JPH01147357A - Enzyme analysis system and analysis by said system - Google Patents

Abstract

PURPOSE:To obtain an analysis system which can make measurement with high-speed response and high sensitivity even with a slight quantity of sample by using a microelectrode of a biofunction material. CONSTITUTION:The microelectrode 1 having a biofunction is the electrode having a conductive fine particle material layer formed by impregnating the biofunction material such as enzyme on the surface of a very small electrode and is produced by electrodepositing the biofunction material directly on the section of a platinum wire or the like and electrolytically reducing a metal salt simultaneously to deposit the conductive fine particle material together with the biofunction material thereon. A micro device 6 is constituted by using this microelectrode as a working electrode 1, a platinum wire as a counter electrode 2 and silver chloride as a reference electrode 3. The microelectrode 1, the counter electrode 2 and the reference electrode 3 are solidified and fixed into a hole of a 'Teflon(R)' resin 5 by a resin 4. The measurement and detection are made with the rapid response and high sensitivity by using such microelectrode.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an analysis system using a biosensor and an analysis method thereof. In particular, microelectrodes immobilized with bovine functional substances (
The present invention relates to an analytical device using a high-speed response, high-sensitivity, and high-output biosensor using microelectrodes) and an analytical method using the same.

[Prior art] Biosensors in which enzymes, antibodies, microorganisms, etc. are immobilized on the surface of platinum or carbon can rapidly detect various chemical substances and biological substances. It is already known that continuous measurement is possible. In such biosensers, biologically functional substances can be produced using the JJ method, in which a film containing a biologically functional substance is attached to the electrode 1-, or by applying an enzyme, etc. to an electrode whose surface has been chemically treated, and then bonding the enzyme, etc. with the surface. In the meantime, it has been identified by methods such as reducing the formation of heavy bonds. However, the performance of Biohin 11 is evaluated by its tTj reactivity, durability, high sensitivity, response speed, etc. +'+ii's Jj method has a problem in terms of response speed, and the latter's)j method has difficulty in increasing the immobilization density. Furthermore, in both methods, immobilization requires several complicated steps.

It was difficult to create a multifunctional senna by attaching several types of biofunctional material electrodes to one senna L.

In addition, conventional immobilized enzyme electrodes have a structure in which an enzyme immobilization membrane is attached to a Clark-type oxygen electrode or a platinum surface in the form of a lower plate. 1-H method for attaching an immobilized enzyme membrane to a platinum electrode 9
There is a method of immobilizing enzymes by coating them on a smooth IE electrode whose surface has been chemically treated.

However, miniaturization is difficult with this various-horn method.

-No.J, the most recent miniaturization technology is conductor integration technology. In the horn method using the knee T conductor technology, an enzyme electrode 4 with a size of several square meters is used.
Made of! However, since it is an 'IE position detection method, satisfactory results have not been obtained in terms of sensitivity and response14.
Miniaturization beyond the current size is considered to be quite difficult.

[Problems to be Solved by the Invention] Under the above-mentioned circumstances, the present inventors have developed an LI approach to overcome the drawbacks of the above-mentioned conventional hinlj, unlike the conventional hinna sysdem. To.

By using miniaturized biofunctional material electrodes (hereinafter referred to as Miku-I'lV, poles), we have developed an analytical system that has high-speed response and high sensitivity and can measure even extremely small samples, and its use. Provide analytical methods.

In addition, the present invention provides an ultra-fine 11'c electrode using a microelectrode as a working electrode.
Get the fee quickly! Providing a high-performance bi-A sensing sensor that can maintain a constant temperature? - to be [1 point]. That is.

The present invention measures minute amounts of substances (e.g., glucose) in a sample by detecting the current generated when a potential is applied to a minute amount of sample. , - It is an object of the present invention to provide a biosensory analysis system and method that does not depend on the liquid to be measured. ,−
The purpose is to , F
FJ's objective is to provide a bioanalysis system that can extremely reduce ripple 11'c (for example, measurement is possible within 1 μp at the minimum Y^) since the electrode (MIG11 electrode) can be placed on a microscopic surface. shall be. A further object of the present invention is to provide a bioanalysis system capable of real-time measurement to directly detect electrode active substances produced by enzyme molecules inside microelectrodes. In addition, since the bioanalysis system of the present invention can apply various detection methods, the bioanalysis system of the present invention can be used to measure the sample in a static IF state without using t). The method is an electrode active substance detection method.

