;

providing means for generating and measuring an electrical response of said electrode;

providing immobilized molecules on said electrode, said molecules binding to at least one molecule of said analyte so as to form a recognition pair complex comprising said immobilized molecule and at least one molecule of said analyte, wherein said recognition pair complex enhances the electron exchange between the redox molecule and said electrode thereby changing said electrical response and allowing the presence or concentration of said analyte to be determined;

the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associating with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attach to, or chemisorption onto said conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im; and

measuring the change in the electrical response and based thereon determining the presence or concentration of said analyte.

13. An electrobiochemical system for determining the presence or concentration of an analyte in a liquid medium comprising:

an electrode;

redox molecules dissolved in the liquid medium for exchanging electrons with said electrode;

means for generating and measuring an electrical response of said electrode;

molecules immobilized on said electrode that bind to said analyte so as to form a recognition pair complex comprising an immobilized molecule and a molecule of said analyte;

the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associating with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attach to, or chemisorption onto a conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im; and

said recognition pair complex being selected from the group consisting of antigen-antibody, sugar-lecithin, ligand-receptor, biotin-aviden, enzyme-substrate, oligonucleotide-DNA, oligonucleotide-protein, and oligonucleotide-cell.

14. The system according to claim 13 wherein said redox molecule is freely tumbling in solution.

15. The system according to claim 13, wherein the electrode material is selected from the group consisting of gold, platinum, silver, copper and conducting glass.

16. The system according to claim 13 wherein the electrode is coated with a material selected from the group consisting of gold, platinum, silver, copper and conducting glass.

17. The system according to claim 13, wherein the immobilized member of the recognition pair comprises a group reactive to exposure to light energy, said group having a first and a second state and converts from the first state to the second state by exposure to irradiation of light of a first wavelength and from the second to the first state by exposure to irradiation of light of a second wavelength, the exposure inducing a change in affinity of the immobilized member for binding to said analyte, whereby in the first state said immobilized member has a high affinity of binding to said analyte such that the analyte remains bound more tenaciously to the immobilized member than in said second state which incurs lower affinity state between said recognition pair.

18. An electrobiochemical system for determining the presence or concentration of an analyte in a liquid medium comprising:

an electrode;

redox molecules dissolved in the liquid medium for exchanging electronic with said electrode;

means for generating and measuring an electrical response of said election;

molecules immobilized on said electrode that bind to said analyte so as to form a recognition pair complex comprising an immobilized molecule and a molecule of said analyte;

said molecule immobilized on said electrode comprises a group reactive to exposure to light energy, said group having a first and a second state and converts from the first state to the second state by exposure to irradiation of light of a first wavelength and from the second to the first state by exposure to irradiation of light of a second wavelength, the exposure inducing a change in affinity of the immobilized member for binding to said analyte, whereby the first in the first state said immobilized member has a high affinity of binding to said analyte such that the analyte remains bound more tenaciously to the immobilized pair than in the second state which has a lower degree of affinity between said recognition pair, and

wherein said recognition pair complex impairs the electron exchange believed said redox molecules and said electrode thereby decreasing said electrical response and allowing the presence or concentration of said analyte to be determined.

19. A method for the determination and, optionally, the concentration of an analyte in a liquid medium comprising the steps of:

(a) providing an electrode, said electrode being made or coated with a metal selected from the group consisting of gold, platinum, silver and copper;

or said electrode being made from conductive glass;

(b) redox molecule for transferring electrons with said electrode, and molecules, immobilized on said electrode, that bind to said analyte so as to decrease electron transfer with the electrode,

(c) the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associating with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attach to, or chemisorption onto said conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im;

(d) reacting said liquid medium, or a fraction thereof, said liquid medium containing an agent, with a reagent either to yield an analyte or to cause a depletion in free analyte concentration,

(e) contacting the electrode with a solution obtained from (b); and

(f) measuring a change in electrical response resulting from contact of the electrode with the analyte, whereby the presence and optionally the concentration of said analyte said liquid medium is determined.

20. The method according to claim 19 wherein said liquid reagent contains an agent which is a molecule which is either (i) broken down by an enzyme into molecules, one of which is said agent (ii) being a precursor molecule which is converted by an enzyme to said agent;

said reagent solution further comprising said enzyme.

