Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
1998-12-15
2001-04-10
Tung, T. (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403060
Reexamination Certificate
active
06214205
ABSTRACT:
FIELD OF THE INVENTION
The present invention is generally in the field of assays for determination of the presence and optionally concentration of an analyte in a liquid medium. More specifically, the present invention concerns such an assay in which the analyte is determined by means of a change in the electrical response of an electrode which occurs in the presence of the analyte.
PRIOR ART
The prior art believed to be relevant as a background to the present invention consists of the following:
1.
Electrochemical Sensors in Immunological Analysis,
Ngo, T.T., Ed.; Plenum Press: New York and London, 1987.
2. Bresler, H. S.; Lenkevich, M. J.; Murdock, J. F.; Newman, A. L.; Roblin, R. O. in
Biosensor Design and Application,
Mathewson, P. R.; Finley, J. W., Eds.; ACS Symp. Ser. 511, Amer. Chem. Soc., Washington, D.C., 1992, pp.89-104.
3. U.S. Pat. No. 4,728,828.
4. U.S. Pat. No. 4,822,566.
5. U.S. Pat. No. 5,057,430.
6. U.S. Pat. No. 4,893,957.
7. Canadian Patent No. 1,256,944.
8. Canadian Patent No. 1,259,374.
9. Weber, S. G.; Purdy, W. C.,
Anal Lett.
1979, 12, 1-9.
10. Doyle, J. M.; Wehmeyer, K. R.; Heineman, W. R.; Halsall, H. B. in
Electrochemical Sensors in Immunological Analysis,
Ngo, T.T., Ed.; Plenum Press: New York and London, 1987, pp. 87-102.
11. Di Gleria, K.; Hill, H. A. O.; McNeil, C. J.; Green, M. J.,
Anal. Chem.,
1986, 58, 1203-1205.
12. Di Gleria, K.; Hill, H. A. O.; Chambers, J. A.,
J. Electroanal. Chem.
1989, 267, 83-91.
13. Chambers, J. A.; Walton, N. J.,
J. Electroanal. Chem.
1988, 250, 417-425.
14. Wright, D. S.; Halsall, H. B.; Heineman, W. R. in
Electrochemical Sensors in Immunological Analysis,
Ngo, T.T., Ed.; Plenum Press: New York and London, 1987, pp. 117-130.
15. Tang, H. T.; Halsall, H. B.; Heineman, W. R.
Clin. Chem.
1991, 37, 245-248.
16. Wehmeyer, K. R.; Halsall, H. B.; Heineman, W. R.; Volle, C. P.; Chen, C.,
Anal. Chem.
1986, 58, 135-139.
17. Aizawa, M., in
Electrochemical Sensors in Immunological Analysis,
Ngo, T. T., Ed.; Plenum Press: New York and London, 1987, pp. 269-291.
18. Franconi, C.; Bonori, M.; Orsega, E. F.; Scarpa, M.; Rigo, A.,
J. Pharm. Biomed. Anal.
1987, 5, 283-287.
19. Uditha de Alwis, W.; Wilson, G. S.,
Anal. Chem.
1985,57,2754-2756.
20. Tsuji, I.; Eguchi, H.; Yasukouchi, K.; Unoki, M.; Taniguchi, I.,
Biosens. Bioelectron.
1990, 87-101.
21. Gebauer, C. R., in
Electrochemical Sensors in Immunological Analysis,
Ngo, T.T., Ed.; Plenum Press: New York and London, 1987, pp. 239-255.
22. Niwa, M.; Mori, T.; Nishio, E.; Nishimura, H.; Higashi, N.,
J. Chem. Soc., Chem. Commun.
1992, 547-549.
23. Willner, I.; Rubin, S.; Cohen, Y.,
J. Amer. Chen. Soc.
1993, 115, 4937-4938.
24. Willner, I.; Blonder, R.; Dagan, A.,
J. Amer. Chem. Soc.
1994, 116, 9365-9366.
25. European Patent Application, Publication No. 668,502.
26. Riklin, A., Katz, C., Willner, I., Stocker A., Backmann, A. F.,
Nature,
1995, 376, 672.
27. Morris, D. L., Buckler, R. T., in
Methods in Enzymology, V.
92, Part E (Eds. J. J. Langone, H., Van Vunakis), Acad. Press, N.Y. 1983, pp. 415-417.