It can also be applied to important enzymes such as oxidases and dehydrogenases. Furthermore, 1 bioanalytical system according to the present invention-1-
t can also be used as an electrode for enzyme immunoassay.

[Structure of the Invention] [Steps to Solve the Problems] In order to solve the problems described in 1-1, the present inventors have solved the problem by avoiding the integration of a biologically functional substance with fine particles of a conductive substance, and 2) A bovine functional substance τI-immobilized microelectrode having a surface layer of microparticles and a conductive material was fabricated as a fl 'ili electrode, and was equipped with a current applying means and a current measuring device for measuring the generated current. The present invention provides an analysis system characterized by determining the concentration of the target substance from the measurement of the generated current.Furthermore, the present invention provides an analysis system characterized by determining the concentration of the target substance from the measurement of the generated current. The biofunctional substance-immobilized microelectrode having the surface layer of the fine particle conductive material was used as the action 1 [pole], and the current value generated when a constant potential was applied was measured.
The present invention provides an analysis method characterized by determining the concentration of a target substance from the value. The processing method that avoids integrating this biologically functional substance with the fine particles of the conductive substance is as follows: - While depositing the fine particles of the conductive substance Y on the surface of the fine conductive substance, the biologically functional substance If1 is deposited on the surface of the fine conductive substance. The idea of adsorption =
It is accomplished in a process. In addition, other additives, conductive fine particles -
After immersing the substance 54 in a solution of the biologically functional substance rI and incorporating the biologically functional substance into the inside of the fine particles or adhering it to the surface, the biologically functional substance can be directly crosslinked with each other. Alternatively, the surface of the fine particles is coated by forming a thin film of 11jj molecules.
1-. 4-ru 2, ni 2'+! done through processing.

The bio-quasi-electrode created by integrating this biologically functional material and a conductive material has the same ability as a super-micropole. 6 This bio-electrode is referred to as a micro-electrode in this invention. In other words, 1) the present invention utilizes such a microelectrode immobilized with a biofunctional substance, measures the current value generated when a constant potential is applied to it, and calculates the microelectrode from (+/i). 3) was discovered by determining the concentration of the target substance.

The present inventors hereinafter 1°111. By using platinum blackening technology and enzyme electrochemical adsorption technology, we succeeded in creating a microscopic and highly efficient enzyme electrode (MIG-11 electrode) and filed a patent application (patent application filed in 1988). 55387 and 56472). This MIG-11 electrode has high-speed response and high sensitivity.

The present inventors further conducted research and discovered a bioanalytical system and analysis method that can analyze trace substances in samples using ultra-low RF using this microelectrode with excellent characteristics. . That is, a biosensing system that can be measured extremely quickly by a constant potential application method using this Miku IJ electrode as a working electrode was obtained.

The Miku l'l electrode with biological functions used in the present invention is
The '1Tt electrode has a structure in which the surface of a minute electrode made of platinum or the like is impregnated with a biofunctional substance such as an enzyme and a layer of conductive material fine particles is formed. As used in the present invention, this electrode uses platinum black as a carrier for biologically functional substances, which has not been done in the past. There is an immobilization method that connects the
In addition, the size of the platinum black particles was increased to 11 tones,
The Jj method, which encases and immobilizes biologically functional substances, is unprecedented.

Therefore, the direct immobilization method of biofunctional substances is not the carrier binding method that conventionally required the use of chemical reagents (crosslinking agents), but can be used without chemical treatment. This allows direct immobilization of biologically functional substances.

One method for manufacturing the microelectrode used in the present invention is 1. For example, a biomechanical material is directly electrodeposited on a conductive material such as a cross section of a platinum wire, and at the same time a metal salt is electrolytically reduced ('I [deposition]). For example, while forming platinum fine particles on a micro platinum electrode, a biologically functional substance is deposited into the pores within the fine particles. It can be created by incorporating. The size of the pores and the amount of biofunctional substances to be immobilized can be controlled by adjusting the current density, electrodeposition time, etc.
I can see it. The function of the immobilized biofunctional substance r1 electrode thus obtained can be maintained for a long period of time.
be.

