Electrochemical and photochemical electrodes and their use

Details Electrochemical and photochemical electrodes and their use Electrochemical and photochemical electrodes and their use

1999-06-30

2002-02-26

Tung, T. (Department: 1743)

Electrolysis: processes, compositions used therein, and methods

Electrolytic analysis or testing

Involving enzyme or micro-organism

C204S403060, C427S002130, C427S058000, C369S121000

Reexamination Certificate

active

06350368

ABSTRACT:

FIELD OF THE INVENTION
The present invention is generally in the field of bioelectronics and concerns electrically conducting solid matrices (to be referred to herein as “electrodes”) carrying redox enzymes such that an electric charge can flow between the surface of the electrode and the enzymes rendering them catalytically active. Also provided by the invention is a process for the preparation of the electrodes as well as devices, systems and methods making use of such electrodes. In accordance with one embodiment, the invention is applied for the determination of the presence and optionally the concentration of an analyte in a liquid medium. In accordance with another embodiment, the immobilized enzymes can be switched by light into two distinct biocatalytic states thus allowing the transduction and amplification of recorded optical signals thus fulfilling “read” and “write” functions, rendering such electrodes useful in optical information storage and processing.
PRIOR ART
The prior art believed to be relevant as a background to the present invention consists of the following:
1. Degani, Y., Heller, A.,
J. Am. Chem. Soc.,
110:2615, 1988.
2. Willner, I., Katz, E., Riklin, A., Kasher R.,
J. Am. Chem. Soc.,
114:10965, 1992.
3. Willner et al., U.S. Pat. No. 5,443,701.
4. Lion-Dagan, M . Katz, E., Willner, I.,
J. Amer. Chem. Soc.,
116791 3, 1994.
5. Willner, I., Lion-Dagan, M., Marx-Tibbon, S., Katz, E.,
J. Amer. Chem. Soc.,
117:6581, 1995.
6. Willner, I. and Rubin, S.,
Angen. Chem. Int. Ed.
Engl. 35: 367 , 1996.
7. Willner, I., Riklin, A., Shoham, B., Rivenson, D., Katz, E.,
Adv. Mater.,
5:912, 1993.
8. Massey, V., Hemmerich, P., in
Flavins and Flavoproteins,
V. Massey & C. H. Williams (Eds.), Elsevier, Amsterdam, 83-96, 1982.
9. Walsh, C., Fisher, J., Spencer, R., Graham, D. W., Ashton, W. T., Brown, J. E., Brown, R. D., Rogers, E. F.,
Biochemistry,
78:1942, 1978.
10. Bückmann, A. F., Erdmann, H., Pietzch, M., Hall, J. M., Bannister, J. V. in K. Kuneoyagi (Ed.), Flavins and Flavoproteins, Gruyter, Berlin, p. 597, 1994.
11. Riklin, A., Katz, E., Willner, I., Stocker, A., Bückmann, A.F.,
Nature,
376:672, 1995.
12. Willner, I., Liondagan, M., Marxtibbon S., Katz, E.,
J. Amer. Chem. Soc.,
117: 6581, 1995
13. Namba, K., Suzuki, S.,
Bull. Chem. Soc. Jpn.,
48:1323, 1975.
14. Katz, E., Schlereth, D. D., Schmidt, H. L.,
J. Electroanal. Chem.,
367:59, 1994.
BACKGROUND OF THE INVENTION
Covalent coupling of redox active groups (ferrocene, bipyridinium, etc.) to amino acid residues of redox enzymes produces biocatalysts that electrically communicate with electrodes electrically “wired” enzymes
(1-3)
. Enzymes modified by photoisomerizable groups (e.g. nitrospiropyran
itromerocyanine) show different enzymatic activities for the different light-induced generated photoisomer states
(4,5)
. The use of photoswitchable biocatalysts as active matrices for optical recordings and optobioelectric devices was recently reviewed
(6)
.
Electrically-wired enzymes were employed for the determination of analytes in electrochemical cells by the attachment of the electrobiocatalyse to electrodes
(3,7)
. In all of the described systems, the functional electroactive or photoactive units are randomly distributed around the protein. The effectiveness of electrical contact between the enzyme redox-center and the electrode is limited. As a result, the rate of electron transfer between the enzyme redox center is relatively slow. This results in competitive electron transfer reactions with co-substrates (e.g. oxygen) or interfering substrates (e.g. oxidation of uric acid or ascorbic acid). As a result the magnitude of the resulting currents that assay the respective analytes are moderately low and the analysis had to be performed in an oxygen free environment. Special care had to be made to eliminate any interfering reagents from the analysis medium.