21. A system for determining the presence or concentration of an agent in a biological sample, comprising:

a reagent solution which in the presence of said agent causes either formation of an analyte or depletion of an a priori present analyte;

an electrode made or coated with a metal or said electrode being made from conductive glass;

redox molecules dissolved in the liquid medium for exchanging electrons with said electrode;

means for generating and measuring an electrical response of said electrodes; and

molecules immobilized on said electrode that bind to said analyte so as to form a recognition pair complex comprising an immobilized molecule and a molecule of said analyte, wherein said recognition pair complex impairs the electronic change between said redox molecules and said electrode thereby decreasing said electrical response and allow to determine the presence or concentration of said analyte, serving as an indication for the presence or concentration of said agent in the biological sample, and

the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associating with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attach to, or chemisorption onto said conducting glass electrode; and

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im.

22. A system according to claim 21, wherein said analyte can bind to said agent, the reagent solution comprising said analyte.

23. A system according to claim 22, wherein said agent is an antigen, said analyte is an antibody which binds to said antigen and the molecules immobilized on said electrode are antigens which bind to said antibody.

24. A system according to claim 22, wherein said agent is an antibody, said analyte is an antigen which binds to said antibody and the immobilized molecules on said electrodes are antibodies which bind to said antigen.

25. A system according to claim 21, wherein said agent is an enzyme which is capable of either breaking down the analyte into products which cannot bind to the molecules immobilized on the electrode, in which case the reagent system comprises the analyte, or is capable of catabolizing the analyte from precursor molecules, in which case said reagent solution comprises the precursor molecules.

26. A system according to claim 21, wherein said agent is a molecule which is either broken down by an enzyme into products, one of which being said analyte, or being a precursor molecule which is converted by an enzyme to said analyte, said reagent solution comprising said enzyme.

27. A method for determining the presence or concentration of an agent in a biological sample, comprising:

(a) reacting said sample or a fraction thereof comprising said agent if present in the sample with a reagent solution, whereby in the presence of an analyte either

i. an analyte forms, or

ii. an analyte already present is depleted so as to form a tested medium;

(b) providing an electrode, said electrode being made or coated with a metal selected from the group consisting of gold, platinum, silver and copper;

or said electrode being made from conductive glass;

(c) redox molecules for transferring electrons to or from said electrodes, and molecules, immobilized on said electrode, that bind to said analyte so as to decrease electron transfer between the redox molecules and the electrode, thereby decreasing electrical response of the electrode;

(d) the immobilized molecule being immobilized on the surface of the electrode by means of a linking group having the following general formula;

space="preserve" listing-type="equation">Z--R'"'"'--Q--Im

wherein;

Z represents a sulfur containing moiety chemically associating with, attaching to, or chemisorption onto the electrode when the electrode is made or coated with said metal or Z represents methoxysilane or alkoxysilane residues which chemically associate with, attach to, or chemisorption onto said conducting glass electrode;

R'"'"' represents a connecting group and Q is a functional group forming a covalent bond with the immobilized molecule represented by Im;

(e) contacting the electrode with a solution comprising the tested medium and the redox molecules dissolved in the solution; and

(f) measuring the decrease in electrical response so as to determine the presence or concentration of the analyte in said liquid solution, and deducing therefrom the presence or concentration of said agent in the biological sample.

28. A method according to claim 27, wherein said analyte can bind to said agent, said reagent solution comprising said analyte.

29. A method according to claim 28, wherein said agent is an antigen, said analyte is an antibody which binds to said antigen and the immobilized molecules on said electrodes are antigens which bind to said antibody.

30. A method according to claim 27, wherein said agent is an antibody, said analyte is an antigen which binds to said antibody and the immobilized molecules on said electrode are antibodies which bind to said antigen.

31. A method according to claim 27, wherein said agent is an enzyme which is capable of either breaking down the analyte into products which cannot bind to the molecules immobilized on the electrode, in which case the reagent system comprises the analyte, or is capable of catabolizing the analyte from precursor molecules, in which case said reagent solution comprises the precursor molecules.

32. A method according to claim 27, wherein said agent is a molecule which is either broken down by an enzyme into products, one of which being said analyte, or being a precursor molecule which is converted by an enzyme to said agent, said reagent solution comprising said enzyme.