In addition, the other Miku 11 'ii poles are manufactured using a two-step process -)■, Miku 1-! For example, a thin platinum wire (a conductive material for boat fishing) is coated with a layer of conductive fine particles such as platinum black, and a biologically functional substance is placed inside it. It is something that becomes fixed. Specifically, the electrode on which the conductive substance fine particle layer has been formed is impregnated by immersing it in an aqueous solution of the biologically functional substance or by coating it, and then preventing the slight elution of the biologically functional substance. In addition, in order to impart antithrombotic properties, a polymeric substance such as albumin or heparin is impregnated thereon, and this is made insoluble with a crosslinking agent to form an insoluble polymer thin film. The microelectrode prepared in this way entrapping and immobilizing the enzyme can be considered to be an application of the so-called entrapment method to a metal electrode system, but the formed polymer film is
It is possible to create a thin film with a thickness of several hundred layers or less, and the presence of the polymer/film does not become a factor that inhibits the expression of the biological function of the biologically functional substance.

Therefore, the diameter of the microelectrode prepared as described in Section 2 depends on the diameter of the platinum wire, and if a thin platinum wire is used, a bioelectrode with extremely small electrodes that cannot be achieved conventionally can be obtained. It is easy to make a high-A'-sized microorganism, and it is convenient for use by implanting it in the body of a dog. Furthermore, with the extremely small size of the electrode, it is possible to create a very handy analyzer with an I-nanzuru chamber, making it possible to analyze a minute 11 ton sample, and providing an analyzer with rapid subdetermination and high responsiveness.

By fabricating electrodes with relatively large sizes, it is possible to fabricate an analytical device with a large detection output.

In the production of the Miku I+TL electrode described below, the initial conductive material and the fine particles to be deposited on it (i.e., porous)
The conductive substance, ie, the fine particle metal, does not have to be the same material; for example, it may have a structure in which platinum black is deposited on graphite. , instead of platinum,
A conductive material such as gold, rhodium, etc. can be used as a porous conductive material, and a fine particle layer of the conductive material can be used as a conductive material.
[Any substance that can be formed on the surface of a chemical substance can be suitably used in the present invention as long as there are no other obstacles.

Furthermore, the electrodes are made of platinum black.

Formed into various shapes such as disk shape, sphere shape, tube shape, etc.''c
,! Because it is a biosenna.

It can be shaped to comply with Measurement 2 and other filling conditions, and can be used for other purposes as well.

Furthermore, the micro TL pole utilized in accordance with the present invention includes proteins,
It is also possible to apply a polymeric substance such as a polysaccharide and form a thin film crosslinked with a crosslinking agent to improve biocompatibility, maintain performance, and minimize elution of biofunctional substances.

Here, "biofunctional substances" are typified by enzymes and antibodies, including various catalysts, microbial cells, proliferating microorganisms, Luganella, antigens, and antibodies.

This includes habten, etc.

In addition, nine bioelectrodes utilized in the present invention were applied.
For i,: ff, polymeric substances that can be used additionally include:
Examples include proteins such as albumin; ion exchange resins; and polysaccharides such as heparin. As a crosslinking agent,
There are crosslinking agents that are suitable for the polymeric material used; for example, for albumin, glutaraldehydride,
Further, carbodiimide, maleimide crosslinking agents, etc. are used.

In 1-2 below, in the MikutJ electrode. The method for depositing porous (fine particle) conductive materials is electrolytic deposition.

Although described, it is clear that electroless methods can also be used.

The analytical method according to the present invention has a fast response and high sensitivity.

A compact and compact biosensing system has been put into practical use, and is useful for clinical chemistry analysis, which requires multi-item measurement such as biosensor integration and multi-functionalization, and for portable health monitoring systems. This will become an extremely important technology for development.

FIG. 1 shows a working electrode 1 of a microelectrode (glucose oxidase) having a diameter of about 1 μm to 500 ffm, and a platinum wire counter electrode 2.
Miku 1 with a configuration using silver and silver chloride as the reference electrode 3. Show device 6, the following three electrodes, Miku 1?electrode 1, counter electrode 2 and reference' north pole 3, def IJ tree!F 5 The MIG-11 device 6 has a structure in which three thin metal wires are sealed and fixed in the hole.

