JPH0321857A - Enzyme sensor and production thereof - Google Patents

Abstract

PURPOSE:To easily, rapidly and accurately measure the substrate concn. of glucose, etc., by measuring an enzyme reaction from the change in the electron transfer reaction by an electron transfer medium effected between the enzyme in a monomolecular film and a conductive base body. CONSTITUTION:An LB (Langmuir-Blodgett) film 3 by an LB technique is formed as the monomolecular film immobilized with the electron transfer medium together with the enzyme on the surface of the conductive base body 4 produced by depositing an iridium oxide film 2a on a glass plate 1 and further a thin film 2b of platinum thereon. The electron transfer is effected in such a manner between the enzyme and the conductive base body so as to cause the indirect oxidation reduction of the enzyme. The enzyme reaction is directly electrochemically detected and the substrate concn. is measured by the increased current value on the conductive base body. The response of the enzyme reaction is accurately measured in this way even if the substrate concn. is low. The response characteristic is thus improved. The construction is simplified and the measurement is facilitated. Further, the time for the measurement is shortened.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to an enzyme sensor that measures substrate concentration by an amperometric method using an enzyme electrode. The present invention relates to an enzyme sensor for direct measurement using a redox reaction, and a method for manufacturing the same.

[Prior Art] Enzyme sensors are mainly used for clinical chemical analysis, and have been put into practical use for measuring glucose, urea, neutrals, phospholipids, and the like. For example, the enzymatic reaction when the measurement target is β1D-glucose is expressed by the following equation.

? (1) That is, β-D-glucose consumes oxygen (0) and converts it into organic acid (gluconolactone) and hydrogen peroxide (H
202). Therefore, the glucose concentration can be measured from the amount of hydrogen peroxide or gluconolactone generated or the amount of oxygen consumed.

By the way, conventionally, when measuring glucose concentration based on the amount of hydrogen peroxide generated, the generated hydrogen peroxide is oxidized with a metal electrode and the oxidation current is measured, or the hydrogen peroxide is reduced and the reduction current is measured. The method was used.

However, these redox currents are easily affected by oxygen, and conventional detection electrodes are also easily affected by changes in their surface conditions. In addition, the measurement principle based on electrochemical methods uses a sensor configuration consisting of an electrode substrate/liquid/enzyme-immobilized membrane/detection liquid, and the liquid existing between the electrode and membrane makes it difficult to miniaturize the sensor. .

Furthermore, other conventional methods for measuring glucose concentration include (1) decomposing hydrogen peroxide into oxygen and water using catalase and measuring the amount of oxygen, or (2) using an enzyme (peroxidase) or an inorganic catalyst ( There was a method of measuring the amount of iodine by oxidizing iodide ions in the presence of molybdenum, etc. and performing the following reaction, and thereby indirectly measuring the amount of hydrogen peroxide.

2H2 0 ... (2) [Problem to be solved by the invention] As mentioned above, conventionally, when measuring the glucose concentration based on the amount of hydrogen peroxide generated, the amount of oxygen consumed or the amount of iodine generated was measured. However, the amount of hydrogen peroxide generated was indirectly determined by the amount.

However, this two-step reaction method requires an enzyme electrode for decomposing glucose, etc. into hydrogen peroxide, as well as a separate electrode for measuring oxygen or iodine, making the measurement difficult. There were problems in that it was extremely complicated and required a long measurement time. On the other hand, in the conventional electrochemical method, since the sensor contains an internal liquid as described above, there are problems in that it is difficult to contaminate the measuring liquid and to miniaturize it.

The present invention has been made in view of such problems, and includes:
It is an object of the present invention to provide an enzyme sensor that can easily and accurately measure the concentration of a substrate such as glucose in a short time, without fear of contamination, and that can be miniaturized, and a method for manufacturing the same.

[Means for Solving the Problems] In order to solve the above-mentioned conventional problems, an enzyme sensor according to the present invention includes an electrically conductive substrate, at least a portion of which is coated, and an electron transfer medium and an enzyme. The invention is characterized by comprising a monomolecular film containing.

In other words, this enzyme sensor causes an electron transfer reaction (oxidation-reduction reaction) to occur between an enzyme and a conductive substrate in a single molecule using an electron transfer medium, and measures the enzyme reaction from changes in this electron transfer reaction. This electron transfer reaction is
It is detected as a change in current when the active potential of the electron transfer medium is constant.

It is preferable that the electron transfer medium and the enzyme are directly bonded to each other and immobilized in a single molecule, and the electron transfer medium is a compound that has a redox center and oxidizes the active center of the enzyme. Alternatively, compounds with a redox potential that can be reduced are preferable, and in particular, when the enzyme is glucose oxidase, specific examples include ferrocene derivatives,
In particular, ferrocenecarboxylic acid is preferred.

