JPS62167465A - Electrochemical enzyme measuring method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] The present invention is based on the electrical
(assay). In particular, the present invention relates to, but is not limited to, techniques and devices useful in immunoassays. In particular, the present invention is useful in a wide range of diagnostic or investigative techniques, e.g. for humans or animals.
The present invention relates to immunoassays used in research and monitoring, for example in food chemistry or process chemistry.

An example of a measurement method using a known enzyme as a label is enzyme-1 labeled
Heterogeneous immunoassay (heterogeneo), known as immunosorbentassay technology
US immunoassay). In the commonly employed sandwich method, the method typically involves three steps separated by washing and rinsing: (a) applying the appropriate antibody to the surface of a support, e.g. polymer beads or, in some cases, plastic; immobilization on the surface of a container wall or a recess of a plastic dish; (b) contacting the surface carrying the immobilized enzyme with the sample to be measured to immobilize the specific analyte in the sample;
) is combined with the antibody at a ratio depending on the sample concentration; and (C) further contacting the surface with an enzyme-labeled antibody, thereby causing the enzyme-labeled antibody to bind only to the antigen, thus It consists of providing a surface with a measurable concentration of enzyme that corresponds to the analyte concentration in the sample.

The Sand-Deutsch method has certain limitations, particularly in that it can only be used when a sample molecule having at least two epitopes is reacted with both an immobilized antibody and an enzyme-labeled antibody.

Another ELISA competitive method for measuring specific analytes involves adding a known concentration of the specific analyte labeled with an enzyme to a sample, and then contacting the sample with a surface carrying a known concentration of antibody, thereby Labeled and unlabeled analytes are competed proportionally for binding positions, and the ratio of labeled analyte/unlabeled analyte is then determined by measuring the yeast concentration.

Most current ELISA and other enzymatic assays use color-forming substrates that release a chromophore at the end of the instrument period.
The concentration of the chromophore can be measured by a spectrophotometer. However, the use of color-forming substrates can have drawbacks in the length of time required to obtain results and the instability of certain chromophores. Therefore, improvement of the measurement method is necessary.

As an alternative to color detection, an electrochemical detection method is described in Analytical Chemistry J (1984) 56.2355, in which electrically inert substances are electrochemically activated by the enzyme label used for measurement. The electroactive compound phenol has a redox potential of +750 mV, and this method cannot be applied as is because the system has a redox potential of +750 mV. This is because other components that oxidize at the 1δ mV position are encountered.Therefore, this system cannot be practically adopted for blood or serum samples.

The present invention particularly relates to assays that make use of response amplification, in particular the measurement of one or more substances in a component mixture where the analyte is present in a particularly low concentration and/or as a mixture with a potential interfering substance. It relates to an assay for detecting the presence or monitoring the level of said substance, wherein the presence or absence of a response is linked to the extent of a particular binding reaction.

EP 78636 uses a mediator compound, such as a ferrocene derivative, to transfer electrons between the enzyme and an electrode when the enzyme catalyzes a redox reaction in a substrate. This is disclosed. This method mainly employs glucose oxidase as a redox enzyme to electrochemically detect glucose as a substrate.
being employed.

Relatively recent European patents, such as εP 125.139, disclose assays in which the substrate concentration is constant and the concentration of the available mediator compound is a variable factor. In this method, contrary to the method of EP 78,636, the combination of Rhodonotus fermentation and substrate allows electrochemical detection of the mediator.

The present invention involves a more advanced measurement method based on electrochemical detection of mediator levels using redox enzymes and redox substrates.

The main objective of the present invention is to provide an invasive assay method using enzymes as labels.

In particular, it is an object of the present invention to provide a lacquered Elisa method.

Another object of the present invention is to provide reagents for use in new methods using enzymes as labels.

The present invention electrochemically detects the mediator level using a redox enzyme and a redox substrate.
The present invention provides an electrochemical enzyme assay method characterized by detecting the conversion of ng compound into an effective mediator.

Accordingly, the present invention uses an electrochemical method to detect active mediator generated from non-mediator compounds under measurement conditions. The active mediator is produced by the enzyme used as a label in the assay. Therefore, the analyte can be measured in a qualitative or quantitative sense. Rapid immunoassays can be achieved with relatively high sensitivity in the nanomolar range.