It can be made into a very small and nice device.

, 2 for example, a micro-181 example λ for a MIG 1 electrode of -.

Even a 1 μP sample can be measured. That is, after dropping a small amount of sample, a potential is applied, and the Tri
By detecting the flow value, trace amounts of substances in the liquid can be detected.

Therefore, in the present invention, real-time measurement is possible in order to directly detect active substances within the 1 electrode produced by biofunctional substances, such as enzyme molecules, inside the 1 electrode.

Furthermore, in the structure below, ′;r! Since various portammetric detection methods can be applied at the poles, the sample can be measured even if it is in a stationary state.

In addition, it is a method for detecting active substances within the TL pole.

This bioanalysis system can also be applied to important biologically functional substances such as oxidases and dehydrogenases.

The present invention will be explained in detail below with reference to Examples, but the present invention is not limited thereto.

[Example 1 Production method Various fine platinum wires with diameters ranging from 1 μm to 500 μm, 2Q
A platinum wire for the counter electrode with a diameter of 1 m, and a platinum wire of 500 μm to 1
After enclosing a silver wire with a diameter of about 100 ml with a wooden finger 4, the surface of the fine platinum wire was polished with alumina powder. Enzymes were immobilized on this polished platinum surface using the following two methods.

(A) Platinum chloride 11 (33+og), lead acetate (0.6m
Glucose oxidase (Pt. was precipitated.

(B) Immersed in a 3% chloroplatinic acid solution containing 300 ppm lead acetate, and electrodeposited platinum black for 10 minutes at a flow value of -50 μA to obtain platinum black with a thickness of several μm. After drying the obtained platinum black deposited electrode at 25°C for 60 seconds,
Maintained at -0.3V for 30 minutes in a 0.5M sulfuric acid aqueous solution,
At 20°C, at zero order to avoid hydrogen generation from the platinum black deposited electrode,
After air drying for 60 seconds, the phosphoric acid 11 solution (pH 6,8) at the 5501 position was added to the imp.
It was immersed for 0 minutes and air-dried again.

Next, in the device 6 with the microelectrode obtained as described above, ma is silver/silver chloride IK.
=j7[i
The microdevice 6 consisting of electrodes was stirred and washed in a 0.1M phosphate buffer solution overnight to obtain a three-electrode microdevice used in the present invention.

[Example 2] Measurement of glucose concentration This three-electrode microdevice was charged with Gluco-3V:
After dropping μ2, a potential of 0.6V was applied. The relationship between the peak current value generated at this time and the glucose concentration was investigated.Next, the relationship between the peak N, flow value, and liquid 1^ was investigated by changing the liquid acid of the sample containing 10mM glucose. That is, various concentrations of Glue:1-sul were added dropwise and a potential of 0.6 V was applied. As a result, responses as shown in FIG. 2 were observed.

That is, when a potential of 0.6V is applied, the result shown in Figure 2 is:! A constant current was generated. When no glucose is contained, a current as shown in the left diagram of Figure 2 is generated, and when a phosphate buffer (50mM, pH 7, 0, 50mN
When aCF was used as a standard sample, a response TrL flow as shown in the right diagram of FIG. 2 was generated.

Immediately, as is clear from Fig. 2, there is an immediate response of 1! upon application of the potential. It can be seen that a flow occurs and quickly decays. This indicates that the hydrogen peroxide produced by glucose oxidase inside the electrode is immediately oxidized as the potential is applied. It was found that by repeating the potential application several times, the peak current becomes constant.

Although similar behavior was observed with phosphate buffer, the peak current value was small. Therefore, the peak current values for various glues: 1-su ρ degree were measured, and the peak '117. f
Take the difference from it 1ft (i.e., blank value) and set it to Guru':? -The relationship with the concentration of gas was investigated. The results are shown in FIG. Therefore, glucose 4 degrees is 1-100
When varying in the mM range.

It was found that a measurement curve as shown in FIG. 3 was obtained between the peak current (/i) and the peak current (/i).