BACKGROUND OF THE INVENTION
Owing to their specificity and high sensitivity, immunoassays are widely used in everyday clinical practice as well as in a variety of other applications such as environmental pollutant analysis and food quality analysis. Such assays are based on a specific binding between an antibody and an antigen.
Immunoassays based on electrochemical detection of binding have been a focus of considerable research and development activity (Ngo, T.T., Ed. 1987). In electrochemical immunoassay, what is detected is a change in electrical properties of an electrode solution interface as a result of antibody-antigen binding, such as changes in capacitance (Bresler, et al, 1992). Capacity affinity sensors have been disclosed in U.S. Pat. Nos., 4,728,822; 4,822,566 and 5,057,430, U.S. Pat. No. 4,893,957 and Canadian Patent Nos. 1,256,944 and 1,259,374. Other electrochemical immunoassay methods are based on a change in the current or potential response as a result of formation of antigen-antibody complexes. Such methods require the presence of a redox probe attached to the antibody or antigen and the response of the electrochemical assay is a result of competitive binding between the analyte and the redox labeled antibody or antigen with the complementary member pair immobilized on the electrode. Such methods are not very sensitive and the amperometric detection of the redox-labeled molcule is limited to the micromolar range (Weber, et al., 1979; Doyle et al., 1987). More recent work has shown that by using a bare electrode and coupling the redox reaction with an enzymatic amplification system (Di Gleria et al, 1986; Di Gleria et al, 1988) there is improvement of the sensitivity. Use of a similar approach, but with an electrode modified with a redox polymer, resulted in an immunoassay that was more sensitive (Chambers, et al, 1988).
Another approach includes direct covalent coupling of antigen or antibody with biocatalytic molecules (usually redox enzymes) for amplification of the electrochemical signal. A number of studies have utilized amperometric or potentiometric detection of electro-active species such as NADPH (Wright, et al, 1987; Tang, et al, 1991), phenol (Wehmeyer, et al, 1986), O
2
(Aizawa, et al, 1987; Franconi, et al., 1987), H
2
O
2
(Uditha, et al., 1985; Tsuji, et al, 1990), NH
3
(Gebauer, 1987), etc., generated analytically by an enzyme label on an antigen or antibody. The high sensitivity of this method makes it competitive with that of radioimmunoassay.
However, an electrical technique without any labeling of antigen/antibody species is even more attractive. Such methods are based on an amperometric detection of the permeability of redox molecules through a monolayer immobilized onto the surface of the electrode. An antigen monolayer provides much higher permeability to a solubilized redox probe than the same electrode reaction with an antibody with high binding affinity to the antigen (for illustration see FIG. 1) (Niwa, et al., 1992; Willner, et al, 1993; Willner et al, 1994; European Patent Application No. 668502).
GENERAL DESCRIPTION OF THE INVENTION
It is an object of the present invention to provide an electrochemical method and system for the determination of the presence and optionally concentration of an analyte in a liquid medium, the analyte being a member of a recognition pair.
It is furthermore an object of the invention to provide electrodes for use in such method and system.
Other objects of the invention will be clarified from the description below.
In the method and system of the invention, the presence of the analyte in the medium gives rise to a change in an electrical response. The term “electrical response” which will be used below denotes the current-voltage behavior of an electrode, e.g. the current response or the flow of charge of an electrode under a certain applied potential, etc. The electrical response may be determined by measuring current or charge flow, under alternating current or direct current conditions.
In the following the term “determine” or “determination” will be used to denote both determination of only the presence or determination of both the presence and concentration of an analyte in a liquid medium.
The electrical response in the method and system of the invention is a result of transfer of electrons between an electrode and an electron transfer chain formed by at least two redox molecules. The term “redox molecule” denotes a molecule which is capable of accepting or donating an electron thereby changing its redox state. The redox molecule may be an electron mediator capable of transferring electrons between an electrode and a redox molecule or between two redox molecules. The redox molecule may also be a catalytic molecule which upon change in its redox state becomes catalytically active following which it acts on a substrate which then changes into a chemically distinct product. A non-limiting example of a catalytically active redox molecule is a redox enzyme. In accordance
Blonder Ron
Cohen Yael
Katz Eugenii
Willner Itamar
Browdy and Neimark
Noguerola Alex
Tung T.
Yissum Research Development Company of the Hebrew University of
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