In addition, as compounds having redox centers, quinone compounds, triphosphoviridine nucleotide compounds, and flavin adenine nucleotide compounds can be used.

As a monomolecular film for immobilizing electron transfer media and enzymes, LB (Langmuir Blood
tt, Langmu? - LB film produced by the Projet method is preferred. According to this LB method, it is possible to easily form a thin film on a conductive substrate in which the orientation of each molecule is aligned and the thickness is equal to the length of a single molecule. Therefore, the reaction occurring on the membrane surface is easily transmitted onto the conductive substrate, that is, a highly sensitive sensor can be obtained. Furthermore, since the thickness of each layer of the LB film has high orderliness, a stable cumulative film can be formed, and the cumulative number, that is, the film thickness or the amount of enzyme in the film can be easily controlled.

Next, the method for producing an enzyme sensor according to the present invention includes the steps of preparing a conductive substrate, spreading a monomolecular film substance on the surface of an enzyme solution containing an electron transfer medium, and holding a certain amount of
A step of applying extra-dimensional pressure to form a monomolecular film with uniform orientation of each molecule, and immersing the conductive substrate in an enzyme solution in which a monomolecular film is formed on the surface, and electrons are formed on the conductive substrate. The method is characterized in that it includes a step of forming a monomolecular film containing a transfer medium and an enzyme.

A fatty acid such as stearic acid CCH3 (cHz)18cOOH) is used to form the monomolecular film,
When this stearic acid is dropped onto one water surface partitioned by a thin Teflon (registered trademark) plate, a monomolecular film with poor coordination properties is formed. In this state, if you drop oleic acid as piston oil onto the other water surface,
Due to the diffusion force of this oleic acid, pressure is applied to the Teflon thin plate, and the Teflon thin plate moves to one side of the water surface until the pressure on both sides reaches equilibrium.As a result, two-dimensional external pressure is applied to the monomolecular film of stearic acid, and Improves distribution. By pouring this monomolecular film with uniform conformation onto the surface of an enzyme solution containing an electron transfer medium placed in an enzyme pot, the electron transfer medium and the enzyme will be placed on the parent group side of each molecule of the monolayer. is adsorbed. Therefore, when a conductive substrate is immersed in this solution and pulled up, a monomolecular film containing an electron transfer medium and an enzyme is deposited on the conductive substrate.

The conductive substrate serves as an electrode, and for example, a metal film such as iridium oxide (I, OK) or platinum thin film, which serves as an electrode, is formed on a glass substrate or a transparent conductive glass substrate (ITO) by sputtering or the like. However, the entire base may be made of a conductive member such as metal.

Moreover, this conductive substrate is a field effect transistor (FE).
In the case of an enzyme sensor using T), if it is formed in the gate portion or an extension of the gate portion, it is possible to downsize the entire sensor including the output portion.

[Function] In the enzyme sensor according to the present invention, since the electron transfer medium and the enzyme are immobilized in the monolayer, the basic principle thereof is as follows. That is, when glucose oxidase (COD) is used as the enzyme, by using a suitable electron transfer medium, the flavin adenine nucleotide (FA D H 2
) can be reoxidized to its oxidized form (FAD), thereby indirectly redoxing the enzyme. By utilizing this phenomenon, electron transfer occurs between the enzyme and the conductive substrate (electrode), and the enzymatic reaction can be directly detected electrochemically. That is, when a Huekouthen derivative is used as an electron transfer medium, electron transfer occurs from the flavin moiety of glucose oxidase to the conductive substrate (electrode).

Specifically, when ferrocene carboxylic acid (FCA), for example, is used as a ferrocene derivative, a reaction as shown in the following formula proceeds.

GOD-FAD +Guru] - GOD-FADH.
10 gluconolactone 2 FC A ” + G O D − F A D
Hz-2FCA+GOD-FAD+2H"2FCA-2FCA"+2eFigure 1 shows the state of electron movement due to the above reaction. That is, such electron transfer causes an increase in current in the conductive substrate (electrode), and the glucose concentration is measured based on this increased current value.

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

(Example 1) First, as shown in FIGS. 2 and 3, a glass plate (15 mm×5 mm×1.2 mm
+m)l was ultrasonically cleaned with a mixed solution of acetone and methanol, washed with water, and then dried. Subsequently, using a high frequency sputtering device (SPF-2 1 0H type, manufactured by Nichiden Anelva Co., Ltd.), a film thickness of 0. A 1 μm iridium oxide film 2a was deposited on the glass plate 1. Furthermore, a film thickness of approximately 0.0000000000000 is applied on this iridium oxide film 2a. A conductive substrate (electrode) was prepared by depositing a 1 μm thick platinum thin film 2b.

Next, an LB film (monomolecular film) 3 on which an electron transfer medium was immobilized together with an enzyme was formed on the surface of the conductive substrate 4 using the LB film manufacturing apparatus shown in FIGS. 4 to 7. 8th
The figure schematically shows the state of formation of this LB film.

That is, first, the enzyme bot 11 is placed deep in a water tank 10 whose entire surface is coated with Teflon, and a lower layer containing barium chloride (BaCl) and potassium hydrogen carbonate (KHCO) in pure water is placed outside of the bot 11. liquid l2
At the same time, an enzyme solution containing an electron transfer medium is placed inside the bot 11. The inside and outside of this enzyme bot 11 are connected only at the notch 11a, so that leakage of the internal enzyme is suppressed to some extent. The barium chloride added to the lower layer liquid 12 is used to form a stable film by combining with stearic acid, which will be described later, and the potassium hydrogen carbonate is used to adjust the pH of the aqueous phase.

On the other hand, a partition plate l3 made of Teflon is floated on the water surface of the water tank IO, and partitions the water surface into two areas A and B.

Next, a solution of stearic acid as a monomolecular film substance is dropped onto the water surface A on the enzyme pot 11 side to develop a monomolecular film on the water surface A, and a small amount of stearic acid is dropped onto the other water surface B. The two partition plates l3 are pushed until an equilibrium state is reached between the water surfaces A and B due to the diffusion of oleic acid. As a result, a two-dimensional external pressure is applied to the single molecule of stearic acid, and the coordinating property between the molecules is improved. At this time, enzyme bot l1
Since the inside is connected to the outside through the notch 11a, a monomolecular film l4 with good distribution is formed on the inside surface as well. The parent tree side of this monomolecular film l4 adsorbs the enzyme-ferrocene bonding substance l5 inside the enzyme pot ti.

In this state, the conductive substrate 4 is lowered into the enzyme bot 11 and gradually pulled up, so that a monomolecular film with good coordination is formed on the surface of the platinum thin film 2a.

This operation was repeated to accumulate (1 to 7 layers) of films adsorbing the enzyme-ferrocene binding substance 15 on the conductive substrate 4, thereby forming the LB film 3 and producing a glucose sensor.

The above enzyme solution was prepared as follows.

Glucose oxidase 400 mg Ferrocenecarboxylic acid 40 mg Cyanamide (binder)
2 mg urea (binder) 3603
．． 6 mg in phosphate buffer (pH = 6.86) l
Dissolve in OmjZ, put this solution into a cellophane paper bag, and let it soak in water for 1 to 2 days. As a result, urea is dialyzed, and an enzyme-ferrocene binding substance 15 can be produced.

In addition, in Fig. 4, 16 is the LB membrane collector, and 5th
In the figure, 17 indicates a surface pressure gauge, and the arrow indicates a constant temperature water circulation path.

(Comparative Example 1) A glucose sensor was produced in the same manner as in Example 1 except that ferrocenecarboxylic acid was not added to the enzyme solution.

The sensor 23 prepared in Example 1 and Comparative Example 1 is immersed as a working electrode in the solution 20 to be measured of the current measurement system shown in FIG.
The output current of the sensor 23 was measured using a reference electrode (sodium chloride Calomero electrode) 21 and a counter electrode (platinum (Pt)) 22. That is, while the electrode potential is always kept constant (0.3 to 0.6 V) using a botensiostat (potential constant device) 24, the specific circuit configuration of which is shown in FIG. The current (I) is manually inputted to the voltage (V) converter 25 through the converter 25, where it is converted to voltage, and further converted into a digital signal by the analog (A) to digital (D) converter 26.
7 was entered. Note that during the measurement, the magnetic stirrer 28 rotated the stirring bar 29 to stir the solution 20 to be measured.

The output current value at each potential between the sensor 23 and the reference electrode 21 is shown in FIG.

As a result, in the sensor of Example 1, the largest output current (approximately 5.0 μA) was obtained at 0.4V. on the other hand,
In the case of the sensor of Comparative Example 1 to which ferrocenecarboxylic acid was not added, 0. The output current gradually increased up to 6V, but even at 0.6V, the output current was about 1.0 μA.

Therefore, it was found that the effect of adding ferrocenecarboxylic acid as an electron transfer medium is large.

(Example 2) A glucose sensor was produced in the same manner as in Example 1 except that the thickness of the LB film 3 of the sensor in Example 1 was changed to five layers.

X Cunning l Using the apparatus shown in Figure 9, the glucose concentration lO,50,1
Changes in output current were measured at 00, 200, and 500 mg/dI2. The results are shown in FIG. According to this, the change in the output current with respect to the glucose concentration is linear at a glucose concentration of 50 mg/dI2 or less, but shows a curve that converges at a concentration higher than this.

