Enzymatic-electrochemical one-shot affinity sensor for the quantitative determination of analytes for aqueous media and affinity assay
First Claim
1. An enzymatic-electrochemical one-step affinity sensor for the quantitative determination of analytes in aqueous media, comprising a support having applied thereon a measuring system or two adjacent measuring systems of identical design, and the contact paths thereof,wherein each measuring system comprises multiple consecutive layers arranged over a redox electrode modified with an electron mediator, and a pseudo-reference electrode, wherein said layers are laterally encapsulated in a liquid-proof fashion by a fixing frame having a top cover which has an area having openings to receive the sample to be measured, and wherein said layers comprise either a) a layer including a phenol oxidase substrate, for that case where a marker enzyme of an affine binding partner is a phenol oxidase, or b) a layer including a hydrolase substrate and an additional layer in the vicinity of the electrode which includes immobilized phenol oxidase, for that case where a marker enzyme of an affine binding partner is a hydrolase, a sample-receiving and reservoir layer, a layer in the vicinity of the electrode, and a) layers which contain affine binding partners, or b) a layer including appropriate immobilized affinity complexes.
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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.
209 Citations
31 Claims
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1. An enzymatic-electrochemical one-step affinity sensor for the quantitative determination of analytes in aqueous media, comprising a support having applied thereon a measuring system or two adjacent measuring systems of identical design, and the contact paths thereof,
wherein each measuring system comprises multiple consecutive layers arranged over a redox electrode modified with an electron mediator, and a pseudo-reference electrode, wherein said layers are laterally encapsulated in a liquid-proof fashion by a fixing frame having a top cover which has an area having openings to receive the sample to be measured, and wherein said layers comprise eithera) a layer including a phenol oxidase substrate, for that case where a marker enzyme of an affine binding partner is a phenol oxidase, or b) a layer including a hydrolase substrate and an additional layer in the vicinity of the electrode which includes immobilized phenol oxidase, for that case where a marker enzyme of an affine binding partner is a hydrolase, a sample-receiving and reservoir layer, a layer in the vicinity of the electrode, anda) layers which contain affine binding partners, or b) a layer including appropriate immobilized affinity complexes.
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23. An enzymatic-electrochemical one-step affinity assay for the quantitative determination of analytes in aqueous media, wherein a
sample fluid is applied to a perforated area of a fixing frame cover of a sensor according to claim 34, which is connected to a potentiostate or a manual measuring instrument of potentiostatic design; - and
said sample initially, diffuses into the sample-receiving and reservoir layer via the spacer, said layer undergoing swelling, thereby pressing the film against the fixing frame cover, thus closing same, and subsequently, the analyte to be determined in the sample fluid undergoing an affine binding reaction when passing the layers of the sensor, wherein a) a phenol oxidase-labelled receptor, a phenol oxidase-labelled ligand, or the corresponding phenol oxidase-labelled affinity complex, together with the analyte, diffuses to a mediator-modified electrode surface, and the phenol oxidase oxidizes a suitable phenol oxidase substrate in the layer in immediate vicinity of the electrode surface to form an electrically active product which is reduced cathodically via the reversibly reduced electron mediator to form a starting substrate of the phenol oxidase, or b) a phenol oxidase on a redox electrode is fixed as a layer, and an educt resulting from the reaction of a hydrolyzing enzyme used as label in the same fashion as in a) with a suitable hydrolase substrate is oxidized in the immediate vicinity of the electrode surface by the phenol oxidase used as a catalytic layer to form an electrically active product which is reduced cathodically via the reversibly reduced electron mediator to form a starting substrate of the phenol oxidase; and
the cyclic substrate regeneration generated in both cases a) or b) resulting in a chemically amplified, analyte-proportional cathodic current which is quantified using per se common voltammetric methods. - View Dependent Claims (24, 25, 26, 27, 28, 29)
a pseudo-homogeneous binding reaction takes place between the analyte and the diffusible phenol oxidase-labelled receptor or the phenol oxidase-labelled ligand, the non-bound fraction of the receptor or ligand conjugate, subsequent to overcoming the diffusion barrier layer is bound to immobilized ligand or immobilized receptor, and the analyte receptor conjugate complex or the analyte-ligand conjugate complex, subsequent to overcoming the diffusion barrier layer, enters the reaction layer in the vicinity of the electrode, being bound to an immobilized receptor or ligand, and the marker phenol oxidase, after mobilization of the phenol oxidase substrate in the substrate layer and its breaking through the diffusion barrier layer into the reaction-layer in the vicinity of the electrode, catalyzes an amplifying reaction between the phenol oxidase substrate and the modified electrode surface, thus providing an analyte-proportional voltammetric current signal. -
25. The assay according to claim 23, wherein the analyte to be determined in the sample fluid mobilizes the phenol oxidase-labelled ligand or receptor,
