JPH02253149A - Enzyme sensor and production thereof - Google Patents

Abstract

PURPOSE:To improve the response speed of the sensor by coating the surface of a conductive base body with an oxygen-reduction catalyst function film and enzyme film. CONSTITUTION:The base body which is a conductive carbon material is coated with an insulating 'Teflon(R)' tube around the base body and an insulating adhesive agent is packed into the spacing to form a capillary electrode. A part of the tip thereof is cut and polished to form the electrode base body 1 of a very small carbon disk type. A Co- TAPP is dropped at 2.4mul (in CH3CN) and a 50mM phosphoric acid buffer soln. contg. GOx and BSA is dropped at 2.0mul onto two pieces of the electrodes 1 by a microsyringe 2 and are mixed by a needle tip. Further, 25wt.% glutaraldehyde is dropped at 1.2mul thereon by a microsyringe 3 and is uniformly mixed by a needle tip 4. The reaction is effected for 15 minutes in atm. The mixture is then brought into reaction in a 10mM phosphoric acid buffer soln. (pH=7.0) for 12hr and the sensor is immersed for 15 minutes into an aq. 10wt.% glycine soln. to remove the unreacted glutaraldehyde. The easy formation of the very small base body is executed in this way and the response speed is extremely speeded up. The measurement with good accuracy is possible.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an enzyme sensor using an oxygen electrode and a method for manufacturing the same, and particularly relates to an enzyme sensor using an oxygen electrode and a method for manufacturing the same.
The present invention relates to an enzyme sensor that measures the concentration of a test substance by detecting a change in a redox response current value proportional to the amount, and a method for manufacturing the same.

[Prior Art] One of the well-known biosensors is an enzyme sensor that measures the glucose concentration in blood by measuring the consumption of oxygen generated by an enzyme reaction.

Conventionally, the detection part of this enzyme sensor was cut with platinum,
The so-called Clark-type oxygen electrode, which uses silver or silver/silver chloride for the anode, encapsulates a standard buffer solution containing chloride as an internal solution, and covers the outside with an oxygen gas selectively permeable membrane, has been widely used.

[Problems to be Solved by the Invention] However, with the above-mentioned Clark type oxygen electrode, it is difficult to miniaturize it, and since it has an internal liquid chamber, it is prone to leakage and contamination problems, and its response speed is low. it was late. Furthermore, although a separate oxygen electrode provides a quick response, the platinum cathode electrode is easily affected by the H''' ion concentration, and therefore in a flow system where the pH concentration changes greatly, the true oxygen partial pressure (PO2 ) It was difficult to distinguish from the concentration.

The present invention has been made in view of such problems, and includes:
Enzyme sensors and sensors that are easy to miniaturize, do not cause contamination problems, are not affected by H ion concentration and oxygen partial pressure, have improved response speed, and are suitable for continuous monitoring measurements in fluid systems. The purpose is to provide a manufacturing method thereof.

[Means for Solving the Problems] In order to solve the above problems, an enzyme sensor according to the present invention includes an electrically conductive substrate and a ligand compound that coats the electrically conductive substrate and has an oxygen reduction catalytic function. It is characterized by comprising an oxygen electrode membrane and an enzyme immobilization membrane covering the oxygen electrode membrane.

According to such a configuration, since it is a solid type, problems such as liquid leakage and contamination unlike conventional sensors do not occur. In addition, it is easy to miniaturize (for example, 0.01 cm2 or less), and H
``It is not easily affected by ion concentration, so it can perform measurements with high accuracy even when used in a fluid system where the pH concentration changes greatly.

The ligand compound having the oxygen reduction catalytic function is as follows:
Those consisting of a cyclic nitrogen-containing compound are preferred, and in particular,
Preferably, they are porphyrin derivatives, phthalocyanine derivatives, cyfram derivatives or phenanthroline derivatives.

Examples of porphyrin derivatives include mesotetra (0
-aminophenyl) cobaltoporphine. Also,
Phthalocyanine derivatives include, for example, tetraminophthalocyanine, and phenanthroline derivatives include diaminophenanthrene.

