JP3631740B6 - Biosensor enzyme electrode and method for producing the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an enzyme electrode of a biosensor, and more particularly to an enzyme electrode of a biosensor that electrochemically measures a test substance concentration in a sample solution by an enzyme reaction between an oxidoreductase and an electron acceptor. The present invention also relates to a manufacturing method thereof.
[0002]
[Prior art]
In recent years, there is a need for an inexpensive and disposable multi-item / simultaneous measurement blood analyzer for blood tests that can be easily performed at home.
[0003]
As a conventional simple test apparatus for the purpose of blood component test, there is a chip-shaped multi-item measurement apparatus. In this method, an ion-selective field effect transistor (ISFET) or the like is incorporated as a sensor on a plastic substrate with a single flow path as a semiconductor electrode so that only a target substance can be measured electrochemically. However, the blood analyzer used for such multi-item measurement requires a large amount of blood necessary for measurement, and the burden on blood collection on the subject is large. Moreover, since the semiconductor electrode is very expensive, the device price is also increased. Furthermore, since the flow path is single, a plurality of sensors having mutual interference effects cannot be mounted in the same device. Against this background, the conventional multi-measurement blood analyzers are often used for examinations in hospitals and the like, and the number of measurement items is limited at present.
[0004]
As an example of a home simple blood analyzer, a blood glucose (glucose) value measuring device can be mentioned. Many commercially available blood glucose level measuring devices enable measurement from a very small amount of whole blood of several μL or less. A so-called electronic mediator type sensor is used as a biosensor of such a measuring instrument (see, for example, Patent Document 1).
[0005]
[Patent Document 1]
Japanese Laid-Open Patent Publication No. 3-202864
In this biosensor, an enzyme reaction layer comprising an oxidoreductase, a hydrophilic polymer and an electron acceptor is provided on the surface of an electrode system on a substrate. When a sample solution such as blood is dropped on the enzyme reaction layer, the substrate in the sample reacts with the oxidoreductase, and the electron acceptor is reduced. After completion of the reaction, a voltage is applied to the electrode system to oxidize the reduced form of the electron acceptor, and the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.
[0007]
In the case of a glucose measuring sensor, glucose oxidase, which is a complex protein with FAD, is used as an oxidoreductase, and ferrocene, for example, is used as an electron acceptor. As shown in FIG. 3, a reaction system using an electron acceptor as a mediator enables measurement of glucose concentration using a trace amount of blood without depending on the oxygen partial pressure in blood. Further, since the electrode system on the substrate can be formed by screen printing using inexpensive carbon, a biosensor can be manufactured at a very low cost.
[0008]
A conventional electron mediator-type enzyme electrode contains an oxidoreductase and an electron acceptor in a hydrophilic polymer medium to perform an enzyme reaction, thereby forming an enzyme-immobilized film. Since the enzyme itself has a large molecular weight, it is relatively easy to hold it in a hydrophilic polymer medium. However, when the hydrophilic polymer itself is a material that is easily eluted by an aqueous sample, the retained enzyme and electron acceptor are eluted together with the hydrophilic polymer or with the swelling of the hydrophilic polymer. Become. In order to prevent such elution of the enzyme, a technique for immobilizing the enzyme by a polyion complex method has also been proposed (see, for example, Patent Document 2).
[0009]
[Patent Document 2]
Japanese Patent Laid-Open No. 8-178886
In this polyion complex method, a polycation aqueous solution, an enzyme solution, and a polyanion aqueous solution are dropped onto a substrate electrode, mixed, and dried to form a polyion complex matrix. Enzyme molecules are firmly incorporated into this matrix structure. I can do it. Due to the dense matrix structure, elution of macromolecules such as enzymes can be prevented almost completely. Further, the polyion complex itself does not dissolve.