In addition, after removing oxygen by bubbling nitrogen gas into the measurement cell shown in FIG.
Approximately 80% or more of the current value shown in Figure 2 flows, and similarly 50f
Good linearity was shown below fig/d12. This indicates that the enzyme sensor of the present invention can be used even in anoxic conditions.

Figure 13 shows the reproducibility experiment results of the above sensor.
As a result, it was found that the second measurement value was considerably lower than the first measurement value, but the values were almost the same from the second measurement onward, indicating that the reproducibility was relatively excellent.

In this way, in the glucose sensor of this example, by adding an electron transfer medium to the LB membrane 3 as an enzyme-immobilized membrane, the glucose concentration can be adjusted to a relatively low concentration of LO.
It was possible to measure up to approximately g/df2, and the sensitivity was high. The main reason for this is that while the S/N ratio in the potential measurement method is around 2, the S/N ratio in the current method is significantly improved to 30, and the signal and noise are separated. it is conceivable that. FIG. 14(a) shows an example of the sensor output in the potential measurement system, and FIG. 14(b) shows an example of the sensor output in the current measurement system.

In the above example, the enzyme sensor of the present invention was used as a glucose sensor to measure the concentration of glucose, but the present invention is not limited to this, and other enzymes may be used to measure the concentration of other substrates. Of course, the present invention can also be applied to sensors for measurement.

[Effects of the Invention] As explained above, according to the enzyme sensor according to the present invention,
An electron transfer reaction (oxidation-reduction reaction) using an electron transfer medium is performed between an enzyme and a conductive substrate (electrode) in a monomolecular film with uniform conformation, and the enzyme reaction is measured from changes in this electron transfer reaction. As a result, the response of the enzyme reaction when the substrate concentration is low, which was hidden by noise in the conventional potential response, can be accurately measured, and the response characteristics are significantly improved. In addition, the structure is simplified, measurement is extremely easy, and measurement time can also be shortened. Furthermore, since the LB method is used to form the enzyme-immobilized membrane, it is possible to coat the electrode substrate with the membrane even if the size is microscopic.Furthermore, the electrode has a solid electrode structure, so there is no need to use the internal liquid Since no chamber is required, there are no problems such as contamination of the measuring solution. It also becomes possible to create ultra-small electrodes,
In particular, it has the effect of greatly increasing its utility as a sensor in the medical field.

[Brief explanation of the drawing]

The drawings show one embodiment of the present invention. FIG. 1 is a schematic diagram showing the electron transfer state in an enzyme sensor, FIG. 2 is a plan view showing the structure of the enzyme sensor, and FIG. 3 is a diagram showing the structure of the enzyme sensor. 4 is a plan view of the LB film production apparatus, and 5 is a cross-sectional view taken along line 1.
The figure is also a side view, Figure 6 is a front view of the enzyme pot, and Figure 7 is a front view of the enzyme pot.
The figure is also a side view, Figure 8 is a schematic diagram showing the state of formation of the LB film, Figure 9 is a configuration diagram of the current measuring device, Figure 10 is a circuit configuration diagram of the boteshitastat, and Figure 11 is a sensor standard electrode. Figure 12 is a diagram showing the relationship between glucose concentration and output current, Figure 13 is a diagram showing the reproducibility characteristics of the sensor, and Figure 14 (a) is the potential measurement system. FIG. 14(b) is a waveform chart showing the sensor output in the current measurement system. l...Glass plate, 2a...Iridium oxide film 2b.
...Platinum thin film, 3...LB film 4...Conductive substrate, 11...Enzyme pot l2...
Lower layer liquid, l4...monolayer l5...enzyme-ferrocene binding substance

Claims (5)

[Claims] (1) An enzyme sensor comprising an electrically conductive substrate and a monomolecular film covering at least a portion of the electrically conductive substrate and containing an electron transfer medium and an enzyme. (2) The enzyme sensor according to claim 1, wherein the electron transfer medium is directly bonded to an enzyme. (3) The enzyme sensor according to claim 1 or 2, wherein the electron transfer medium is a compound having a redox center. (4) The enzyme sensor according to any one of claims 1 to 3, wherein the compound having a redox center is a ferrocene derivative. (5) A manufacturing method for manufacturing the enzyme sensor according to claim 1, which includes the step of manufacturing a conductive substrate, spreading a monomolecular film substance on the surface of the enzyme solution containing an electron transfer medium, and A step of applying two-dimensional external pressure to form a monomolecular film with uniform orientation of each molecule, and immersing the conductive substrate in an enzyme solution in which a monomolecular film is formed on the surface of the conductive substrate. A method for producing an enzyme sensor, comprising the step of forming a monolayer containing an electron transfer medium and an enzyme.