Conditions are selected so that the substrate for the labeled enzyme used in the measurement is not effective as a mediator, and the labeled enzyme used in the measurement acts to convert the substrate into an effective mediator. A compound that does not act as a mediator can itself be a potential mediator, such that the compound is not effective as a mediator under the selected conditions and thus is "not a mediator."

Therefore, in performing electrochemical enzyme measurement using a labeled enzyme, the present invention uses a redox enzyme and a redox substrate, and the labeled enzyme converts the substrate that does not act as a mediator from the ineffective mediator to the redox enzyme and the electrode. An electrochemical enzymatic assay method is provided, which comprises electrochemically detecting the 7-dried fermentation product as a result of its conversion into an effective mediator that mediates electron transfer between the enzymes.

By using labeled enzymes for assays together with mediator/redox enzyme systems, known specific binding assays can be extended to levels even higher than previously achieved, yet without interference. Pretreatment of the sample to remove substances is no longer necessary.

In the method of the present invention, the above-mentioned systems and PCT/GB
) The system described in Patent No. 8,600,095 is that, in the present invention, the redox substrate diffuses freely before being converted into an effective mediator, whereas in the conventional system, the active ingredient is one part of the immobilizing agent. They differ in that they become inactive by binding to large target molecules.

The method of the present invention is typically an immunodetection method.

Suitably, this method uses the Sanderuch method or the competitive method. Therefore, preferably the method of the invention is a sandwich ELISA or a competitive ELISA.

In the Sand-Deutsch method, a mono- or polyclonal antibody is immobilized, the sample is contacted with the immobilized antibody, the system is then contacted with an enzyme-labeled antibody, and the substrate is subjected to system conditions such that this substrate does not act as a mediator. The redox enzyme and redox enzyme substrate are then used for electrochemical determination of the effective mediator.

In the case of a competitive method, a sample and an enzyme-labeled specimen are brought into contact with an immobilized antibody, a substrate that does not act as a mediator is added, and electrochemical measurements are performed.

Normally, for electrochemical detection, it is preferred that the non-mediating substrate itself be a low-potency mediator that has become non-mediating, the reason for this being the operating conditions. To this end, the electrode must be placed between the redox potential of the substrate and the redox potential of the product for the redox enzyme, lower than the potential maintained at the electrode, so that only products with a redox potential are at the electrode. is maintained at a predetermined potential such that it can undergo a redox reaction.

the transfer of said substrate to said product by using as a mediator a product which exhibits electron transfer at a given potential and which can be formed by an enzymatic reaction from a substrate which does not exhibit mediator activity at said given potential. The degree of conversion and thus the presence or activity of fermentation can be determined. A variety of assays can be performed in which the conversion of substrate to product is achieved by increasing mediator activity under the assay conditions.

The substrate for the labeled enzyme is a mediator derivative that does not act as a mediator under the measurement conditions. The nature of this derivative depends on the nature of the labeled enzyme.

Examples of mediator compounds that can form the basis of the substrate are metallocenes; ruthenium compounds; carboranes;
Conductive salt of TCNQ; haloanil
) and their derivatives; viologens; quinones; alkyl-substituted phenazine derivatives; transition metal bis-cyclopentadienyl<C9) 2MX, complexes: and phenol derivatives, including heerocene-phenol and indophenol compounds. Such compounds can be easily derivatized to provide suitable substrates.

By using water-soluble derivatives that are not sensitive to air, measurements can be performed on biological fluid samples that have undergone minimal pretreatment.

Appropriate derivatives that provide substrates for a given enzyme can be easily synthesized. For example, phosphate derivatives are recognized by acidic or alkaline phosphatases.

The substrate for the phosphatase-labeled enzyme is preferably a metallocene derivative, preferably a ferrocene derivative.

An example of a suitable phosphate derivative of ferrocene is represented by the following formula (I
) and (■): Represented by ○. In a particularly preferred embodiment of the invention, the redox substrate is a phosphate compound (1), which can also be represented by the following formula; This compound is a new compound and forms part of the present invention.

Compound 'IM [N-ferrocenoyl]-4-aminophenyl phosphate has an E172 of 390 mV vs. standard glucose electrode (rscEJ). When this compound is treated with a phosphatase, e.g. acidic or alkaline phosphatase, this compound is converted into a phenol derivative with the following formula (): %formula %();
It has an oxidation potential of +180 mV vs. CE.

Therefore, when current measurements are made at about +230 mV vs. SCE, only the contact current due to compound 'proboscis (8) is detected, and compound ([) shows no electrode response at this potential.

In another preferred embodiment of the invention, the substrate is one phenolic derivative;
For example, 2,6-dichloroindophenyl phosphate, this compound is represented by the following formula (■):

Phosphatases are only one example; the invention is applicable to any enzyme substrate coupled to electroactive species. Therefore, even when phosphatase is used as a labeling enzyme, the present invention is not limited to such phosphate compounds. Mediator compounds can be easily synthesized and coupled to pond enzyme systems;
Therefore, the nature of the reaction catalyzed by the labeled fermentation used in the measurement is not important. For example, the enzyme can be a protease, amidase or hydrolase.

In another aspect of the invention, the invention provides a system for detecting the activity of a first enzyme, which system comprises: a) convertible by said first enzyme into a product exhibiting mediator activity; the presence of said first enzyme in active form, comprising: 1) a freely spreading reagent which allows the detection of said product; and b) detection means consisting of a redox enzyme, a substrate for said redox enzyme, and an electrode. wherein said reagent is 'converted to said product and a detectable change occurs during mediated electron transfer to said electrode. Primarily, the characteristic mediator property of the product is electron transfer at a given electrode potential.

Such a system can be used to provide a first enzyme assay, where the first enzyme may or may not be a labeled enzyme.

In the pond aspect of the invention, the invention provides a bioelectrochemical cell (BEC), which cell comprises a standard Mf used to measure
! A substrate for the enzyme is incorporated into the BEC, a labeled enzyme used in the measurement is configured to detect the enzyme by consuming the substrate and causing a change in the output from the BEC, and converting the substrate into a mediator. The second enzyme reaction system is characterized in that it includes a second enzyme reaction system that provides electrical output through the second enzyme reaction system.

In the second enzymatic reaction used in BEC, a ferrocene mediator is preferred to mediate electron transfer in the reaction between glucose and glucose oxidase. In such systems, due to the mediator action of the ferrocene compound,
The effect of changes in redox potential on ferrocene compounds is amplified.

Changes in the ferrocene compound or other substrate that occur as the enzyme consumes it can significantly affect the mediator properties and thus the output from the BEC.

In yet another aspect of the invention, the invention provides a method for detecting the activity of a non-redox enzyme in the presence of at least one electrode maintained at a constant potential, the detection method comprising: a) said non-redox enzyme; A sample suspected of containing a redox enzyme is treated with a reagent having a redox potential higher than the potential maintained at the electrode, wherein the reagent is a substrate for a non-redox enzyme, and the activity of the redox enzyme is b) treating the sample with a redox enzyme and a substrate for the redox enzyme; , characterized in that it causes measurable electron transfer to said electrode in the presence of a mediator compound having a redox potential lower than the potential maintained at said electrode.

The present invention detects the activity of an enzyme that does not exhibit redox activity, but is capable of catalyzing the conversion of a substrate to a product having mediator properties under the conditions of measurement. enable.

Although the invention is described below with reference to phosphatase enzymes, the scope of the invention extends to Ike assay systems and other reagents. For example, one can use a labeled enzyme that can react with the reagent to produce a product with mediator properties.

Note that although the present invention is described for labeling antigens, the labeling enzyme can be used in other types of specific binding reactions, such as between complementary strands of acetic acid.

In FIG. 1, the mediator compound 0.1 i ) is, for example, compound (I). This compound is a water-soluble reagent that makes the electrode +230 relative to SCE.
When maintained at a potential of mV, glucose oxidase (
COD) and its substrate glucose.

In the presence of a phosphatase enzyme (E) present as a label, compound (1) is converted to compound (A).

Compound (8) is +230 mV + for glucose oxidase (COD) and its substrate glucose and SCE;
: Mediator activity (Ledl) between maintained electrodes
oj not shown.

The system of Figure 1 therefore allows the detection of the activity of phosphatase enzymes. The current detected at the electrode is
8) is attributable only to the existence of

In the xrx diagram, the phosphatase enzyme is a label for the antibody (8b), so the concentration of the phosphatase enzyme is influenced by the specific binding reaction between the antibody and its corresponding antigen. The steps of exposing the antibody to the antigen and separating the bound and free portions can be performed by known immunoassay techniques. Alternatively, a phosphatase enzyme is directed against the antigen. It can be a sign.

Next, the present invention will be described with reference to the drawings and experimental examples.

Experimental Example 1 (a) Production of compound represented by formula (A) “Journal・
Of Organic Chemistry”, You, 2-8
0-281 (1959), ferrocene monocarboxylic acid was converted to ferrocenoyl chloride.

Ferrocenoyl chloride (1 mM) was then dissolved in 5d water-cooled dry pyridine. Add 5 ml of p-aminofairfur (1.1 mM) to this solution! of dry pyridine was added in one portion. The reaction was allowed to proceed for 2 hours at 4°C, then the reaction solution was warmed to room temperature and stirred for an additional 2 hours. Afterwards the reaction solution was filtered and the solvent was removed under vacuum. The residue was then dissolved in a minimum amount of acid-resistant ethyl, and the compound was purified by column chromatography using silica as the packing and one volume of ethyl acetate/hexane (3: lv/v) as the eluent. Purified. The purified compound crystallized out as orange needles by slowly diffusing hexane into a solution of the compound in a minimum amount of ethyl acetate.

A parent ion peak was obtained at m/e=321 by mass spectrometry. This peak corresponded to the desired compound (8).

(b) Production of the compound represented by formula (I) Substrates used in the alkaline phosphatase measurement method are described in The Biochemical Journal J47, 93-95 (1950) and 33.1182-1184 (1939) It was manufactured by an improved method of the previously described method.

Compound (A) (100mg, 0.31m), 4) was dissolved in 5me dry pyridine and the mixture was cooled in a water bath.

Add freshly distilled phosphorus oxychloride (30μ!) to this solution.

0.32mM) was added and the purified mixture was stirred at 0°C until the reaction was complete. After the reaction, thin layer chromatography was performed using 1 portion of ethyl acetate as an eluent.

After the reaction is completed, add a small amount (about 5 m2) of water.
Then, a saturated aqueous barium hydroxide solution was added at 0° C. until the solution became alkaline.

After the addition of barium hydroxide, an equal volume of water-cooled ethanol was added to precipitate the barium salt of the phosphate ester. The barium salt was collected by filtering, washed with ethanol, and dried under vacuum.

The crude barium salt was slightly water soluble. This was treated with a stoichiometric amount of 1I23O4. The resulting solution is filtered and then lyophilized to remove the free acid.

Experimental Example 2 Preparation of Pond Substrate of Formula (II) Hydroxymethylferrocene (216 mg) was dissolved in 5i of dry pyridine and then 111 μβ of fresh phosphorus oxychloride was added in one portion. The bright yellow solution immediately turned dark red and aqueous chloride gas was evolved. This solution was stirred for approximately 2 hours. Is the reaction of hydroxymethylferrocene soluble? Ethyl acetate was used as an i agent (6'N was confirmed by thin layer chromatography on a glass plate). 5
After a minute of reaction, no hydroxymethylferrocene was repelled into the mixture.

After 2 hours, the solution was diluted with slightly basic distilled water (p11
8) was added and then extracted four times with ethyl acetate to remove the pyridine. The aqueous layer was further extracted with chloroform 11 times and then evaporated to dryness on a rotary evaporator. The residue was dissolved (4 times) in slightly acidic distilled water (pH 5,5).
Then, the remaining pyridine was removed by repeated evaporation to dryness.

Dissolving the product in ethanol to precipitate inorganic impurities;
This impurity was then filtered off.

Finally, to dry the product, it was dissolved in a minimum amount of distilled water and then freeze-dried for 48 hours.

When left standing at 4°C for 48 hours, the crystalline substance compound (I
I) was obtained.

Experimental Example 3 Measurement of Redox Potential The redox potentials of compounds (1) and (II) were measured.

An electrochemical cell was assembled using a total working electrode, a saturated reference electrode (SEC), and a platinum counter electrode and connected to a potentiostat. Ferrocene derivative (2mM) and glucose (8mM) were added to this clean electrochemical cell.
A solution of 0mM) dissolved in lQQm 1.1 of Tris at pH 10.15 was filled. Final volume is 400μ
It was β. Solutions were made using two different buffers. However, no effect was observed on the results.

The voltage was varied from OmV to 600mV at a rate of 5mV/sec. The redox potentials of compounds (I) and ([) were calculated from the obtained portamograms.

In the case of compound (I), the redox potential is approximately +390 m
It was V.

In the case of compound (II), the redox potential is approximately +370
It was mV.

The redox potentials of compounds (I) and (II) were independent of pH in the range studied.

Experimental Example 4 Production of pond substrate represented by formula (III) I 5 g of sodium salt of 2.6-dichloroindophenol
was dissolved in water, and hydrogen chloride gas was added until a deep blue precipitate was deposited. The resulting solid was filtered off, dissolved in dichloromethane and dried over magnesium sulfate. The solution was filtered and the solvent was removed to yield 2,6-dichloroindophenol.

Dissolve this 2,6-dichloroindophenol in dry pyridine and add 1 equivalent of phosphorus oxychloride while stirring.
I did business. Add ice after 15 minutes to carbonate the solution) tJ
Neutralized with um. The solvent was removed and the residue was extracted with boiling ethanol. The ethanol layer was filtered and evaporated. The residue was washed with ethanol to obtain the desired compound (III>).

Practical Example 5 The activity of alkaline phosphatase was 7j, Hl. An electrochemical cell with two compartments was used.

In addition to a working electrode of a graphite disk with a diameter of 4 mm, the cell contained a counter electrode of 1 cm 2 platinum wire mesh and a reference electrode of saturated dimension (rscEJ).

Circular porcunmetry experiments in direct current were performed using a Beanie Nis (BΔ5)-100 Electrochemical Analyzer (E
electrochemical analyzer).

Calf intestinal alkaline phosphatase (cardim ((a)
lzyme) at pH 10.1.
5 in 0.1 M) +) buffer. The final protein concentration was 0.86 mg/ml.

MgC (! 2mM at pH 10,15 in 0,1-1 Tris containing 210mM and NaCj25QmM
Substrate solution 0.6mjl! was placed in the sample chamber of an electrochemical cell and the annular polumogram was recorded in direct current. The peak current, if any, was measured at 180 mV and then an appropriately diluted alkaline phosphacuse stock solution of 100 mV was added.
µg was added and the contents of the cell were incubated at room temperature for 15 minutes. Warm; write the circular portamogram after 1'
p, TJ: I, , the current was measured at 180 mV. This operation was repeated over a range of concentrations of alkaline phosphatase.

The activity of alkaline phosphatase was evaluated using the peak current at 18θ[IIV] by oxidation of ferrocene to the phenolic product IJ miniram ion. The peak current at 180 mV as a function of alkaline phosphatase concentration is shown in FIG.

Alkaline phosphatase in-cell
.

Concentration 1.23XLO-12 mol/! corresponded to 8.6×10 −16 moles of enzyme in the electrochemical cell. Therefore, when using a cyclic voltamme under relatively mild conditions (IJ-) and incubation at room temperature for 15 minutes (incubation at 37°C for at least 30 minutes, conditions fairly commonly used in immunoassays) ), it was possible to measure extremely small amounts of alkaline phosphatase.

Experimental Example 6 E N by current measurement (amperOmeLry)
D A B Enzyme Immunoassay To demonstrate the inherent sensitivity of the electrochemical alkaline phosphatase assay, a detection system for the enzyme assay was investigated. A kit for the ENDAB enzyme assay for unconjugated estriol in serum was purchased from CMD (UK).

The second measurement welding when using the Estriol kit.
The antibody precipitation step was performed, and then the measurement method of the present invention was carried out by changing the procedure. 50μ! Each estriol standard solution (0, 1, 3, 10, 20 and 40 ng/me
) into a suitable glass tube.

To perform non-specific binding (rNsBJ), NSB
The tubes were filled with 50 μβ of Q ng/me standard solution.

Then, 25 μa of estriol-enzyme conjugate (co
n, iugate) to all glass tubes, and 100
μl of estriol antiserum was added. (varnish to the NSB tube) Do not add IJ all antiserum, instead add 100AtR.
background reagents were added.

These reagents were mixed and incubated at room temperature for 20 minutes.

1 ml of second antibody separation reagent was then added to all tubes. The tubes were centrifuged at 3000 rpm for 10 minutes and the supernatant was discarded. The tubes were then placed on blotting paper to remove excess liquid.

At this point, instead of following the specific method of using a kit for spectrophotometric estriol determination, the centrifuged pellet containing the estriol-alkaline phosphatase conjugated to the antibody is replaced with a ferrocene-conjugated substrate. Solution 0. IgCβ21QmM and NaCj25
Suspended in a 2mM solution at pH 10,15 in 0,6me) containing 0mM).

The solution was incubated at room temperature for 15 minutes. After 15 minutes of incubation, the solution was transferred to the sample chamber of the electrochemical cell.

The portamogram of this solution is O~+65 relative to SCE.
Recordings were made over a potential range of 0 mV and the peak current at 180 mV was measured.

This operation was performed for each serum estriol, 1% 2% D solution, ie 'E, O, l, 3.20 and 4Qng/mf of the above standard 2<q solution.

The group τ′j in the absence of alkaline phosphatase
Background current at 1.80 mV,
Zuna4's non-specific binding current was 1.15μ8.

This value was subtracted from the results obtained using one volume of blood standard solution.

Corrected peak currents at 180+y+V for each serum 7 standard are provided in the kit. Plotted on marking curve paper.

When Nn +/io is plotted against the estriol concentration (ng/mg>), a straight line (y=0.04X +0.4
3°r=0.98).

Such an ENDAB immunoassay with amperometric detection could be performed using shorter incubation times and lower temperatures than the standard kit method.

Experimental Example 7 (a) Use of Compounds (I) and (I[) The same apparatus as in Experimental Example 3 was used, and the voltage was changed in the same way.

In this experiment, 1 μl of the enzyme glucose oxidase (COD) at 175 mg/me was added to the same solution as in Experimental Example 3 to give a maximum volume of 0.35 mff.

As before, we changed the voltage and recorded the resulting relationship between current and voltage.

It was found that the proboscis (I) and ((I) act as mediators for the glucose oxidase enzyme reaction and that the current generated at the oxidation potential is amplified. This is due to catalytic reduction of the ion and then re-oxidation of ferrocene that occurs at the electrode.

Current versus time measurements at a constant potential of 450 mV are carried out in a three-electrode system to detect the hydrolysis of the phosphate group of ferrocene in the presence of a crude acid phosphatase solution using compound (■) (50 μM in ethanol) 8
μβ and glucose in 100 mM citrate buffer at pH 4,66 (IM > 50 μβ were used, with a final volume of 400.d. All samples were stirred during the experiment.

Measurement results were recorded for the following four groups: (a) Compound (
II) Plus no glucose, acid phosphatase or glucose oxidase.

(b) Same as (a), except that compound (II) and glucose were incubated at 37° C. for 10 minutes, and then 25 μβ of 2 mM glucose oxidase was added. This preservation is due to the decrease in current that occurs after the addition of acid phosphatase.
) This was to show that this was not caused by breakdown in the solution.

A clear difference in current is observed between (a) and (b), and this difference is due to the oxidase acid accumulation reaction acting through the mediator compound (II).

(c) Same as (b), but before addition of glucose oxidase, 10 μβ crude acid phosphatase solution was warmed together with compound (II) and glucose at 37° C. for 10 minutes.

A decrease in current was observed compared to (b) above. This is because acid oxidase acts on compound (n) to change its mediator properties.

(d) Same as (b), but 30μβ acid phosphatase solution was added.

A further decrease in current was observed. This indicates that there is an inverse relationship between acid phosphatase concentration and contact current.

(b) Use of compound (III) In the same manner as in Experimental Example 7(a), compound (III) was added with 0.
The annular portamograms of the 114 glucose-loaded samples were measured at pH 7,8 using a scan rate of 20 mV/s and the curves (a), (b) and (C
) is the first prize.

Curves (a), (b) and (C) are for the compound (■
), 5 minutes after addition of alkaline phosphatase to compound (I), and for compound ([[I) plus alkaline phosphatase and glucose oxidase. Amplification was also significant in this case.

[Brief explanation of drawings]

Figure 1 is a diagram explaining the principle of the measurement method of the present invention, Figure 2 is a graph showing the relationship between the peak current at 180 mV obtained by an example of the method of the present invention and the phosphatase concentration, and Figure 3 is the method of the present invention. A graph showing an example of the relationship between the t-corrected peak current at 180 mV obtained in the pond example and the estriol concentration. It is a line diagram. Ab Antibody E Phosphatase enzyme G
OD glucose oxidase Mi mediator compound Mred/. 8 Mediator active estriol, 2 degrees (7+f/″J) Flo, 3 degrees

Claims (1)

[Claims] 1. When performing electrochemical enzyme measurements, by electrochemically detecting the mediator level using a redox enzyme and a redox substrate, the level of the mediator is detected by the labeled enzyme used in the measurement. , an electrochemical enzyme assay method characterized by detecting the conversion of a compound that does not act as a mediator into an effective mediator. 2. The method according to claim 1, which is an immunoassay method. 3. The method according to claim 2, which is ELISA. 4. For electrochemical detection, the electrode has a redox potential intermediate between the redox potential of the substrate and the redox potential of the product for the redox enzyme, but lower than the potential maintained at the electrode. 4. A method as claimed in any one of claims 1 to 3, in which a predetermined potential is maintained such that only the product can undergo a redox reaction at the electrode. 5. The method according to any one of claims 1 to 4, wherein the redox substrate is a phenol derivative. 6. The method according to any one of claims 1 to 5, wherein the labeling enzyme is a phosphatase. 7. The method according to claim 5, wherein the redox substrate is a compound represented by the following formula (I): ▲Mathematical formula, chemical formula, table, etc.▼(I). 8. When performing electrochemical enzyme measurement using a labeled enzyme, a redox enzyme and a redox substrate are used, and the substrate that does not act as a mediator is transferred from the ineffective mediator to the redox enzyme and the electrode by the labeled enzyme. An electrochemical enzyme assay method, characterized in that the labeled enzyme is electrochemically detected as a result of its conversion into an effective mediator that mediates migration. 9. The method according to claim 8, which is an immunoassay method. 10. The method according to claim 9, which is ELISA. 11. For electrochemical detection, the electrode has a redox potential intermediate between the redox potential of the substrate and the redox potential of the product for the redox enzyme, but lower than the potential maintained at the electrode. 11. A method as claimed in any one of claims 8 to 10, in which the product is maintained at a predetermined potential such that only the product can undergo a redox reaction at the electrode. 12. The method according to any one of claims 8 to 11, wherein the redox substrate is a phenol derivative. 13. The method according to any one of claims 8 to 12, wherein the labeling enzyme is a phosphatase. 14. The method according to claim 12, wherein the redox substrate is a compound represented by the following formula (I): ▲A mathematical formula, a chemical formula, a table, etc.▼(I). 15. A system for detecting the activity of a first enzyme, comprising: a) a freely diffusing reagent that can be converted by said first enzyme into a product exhibiting mediator activity, allowing said product to be detected; b) ) a redox enzyme, a substrate for the redox enzyme,
and a detection means consisting of an electrode, such that in the presence of the first enzyme in active form the reagent is converted to the product and a detectable change occurs during mediated electron transfer to the electrode. 1. A system for detecting the activity of a first enzyme, characterized in that the system is configured. 16. In a bioelectrochemical cell, a substrate for a labeled enzyme used in the measurement is incorporated, and the labeled enzyme used in the measurement consumes the substrate and causes a change in the output from the biochemical cell, thereby causing a change in the output from the biochemical cell. 1. A bioelectrochemical cell, further comprising a second enzymatic reaction system configured to detect said substrate and providing electrical output through conversion of said substrate to a mediator. 17. In detecting the activity of a non-redox enzyme in the presence of at least one electrode maintained at a constant potential, a) a sample suspected of containing said non-redox enzyme is placed at a potential maintained at said electrode; treatment with a reagent having a higher redox potential, wherein said reagent is a substrate for a non-redox enzyme which has mediator activity by the activity of said redox enzyme and which is lower than the potential maintained at said electrode. b) treating said sample with a redox enzyme and a substrate for said redox enzyme to have a redox potential lower than the potential maintained at said electrode; A method for detecting the activity of a non-redox enzyme, characterized in that in the presence of a mediator compound, a measurable electron transfer to said electrode occurs.