The transition metal incorporated into the above-mentioned ligand compound may be any metal as long as it has an oxygen reduction catalytic function, and examples thereof include cobalt, iron, nickel, chromium, molybdenum, and ruthenium.

Further, the conductive substrate is preferably formed of a material that is not affected by oxygen concentration or H ion concentration, such as a conductive carbon material.

The enzyme sensor described above is manufactured by coating the surface of a conductive substrate with an oxygen electrode film by applying a solution containing a ligand compound having an oxygen reduction catalytic function, and then coating the surface of the oxygen electrode film with an enzyme. It can be manufactured by coating an enzyme-immobilized membrane by applying an immobilization reagent.

In addition to the above-mentioned coating properties, the oxygen electrode membrane may be manufactured by a dropping method or a resist method, and furthermore, by an electrolytic polymerization method, a vapor deposition method, etc., NH2 groups, OH groups,
Polymerization of C0OH groups, vinyl groups, etc. (pendant type polymers, ladder type bombers, vinyl type polymers, etc.)
It may also be done by doing so.

In addition, the above-mentioned ligand compound having an oxygen reduction catalytic function,
It may also be contained in the same membrane together with the enzyme, in particular insolubilized and immobilized together with the enzyme, and in this case as well, the same effect as the above sensor can be obtained.

In this enzyme sensor, a solution containing a ligand compound having an oxygen reduction catalytic function, a solution containing an enzyme, and an immobilization reagent are sequentially applied or dropped onto the surface of a conductive substrate, and these are mixed and reacted. It can be produced by insolubilizing and immobilizing a ligand compound together with an enzyme.

[Example] Hereinafter, an example of the present invention will be specifically described with reference to the drawings.

(Example 1) Conductive carbon material (BPG: Ba5al pla
A cylindrical (or disk-shaped) base body (diameter: 0.0 mm) made of nepyroLytic carbon (manufactured by UCC).
525 mm), connect the lead wire to one end using conductive adhesive, cover it with an insulating Teflon tube, and fill the gap with insulating adhesive (manufactured by Three Pond,
TB2067) and electrically insulated to prepare a capillary electrode. Next, a part of the tip of this capillary electrode was cut off and polished to produce a micro carbon disk-shaped electrode base l as shown in FIG. 1(a). Prepare two of these electrodes 1, and place the following (A) and (B) on each electrode.
) solution was added dropwise using microsyringe 2 and mixed with the needle tip.

(A) 5mM mesotetra(0-aminophenyl)cobaltporphyrin (Co-TAPP)
2. 4u12 (in CH, CN (solvent)) + 50mM phosphate buffer containing 15% bovine serum albumin (BSA) 2.0μ℃ (B) 5m
M Mesotetra (0-aminophenyl)cobaltoporphyrin (Co-TAPP) 2. 4u, e(
CH3CN (solvent)) + 0, 50 mM phosphate buffer containing 2 mg/mI2 glucose oxidant ffGo 25 with syringe 3
1.2 μC of glutaraldehyde (wt%) was dropped thereon, and these solutions were again mixed uniformly with the needle tip 4 as shown in the figure (C), and allowed to react in the atmosphere for 15 minutes.

Then, 1% in 10mM phosphate buffer (pH=7.0)
The mixture was reacted for 2 hours and further immersed in a 10% by weight aqueous glycine solution for 15 minutes to remove unreacted glutaraldehyde.

Using the enzyme electrode prepared in Example 1, a cyclic portamogram was determined for oxygen concentration in 10 mM phosphate buffer (pH=7.0).

FIG. 2(a) shows the results for electrode A not containing glucose oxidase (GoX), and FIG. 2(b) shows the results for electrode B containing glucose oxidase. As a result, it was revealed that at a constant potential (0.8 V vs. saturated sodium chloride Romero electrode (SSCE)), electrode B containing glucose oxidase had a larger current change than electrode A.

In addition, the current density (A/cm") is 2 for electrode B, 1
2X] 0-3A/cm2. Electrode A is 1.34X
10-''A/cm2, which was 1.58 times larger for electrode B.

