JPS63246651A - Biosensor - Google Patents

Abstract

PURPOSE:To determine NADH or NADPH in a sample handily and accurately, by employing diaphorase as enzyme and an immobilized cytochrome C electrode as electrode. CONSTITUTION:This biosensor is made up of diaphorase, a mediator to receive electrons being conjugated with an oxidation/reduction reaction by diaphorase and a transducer to convert the reception of the electrons into an electrical signal and the mediator of the biosensor is made of an immobilized cytochrome C. The cytochrome C herein used is preferably of a mammal type and the diaphorase herein used is preferably produced by an microorganism with the optimum growing temperature of 50-85 deg.C. The cytochrome C is used as immobilized on a water insoluble carrier in a film and preferably covers a sensitive surface of an electrode directly. In the application of the diaphorase, a water insoluble carrier is further placed on a cytochrome C electrode to immobilize it thereon.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a biosensor used for measuring the concentration of N A D H or NADPH in a sample.

(Prior Art) Quantification of NADH or NADPH provides an important means for biochemical analysis techniques in clinical chemistry analysis 1 food analysis and the like. Particularly in clinical chemistry analysis, the quantification of NADH or NADPH is an essential tool.
For example, measurement of the amount of glucose in biological samples such as blood and urine, measurement of taleatin kinase activity, etc. are performed by quantifying NADPH.

For example, in such an analysis, the substance to be measured is treated with an appropriate substance to lead it to a form that is easy to quantify.Recently, the use of enzymes has attracted attention as an appropriate substance, and many Enzymes are now playing a leading role in the analysis items.H202 or NADH (or NADPH) are used as forms that are easy to quantify.

When oxidase is used as the enzyme, H20□ is quantified, and when dehydrogenase is used, NA
DH or NADPH will be quantified. Comparing both methods, the latter method is said to be superior because the former has problems with stoichiometry, is easily affected by the amount of dissolved oxygen, and is easily affected by reducing substances. Ru.

In the latter method, NADH or NADPH is 34
Since it has a characteristic absorption spectrum at 0 nm, 340
Quantification is performed by measuring absorbance at nm. However, since an expensive spectrophotometer is required, development of a simpler measurement method is desired.

For this reason, expectations are high for the development of a biosensor for quantifying NADH or NADPH, but the reality is that no practical sensor has been obtained yet.

For example, it is known that diaphorase is extremely useful as an oxidoreductase that acts on NADH or NADPH, and there are research examples of biosensors using this. Specifically, Cheno et al. are researching a biosensor that uses diaphorase and ferricyanide as a mediator to detect NADH with a graphite electrode.
ng, 21 + 1905 (1979)) using diaphorase.

Dichlorophenolindophenol as mediator
We are currently researching a biosensor that detects NAD II using a rotating platinum electrode (Anal, Chem.

54, 1987 (19B2)).

(Problem to be solved by the invention) However, from the standpoint of practicality of the biosensor, it is desirable to integrate and immobilize all the enzymes and mediators necessary for measurement on the electrode. All of them had problems in practical application. That is, ferricyanide and dichlorophenol indophenol are extremely difficult to immobilize because the former is an inorganic salt and the latter is a low molecular weight substance.

As mentioned above, although an excellent enzyme called diaphorase is known, in a practical sense, NADH or NA
There is currently no known biosensor that allows the quantification of DPH. This is NADH or NADPH
This seems to be due to the fact that an appropriate mediator for converting the amount of diaphorase acting on the diaphorase into an electrical signal has not been obtained.

(Means for Solving the Problems) The present inventors have conducted intensive research to solve these problems, and found that using diaphorase as an enzyme,
We have completed the present invention by discovering that by using an immobilized cytochrome C electrode as an electrode, it is possible to construct a biosensor that can easily and accurately quantify NAD H or NADPH in a sample.

That is, 1. the present invention is a biosensor consisting of diaphorase, a mediator that transfers and receives electrons by conjugating with the redox reaction of diaphorase, and a transducer that converts the transfer of electrons into an electrical signal, in which the mediator is immobilized. The gist of the present invention is a biosensor characterized by being composed of cytochrome C.

The transducer used in the present invention is as follows.

Both electrochemical devices and semiconductor devices can be used, but it is preferable to use electrochemical devices, such as indium oxide/tin oxide evaporated glass electrodes, platinum electrodes, glassy carbon electrodes, gold electrodes, etc. be.