For many enzymes (e.g. flavoenzymes) the FAD-cofactor can be removed from the native protein to yield the unfolded apo-protein which can be reconstituted back with the natural cofactor or chemically modified FAD cofactors to yield the bioactive enzyme
(8-10)
. The reconstitution of apo-flavoenzymes with a FAD-cofactor bound to an electron mediator group generated an “electro enzynze” that exhibited electrical contact with electrode surfaces. Mediated electron transfer activates the reconstituted enzymes for the electrocatalytic oxidation of their substrates
(11)
.
Enzyme-electrodes for electrochemical determination of an analyte can operate as non-invasive or invasive analytical devices. For invasive analyses the electrodes must be constructed of bio compatible non-hazardous substances, and the electrodes must be fabricated as thin needles to exclude pain upon invasive penetration. The low surface area of the electrodes must be compensated by a high electrical activity of the sensing biocatalysts to yield measurable current responses.
The functions of enzymes modified by randomly substituted photoisomerizable units are only incompletely switched by external light signals. The perturbation structure of the protein environment of the active redox center of enzymes is only partially affected by remote photoisomerizable units. This yields only to partial, incomplete, deactivation of the photoisomerizable enzyme
(12)
.
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 the concentration of an analyte in a liquid medium.
It is furthermore an object of the invention to provide electrodes for use in such method and system. It is particularly an object of the invention to provide such electrodes comprising a solid, electrically conducting matrix carrying immobilized enzymes such that electric charge and flow between the electrode to the enzymes renders the enzyme catalytically active whereby they catalyze a reaction in which the analyte to be assayed is converted into a product.
It is furthermore an object of the invention to provide such electrodes with high and efficient electron transport between the electrode and the enzymes such that the electrode is essentially insensitive to the presence of otherwise interfering redox reagents, i.e. there is a minimum of non-specific redox reactions.
It is furthermore an object of the invention to provide enzyme-electrodes where the entities immobilized on the electrodes are non toxic and non immunogenic enabling the use of the electrode in invasive analysis.
It is furthermore an object of the invention to provide enzyme-electrodes with photoswitchable enzymes immobilized on the electrode surface, for use in the recordal of optical signals and transduction of recorded optical signals.
It is another object of the invention to provide uses of the electrodes of the invention as well as processes for their preparations.
Other objects of the invention will be clarified from the description below.
The present invention has two aspects: one aspect, to be referred to herein as the “first aspect” in which the electrode is useful for the determination of the presence and optionally the concentration of an analyte in a liquid medium; and another aspect, to be referred to herein as the “second aspect”, in which the electrical response of the electrode is photoregulated (i.e. the degree of electrical response is controllable by irradiation of light at a specific wavelength) allowing the use of the electrode in recordal of optical signals and the electrical transduction of recorded optical signals. Both aspects of the present invention share a common denominator in that the electrodes carry immobilized enzymes, and in that the enzymes have functionalized cofactors, i.e., cofactors modified by the addition of a functional group or moiety (such enzymes to be referred to at times as “functionalized enzymzes”).
In accordance with the invention a functionalized enzyme may be obtained by reconstituting an apo-enzyme (an enzyme without its cofactor) with a FAD modified by the addition of a functional group

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