[0011]
Thus, elution of the oxidoreductase can be prevented, but the low molecular weight electron acceptor easily flows out of the matrix structure even if it is held in the polyion complex membrane. The measurement current value of this enzyme electrode depends not only on the amount of immobilized enzyme and enzyme activity, but also on the amount of electron acceptor in the enzyme-immobilized membrane, so it is measured when the electron acceptor flows out of the enzyme-immobilized membrane. The value is low. That is, there is a problem that the measured value (output current value) gradually decreases due to repeated use.
[0012]
In particular, since the enzyme electrode does not always have the same performance, it is necessary to calibrate the enzyme electrode using a calibration solution containing a test substance having a known concentration. At this time, if the electron acceptor in the enzyme-immobilized membrane is eluted by the calibration solution, the meaning of calibration is lost. In addition, when multiple biosensors are installed in the same channel to analyze multiple items of test substances, if the electron acceptor elutes from the enzyme electrode of each adjacent sensor, an electron using the same electron acceptor is used. The output of other sensors of the mediator type will be affected.
[0013]
If the enzyme-immobilized membrane itself is made of a hydrophobic polymer medium, such elution of the electron acceptor can be prevented, but in this case, the enzyme reaction itself is performed in a non-aqueous environment. As a result, the detection sensitivity itself is lowered due to an extremely slow reaction.
[0014]
OBJECT OF THE INVENTION
The present invention has been made in view of such circumstances, and provides an enzyme electrode of a biosensor that enables highly sensitive measurement of a test substance concentration without eluting an electron acceptor into an aqueous sample. Is the first purpose. Moreover, it aims at providing the manufacturing method of the enzyme electrode of this biosensor.
[0015]
According to the present invention, a first object is to provide an enzyme of a biosensor that electrochemically measures the concentration of a test substance in a sample solution by an enzyme reaction between an oxidoreductase using the test substance as a substrate and an electron acceptor. In the electrode:
An insulating substrate;
Electrodes provided on the substrate;
A porous charge transfer medium layer provided on the electrode and containing an electron acceptor dispersed in a hydrophobic polymer medium;
An enzyme electrode of a biosensor comprising an enzyme reaction layer provided on the charge transfer medium layer and having an oxidoreductase immobilized in a hydrophilic polymer medium;
Is achieved.
[0016]
That is, in the enzyme electrode of the present invention, the enzyme-immobilized membrane is divided into an enzyme reaction layer and an electrolysis transfer medium layer, and the oxidoreductase is immobilized and held in the hydrophilic polymer medium layer. On the other hand, the electron acceptor which is a low molecular weight substance is held in a hydrophobic polymer medium to form a charge transfer medium layer. Electron acceptors in a hydrophobic environment do not easily elute into aqueous samples. On the other hand, since the charge transfer medium layer is a porous layer, it can easily receive electrons generated by the oxidation-reduction reaction in the enzyme reaction layer. The received electrons are finally transferred to the electrodes by the Red-Ox conversion of the electron acceptor and output current (see FIG. 3).
[0017]
If a counter electrode is provided on the substrate in addition to the enzyme electrode, an electrode-type biosensor using the enzyme electrode as a working electrode can be configured. Moreover, you may provide a reference electrode on a board | substrate as needed.
[0018]
As the hydrophobic polymer constituting the charge transfer medium layer, polyvinyl butyral, polyvinyl chloride, polyvinyl formal, epoxy polymer, and the like can be used, and those that can be firmly bonded to the lower electrode are preferable.
[0019]
As a hydrophilic polymer constituting the enzyme reaction layer, it is preferable to use a polyion complex composed of a polycation polymer and a polyanion polymer. The oxidoreductase can be included and immobilized in this polyion complex to prevent elution into the aqueous sample.
[0020]
The enzyme reaction layer may be coated with a biocompatible (blood compatible) polymer material to prevent nonspecific adsorption of blood proteins and blood cell components (particularly platelets). it can. As such a polymer material, 2-methacryloyloxyethyl phosphorylcholine polymer is preferably used.
[0021]
A second object of the present invention is a porous charge transfer medium comprising an insulating substrate, an electrode provided on the substrate, and an electron acceptor provided on the electrode and dispersed in a hydrophobic polymer medium. And an enzyme reaction layer in which a oxidoreductase is immobilized in a hydrophilic polymer medium provided on the charge transfer medium layer, and the test substance concentration in the sample solution is determined using the test substance as a substrate. In a method for producing an enzyme electrode of a biosensor that electrochemically measures an oxidoreductase, the electron acceptor and an enzyme reaction:
A biosensor characterized in that the porous charge transfer medium layer is provided by applying and drying a hydrophobic polymer solution in which the hydrophobic polymer and the electron acceptor are dissolved in an organic solvent on the electrode. This can be achieved by the electrode manufacturing method.
[0022]
That is, in the production method of the present invention, a porous charge transfer medium layer made of a hydrophobic polymer is formed on an electrode by a very simple process of evaporating and removing an organic solvent. The enzyme reaction layer can be provided by comprehensively immobilizing oxidoreductases using electrostatic interaction in a polyion complex comprising a polycation polymer and a polyanion polymer.
[0023]
Embodiment
FIG. 1 is a schematic cross-sectional view of a biosensor according to an embodiment of the present invention. Reference numeral 10 denotes an insulating substrate made of a resin such as polyester, PET (polyethylene terephthalate), or polycarbonate (PC), and a base lead electrode 12 is formed thereon. On the lead electrode 12, electrodes 14 and 16 formed of a conductive carbon paste or the like are provided. One electrode 14 is used as a working electrode, and the other electrode 16 is a counter electrode. A charge transfer medium layer 20 and an enzyme reaction layer 22 are provided on the working electrode 14. Reference numeral 18 denotes an insulating layer that insulates between the electrodes 14 and 16.
[0024]
The charge transfer medium layer 20 is a porous hydrophobic polymer medium layer and contains an electron acceptor that undergoes an oxidation-reduction reaction coupled with an enzyme reaction of an oxidation-reduction enzyme. As the electron acceptor, quinone derivatives such as p-benzoquinone, ferrocene derivatives such as ferrocene and dimethylferrocene, and viologens can be used.
[0025]
As the hydrophobic polymer, polyvinyl butyral, polyvinyl chloride, polyvinyl formal, epoxy polymer, and the like can be used, and those capable of firmly bonding to the lower electrodes 14 and 16 are preferable. This hydrophobic polymer is dissolved in an organic solvent (dichloromethane, tetrahydrofuran, acetone, isopropyl alcohol, ethanol, etc.) together with an electron acceptor, and this mixed solution is uniformly dropped on the electrode (working electrode) 14 and dried. Thus, a porous charge medium moving layer can be formed. An electron acceptor such as ferrocene can be uniformly and densely contained in the formed hydrophobic medium layer.
[0026]
The enzyme reaction layer 22 is a hydrophilic polymer medium layer and contains an oxidoreductase enzyme corresponding to the test substance. For example, glucose oxidase (GOD) is used when measuring the glucose concentration. When the test substance is alcohol, cholesterol, lactic acid or the like, an oxidoreductase such as alcohol oxidase, cholesterol oxidase, or lactose oxidase can be used. These oxidoreductases contain electron acceptors such as FAD and NAD as coenzymes and prosthetic groups.
[0027]
As the hydrophilic polymer constituting the enzyme reaction layer 22, a polyion complex composed of a polycation polymer and a polyanion polymer can be used. When a polycation aqueous solution, an enzyme solution, and a polyanion aqueous solution are successively dropped and mixed on the charge transfer medium layer 20 and dried, an enzyme-containing polyion complex film that takes in enzyme molecules present in the vicinity at the time of forming the polyion complex is formed. . The enzyme is firmly immobilized in the matrix of the complex by the electrostatic interaction of the polycation and polyanion. The polycation polymer is a hydrophilic polymer having a cationic group in a repeating unit, and examples thereof include poly L lysine, chitosan, chitin, and cellulose. The polyanion polymer is a hydrophilic polymer having an anion group in the repeating unit, and examples thereof include polystyrene sulfonate, polyglutamic acid, and polyacrylic acid.
[0028]
FIG. 2 shows a cross-sectional view of a blood analyzer covered with an upper substrate 24 in which a channel structure is formed on the produced biosensor. By introducing a blood sample into the channel 26, the concentration of the test substance in the blood is electrochemically analyzed.
[0029]
Here, if the inner wall of the flow path 26, the surface of the enzyme reaction layer 22 and the counter electrode 16 are coated with a polymer material having biocompatibility (blood compatibility), blood cell components such as platelets are adsorbed and aggregated. In addition to preventing, nonspecific adsorption of plasma / serum proteins can be prevented. As such a polymer material, there is a phospholipid polymer, and 2-methacryloyloxyethyl phosphorylcholine polymer (MPC polymer) is particularly preferable.
[0030]
[Example 1]
A biosensor was produced as follows. The base lead electrode 12 was printed on the polyester resin substrate 10 by a screen printing technique and then dried by heating. On the lead electrode 12, a conductive carbon paste was printed by a screen printing technique to provide an electrode system of a working electrode 14 and a counter electrode 16. An insulating film 18 in which an opening having a diameter of 400 μm was provided in advance at the position of the working electrode 14 and the counter electrode 16 by lithography was superimposed on the substrate 10 and thermocompression bonded.
[0031]
Polyvinyl butyral and ferrocene are dissolved in isopropyl alcohol so as to be 1% (W / W) and 2% (W / W), respectively, and 0.05 μL of this mixed solution is uniformly dropped into the opening on the working electrode 14. The charge transfer medium layer 20 was provided by drying. Next, a 25 mM polystyrene sulfonate aqueous solution, a glucose oxidase (GOD: 1500 unit / mL in PBS) solution, and a 20 mM poly-L lysine aqueous solution are sequentially dropped onto the charge transfer medium layer 20 and dried, and then the enzyme reaction layer 22 is dried. A biosensor for glucose analysis was prepared.
[0032]
As a comparative example, when preparing the charge transfer medium layer, only ferrocene was dissolved in isopropyl alcohol without using polyvinyl butyral, and 0.05 μL of this solution was uniformly dropped into the opening on the working electrode 14 and dried to obtain a ferrocene layer. Was established. On top of this, an enzyme reaction layer 22 was provided in the same manner as in the example. In the comparative example in which the ferrocene layer was produced with a solution in which only ferrocene was dissolved, ferrocene was not uniformly deposited on the carbon electrode 14, and partial peeling was observed. The same was true when ferrocene was dissolved in another organic solvent such as tetrohydrofuran or dichloromethane. On the other hand, in the charge transfer medium layer 20 of the example in which ferrocene is included by a porous polymer having high adhesion and porous properties, a high-density and uniform ferrocene-containing polymer layer was obtained.
[0033]
1 to 3 μL of a D-glucose standard solution is dropped as a sample solution so as to cover both the enzyme reaction layer 22 and the counter electrode 16, and the ferrocene is applied to the working electrode 14 with reference to the counter electrode 16. A constant voltage of +350 mV, which is the oxidation-reduction potential, was applied, and the oxidation current after 30 seconds was measured. When glucose is oxidized by the enzyme reaction layer 22, ferrocene is reduced. Since the oxidation current of the reduced ferrocene corresponds to the glucose concentration, measurement is possible even if the amount of glucose in the sample solution is extremely small. The measurement was performed by first measuring the oxidation current in a 0 mM glucose solution, washing the surfaces of the enzyme reaction layer 22 and the counter electrode 16 with pure water, and then measuring the oxidation current using a 10 mM glucose solution. Thereafter, the oxidation current was measured with each glucose solution of 20 mM to 50 mM while repeating the surface cleaning of the enzyme reaction layer 22 and the counter electrode 16. The results are shown in FIG.
[0034]
As shown in FIG. 4, the glucose sensor of the example showed high linearity output characteristics up to a high concentration with respect to the glucose concentration. It is said that the blood glucose concentration of healthy people is 3 to 6 mM and that of diabetic patients is 30 to 50 mM, and the biosensor of this example has been shown to be capable of highly sensitive measurement in a wide concentration range. On the other hand, in the comparative example in which ferrocene was not immobilized, the output value did not increase after the glucose concentration of 20 mM. This is considered to be because ferrocene was eluted in the washing solution or the aqueous sample by repeated measurement. Thus, in comparison with the conventional glucose sensor using non-immobilized ferrocene, in the example in which ferrocene was fixed in the hydrophobic medium layer, repeated measurement was possible several times, and blood after calibration with a calibration solution was possible. It was shown that it can be used for measurement.
[0035]
[Example 2]
Four glucose sensors exactly the same as in Example 1 were prepared, and 0.3 wt% of MPC (2-methacryloyl) was formed on the insulating film 18 of the two glucose sensors including the surface of the enzyme reaction layer 22 and the counter electrode 16. Sensor 1 and 2 were prepared by coating with an oxyethyl phosphorylcholine) polymer solution and drying to provide an MPC polymer coating layer. On the other hand, as a comparative example, sensors 3 and 4 were not covered with the MPC polymer.
[0036]
As an initial measurement, 10 μL of 10 mM glucose solution (PBS) was dropped onto each sensor, a constant voltage of +350 mV was applied to the working electrode 14, and the oxidation current after 30 seconds was measured. In the second measurement, the oxidation current output was measured with human serum (containing 5.6 mM glucose). The electrode was washed again, glucose-free PBS was dropped, and the oxidation current after 30 seconds was measured. The results are shown in FIG.
[0037]
As shown in FIG. 5, sensors 3 and 4 without MPC polymer treatment did not provide sufficient output sensitivity in human serum containing protein. On the other hand, in the sensors 1 and 2 subjected to the MPC polymer treatment, output characteristics equivalent to the measured values in the glucose standard solution in which no protein coexists were obtained. In addition, in the sensors 3 and 4 without MPC polymer treatment, the adsorbed protein cannot be completely removed by washing, so the output value in the 0 mM glucose solution (PBS) is higher than that of the sensors 1 and 2 treated with MPC polymer. It became low. In a chip blood analyzer that analyzes multiple items from a small amount of sample, it is necessary to reduce the size of the electrode area in order to increase the density of the electrodes in the chip. In such a case, it was found that an MPC polymer coating that suppresses the adsorption of plasma proteins is effective.
[0038]
【The invention's effect】
As described above, in the enzyme electrode of the biosensor of the present invention, the enzyme-immobilized membrane is divided into the enzyme reaction layer and the electrolytic transfer medium layer, and the oxidoreductase is immobilized and retained in the hydrophilic polymer medium layer. Since the electron acceptor is held in a hydrophobic polymer medium and used as a charge transfer medium layer, the electron acceptor in a hydrophobic environment can be easily eluted in an aqueous sample. There is no. This makes it possible to measure the concentration of the test substance with high sensitivity and high accuracy. Even when used repeatedly, the measurement sensitivity does not decrease. Further, even if calibration is performed using a calibration solution, since the electron acceptor is not eluted, more accurate calibration can be performed. Even if a plurality of biosensors are provided in the same flow path, the electron acceptor does not elute from the enzyme electrode of each adjacent sensor, so that the output of other sensors is not affected.
[0039]
If the enzyme reaction layer is formed of a polyion complex consisting of a polycation polymer and a polyanion polymer, and the oxidoreductase is comprehensively immobilized in this polyion complex, elution of the oxidoreductase can be almost completely prevented. Highly sensitive measurement is possible.
[0040]
If the enzyme reaction layer is coated with a polymer material that has biocompatibility (blood compatibility) such as MPC polymer, it prevents nonspecific adsorption of proteins in blood and adsorption of blood cell components (particularly platelets). Thus, a more sensitive analysis can be performed.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view of a biosensor according to an embodiment of the present invention.
FIG. 2 is a cross-sectional view of a blood analyzer covered with an upper substrate on which a flow channel structure is formed on the biosensor of FIG.
FIG. 3 is an explanatory diagram of a reaction system using an electron acceptor as a mediator.
4 is a diagram showing measurement results of Example 1. FIG.
FIG. 5 is a diagram showing the measurement results of Example 2 in which MPC polymer coating was performed.
[Explanation of symbols]
10 substrate 12 ground lead electrode 14 working electrode 16 counter electrode 18 insulating film 20 charge transfer medium layer 22 enzyme reaction layer 24 upper substrate 26 channel

Claims (11)

At the biosensor enzyme electrode, where the analyte concentration in the sample solution is measured electrochemically by the enzymatic reaction between an oxidoreductase with the analyte as a substrate and an electron acceptor:
An insulating substrate;
Electrodes provided on the substrate;
A porous charge transfer medium layer provided on the electrode and containing an electron acceptor dispersed in a hydrophobic polymer medium;
An enzyme electrode for a biosensor, comprising: an enzyme reaction layer provided on the charge transfer medium layer and having an oxidoreductase immobilized in a hydrophilic polymer medium. 2. The enzyme electrode for a biosensor according to claim 1, wherein the hydrophobic polymer is any one of polyvinyl butyral, polyvinyl chloride, polyvinyl formal and epoxy polymer. 2. The enzyme electrode for a biosensor according to claim 1, wherein the electron acceptor is ferrocene or dimethylferrocene. The hydrophilic polymer is a polyion complex comprising a polycation polymer and a polyanion polymer, and the oxidoreductase is included and immobilized in the polyion complex to constitute the enzyme reaction layer. The enzyme electrode of the biosensor according to claim 1. 5. The enzyme electrode for a biosensor according to claim 4, wherein the polycation polymer is any one of polylysine, chitosan, chitin, and cellulose. 5. The enzyme electrode for a biosensor according to claim 4, wherein the polyanion polymer is polystyrene sulfonate or polyacrylic acid. 2. The enzyme electrode for a biosensor according to claim 1, wherein a biocompatible polymer is coated on the enzyme reaction layer. The biosensor enzyme electrode according to claim 7, wherein the biocompatible polymer is 2-methacryloyloxyethyl phosphorylcholine polymer. In a biosensor that electrochemically measures the concentration of a test substance in a sample solution by an enzyme reaction between an oxidoreductase and an electron acceptor using the test substance as a substrate:
An insulating substrate;
An electrode system including a working electrode and a counter electrode provided on the substrate;
A porous charge transfer medium layer provided on the working electrode and containing an electron acceptor dispersed in a hydrophobic polymer medium;
A biosensor comprising an enzyme reaction layer provided on the charge transfer medium layer and having an oxidoreductase immobilized in a hydrophilic polymer medium. An insulating substrate, an electrode provided on the substrate, a porous charge transfer medium layer containing an electron acceptor provided on the electrode and dispersed in a hydrophobic polymer medium, and the charge transfer medium layer And an enzyme reaction layer in which an oxidoreductase is immobilized in a hydrophilic polymer medium, and the concentration of the test substance in the sample solution is determined using the test substance as a substrate, the oxidoreductase and the electron acceptor In a method for producing an enzyme electrode of a biosensor for electrochemical measurement by enzymatic reaction of:
A biosensor characterized in that the porous charge transfer medium layer is provided by applying and drying a hydrophobic polymer solution in which the hydrophobic polymer and the electron acceptor are dissolved in an organic solvent on the electrode. A method for producing an enzyme electrode. 11. The biosensor electrode according to claim 10, wherein the enzyme reaction layer is provided by comprehensively immobilizing the redox enzyme in a polyion complex comprising a polycation polymer and a polyanion polymer. Manufacturing method.

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