Cytochrome C includes #mammalian cytochrome C (animal,
Various types of cytochrome C can be used, such as yeast, higher plants, etc.), microbial cytochrome C (bacteria, etc.), but among these, mammalian cytochrome C1, particularly cytochrome C derived from horse heart, is preferably used. Various types of diaphorase can be used, including those derived from microorganisms and those derived from animals, but diaphorases produced by microorganisms with an optimum growth temperature of 50°C to 85°C are particularly preferred. Such microorganisms include, for example, microorganisms of the genus Bacillus such as Bacillus stearothermophilus, Bacillus thermobrotheoliticus, and Bacillus acidocaldarius, as well as microorganisms of the genus Thermoactinomyces, Thermus, and Thermomicrobium. can give. Among these, a particularly preferred microorganism is Bacillus stearothermophilus, and a specific example thereof is ATCC 793.
3゜7954.10194.12980. NCA 15
03. UK 563 strain (Feikoken Bacteria No. 7275, F
CRM P-7275, deposited on September 29, 1982)
and so on.

Cytochrome C in the present invention is in the form of a membrane, and includes 1 a natural polymeric substance 1 a water-insoluble carrier such as a synthetic polymeric substance 2 such as T-benzyl-thi-glutamic acid partial hydrolyzate, polymethacrylic acid ester partial hydrolyzate, succinylated It is used immobilized on a water-insoluble carrier such as chitosan. In this way, it is preferable to directly coat the sensitive surface of the electrode with the water-insoluble carrier on which cytochrome C is immobilized, in order for the cytochrome C to exhibit its functions. It is also a preferable method to cover the sensitive surface of the electrode in advance with a water-insoluble carrier such as a protein 1 synthetic polymer and to immobilize cytochrome C thereon.

In order to immobilize cytochrome C on a water-insoluble carrier in the present invention, for example, "Immobilized Enzymes" edited by Chibata, Kodansha (197
Conventionally known covalent bonding methods and adsorption methods as described in 5) can be employed. Further, methods such as a crosslinking method or an enclosing method can also be employed.

The diaphorase of the present invention is further coated on a cytochrome C electrode with a water-insoluble carrier such as a natural polymer substance 9 or a synthetic polymer substance, such as the water-insoluble carrier mentioned above for immobilization of cytochrome C, preferably a polycationic polymer substance (chitosan). , polylysine derivative.

Polyallylamine complex, etc.) or aniline electrolytically polymerized membrane are used for immobilization. The above-mentioned method can also be used for immobilizing diaphorase.

It is also possible to use a mixture of cytochrome C and diaphorase, immobilized on the same water-insoluble carrier, and coated on the sensitive surface of the electrode.

Using the biosensor of the present invention, NADI (or N
To measure ADPH, for example, a multilayer-coated transducer in which immobilized cytochrome C is directly coated on the transducer sensitive surface and further coated with immobilized diaphorase is immersed in a buffer solution.

A method of measuring the change in the amount detected by a transducer caused by the addition of a measurement sample. A mixed immobilized product of cytochrome C and diaphorase is directly coated on the transducer sensitive surface, and the resultant mixture is immersed in a buffer solution, It is possible to adopt a method of measuring the resulting change in the amount detected by the transducer.

The solution for measurement is 1, for example 0. A solution of O1 to 0.1M phosphate buffer (pH 5 to 10, preferably 7 to 8) may be used.

The measurement temperature used is 0 to 60°C, preferably 20 to 40°C.

(Function) The biosensor of the present invention performs the first-order reaction N A D H or N A D ' or
It is constructed by detecting the amount of electron transfer generated by cytochrome C(Fe)NADP with an electrochemical device or a semiconductor device.

NADH or NADP H originally present in the sample
Not only can the amount be measured, but also the amount of the substance to be measured other than NADPH in the sample can be determined by appropriate means, such as rQ A D H or N A D by the action of dehydrogenase.
As a result of the amount of PH, NADH or N in the sample
The original measurement object' by measuring ADPH3l
The amount of It can be measured.

(Example) Next, the present invention will be specifically explained with reference to Examples.

