JPS6017346A - Inspection method using one kind or more of enzyme - 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 relates to a method for detecting or measuring an enzyme or its specific substrate, and a sensor used in the method.

In European Patent Application No. 82805597, the inventors propose a sensor electrode comprising, at least on the external surface, a combination of an enzyme and a mesoieta compound that transfers charge to the electrode when the enzyme becomes catalytically active. did. Such an electrode, when contacted with a specific substrate for the enzyme in question balanced at an appropriate potential, produces a signal responsive to the presence of an enzyme/substrate reaction, even in a complex mixture of substrates, and thus a signal indicative of the extent of the reaction. .

This is because the enzyme is specific for the desired substrate component.

The practical operation of such systems depends on the incorporation of mesoieta compounds. Various examples of such compounds are disclosed in the aforementioned patent application Al, including polyviologens, fluoranyl, chloroanyl, etc., but the most characteristic mesoieters are metallocenes.

Ferrocenes (biscyclopentagenyl iron and its derivatives) belong to the last group mentioned above and have a number of advantages over other mesoieters used for charge transfer in enzyme/substrate reactions. There has been a considerable amount of theoretical and experimental research into the unique structure and properties of ferrocene and its derivatives. Ferrocene, first synthesized in 1951, was the earliest known example of metallocene compounds. Although ferrocenes have poor solubility in aqueous solutions and low extinction coefficients, making them limited in spectrophotometric assays, they have been found to be more suitable for bioelectrochemical systems. Ferrocenes are characterized by fa) a wide range of redox potentials that can be varied by V-conversion of the functionalizable cyclopentadienyl ring, (b) electrochemically reversible one-electron redox properties, and (cl) pH-independent redox potentials. Has slow autoxidation of potential and reducing conditions.

These compounds serve for example for the formation of derivatives by substitution and/or polymerization of one or both cyclopentadienyl rings. The inventors have investigated a number of ferrocene derivatives as listed in the table below.

1.1'-dimethylferrocene 100 I, D -ferrocene acetic acid 124 S No. 7゜hydroxyethylferrocene 161 S -ferrocene 165 I, D 81351.1'-bis(hydroxymethy#) 224 3 885 ferrocene ferrocene monocarbon Acid 275 S 420 Ferrocene 1. i'-dicarboxylic acid 885 S -chloro ferrocene 845 I, D - In the above table, S is water-soluble,
I and D are insoluble in water and soluble in 8% detergent (Tween-20) solution, and Eo is a labeled calomel electrode (hereinafter referred to as SOE).
), E is the measured value in mV for crn-
The measured value is indicated by 'M-'.

The E0 candy for ScE in phosphate buffer at pH = 7.0 for various ferrocenes shown in the above table is 100
It is within the potential range of ~400 mV. This trend in E0 values is consistent with what would be expected based on substitution effects. Generally, electron donating groups stabilize the positive charge and therefore promote oxidation more than electron accepting groups.

Among the ferrocenes mentioned above, 1,1-dimethylferrocene and ferrocene monocarboxylic acid were found to be generally preferred due to the wide range of enzymes that can be used.

The invention described in the above-mentioned patent application No. 6 is particularly adapted to the use of glucose as substrate and glucose oxidase or dehydrogenase as enzyme (thus, for example, as a dulcose sensor for diagnosing diabetic conditions). However, the electrochemical behavior of other enzymes whose electrochemical behavior was studied in collaboration with mesoieta compounds by the present inventors/
Substrate combinations include the following.

Pyruvate oxidase Pyruvate L-amino acid oxidase L-amino acid aldehyde oxidase
Aldehydes xanthine oxidase Xanthines glucose oxidase Glucose glucorate oxidase Dalicholate sarcosine oxidase Sarcosine lactate oxidase Lactate glutathione reductase NAD(P)H riboamide dehydrogenase NADH t L! Glucose Dehydration Enzymes Glucose Methanol Dehydrogenase Methanol and Other Alkanols Methylamine Dehydrogenase Methylamine HAEM Containing Enzymes Lactate Dehydrogenase Lactate Horseradish Beroxidase Hydrogen Peroxidase Nonmetallic Yellow Proteins Carbon Monoxide Oxide reductase carbon monoxide Of these, enzyme/substrate combinations that have the most well-established behavior and produce good, preferably linear, responses over the expected measurement range are preferred for use in 4-IJ. I found it to be clearly advantageous.

The invention of the above-mentioned patent application primarily relates to a sensor in which both the mesoieter and the enzyme are present on the electrode for contacting the substrate. All useful sensors, their characteristics and manufacture, and equipment for facilitating their use are described in the "Analysis I" filed on the same date as this application.
f and sensor electrodes used therewith", which are incorporated herein by reference.

By the way, the system of the present invention is the same as that of the above-mentioned patent application when mesoieta, enzyme and substrate are all in solution form, or when the sensor carries only mesoieta and enzyme, or only mesoieta alone. It is.

Additionally, other general patent applications dated the same date as this application titled "Assay Techniques Using Specific Binding Systems" use a solution-based basic system to analyze the electrochemistry of enzymes or mesoieters, or both. Specific binding reactions (eg, antigen/antibody reactions or nucleic acid probe/target sequence reactions) are assayed for effect on target conditions. In particular, descriptions of such liquid-based systems are included herein by reference.

All the patent applications mentioned above primarily concern single-enzyme systems. In addition, another patent application dated the same date as this patent application titled "Assay System Utilizing One or More Enzymes" also states that an additional enzyme (in a liquid or on an electrode) acts on its specific substrate. It has been described to influence the levels of mezoieta-conjugated enzyme substrates. This can be accomplished, for example, by the complete conversion of a substrate such as creatinine through creatine to sarcosine in one or more steps; The mesoieta/conjugated oxidase can be performed to give a readout that can be taken to determine the creatinine level.Also, a more or less complex mechanism of competitive reactions for the same substrate, e.g. mesoieta-conjugated glucose oxidase and the ATP-driven kinase producing glucose phosphate. The extent of the competitive reaction is an unknown proportion of ATP or kinase.

The descriptions of the above-mentioned patent applications which discuss multiple enzymes linked by substrate changes are also included herein by reference.

Although the present invention relates to a multi-enzyme system, -F
It has a different form of internal system linkage than the substrate binding chain of the reaction described in the patent application.

The first feature of the present invention is to apply a charge to a first enzyme, a cofactor bound to the enzyme, and the electrical state of the first enzyme by a reaction of the cofactor substance, using an electrode balanced at an appropriate potential. and a mesoieta compound that is transferred to an electrode.

NAD and NADP (collectively referred to as NAD(P)), cAMP, ATP as cofactors
, GTP.

There are TTP and CTP.

A second feature of the present invention is that ten electrodes balanced at appropriate potentials are connected to the first enzyme, the nicotinamide adenine dinucleotide compound bound to the enzyme, and the electrical state of the first enzyme, NAD(P)-NAD( P) A mesoieta compound that transfers charge to an electrode when transformed by a H reaction.

In practicing the invention, a second enzyme is coupled to an NAD(P) compound, a substrate for the second enzyme is present in the system, and the substrate/second enzyme reaction results in the NAD(P) compound. To undergo the reversible reaction and thus affect the first enzyme, the charge is transferred to the 'electrode in an amount correlated to the extent of the second enzyme/substrate reaction and assay for the other when one is known. It is preferable to operate it so that it does.

With respect to said other patent applications referred to herein, there are a number of modes of operation in which the active ingredient is distributed in solution or on electrodes. That is, the metal electrode may be immersed in a solution containing the mesoieter, the first and second enzymes, the NAD(P) compound, and the substrate. Or a trench.
An electrode may be coated with mesoieter, both enzymes, and the NAD(P) compound and immersed in a solution containing the substrate to detect the substrate or measure its coherency. Alternatively, an electrode may be coated with a mesoieta, NAD(P) compound, and a substrate and immersed in a solution containing a second enzyme to detect the enzyme or measure its concentration.

Specific enzymes, cofactors, mesoietors and substrates are illustrated below. In addition, when the electrode is made of a noble metal such as gold,
This can be combined with thiol (t or similar valency) substituted ferrocenes, and the large mesoieta can also be chemically combined with the enzyme, both of which are published on the same date as the present application. Examples are provided in detail in other patent applications entitled "Assay Systems Using Specific Binding Agents," which are incorporated herein by reference.

The invention will now be explained with reference to the drawings.

The mechanism shown in Figure 1 consists of an electrode 1 and four molecular species, namely ferrocene, preferably 1, 1'-
Mesoieta F, such as dimethylferrocene or ferrocene monocarboxylic acid in a free diffusion system, enzyme E1, which can be chemically combined with ferrocene to transfer charge from the enzyme to the electrode by the ferrocene, and nicotinamide, as detailed below. Adenine dinucleotide substance N and a second enzyme E2 with a different substrate SK% converted to reaction substrate R8 are shown.

The difference between the invention shown in No. 1M and the invention described in other patent applications other than the present application lies in the bond between El and N. In the invention of this other patent application, charge is transferred from El to the electrode 1 as long as the enzyme E1 becomes catalytically active towards its specific substrate. In the present invention, there is no substrate for El, but E
When l is bound to the enzyme E2 by compound N as part of the charge transport chain, and thereby E2 acts on its group fMS,
Charge will be transferred to the electrode 1 via the chain.

This system can be implemented in many different ways. For example, a simple gold electrode l can be used as F, El, N, E2
and S, and when balanced against the reference electrode, a current corresponding to the degree of the enzyme-catalyzed 5-R8 reaction can be obtained.

In another example, F, El, N, and E2 are all present on the surface of a composite electrode to form a sensor electrode to detect or measure the level of substrate S in solution. can do. Combined electrodes F and E as required
1, N and S form a sensor by which E2 can be calibrated. In addition, it is possible to obtain an assay for the presence of the E2-catalyzed S-SR reaction by constructing only the electrodes F, El and H. Other combinations of immobilized and dissolved components can be envisioned by those skilled in the art.

This system can also be simplified as an assay for compound N by omitting <E2 and S as shown in Figure 1a.

FIG. 2 shows a specific example of the invention. In this example, F is <1.1'-dimethylferrocene as described above. Enzyme E1 in Figure 2 is glutathione oxidoreductase G
R(E,O,,1,6,4,2).

Compound N is nicotinamide adenine dinucleotide phosphate (NADP). Enzyme E2 is D-incitrate dehydrogenase CD (EC1,1,1゜42
), whose substrate S is therefore D-isocitrate (CD), which is converted enzymatically to α-ketoglutarate (KG).

This system uses a gold electrode balanced for SCE and can be implemented in a solution containing ferrocene monocarboxylic acid, glutathione reductase, D-incitrate dehydrogenase, and NADP. Since such solutions do not generate electrode currents, the gold does not produce any detectable side reactions. When Kashi and D-Incitrate are added,
A dehydrogenation reaction takes place to give α-ketoglutarate and produce the reduced state of NADP, or NADPH. This is then reoxidized by glutathione reductase to give the reduced state GR enzyme, which in turn
Reduces the ferridinium mesoieta ion, which transfers charge to the electrode for indication of isocitrate concentration.

Conversely, forming a system containing F 1 GR 0 NADP 0 Substrate C provides an assay system for D-isocitrate dehydrogenase.

In addition, the assay system was configured with a cholesterol solution and
- It can also be an enzyme assay for dihydrocholesterol reductase.

Similar choices for enzyme or substrate detection are possible with any of the enzyme/substrate combinations listed below.

Enzyme Substrate D-isocitrate dehydrogenase D-isocitrate glutamate dehydrogenase Glutamate glucose-6-
Phosphate Glucose-6-phosphate dehydrogenase 20-β-hydroxysteroid 20-α-hydroxy dehydrogenase Steroids Glycerol dehydrogenase Glycerol Selected specific enzymes can be used in solution, and electrodes may be chemically bonded to the surface of In addition, if present, glutathione reductase can be chemically bonded to the surface of the electrode.

The assay can be extended to a wide range of NADP-binding enzymes as well as other cofactor-binding systems, making it possible to construct sensors for a wide range of enzyme-catalyzed reactions and to accommodate a wide range of equipment and end uses. can.

That is, since many of the enzymes listed involve substrates other than the native substrates, the use of ferrocene mesoietors is particularly useful in the production of sensors for process control, including conventional fermentation control, for incorporation into side flow monitoring and control systems.

FIG. 8 shows another example of the invention.

F is ferrocene monocarboxylic acid as described above. E
The enzyme in l is dihydroriboamide dehydrogenase (E, 0.1
．． Diaphorase (6.4.8) also known as
D), which is Clostridium krugbinii (Gl
ostridium Klugvini) and is commercially available from Boehringer. N is nicotinamide adenine dinucleotide (NAD)
and E2 is glycerol dehydrogenase (CD). Additionally, the system of Figure 8 includes the necessary glycerol substrate for an enzyme-catalyzed reaction whereby triglycerides T are reacted with lipase (glycerol ester hydrolase GEH) to form a glycerol/fatty acid mixture.

Soluble ferrocene monocarboxylic acid, diaphorase, N
A mixed solution containing AD, glycerol dehydrogenase and glycerol ester humanylase was formed. No current was observed when the solution was contacted with a gold N-electrode balanced at 150 mV to the SOE. After adding triglyceride and converting it into glycerol and fatty acids by the GEH enzyme, the glycerol component of the resulting mixture was oxidized to dihydroxyacetone (DHA) by the enzyme CD. As the latter reaction progresses, the charge generated is transferred through the chains of the components, resulting in a current at the electrode that can be measured in relation to the original triglyceride level.

It is recognized that the reaction of triglycerides to glycerol/fatty acids and further to dihydroxyacetone (DHA) itself is an example of the invention in another patent application entitled "Assay System Using One or More Enzymes." In the invention of this patent application, the two enzymes are substrate bound, ie the product of the first reaction becomes the substrate of the next reaction.

In the present invention, two enzymes are linked, for example by NAD or NADP, which produces a cyclic reaction, whereby El and E2 are electrically linked.

1. Deposit 1'-dimethylferrocene from a toluene solution onto a carbon foil (GRAPHOIL) and apply the diaphorase enzyme onto this ferrocene with the carbodiimide substance DOO.
(1-cyclohexyl-8(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate). This composite electrode is +150 against SOE.
It was immersed in a NAD/glycerol dehydrogenase solution balanced in mV and quantitatively sensitive to glycerol addition as the current reading at the electrode.

Other NAD-binding enzymes for use in the invention include the enzymes listed below with corresponding substrates.

Enzyme Substrate formate dehydrogenase Formate β-hydroxybutyrate White liquor ketones dehydrogenase Lactate dehydrogenase Lactate (NAD bond or cytochrome bond) Alcohol dehydrogenase Alcohol malate dehydrogenase Malates glycerate-1.8-phosphate Glycerate Dehydrogenation, 8- Theory Hydrogenases Phosphate Galactose Dehydrogenase Galactose Sorbitol Dehydrogenase Sorbitol Glucose Dehydrogenase Glucose (NADPH dependent) Cholesterol Reductase Cholesterol This example always contains mesoieta compounds and enzymes in the system.

% K provides a chemically modified enzyme, ie, an enzyme whose mesoieta group is chemically bonded to the complex structure so as not to disrupt enzyme activity. ...For example, it is possible to introduce ferrocene groups at 8 or exactly 12 times into the glucose oxidase enzyme, and by analogy chemical modification of the enzyme can occur in the valleys in other enzymes that can be used in the present invention. I found out that I can do it.

[Brief explanation of drawings]

Figure jA1 is a diagram showing the general mechanism of the binding enzyme used in the method of the present invention, Figure 1a is a diagram showing the mechanism of the assay system as an example of the method of the present invention, Figure 2 is a diagram showing the mechanism of the assay system as an example of the method of the present invention, and Figure 2 is a diagram showing the mechanism of the binding enzyme used as the binding enzyme. Diagram showing an example of a mechanism. FIG. 8 is a diagram showing an example of a mechanism using diaphorase as a binding enzyme. Patent applicant Genetics International
Continuing from page 1 of Inc. Priority claim September 6, 1983 ■ United Kingdom (GB)
■8323801 ■February 29, 1984 ■United Kingdom (GB) ■8405262 02/29, 1984 [Stock] United Kingdom (GB) ■84
05263 Procedural amendment (method) % formula % 1 Indication of case Patent application No. 90833 of 1982 2 Title of invention Assay method using one or more enzymes 5 Date of amendment order July 3, 1988] Day 6, Subject of amendment: "Patent applicant" column of the application, power of attorney, and drawings

Claims (1)

[Claims] L An electrode balanced at an appropriate potential is connected to a first enzyme, a nicotinamide adenine dinucleotide compound bound to the enzyme, and the electrical state of the first enzyme to NAD(P)/
An assay method that involves contacting a system consisting of a mesoieta compound that transfers charge to an electrode when transformed by a NAD(P)H reaction. A second enzyme is combined with a NAD (P) compound,
A substrate for the second enzyme is present in the system so that the substrate/second enzyme reaction yields the NAD(P) compound and undergoes its reversible reaction, thus affecting the second enzyme and transferring a charge to the electrode. 2. The method according to claim 1, wherein when one of the two enzymes/substrate reactions is known, the other is assayed by transferring amounts that correlate with the extent of the enzyme/substrate reaction. A metal electrode is used as a mesoieta, the first and second enzymes,
3. The method according to claim 2, wherein the method is immersed in a solution containing the NAD(P) compound and the substrate. 4 Connect the electrodes to the mesoiator, the first and second
solution 'VC coated with AD(P) compound and containing substrate;
3. The method according to claim 2, wherein the substrate is detected or its concentration is measured by immersion. (v) An electrode is coated with a mesoieter, a first enzyme, an NAD(P) compound, and a substrate, and is immersed in a solution containing a second enzyme to detect the enzyme or measure its rigidity. The method described in Section 2. a The first enzyme is glutathione oxidoreductase,
A patent in which the NAD(P) compound is nicotinamide adenine dinucleotide phosphate, the second enzyme is D-isocitrate dehydrogenase, the substrate is D-isocitrate, and the degree of catalytic conversion of the substrate to α-ketodalglate is measured The method according to claim 2. The first enzyme is diaphorase (i.e. dihydroriboamide dehydrogenase), the NAD (P) compound is nicotinamide adenine dinucleotide, the second enzyme is dalicerol dehydrogenase, the substrate is glycerol, and the conversion of glycerol to dihydroxyacetone 3. A method according to claim 2 for determining the degree of catalytic conversion. &Crycerol ester The method according to claim 7, wherein fatty acids and glycerol as a substrate for the second fermentation purple are obtained by an enzyme-catalyzed reaction in the presence of hydrolaseol together with a triglyceride substance. 9. The method according to any one of claims 1 to 8, wherein the mesoieta compound is ferrocene. 9. The method according to claim 1, wherein the 1α mesoieter is 1,1'-dimethylferrocene, which is coated on the electrode. 11. A process according to any one of claims 1 to 8, wherein the mesoieter is present in solution as carboxy-substituted ferrocene. 11 Sensor electrode phased with ferrocene mesoieta, glutathione reductase and nicotinamide adenine dinucleotide phosphate. 1 & Sensor electrodes disrupted with ferrocene mesoieta, diaphorase and nicotinamide adenine dinucleotide. 14 Ferroceneka 1. The sensor' electrode according to claim 12 or 18i, which is 1'-dimethylferrocene. 19. The sensor electrode according to claim 12 or 18, wherein both 1F-ferrocene and the enzyme are chemically bonded. 1a A first enzyme, a cofactor bound to the enzyme, and a charge is transferred to the electrode when the electrical state of the first enzyme is changed by a reaction of the cofactor substance, which is balanced at an appropriate potential. An assay method that involves contacting a system consisting of a mesoieta metal object.