Using electrode B containing glucose oxidase and electrode A not containing glucose oxidase in Experimental Example 1, changes in current value with respect to glucose concentration were investigated.

In the experiment, 1 g/i glucose solution was added to 10 mM phosphate buffer (pH 7,00), air bubbling was performed (rate 0.21!/min), and Stark (
Stir with a stirrer) and keep at a constant potential (-0,6V vs. 5SCE)
We investigated the change in current value when . The results are shown in Figure 3(a).
Shown in (b). The same figure (a) is 4 days after preparation, and the same figure (a)
b) shows the results 200 days after production.

As a result, the current value of the electrode 4 days after preparation was approximately 100 to 39.2 when the glucose concentration was 5 to 100 mg/dβ.
9nA, further 20 days after production, about 110-33.3
It has been found that a good enzyme sensor that varies linearly with nA can be obtained.

(Example 2) Mesotetra(0-aminophenyl) of electrode B of Example 1
An enzyme electrode containing glucose oxidase was prepared in the same manner as in Example 1 except that the concentration of cobalt porphyrin was changed to lomM.

Experimental Example 3 The electrode produced in Example 2 was examined for changes in current value with respect to glucose concentration (5-100 mg/dj2) after 20 days in the same manner as in Experimental Example 2. As a result, the current value is 110~10
It was revealed that the value changes linearly in the nA range, and that good enzymes and sensors can be produced. In addition, the current density is 1,62X at a constant potential (-0,8V vs. 5SCE)
It was 10-3A/cm2.

(Example 3) One end of carbon fiber (cross-sectional area 7.85 x 100 m2) was connected to a lead wire using conductive adhesive, and the tip was inserted into a thinly stretched glass capillary, and then an insulating adhesive was applied in the gap. The capillary electrode was filled with a capillary agent and electrically insulated. A portion of the tip of this capillary electrode was cut off and polished to create an ultra-fine disk-shaped carbon fiber electrode.

Next, three-electrode electrolytic polymerization was performed using a saturated sodium chloride Romero electrode (SSCE) as a reference electrode, a platinum wire as a counter electrode, and a carbon fiber electrode as a working electrode. For experiments, use as an electrolyte.

1mM mesotetra(0-aminophenyl)cobaltoporphyrin (Co TAPP)
2. 4uQ (in CH3CN (solvent)) using 0.1 M NaCl204 (in CH3CN), 0-1.8 V (vs. 5 SCE) (7) three times in the potential range;
After sweeping the potential at a potential sweep rate of 50 mV/sec, 1,8
Constant potential electrolysis was performed at V for 10 minutes to form an electrolytic polymer film with a thickness of 0.5 um on the carbon fiber electrode.

Next, 100 mg/mI2. The membrane electrode was immersed in a phosphate buffer (pH=8.00) containing glucose oxidase and 15% by weight bovine serum albumin and dried. After repeating this soaking and drying process about 5 times, 50%
The sample was placed in the steam of a glutaraldehyde (GA) solution for 12 hours to perform a crosslinking reaction to immobilize glucose oxidase.

Thereafter, unreacted glutaraldehyde was removed by washing with a 20% gutsin solution to prepare an enzyme electrode.

Subsequently, 05% poly-hexaethyl methacrylate
(P-HEMA) in a methanol-DMF (dimethylformamide) solution and covered with a protective film having a thickness of about 30 μm.

Experimental Example 4 A 18 g/d 50 mM phosphate buffer containing an f2 glucos solution was dropped onto the enzyme sensor of Example 3, and the response speed at that time was examined in nitrogen, air, and oxygen atmospheres, respectively.

Figure 4 shows the results, and the response speed was several seconds. The potential was measured using a saturated sodium chloride calomel electrode (SSCE).
), and the current value as a function of glucose concentration is -0.6v when the glucose concentration is 2-22mM (36-396g/cH2), as shown in Figure 5.
), the current varied linearly from about -200 to about -10 pA.

(Example 4) To aim at application to so-called flow injection analysis for monitoring the current generated by reacting the active material in the specimen (0□ in the present invention) on the surface of the dual working electrode in the thin layer cell. , Bioanalytical Co., Ltd. (Japan distributor, Bioanalyti) shown in Figures 6 and 7.
cal System Inc, Bee Ninis (
EC-Detector (Model LC-48) manufactured by
l O working electrode (size 30mm x 30mm x 5
mm, in which there is a glassy carbon electrode 1 with a diameter of 3 mm
2 are buried. ) 11 was coated with two layers of cobalt porphyrin electropolymerized thin film under the following conditions. Note that FIG. 7 is an enlarged view of section A in FIG. 6.

The above glassy carbon electrode 12 was used as a working electrode, commercially available silver/
Using a three-electrode cell with a silver chloride electrode as a reference electrode and a platinum winding as a counter electrode, the voltage was 0.0-+1.8V (
(vs. 5SCE), two sweeps were performed at a sweep rate of 50 mV/sec to complete the film deposition.

1 m M mesotetra (0-aminophenyl) cobalt porphyrin (Co-TAPP)
2. 4u, l2 (in CH3CN (solvent)) 0.1 M NaC, 904 (in CHj CN) A polymesotetra(0-aminophenyl)cobaltoporphine film having a film thickness of 01 μm was thus obtained.

(Example 5) Carbon electrode/polymethotetra(0
-aminophenyl) cobalt porphine membrane electrode surface,
The enzyme-immobilized membrane was coated by the following method. That is, 100 mg/n+j2 of glucose oxidase and 15 mg/n+j2 of bovine serum albumin were added in phosphate buffer (pH=8.0).
The membrane electrode of Example 4 was immersed in a solution dissolved in a proportion of % by weight and dried, and this process was repeated about 5 times. Thereafter, a crosslinking reaction was performed with a 50% curcuraldehyde solution to immobilize glucose oxidase, and unreacted substances were subsequently removed by washing with a 20% glycine solution.

Enzyme immobilization with a film thickness of about several + μm was performed in this manner to obtain a carbon electrode/polymesotetra(0-aminophenyl)cobaltoporphine thin film/enzyme-immobilized membrane electrode.

Working electrode in flow injection analyzer (glassy carbon electrode: diameter 3mmφ electrode area 7,065'X
10-2 cm2) of the polymethotetra of Example 5 (0
-aminophenyl) cobalt porphine thin film/enzyme-immobilized membrane electrode, and a silver/silver chloride electrode as the reference electrode, current characteristics were investigated. The experimental conditions were: Current value change during constant potential of 11-0.4V (eye/silver chloride) 2) Carrier solution 50mM phosphate buffer (pH =
7.0) 3) Injection solution With 100 mg/df2 glucose and a constant injection amount of 20 μC, the reproducibility of flow rate changes was examined at three flow rates: 1, 1, 5, and 0.5 mg/min. As a result, it was found that the reproducibility between constant flow rates was excellent and that flow injection analysis could be performed with high accuracy.

In the above embodiments, the conductive base portion is a disk electrode, but the present invention is not limited to this, and may be a minute band-type electrode, an array-type electrode, or a columnar or groove-type electrode. Of course, electrodes may also be used.

In this example, glucose oxidase was explained as an enzyme, but enzymes that use oxygen as an acceptor and produce hydrogen peroxide as a reaction product, such as glucose oxidase, malate oxidase, hexose oxidase,
Cholesterol oxidase, allyl alcohol oxidase, L-gluconolactone oxidase, galactos oxidase, vilanose oxidase, L sorbose oxidase, pyridoxine 4-oxidase, alcohol oxidase, pyruvate oxidase, oxalate oxidase, glyoxylate oxidase, Dihydroorotate oxidase, radosterol oxidase, D-alcohol oxidase, D-amino acid oxidase, L-amino acid oxidase, amine oxidase, D-glutamate oxidase, L-glutamate oxidase, ethanolamine oxidase,
Tyramine oxyguse, putrescine oxidase, cyclohexylamine oxidase, L-lysine α-oxidase, N-methylamino acid oxidase, N6-methylnosine oxidase, 6-hydroxy-D-nicotine oxidase, dimethylglycine oxidase, nitroethane oxidase It is clear that the same can be said for sulfide oxidase.