Example 1 2 g of poly-T-benzyl monoglutamic acid (molecular weight 150,000-350,000, manufactured by Sigma) was added to 50 ml of acetic acid to swell it, and then added to 10 mj+ of an acetic acid solution containing 30% hydrogen bromide. The mixture was stirred at 30°C for 25 minutes. Pour the reaction solution into 200mj of ether! The resulting precipitate was collected by filtration, further washed with ether, and dried to obtain about 1.7 g of a partial hydrolyzate of poly T-benzylic monoglutamic acid (γ-benzylic monoglutamic acid and glutamic acid). copolymer) was obtained. The proportion of benzyl groups hydrolyzed was approximately 65%.

The obtained copolymerized 4*o,sg of γ-benzylic monoglutamic acid and glutamic acid was dissolved in 100 m# of a mixed solution of dimethylformamide and ethanol (2:1), and indium oxide glass doped with tin oxide during this dissolution. An electrode (manufactured by Nippon Sheet Glass Co., Ltd., hereinafter referred to as InzOs/Sn○2 evaporated glass electrode) was immersed for 5 minutes and then air-dried.

As a result, about 20 μg of the copolymer of γgovendy-L-glutamic acid and glutamic acid was coated on the 1B20*/SnO□ vapor-deposited glass electrode per lcA.

This electrode was then treated with 100mM aniline, 40μM
Cytochrome C (derived from horse heart, type Vl manufactured by Sigma)
), 20 units/m1 of diaphorase (derived from Bacillus stearothermophilus, manufactured by Seikagaku Corporation), 10 m
The sample was immersed in a solution consisting of M phosphate buffer (pH 7.4), and electrolytically polymerized for 5 minutes. In this way, cytochrome C and diaphorase were immobilized by directly coating the electrode surface. A so-called cytochrome C-diaphorase immobilized electrode was prepared.

Using this electrode, an electrochemical measurement system as shown in FIG. 1 was assembled to measure the oxidation current value caused by the presence of NADH.

That is, FIG. 1 shows a three-electrode system consisting of a reference electrode 11, a working electrode 4, and a counter electrode 5 (2 indicates an agar salt bridge, 3 indicates a capillary, and 7 indicates a saturated potassium chloride aqueous solution), and a constant potential is applied. The potential of the working electrode 4 was controlled to +0.2■ with respect to the reference electrode 1, which was guaranteed to exhibit a positive effect. NPGFZ-2501A potentiostat 6 (manufactured by Nichijo Keizoku Co., Ltd.) was used to control the potential. In addition, a saturated calomel electrode is used as the reference electrode 1, a platinum electrode (platinum wire) is used as the counter electrode 5, and 1 working electrode 4 is used.
For this purpose, the I n20 :l/S n O□ vapor-deposited glass electrode prepared above was coated and immobilized with cytochrome C and diaphorase. At the working electrode surface, the added NA
An amount of electrons proportional to DH is transferred to cytochrome C via diaphorase, and the oxidized cytochrome C becomes a reduced form, but the electrons are taken away by the electrode.

It becomes the oxidized form again, and the oxidation current of cytochrome C at this time is measured.

The concentration of NADH aqueous solution 8 used as a sample was 4°8.1
The test was carried out at 6.32 and 64 μM, and the relationship between the obtained oxidation current values is shown in FIG.

As shown in Figure 2, NADH with one sensor showed good response in the NADH concentration range of 4 to 64 μM.
It was found that quantitative determination was possible.

(Effect of the invention) The biosensor of the present invention has diaphorase, cytochrome C
and a transducer that converts the amount of redox into an electrical signal.
It has an excellent ability to measure A D P H concentration easily, quickly, and with high selectivity.

[Brief explanation of drawings]

Figure 1 shows a saturated calomel electrode, a platinum electrode, and InzO=/5nOz with immobilized cytochrome C and diaphorase.
FIG. 2 is a diagram showing a three-electrode system consisting of vapor-deposited glass electrodes, and FIG. 2 is a diagram showing the relationship between N A D H>H degree and the oxidation current value of cytochrome C. 1. Reference electrode, saturated calomel electrode 2, agar salt bridge 3, capillary 4, working electrode, Intox/5n02 vapor-deposited glass electrode with immobilized cytochrome C and diaphorase. 5. Counter electrode, platinum electrode. 6. Potentiostat. 7. Saturated potassium chloride aqueous solution. 8. NADH aqueous solution.

Claims (1)

[Claims] (1) In a biosensor consisting of diaphorase, a mediator that transfers and receives electrons by conjugating with the redox reaction of diaphorase, and a transducer that converts the transfer of electrons into an electrical signal, the mediator was composed of immobilized cytochrome C. A biosensor characterized by: