Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
1999-02-16
2001-04-24
Tung, T. (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403060
Reexamination Certificate
active
06221238
ABSTRACT:
The invention relates to an enzymatic-electrochemical affinity sensor and a one-step affinity assay for the quantitative determination of analytes in aqueous media. More specifically, the invention relates to an enzymatic-electrochemical signal amplification system for a highly sensitive indication of affinity reactions and is particularly suitable in the form of a one-step affinity sensor for in situ analytics. The invention is also directed to the use of phenol oxidase as a marker enzyme for the affine binding partners in an electrochemical affinity sensor or assay, and to the use of an enzyme hydrolyzing phenolic compounds as marker enzyme for the affine binding partners, in combination with a phenol oxidase as catalyst for the amplifying reaction in an electrochemical affinity assay.
In order to detect immunochemical reactions or affinity reactions in general, a number of electrochemical or enzymatic-electrochemical indication systems are known which are based on the indication of an electrochemically active marker (Heinemann and Halsall, Anal. Chem. 57 (1985), pp. 1321A-1331A; Patent Specification DE 42 16 696; Le Gal La Salle, J. Electroanal. Chem. 350 (1993), 329-335) or an electrochemically active product of the marker enzyme. Above all, alkaline phosphatase (Doyle et al., Anal. Chem. 56 (1984), pp. 2355-2360; McNeil et al., Biosensors 3 (1987), pp. 199-209; Duan et al., Anal. Chem. 66 (1995), pp. 1369-1377; Meusel et al. Biosens. & Bioelectron. 10 (1995), pp. 577-586) or galactosidase (Masson et al., Anal. Chim. Acta 304 (1995), pp. 353-359) is used as marker enzyme. A variety of other enzymatic-electrochemical indication systems are based on the cyclic regeneration of redox-active reactants.
The most frequently described assays are those where either a redox enzyme or a redox mediator is used as a marker for an antigen or an antibody. Following the corresponding immunochemical reaction, an enzymatic redox reaction sequence is completed by the marker in the presence of the enzyme substrate, wherein a redox mediator, a redox-active prosthetic group of the enzyme, or a redox-active enzymatic co-factor, or a redox-active co-substrate is reduced or oxidized. Using an amperometric redox electrode, the redox system is regenerated directly or via a mediator. The current resulting therefrom depends on the analyte concentration, i.e., the redox-active conjugates which, depending on the analyte, is provided by the immune reaction at just a low concentration, causes a cyclic enzymatic-electrochemical regeneration reaction resulting in an amplified signal generation, with a correspondingly high measurement current. The redox marker may be a component of a homogeneous or a heterogeneous immunoassay or an immunoassay according to the competition, titration or displacement principle.
Weber and Purdy (Anal. Letters 12 (1979), pp. 1-9) have been the first to accomplish a homogeneous immunoassay using ferrocene as redox-active label of an antigen and detect the direct oxidation of the ferrocene conjugate at −500 mV (vs. SCE).
One enzymatic-electrochemical immunoassay (diGleria et al., Anal. Chem. 58 (1986), pp. 1203-1206) also uses ferrocene as antigen label, wherein glucose oxidase is used in the mediated indicator reaction of the antigen-ferrocene conjugate which is displaced from the binding site of the antibody in the presence of analyte and thus, may assume the electron transfer between said oxidase and the electrode.
The perfection of the above enzymatic-electrochemical immunoassays has been described in the Patent Specification EP 125,139. Again, a redox mediator is used as label for the antigen or the antibody. In the publication by Suzawa et al. (Anal. Chem. 66 (1994), pp. 3889-3894), ferrocene is used as multi-label in combination with glucose oxidase. Another well-known immunoassay (Gyss and Bourdillon, Anal. Chem. 59 (1987), pp. 2350-2355) uses glucose oxidase as marker, wherein benzoquinone serves as mediator.
Increasing the sensitivity of amperometric indication systems on the basis of redox enzyme/mediator sequences in immunoassays is the aim of Patent Specification EP 241,309. Therein, a second electron acceptor (ferricyanide, polyvinylferrocene or Berlin blue) is introduced into the measuring solution or used to modify the electrode surface and accumulate reduction equivalents from the enzymatic glucose oxidation the via hapten-ferrocene conjugate. Following an accumulation period, the amperometric measurement of the electron acceptor is effected, which has been reduced during this accumulation phase.
In the Patent Specification EP 223,541, use is made of redox mediators shifted in negative direction in their formal potential by coupling a phosphate group or a phenol derivative so that, in contrast to their non-derivatized form, no electron transfer from glucose oxidase to the electrode surface via mediator can occur. Ferrocene or dichlorophenol-indophenol are used as mediators. In the presence of a hapten conjugate which has alkaline phosphatase as marker enzyme and results from a competitive reaction with the analyte, cleavage of the derivatizing group occurs so that the mediator may assume the electron transfer between glucose oxidase and the redox electrode. Depending on the concentration of the available marker enzyme-antigen/antibody conjugate, an anodic measurement current will occur.
Furthermore, a measuring system is known (PCT 86/03837) wherein the marker enzyme itself does not catalyze the indicative redox reaction but rather, generates a redox-active “trigger substance” which either may be detected directly by amperometry or completes an enzymatic-electrochemical amplification sequence as a result of its reversible redox behavior. Alkaline phosphatase or &bgr;-galactosidase is used as marker enzyme. In the event of alkaline phosphatase, NADP
+
is used as “trigger substrate” which is hydrolyzed to NAD
+
by elimination of a phosphate group and, as a co-factor in an ethanol/alcohol dehydrogenase/diaphorase, ethanol/alcohol dehydrogenase/ferricyanide, or ethanol/alcohol dehydrogenase/ferrocene/ferricyanide redox electrode sequence, results in the completion of this redox cascade. In analogy, electrochemical-enzymatic assays on the basis of the direct or mediator-coupled indication of the NADH co-factor have been described by Eggers et al. (Clin. Chem. 28/9 (1982), pp. 1848-1851) and Cardosi et al. (Electroanalysis 1 (1989), 297-304). Alternatively, the alkaline phosphatase marker enzyme may be used to cleave the phosphate group of an SH-containing compound which then acts as an electron donor for a glutathione reductase or diaphorase and is regenerated cathodically. In addition, this Patent Specification also describes the use of &bgr;-galactosidase as marker enzyme hydrolyzing the p-hydroxyphenyl-&bgr;-galactoside. The p-hydroxyquinone having formed serves as substrate for laccase and again, is regenerated cathodically, so that the resulting reduction current is proportional to the concentration of the anti-body/enzyme conjugate.
An enzymatic electrochemical indication system for immunochemical reactions is known from Patent Specification PCT 86/04926, which system has a depolymerase/ligand conjugate and a redox sequence consisting of an oxidoreductase, a mediator and a redox electrode spatially separated from each other by a polymer, or which contains one of the above-mentioned redox components bound in a polymer or incorporated in a polymer. Preferably, a polysaccharide or a liposome matrix is used as polymer, amyloglucosidase, &agr;-amylase or phospholipase is used as depolymerase, ferrocene or a ferrocene derivative is used as mediator, and glucose oxidase or glucose dehydrogenase is used as redox enzyme.
The depolymerase used as marker enzyme causes cleavage of monomers from e polymer, which serve as substrates for an oxidoreductase or become diffusible in the form of mediator-coupled monomers so that in either case, an enzymatic-electrochemical reaction is generated. Other possible depolymerase reactions result in liberation of the red
Grundig Bernd
Kopinke Holm
Renneberg Ilka
Strehlitz Beate
Birch Stewart Kolasch & Birch, LLP.
Noguerola Alex
Tung T.
UFZ-Umweltforschungszentrum Leipzig-Halle GmbH
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