Furthermore, the enzyme analysis system according to the present invention has been shown to be able to measure the concentration of minute amounts of static samples in real time (it is possible to measure specific substances from minute amounts of samples in millisecond order).

[Example 3] Solution '4 Dependency Test 10mM glucose standard solution (2.5°10.
15.20μ! 9 when a constant potential of 0.6 V was applied after dropping it into the microbiosenner system.
The generation M, flow was measured. As a result, 4゜ as shown in Fig. 4 was obtained. Therefore, the obtained peak '+'rj, BE
was found to be independent of the sample termination.

Based on the following results, the analysis system and method according to the present invention detects only a part of the sample to be measured in the measurement. ! It was shown that I+ can be determined and information on the entire sample in the static 11- state can be extracted.

Here we go! As described above, the analysis system and method according to the present invention does not require stirring the sample, and therefore has the potential to be used as a two-in-body sensing system or a portable biosensor. It shows.

In the bioanalysis system according to the present invention, even in the convection container,
It goes without saying that even with a batch method, measurements can be made quickly and with high sensitivity with the same high-speed response as with a stationary method.

[Effects of the Invention] The analysis system and method of the present invention have the following remarkable technical effects.

First, it is possible to provide high-performance biosensing that has a very quick response, high sensitivity, and can measure and detect even static samples.

Second, tiny enzyme electrodes (i.e., microelectrodes).

Since the counter electrode and the reference electrode can be arranged on a microscopic surface, the number of nanometers and loops can be extremely reduced.Therefore, an analytical system and a horn method are provided that can quickly and responsively measure even extremely warm samples. Ru.

Third, it is possible to provide a system that can be used in real time to directly detect electrode active substances produced by internal enzyme molecules.

Fourthly, since two different portammetric detection methods can be applied, it is possible to provide an analytical system that can detect samples with high sensitivity even in a stationary state.

Fifth, since it is an electrode active substance detection method, it can also be applied to important enzymes such as oxidases and dehydrogenases, and biologically functional substances.

Sixthly, it is possible to provide a bioanalysis system that can be used as an in-vivo sensing system or a portable biosensor.

[Brief explanation of the drawing]

Figure 1 is an explanatory diagram illustrating the structure and manufacturing method of the three-electrode MikuJ device used in the analytical system according to the present invention. Ti
This is a graph showing the flow. FIG. 3 shows 9 microanalysis systems according to the present invention;
1 is a graph showing the relationship between the 1-phase concentration and the generated current value. Figure 4 shows 1-
3 is a graph showing the relationship between the capacity of a sample and the peak TL flow value. [Explanation of symbols for convenient parts] 1111 Working electrode (micro electrode) 211. Counterpoint 309. Reference electrode 40. , resin 505. Teff [7n698. Microdevice special rF'f Applicant National Rehabilitation for the Disabled Hinter Agent jF Physician Hiroshi Kuramochi (1 other person) Figure 1 1-→1h Figure 2 Figure 3 Figure 4 Procedure amendment (method) % formula % 2. Name of the invention Enzyme analysis system and analysis method using the same 3. Supplementary IF -
Relationship with the 11 cases Applicant address: 4-1 Namiki, Sawa City, Saitani Prefecture 151 Name: President of Tonter, Rehabilitation for the Physically Disabled Nao Yama - 4, Flj Address: 101 Chiyo III, Tokyo Ward God III Su III
Town I-chome 2-5, Supplementary W Order No. 11, 1986 2
7131] (Showa 63 2 Jl 23 [(Sent) 6
Subject of amendment (1) Applicant's column in face 11 (Fig. 2 of the drawings)

Claims (2)

[Claims] (1) The working electrode is a biofunctional substance-immobilized microelectrode that has a surface layer of a particulate conductive material produced by integrating a biofunctional substance with fine particles of a conductive substance, and a potential applying means and a generated current. An analysis system comprising a current measuring device for measuring, and measuring a target substance by measuring the generated current. (2) When a constant potential is applied using a biofunctional substance immobilized microelectrode having a surface layer of a microparticle conductive material produced by integrating a biofunctional substance with fine particles of a conductive substance as a working electrode, An analysis method characterized by measuring the generated current value and determining the concentration of the target substance.