[Effects of the Invention] As explained above, the enzyme sensor according to the present invention has an oxygen reduction catalytic function membrane and an enzyme membrane coated on a conductive substrate such as conductive carbon, so that it is different from conventional sensors. In comparison, problems such as contamination do not occur, and it is easy to manufacture and miniaturize. Therefore, the response speed is extremely fast, and it is not affected by the concentration of H+ ions, etc., making it possible to accurately measure even in a fluid system.

In addition, when a conductive substrate is coated with a film containing a mixture of an oxygen reduction catalyst substance and an enzyme, it is easier to manufacture and ultra-miniaturize it, and the mixing ratio of the catalyst substance and the enzyme is By changing , substrate permeation control, response speed, etc. can be easily controlled.

Furthermore, according to the method for manufacturing an enzyme sensor according to the present invention,
The enzyme sensor described above can be easily manufactured.

[Brief explanation of drawings]

FIGS. 1(a) to (C) are perspective views showing a method for manufacturing an enzyme sensor according to Example 1 of the present invention, FIG. 2(a), and FIG.
, (b) are diagrams showing cyclic portamograms of the sensor manufactured by the method shown in Figure 1. Figure (a) is a characteristic diagram for an electrode that does not contain glucose oxidase, and Figure (b) is a characteristic diagram for an electrode that does not contain glucose oxidase. Figures 3 (a) and 3 (b), which are characteristic diagrams of electrode B, respectively show changes in current value with respect to glucose concentration of electrode B.
The characteristic diagram of Sun and Moon, the same figure (b) is the characteristic diagram 20 days after production,
FIG. 4 is a diagram showing the response characteristics to glucose of the enzyme sensor of Example 3, FIG. 5 is a diagram showing the relationship between glucose concentration and current value based on the characteristic values in FIG. 4, and FIG. 6 is a diagram showing the example. FIG. 7 is a cross-sectional view showing a schematic configuration of the apparatus used in FIG. 4, and FIG. 7 is an enlarged cross-sectional view showing the main part of FIG.

Claims (8)

[Claims] (1) A conductive substrate, an oxygen electrode membrane covering the conductive substrate and containing a ligand compound having an oxygen reduction catalytic function, and an enzyme immobilization membrane covering the oxygen electrode membrane. Features of the enzyme sensor. (2) An enzyme sensor comprising an electrically conductive substrate, and a solid coating film that covers the electrically conductive substrate and contains a ligand compound having an oxygen reduction catalytic function and an enzyme, respectively. (3) The enzyme sensor according to claim 1 or 2, wherein the ligand compound comprises a cyclic nitrogen-containing compound. (4) The enzyme sensor according to claim 3, wherein the ligand compound is a porphyrin derivative, a phthalocyanine derivative, or a phenanthroline derivative. (5) The ligand compound having an oxygen reduction catalytic function is
The enzyme sensor according to claim 2, wherein the enzyme sensor is insolubilized and immobilized together with the enzyme. (6) A step of coating the surface of the conductive substrate with an oxygen electrode film by applying a solution containing a ligand compound having an oxygen reduction catalytic function, and an immobilized reagent containing an enzyme on the surface of the oxygen electrode film. A method for producing an enzyme sensor, comprising the step of coating an enzyme-immobilized membrane by coating. (7) An enzyme characterized by comprising a step of forming a solid coating film by insolubilizing and immobilizing a ligand compound having an oxygen reduction catalytic function together with an enzyme using an immobilization reagent on the surface of a conductive substrate. How to manufacture the sensor. (8) A step of coating the surface of the conductive substrate with an oxygen electrode membrane by polymerizing a ligand compound having an oxygen reduction catalytic function, and a step of coating the surface of the oxygen electrode membrane with an enzyme-immobilized membrane. A method for manufacturing an enzyme sensor, comprising: