JP4089975B2 - Electrode for electrochemical measurement, method for producing the same, and electrochemical biosensor - Google Patents

Description

  The present invention relates to an electrode for electrochemical measurement, a method for producing the same, and an electrochemical biosensor.

  Since electrochemical measurement methods can measure the concentration of biomolecules easily and with high sensitivity, research on electrochemical biosensors has been actively conducted. Electrochemical biosensors can be used to measure substances that have electrochemical activity in the blood and body fluids of animals including humans as they are, or for substances that have electrochemical activity, such as enzymes that are biocatalysts. It is a general term for technologies that are detected by direct oxidation or reduction on the electrode after conversion.

  Many biologically active substances exist in biological samples and react at the measurement potential of the substance to be measured, or there are substances that inhibit the electron transfer of the enzyme-electron transfer mediator composite electrode. It is difficult to obtain a response that can only be obtained from. Examples of such substances that interfere with measurement (hereinafter, these substances are collectively referred to as contaminants) include L-ascorbic acid and uric acid.

  As a method for removing these influences, there is a method in which the electrode is modified with a polymer film. Until now, Nafion films, excessively oxidized polypyrrole films (peroxidized polypyrrole films), polyaniline films, polyion complex films, and the like have been used as polymer films.

The Nafion membrane has a sulfonic acid group, and the polypyrrole peroxide membrane has a carboxyl group, and can suppress the diffusion of anionic molecules such as L-ascorbic acid and uric acid onto the electrode due to electrostatic repulsion (non- Patent Document 1). In the polyion complex membrane, it is possible to suppress the diffusion of anionic molecules on the electrode by utilizing the difference in molecular size (Non-patent Document 2).
M.M. N. Szentirmay and C.I. R. Martin, Anal. Chem. , 1984, 56, 1898-1902 S. Yabuki, F .; Mizutani, Y. et al. Hirata, Sensors and Actuators B76 (2001) 142-146

  When Nafion is used as the diffusion control film, the Nafion film is formed by spreading the polymer solution and drying the organic solvent, so the pore size inside the polymer film remaining after volatilization of the organic solvent is compared. Since anionic impurities are also easily diffused, the thickness required to obtain a sufficient diffusion control effect has to be formed with a thickness on the order of several μm.

  As a result, there is a problem that the diffusion distance for the measurement target substance to reach the electrode is increased and the measurement sensitivity is lowered. Moreover, since the adhesiveness with the electrode body is not good, there is a problem that long-term stability is lacking. Also, since the polypyrrole peroxide film is formed in a very thin state, when the concentration of the measurement target substance is high, measurement can be performed even in the presence of a measurement interfering substance, but the concentration of the measurement target substance is low. In some cases, the selection detection has not been achieved.

  As described above, high-sensitivity or high-selection detection of biomolecules can control the diffusion of only the contaminant substance without inhibiting the diffusion of the analyte substance on the electrode. An electrode modification film capable of separating the measurement potential from the measurement potential of the contaminant and having good adhesion is required.

To solve the problems described above, an electrochemical measurement electrode according to the present invention, the surface of the electrode body for electrochemical measurements are polymeric films made by high-frequency sputtering method modified as a target organic solid materials The target is obtained by developing and drying a polymer B solution having a molecular structure different from that of the polymer A on the solid polymer A.

  Further, the method for producing an electrode for electrochemical measurement according to the present invention is a composite material in which a polymer B solution having a molecular structure different from that of the polymer A is spread on a solid polymer A and dried on the electrode body. And a polymer film having a structure in which fine grains are densely condensed on the electrode body by high-frequency sputtering.

  An electrochemical biosensor according to the present invention comprises a channel for circulating a sample, an electrode for electrochemical measurement according to claim 1 provided so as to be in contact with the sample flowing through the channel, a reference electrode for the electrode, And a counter electrode.

  The polymer membrane modified electrode (electrochemical measurement electrode) according to the present invention and the biosensor using the same are anionic molecules such as L-ascorbic acid and uric acid that interfere with the measurement of cationic or nonionic molecules. It can be excluded. For this reason, it has the effect that the in-vivo substance or the substance released from the cell can be measured with high selectivity.

  ADVANTAGE OF THE INVENTION According to this invention, the diffusion control film | membrane which contains the functional group which exhibits anionic property in the neutral solution in a living body level in a high-density state, has high adhesiveness with a reaction electrode, and was excellent in film thickness controllability. To do.

  The electrode for electrochemical measurement according to the present invention is obtained by forming a polymer thin film produced by a high frequency sputtering method using an organic solid material as a target on an electrode substrate.

  The electrode substrate has a structure in which an electrode body for electrochemical measurement is provided on the substrate. For example, a thin film of titanium 2 and gold 3 electrode bodies is formed on a substrate 1 such as glass by sputtering. (See FIG. 1). At this time, an electrode pattern may be provided on the electrode body.

  A polymer thin film prepared by high-frequency sputtering using an organic solid material as a target is provided on the surface of the electrode body of such an electrode substrate.

  The high-frequency sputtering method using an organic solid material as a target material has a thin film with the basic structure of the target material, a high element density compared to the target material, and a film with good adhesion to the substrate material with excellent film thickness controllability. It is a method that can be formed.

  For example, a target material produced by developing a polymer solution (B polymer) on a bulk polymer material (organic solid material: A polymer) and drying the solvent is produced. Next, by high-frequency sputtering using this target material, the particles released into the plasma caused by both the A and B polymers are combined by a reaction in the plasma environment, and further deposited on the electrode substrate. A polymer thin film is formed on the surface of the electrode body of the substrate. Moreover, since the monomer which became high energy particles collides with the said board | substrate and contributes to film formation, the thin film of a composite polymer with high adhesiveness with the said board | substrate can be formed.

  As such an A polymer, for example, a hydrocarbon polymer can be used, and as the polymer B, a polymer having a cation exchange group, for example, a fluoride polymer can be used. More specifically, polyethylene can be used as the hydrocarbon polymer, and Nafion can be used as the fluoride polymer.

  Furthermore, examples of the polymer film modified on the electrode body include amino acids, nucleobases, biological gels, and the like. However, the present invention is not limited to this.

  For example, by applying anionic Nafion as described above as a target for high-frequency sputtering, a thin film material having an anionic functional group, high adhesion, and an appropriate film thickness is developed and reacted. An electrode for electrochemical measurement can be formed by forming on the electrode body.

  Such a polymer film can be made into a polymer film in which an enzyme is immobilized, the property of the modified polymer film is modified, and contaminants different from the modified polymer film are diffused. For example, glucose oxidase can be immobilized on the polymer membrane, glucose can be changed to a substance having electrochemical activity, and the substance can be oxidized for measurement. In this case, the reaction of other substances (contaminating substances) is not performed at the electrodes.

  The electrochemical biosensor according to the present invention uses the electrode for electrochemical measurement as described above. As shown in FIG. 2, the electrochemical biosensor according to the present invention includes a flow channel 16 for circulating a sample 21, an electrode 13 for electrochemical measurement according to the present invention provided in contact with the flow channel 16, The counter electrode 12 of the electrode and the reference electrode (silver paste) 15 are provided.

  In such an electrochemical biosensor, prior to introducing the sample 21 into the flow channel 16 (and therefore upstream of the flow channel 16 with respect to the sample flow), the sample 21 is placed in the microdialysis probe 20 or glass capillary. It is possible to manufacture an electrochemical biosensor having a structure in which a gas is passed. By providing such a microdialysis probe 20 or a glass capillary upstream of the flow path, it is possible to prevent response deterioration.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited to a following example.

  A schematic view of a modified electrode for electrochemical measurement according to the present invention and a measurement system are shown in FIG. Gold was used as the electrode body used in the modified electrode for electrochemical use. On the glass substrate 1, titanium 2 and gold 3 as an electrode body were sputtered to obtain an electrode substrate. An organic plasma thin film 4 was produced on the electrode substrate using a high frequency sputtering apparatus.

At this time, a high-frequency sputtering target was manufactured as follows. First, an etching process using a rare gas was performed as a pretreatment on the surface of a disk-shaped bulk polymer material serving as a target material substrate. Next, a Nafion isopropyl alcohol solution is developed in the center of a polyethylene disk (disc-shaped bulk polymer material) (diameter 13.5 mmφ, thickness 15 mm) and in a range of 50 mm in diameter, and the solvent component is volatilized in an atmospheric environment. It was. The basic skeleton of polyethylene, which is a disk-shaped polymer, is CH ₃ — (CH═CH) _x —CH ₃ , while the basic structure of Nafion, which is a polymer developed on a disk, is-[(CF _{2 CF 2) n -C = CF- (} OCF 2 CF (CF 3)) m OCF 2 CF 2 SO 3 H] x - is.

  When the film thickness of the produced polymer thin film was measured, it was about 100 nm. Further, from observation by SEM, a structure in which grains of 100 nm or less aggregated at a high density was observed.

  This was cut using a dicing saw (Disco DAD521) and processed into a length of 40 mm and a width of 10 mm. In order to use as a working electrode, the cut adhesive sheet 5 was used to form a circular electrode 6 having a diameter of 3 mm. Moreover, the part which is not covered with the adhesive sheet was used as the pad 7 for terminal connection. The modified electrode for electrochemical measurement was immersed in the solution 10 together with the silver / silver chloride reference electrode 8 and the counter electrode 9 (platinum). The sample used in this example was prepared using a phosphate buffer solution. These electrodes were connected to a potentiostat 11 (ALS Model 1000).

  First, it was immersed in a phosphate buffer solution, the natural potential was measured, and it was confirmed that the electrode and the solution were not insulated by the polymer film. Next, cyclic voltammetry was performed using a 10 μM dopamine solution. The potential sweep range is -0.2 to 0.8 Vvs. The potential sweep rate was set to 10 mV / s for Ag / AgCl. As a result, an oxidation current peak was observed near 0.3 V, and the current value at that time was about 250 nA. Thereafter, as the potential increased, the current value gradually decreased. Subsequently, when a similar experiment was performed using a 10 μM ascorbic acid solution, the current value did not increase even when the potential increased. This result shows that catecholamines such as dopamine which is a cationic molecule can be detected, and L-ascorbic acid which is an anionic molecule cannot be detected.

  As a comparative experiment, when the same measurement was performed using a gold electrode, the current value obtained from 1 μM dopamine was about 250 nA, which was the same current value as the electrode according to the present invention. On the other hand, the current value obtained from the same concentration of L-ascorbic acid was about 200 nA. Further, when a Nafion film was applied to the carbon electrode and the same measurement was performed, no response was obtained from 1 μM L-ascorbic acid, but the current value obtained from 1 μM dopamine was about 220 nA. .

  In addition, when the same measurement was performed using an organic plasma thin film formed after forming a microelectrode pattern on a gold electrode, the current density was increased approximately 20 times by forming the electrode into a microarray. As compared with the case where the electrode was not modified with the polymer film, an equivalent S / N was obtained. This is because the polymer thin film has a thin film thickness of 100 nm and does not hinder the diffusion of dopamine as a measurement target substance.

  As described above, the electrode for electrochemical measurement according to the present invention can be selectively detected in the presence of L-ascorbic acid without inhibiting the sensitivity to catecholamines which are measurement target substances.

  A modified electrode (electrochemical measurement electrode) similar to that of Example 1 was prepared, and the following experiment was performed. In this example, a sodium chloride solution was used in place of the phosphate buffer as the measurement sample. First, it was immersed in a sodium chloride solution, the natural potential was measured, and it was confirmed that the electrode and the solution were not insulated by the polymer film. Next, cyclic voltammetry was performed using a 10 μM dopamine solution. The potential sweep range is -0.2 to 0.8 Vvs. The potential sweep rate was set to 10 mV / s for Ag / AgCl. As a result, an oxidation current peak was observed near 0.1 V, and the current value at that time was about 250 nA. Thereafter, as the potential increased, the current value gradually decreased.

  Subsequently, when a similar experiment was performed using a 10 μM ascorbic acid solution, the current value increased as the potential increased, and an oxidation current peak was observed in the vicinity of 0.3V. The current value at that time was about 100 nA. From this result, it was found that the oxidation potential of dopamine and L-ascorbic acid can be separated by using a sodium chloride solution as a sample preparation solution.

  As described above, the electrochemical measurement electrode according to the present invention can separate the oxidation potential of catecholamines and L-ascorbic acid, and can selectively detect catecholamines without being affected by L-ascorbic acid. it can.

  An organic plasma thin film modified electrode similar to that in Example 1 was prepared, and an electrochemical measurement electrode in which glucose oxidase was immobilized on the organic plasma thin film was prepared. A mixed solution of 1 mM glucose and L-ascorbic acid was prepared using a phosphate buffer solution, and an electrode was inserted into the solution to perform electrochemical measurement. The applied potential was 600 mV. The glucose concentration is quantified from the current value observed by oxidizing this hydrogen peroxide because hydrogen peroxide is produced by glucose oxidase. As a result of the measurement, a response current value of about 100 nA was obtained. Further, even when only glucose was measured, a response current of about 100 nA was obtained.

  On the other hand, a current value of about 200 nA was obtained for the organic plasma thin film unmodified electrode for comparison. The reason why the response current was higher in the electrode without the organic plasma thin film is that L-ascorbic acid also reacted on the electrode, and it can be measured by an enzyme reaction. In contrast, the polymer thin film-modified electrode of the present invention is effective.

  FIG. 2 shows a sketch of a chip-type sensor to which a microdialysis probe is connected and a polymer thin film modified electrode is mounted. This sensor consists of two glass substrates, and three gold thin film electrode bodies 12, 13, and 14 are formed on one glass substrate. The electrode 13 was used as a detection electrode, and when the polymer thin film was formed by sputtering, the electrode bodies 12 and 14 other than the electrode 13 of Example 1 of the present invention were covered. The electrode 14 was coated with a silver paste 15 (Fujikura Kasei) and used as a reference electrode. A flow path 16 having a width of 1 mm and a depth of 50 μm is formed on the other substrate by a double-sided tape 17, and a sample introduction tube 18 and a discharge tube 19 are connected to the other substrate. These two substrates are bonded by a double-sided tape 17.

  Next, the microdialysis probe 20 was connected to the sample introduction tube 18. As sample 21, a sample obtained by mixing equimolar serum with 0.1 mM dopamine and dopamine and L-ascorbic acid to 0.1 mM each was used. In addition, a phosphate buffer solution is placed in the syringe 22, and a flow rate of 2 μl / min. The liquid was sent. The measurement potential was 0.2 V with respect to the reference electrode.

  First, when a sample in which only dopamine was mixed with serum was measured, the response current was 200 nA. Next, when a sample in which serum was mixed with dopamine and L-ascorbic acid was measured, a current value almost equal to the response obtained from the sample in which serum was mixed with only dopamine was obtained. This is thought to be because the oxidation potential of L-ascorbic acid shifts to a noble potential and is hardly oxidized at 0.2V. Moreover, even when measured for a long time, no deterioration in response was observed. This is considered to be because the protein was not introduced into the electrochemical biosensor and the adsorption onto the electrode was suppressed by connecting the microdialysis probe.

  As described above, by applying the polymer thin film modified electrode according to the present invention to an on-line sensor connected to a microdialysis probe, it is possible to measure a molecule present in a blood sample without being affected by L-ascorbic acid or protein. it can.

  In the present application, an electrode modification film for removing substances other than the measurement target of an electrochemical biosensor is manufactured by a high-frequency sputtering method using anionic Nafion as a target in a neutral solution at an in vivo level. It is characterized in that a film containing an anionic functional group in a high density state, having high adhesion to the reaction electrode, and excellent in film thickness controllability is obtained.

The figure which shows the schematic diagram and measurement system of the modified electrode for electrochemical measurements by this invention. FIG. 2 is a sketch of a chip-type electrochemical biosensor to which a microdialysis probe is connected and an electrode for electrochemical measurement according to the present invention modified with a polymer thin film is mounted.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Glass substrate 2 Titanium 3 Gold 4 Organic plasma thin film 5 Adhesive sheet 6 Circular electrode 7 Pad 8 Reference electrode 9 Counter electrode 11 Potentiostat 12 Gold thin film electrode 13 Gold thin film electrode 14 Gold thin film electrode 15 Silver paste 16 Flow path 17 Both sides Tape 18 Tube for sample introduction 19 Tube for discharge 20 Microdialysis probe 21 Sample 22 Syringe 23 Syringe pump

Claims (9)

The surface of the electrode body for electrochemical measurement is modified with a polymer thin film produced by a high frequency sputtering method using an organic solid material as a target, and the target is formed on the solid polymer A and the polymer A. An electrode for electrochemical measurement, wherein the polymer B solution having a molecular structure different from that of the polymer B is developed and dried .   The electrode for electrochemical measurements according to claim 1, wherein an electrode pattern is formed on the surface of the electrode body. The electrode for electrochemical measurements according to claim 1 or 2, wherein the polymer A is a polymer hydrocarbon, and the polymer B is a polymer having a cation exchange group. The electrochemical measurement electrode according to any one of claims 1 to 3, wherein the polymer B is Nafion, and the polymer A is polyethylene.   The electrode for electrochemical measurements according to claim 1 or 2, wherein the polymer thin film modified on the electrode body is any one of amino acids, nucleobases, and biological gels. The electrode for electrochemical measurement according to any one of claims 1 to 5 , wherein a polymer thin film on which an enzyme is immobilized is laminated on the electrode body.   A structure in which a polymer B solution having a molecular structure different from that of the polymer A is developed and dried on a solid polymer A, and fine grains are densely condensed on the electrode body by high-frequency sputtering. The manufacturing method of the electrode for electrochemical measurements characterized by forming the polymer film which has this.   A flow path for circulating a sample, an electrode for electrochemical measurement according to claim 1 provided so as to be in contact with the sample flowing through the flow path, a reference electrode of the electrode, and a counter electrode are provided. Electrochemical biosensor. 9. The electrochemical biosensor according to claim 8, wherein the sample passes through a microdialysis probe or a glass capillary prior to introducing a sample into the flow path.

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