the analyte and the phenol oxidase-labelled ligand or the phenol oxidase-labelled receptor, subsequent to overcoming the diffusion barrier layer, compete with immobilized ligand or receptor for the available binding sites, and the non-bound phenol oxidase-labelled ligand or phenol oxidase-labelled receptor, subsequent to overcoming the next diffusion barrier layer, enters the reaction layer in the vicinity of the electrode, being bound to an immobilized receptor or ligand, and the marker phenol oxidase, after mobilization of the phenol oxidase substrate in the substrate layer and its breaking through the diffusion barrier layer into the reaction layer in the vicinity of the electrode, catalyzes an amplifying reaction between the phenol oxidase substrate and the modified electrode surface, thus providing an analyte-proportional voltammetric current signal. -
26. The assay according to claim 23, wherein the analyte to be determined in the sample fluid reaches layer which contains immobilized ligand or receptor, the binding sites thereof being saturated with phenol oxidase-receptor conjugate or phenol oxidase ligand conjugate,
the analyte displaces part of the phenol oxidase-ligand conjugate or the phenol oxidase receptor conjugate, and the phenol oxidase ligand conjugate complexed with analyte or the phenol oxidase receptor conjugate complexed with analyte, subsequent to overcoming the diffusion barrier layer, enters the reaction layer in the vicinity of the electrode, being bound to the receptor or ligand immobilized therein, and the marker phenol oxidase, after mobilization of the phenol oxidase substrate in the substrate layer and its breaking through the diffusion barrier layer into the reaction layer in the vicinity of the electrode, catalyzes an amplifying reaction between the phenol oxidase substrate and the modified electrode surface, thus providing an analyte-proportional voltammetric current signal. -
27. The assay according to claim 23, wherein the analyte to be determined in the sample fluid mobilizes the hydrolase-labelled receptor or ligand,
a pseudo-homogeneous binding reaction between the analyte and the diffusible hydrolase-labelled receptor or ligand takes place, the non-bound fraction of the receptor conjugate or ligand conjugate, subsequent to overcoming the diffusion barrier layer, is bound to immobilized ligand or receptor in layer, and the analyte-receptor conjugate complex or the analyte-ligand conjugate complex, subsequent to overcoming the diffusion barrier layer, enters the reaction layer in the vicinity of the electrode, being bound to an immobilized receptor or ligand, and the marker hydrolase, after mobilization of the hydrolase substrate in the substrate layer and its breaking through the diffusion barrier layer into the reaction layer in the vicinity of the electrode, hydrolyzes an educt which penetrates a layer additionally arranged in front of the electrode surface containing immobilized phenol oxidase and, being a substrate of phenol oxidase, triggers a phenol oxidase-catalyzed amplifying reaction between the phenol oxidase substrate and the modified electrode surface, which provides an analyte-proportional voltammetric current signal. -
28. The assay according to claim 23, wherein the analyte to be determined in the sample fluid mobilizes the hydrolase-labelled receptor or ligand,
the analyte and the hydrolase-labelled ligand or receptor, subsequent to overcoming the diffusion barrier layer, compete with immobilized ligand or immobilized receptor for the binding sites in layer, and the non-bound fraction of hydrolase-labelled ligand or hydrolase-labelled receptor, subsequent to overcoming the diffusion barrier layer, enters the reaction layer in the vicinity of the electrode, being bound to an immobilized receptor or ligand in said reaction layer, and the marker hydrolase, after mobilization of the hydrolase substrate in the substrate layer and its breaking through the diffusion barrier layer into the reaction layer in the vicinity of the electrode, hydrolyzes an educt which penetrates a layer additionally arranged in front of the electrode surface containing immobilized phenol oxidase and, being a substrate of phenol oxidase, triggers a phenol oxidase-catalyzed amplifying reaction between the phenol oxidase substrate and the modified electrode surface, which provides an analyte-proportional voltammetric current signal. -
29. The assay according to claim 23, wherein the analyte to be determined in the sample fluid reaches a layer which contains immobilized ligand or receptor, the binding sites thereof being saturated with hydrolase-receptor conjugate or hydrolase-ligand conjugate,
the analyte displaces part of the hydrolase-ligand conjugate or the hydrolase-receptor conjugate, and the hydrolase-ligand conjugate complexed with analyte or the hydrolase-receptor conjugate complexed with analyte, subsequent to overcoming the diffusion barrier layer, enters the reaction layer in the vicinity of the electrode, being bound to the receptor or ligand immobilized therein, and the marker hydrolase, after mobilization of the hydrolase substrate in the substrate layer and its breaking through the diffusion barrier layer into the reaction layer in the vicinity of the electrode, hydrolyzes an educt which penetrates a layer additionally arranged in front of the electrode surface containing immobilized phenol oxidase and, being a substrate of phenol oxidase, triggers a phenol oxidase-catalyzed amplifying reaction between the phenol oxidase substrate and the modified electrode surface, which provides an analyte-proportional voltammetric current signal.
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- 30. A method of determining chemical affinity comprising using a hydrolyzing phenolic compound as a marker enzyme for affine binding partners, and a phenol oxidase as a catalyst for an amplifying reaction between a phenol oxidase substrate and a redox mediator and determining the bond formed between affine partners in an electrochemical assay.
Specification