JPH07101215B2 - Analytical method using biofunctional substance-immobilized electrode - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION “Object of the Invention” “Industrial Application Field” The present invention relates to a biosensor analysis method for detecting a biological substance by utilizing the molecular identification function of the biological functional substance. In particular, the present invention relates to a rapid analysis method for a trace amount sample having excellent reproducibility and reliability, which uses an immobilized electrode produced by directly entrapping a biofunctional substance on a porous electrode.

"Prior Art" The present inventors have already found that a biofunctional substance can be comprehensively immobilized on a platinum black surface, succeeded in producing an immobilized electrode using this, and applied for a patent. (Patent application No. 55387 (Japanese Patent Laid-Open No. 63-222256) and No. 56472 (Japanese Patent Publication No. 6-75054) in 1987). Furthermore, we developed and applied a patent for an analysis system based on an electrochemical unsteady method using an analysis system that can measure a stationary minute sample using an immobilized electrode (patent application No. 3045 in 1987).
No. 23 (JP-A-1-147357).

This method does not require a strict regulation of the amount of sample solution to be taken, and is capable of measuring a small amount of static sample and undiluted measurement, and is characterized by rapid measurement on the order of milliseconds, and It greatly expanded the possibilities of application. However, a blank measurement is performed in advance with a buffer solution that does not contain the substance to be measured (hereinafter referred to as a blank solution),
It is necessary to subtract the value from the response to the target sample solution, which makes the operation complicated. Therefore, although this method is a rapid analysis method utilizing a response on the order of milliseconds, the advantage cannot be sufficiently exhibited in the entire measurement operation including the operation before the sample solution measurement.

"Problems to be solved by the invention" When an unsteady response current to a constant potential simple pulse was recorded by applying an electrochemical unsteady method to an enzyme electrode in which an enzyme was entrapped and immobilized on a platinum black electrode, its response The current does not become zero even for the blank solution as shown in Example 2. This unsteady current includes a charging current of the electric double layer capacitance immediately after the pulse application, and a faradaic current derived from the electrochemical redox response of the impurity molecules and the electrode surface. Therefore, it is necessary to subtract the unsteady current value observed for the blank solution from the unsteady current value observed for the specimen sample. Further, when the response to the blank solution is not so small as to be negligible as compared with the response current to the analyte sample, the reproducibility and reliability of the sensor response are also impaired. Therefore, the present invention is intended to provide a method for measuring a response regarding a blank solution, that is, an analytical method that does not require so-called blank measurement, and by this, good reproducibility,
It is intended to provide a highly reliable rapid analysis method.

[Structure of the Invention] [Means for Solving Problems] In order to solve the above problems, the present invention applies a preliminary pulse immediately before applying a measurement pulse, and measures an object to be measured by electrochemical pretreatment. Provided is an analysis method, which comprises reducing a response not derived from a substance, and thereafter maintaining an open circuit state for a certain period of time, and then measuring a sensor response by a subsequent measurement pulse.

The electrode and the analysis system in which the biological functional substance is comprehensively immobilized used in the present invention uses the non-steady response described in the patent application (Japanese Patent Application No. 304523 of 1987 (JP-A-1-147357)). The analysis system That is, as described in Japanese Patent Application No. 55387 (Japanese Patent Application Laid-Open No. 63-222256) and Japanese Patent No. 56472 (Japanese Patent Publication No. 6-75054) in 1987, fine particles composed of fine particles such as platinum. The electrode having a structure having a conductive fine particle layer in which a biofunctional substance such as an enzyme is entrapped and immobilized on the surface of the electrode is used as a working electrode, and silver
It is an electrochemical system having a reference electrode such as silver chloride and three electrodes provided with a counter electrode, and an example of the structure is shown in FIG. In FIG. 1, the working electrode 1 is a microelectrode in which a biological functional substance (eg glucose oxidase) is entrapped and immobilized, and the diameter thereof is, for example, about 1 μm to 500 μm. The sensor element 6 is composed of a counter electrode 2 of a platinum wire and a silver / silver chloride-based reference electrode 3. The above three electrodes, that is, the micro-immobilized electrode 1, the counter electrode 2, and the reference electrode 3 are embedded in the holes of the Teflon (registered trademark) form 5 with the polyester resin 4. Since such a sensor element 6 has a structure in which only three thin metal wires are encapsulated and fixed, it is possible to configure all of them into a very minute sensor by using a fine processing technique.

If this sensor element is used, it is possible to measure a minute amount of sample, for example, about 1 μ1. That is, the substance in the trace sample can be detected by the method of applying a potential after dropping the trace sample and detecting the current value generated at this time.

The sensor response is obtained by recording the unsteady current response to a constant potential pulse using the analysis system described above, but the response to a simple pulse is shown in Example 2 even for a solution containing no measurement target substance. Thus, a faraday current that cannot be ignored is observed. Traditionally, blank measurements were required because this current had to be subtracted.

As shown in FIG. 2, the present invention applies a preliminary constant-potential pulse 7 immediately before applying a constant-potential pulse 9 for measurement and does not apply a potential for a certain period of time to a so-called open circuit state 8. The above problem is solved by applying the measurement pulse 9 after the temperature is maintained.

"Action" The unsteady current response observed when a potentiostatic pulse is applied to an electrochemical system is generally derived from the capacitive current resulting from the charging of the electric double layer capacity and the electrochemical redox reaction at the electrode. It consists of two components of the so-called Faraday current. Since the capacitive current is attenuated with a time constant of several tens of microseconds to several hundreds of microseconds, the problem with the non-steady current of several milliseconds to several tens of milliseconds, which is a problem in the present invention, is the problem of the Faraday current. You can think that it is only.

It can be considered that the Faraday current for the blank solution mainly consists of an electrode reaction of impurities contained in the solution and an electrochemical redox reaction of the electrode surface. In the following description and examples, the case of application to glucose detection will be described as an example. In this case, glucose is oxidized by the action of glucose oxidase immobilized on the electrode surface, but the one that tries to detect glucose by detecting the current required for electrochemical oxidation of hydrogen peroxide produced at this time Is. Therefore, in the following description, the oxidation pulse is used for both the preliminary pulse and the measurement pulse, but the reduction pulse may be applied depending on the combination of the biofunctional substance and the measurement target substance.

Oxidation current observed when a 0.6V constant potential pulse was applied to the silver / silver chloride reference electrode was observed in addition to the oxidation current of hydrogen peroxide, the oxidation current of reducing impurities near the electrode, and the platinum black surface. It may include an oxidation current that oxidizes the to produce surface oxides. It is considered that the unsteady current observed in the blank solution is mainly derived from the oxidation current of the reducing impurities and the oxidation current of the platinum black surface. Therefore, by applying the preliminary pulse prior to the measurement pulse, for the blank solution, the reducing impurities existing near the electrode surface were oxidized, and the oxidation state of the platinum black surface was the same as when the measurement pulse was applied. The response of the blank solution can be made negligibly small due to the effect of adjusting to.

For the sample solution containing glucose, the hydrogen peroxide that has already been generated is similarly oxidized by the preliminary pulse, but since hydrogen peroxide is again generated by the reaction of glucose during the open circuit state of 8, The hydrogen peroxide generated at this time is detected by the measurement pulse 9. Such an open circuit state is a technique that has not been used because the electrode potential becomes indefinite in the electrochemical measurement such as the conventional constant potential pulse application method, but it is an essential technique in the biofunctional electrode of the present invention. is there.

“Example” Example 1 Preparation of Sensor Element The sensor shown in FIG. 1 was prepared by the following procedure.
In the Teflon mold 5, a minute platinum wire with a diameter of 1 μm to 500 μm, a platinum wire for a counter electrode with a diameter of 200 μm, and 500 μm
Each of the silver wires having a diameter of about 1 mm was sealed with polyester resin 4 and then polished with an alumina abrasive. Immobilization of the enzyme on the surface of the platinum working electrode was performed by the following method.

In a 3% chloroplatinic acid solution containing 300 ppm of lead acetate,
The silver chloride reference electrode was subjected to constant potential electrolysis at a potential of -0.1 V for 5 minutes to perform electrolytic deposition of platinum black to obtain platinum black having a thickness of about several μm. Next, the obtained platinum black deposition electrode was dried at room temperature for 60 seconds, and then held at −0.3 V for 30 minutes in a 0.5 M sulfuric acid aqueous solution to generate hydrogen from the platinum black electrode. After air-drying for 60 seconds, a constant potential of 1.2 V was applied for 15 minutes to oxidize the electrode surface, and then immersed in 1 ml of 5500 glucose oxidase-containing phosphate buffer (pH 6.8) for 30 minutes, Air dried again.

Next, in the sensor element 6 having the microelectrodes obtained as described above, the silver wire was used as a silver / silver chloride reference electrode.
The sensor element 6 composed of three electrodes produced in this way
The mixture was stirred and washed in a 0.1 M phosphate buffer solution for 24 hours to obtain a three-electrode type sensor element used in the present invention.

Example 2 Measurement of Glucose Concentration Using Simple Pulse The measurement system shown in FIG. 3 was used to measure the glucose concentration. That is, the working electrode 1, the counter electrode 2, and the reference electrode 8 of the sensor element 6 were respectively connected to the potentiostat 10, and the application of the pulse actuated the potentiostat by the signal from the function generator 11.
The unsteady current was recorded in the digital memory scope 12. The unsteady current response when a simple constant potential pulse of 0.6 V was applied to the silver / silver chloride reference electrode was used as the working electrode with the immobilized enzyme electrode prepared using a platinum wire of 50 μm in diameter.
Shown in the figure. Curve 13 is the response to a phosphate buffer solution containing 20 mM glucose, 14 is the response to a blank solution of phosphate buffer solution only, and 15 is the response to a phosphate buffer solution containing 20 mM fructose.

In both responses, it is considered that the initial peak current, which is considered to be due to charging of the double-layer capacity, attenuates in about 1 millisecond, and the Faraday current is observed after 2 milliseconds. Since the fructose solution gave almost the same curve as the blank solution, the difference between curves 13 and 14 flowed during the electrochemical oxidation of hydrogen peroxide produced by the presence of glucose at a concentration of 20 mM. It is considered to be derived from the electric current. Therefore, the difference in current response between the glucose solution and the blank solution after 2 milliseconds was defined as the sensor response, and the sensor response at various concentrations was measured. The results are shown in FIG. 5, and the sensor response uniquely corresponds to the concentration, and the glucose concentration can be known from this response.
However, in order to obtain the data shown in FIG. 4 with good reproducibility, the time from the contact between the sensor and the solution to the start of measurement must be kept constant, and the response to the blank solution must be kept constant. It was necessary to measure.

Example 3 Measurement of Glucose Concentration Using Preliminary Pulse Using the same apparatus as in Example 2, after applying the preliminary pulse 7 shown in FIG. 2, the open circuit state 8 was maintained for a certain period of time, and then the measurement pulse 9 was used. The measurement was performed. After applying a 0.6V constant-potential preliminary pulse to the silver / silver chloride reference electrode for 60 seconds,
Fig. 6 shows the unsteady current response obtained by keeping the circuit open for a second and then applying a constant potential measuring pulse of 0.6V. Curve 16 is the response to 5 mM glucose and 17 is the response to the blank solution.

Response to blank solution is 1.5 μA in about 5 ms
However, after 10 ms, it is negligibly small compared to the response current to the glucose solution. That is, when the preliminary pulse is used, the response of the blank solution is very small, and therefore it is not necessary to measure the response to the blank solution each time and subtract the current value.

Example 4 Glucose Concentration Dependence of Sensor Response In order to determine the glucose concentration from the unsteady current response obtained in Example 3, the relationship between the current value and the glucose concentration at a fixed time after the application of the measurement pulse was obtained.

Simultaneously with Example 3, a preliminary pulse was applied to sample solutions having various glucose concentrations for 20 seconds, then kept in an open circuit state for 10 seconds, and then a non-steady-state current was measured with respect to a potentiostatic measurement pulse. FIG. 7 shows the results of measuring the glucose concentration dependence of the current value as the sensor response. It shows good linearity between glucose concentrations of 0.1M and 10mM, and that the glucose concentration can be directly determined from the unsteady current value for the measurement pulse by using the preliminary pulse without measuring the response to the blank solution. Shows.

Example 5 Pulse application time and sensor response The same apparatus as in Example 2 was used to examine the relationship between the pulse application time and the sensor response in FIG. 2 under the same potential conditions as in Examples 3 and 4. Pre-pulse application time from 1 second
Fig. 8 shows the non-steady-state current value after 10 milliseconds measured after changing to 60 seconds and keeping the circuit open for 10 seconds. Curve 18
Indicates a response to a phosphate buffer solution containing 5 mM glucose, and 19 indicates a response to a blank solution.

The response to the blank solution rapidly decreases to about 15 seconds of the preliminary pulse, and about 60 seconds are required for sufficient decay. On the other hand, the response to glucose takes a nearly constant value in about 30 seconds.

On the other hand, in order to investigate the relationship between the open circuit time and the sensor response, the unsteady current after 10 milliseconds when the preliminary pulse time was kept at 20 seconds and the open circuit time was changed from 1 second to 30 seconds FIG. 9 shows the result of examining the sensor response defined by the value. Curves 20, 21, and 22 are the responses to 10 mM, 5 mM, and 2 mM glucose solutions, respectively. From these results, it is considered that in the open circuit state, the oxidation reaction of glucose mainly by glucose oxidase proceeds, and the production of hydrogen peroxide proceeds on the electrode surface.

Summarizing the above results, it can be seen that the longer the preliminary pulse time, the smaller the response given by the blank solution, and the longer the open circuit time, the larger the sensor response. On the other hand, from a practical point of view, the shorter time is more advantageous for rapid analysis. Therefore, FIG. 10 shows the results of measuring the sensor response to glucose solutions of various concentrations under the conditions of a preliminary pulse of 5 seconds and an open circuit time of 5 seconds.

The glucose concentration is zero, that is, the response to the blank solution is not zero, but the reproducible sensor response is obtained for each glucose concentration, and when the preliminary pulse is given, the response to the blank solution is completely reduced. It is shown that the measurement can be performed with good reproducibility even when it does not wait until it reaches zero, and it is possible to speed up the measurement under this condition if necessary.

Example 6 Examination of Reproducibility of Response In order to examine the improvement of reproducibility by applying the preliminary pulse, the sensor response was repeatedly measured for the case of using the preliminary pulse and the case of using the simple pulse. The measuring apparatus was the same as in Example 2, using a 10 mM glucose solution as a sample, the preliminary pulse time was 20 seconds, and the open circuit time was 30 seconds. An example of the results is shown in FIG.

The white circles in FIG. 11 show the sensor response in the case of a simple pulse that does not use the preliminary pulse, and the black circles show the results when the preliminary pulse is used.

In the case of a simple pulse, the first response always gave an abnormally large value, and a variation with a standard deviation of about 3% was also observed after the second response. On the other hand, when using the preliminary pulse,
The reproducibility was also improved, and the standard deviation was about 0.4%.

"Effects of the invention" Applying an electrochemical unsteady method to an enzyme-encapsulated platinum black electrode and using it for the detection of biological substances will open the way for establishing a rapid analysis method in which the sensor response does not depend on the sample amount. However, there is a difficulty in convenience such as requiring a blank measurement, and it is difficult to put the sensor element into practical use as it is as a sensor element for use in self-management of blood glucose level of diabetic retinopathy patients and the like. there were. The combined use of the preliminary pulse and the open circuit state according to the present invention makes it possible to remarkably simplify the operation as shown in Examples 3, 4 and 5, and the reliability is remarkably improved as shown in Example 6. did.

In short, the technical effect of the present invention is that it enables simple, reliable, highly sensitive and rapid analysis of a small amount of sample.

[Brief description of drawings]

FIG. 1 is a structural example of a sensor element used in the present invention. FIG. 2 is an example of a preliminary pulse and an open circuit state program used in the present invention. FIG. 3 is a conceptual diagram of a measurement system used in Examples 2 to 6. FIG. 4 is an example of an unsteady current response when a constant potential simple pulse is applied. FIG. 5 shows the dependence of the sensor response obtained by using a non-stationary current response to a constant potential simple pulse on the glucose concentration. FIG. 6 is an example of an unsteady current response when a preliminary pulse is used. FIG. 7 shows the glucose concentration dependence of the sensor response obtained from the unsteady current response when using the preliminary pulse. FIG. 8 shows the dependence of the sensor response on the preliminary pulse time. FIG. 9 shows the dependence of the sensor response on the time of the open circuit state. Figure 10 shows the sensor response when the preliminary pulse and open circuit state are both set to 5 seconds. Fig. 11 is a comparison of the sensor response obtained from the response to a simple constant potential pulse and the reproducibility of the sensor response when a preliminary pulse is used. “Explanation of symbols for main parts” 1 …… Biofunctional substance-immobilized electrode 2 …… Counter electrode 3 …… Silver / silver chloride reference electrode 4 …… Polyester resin 5 …… Teflon mold 6 …… Sensor element 7 …… Preliminary pulse 8 …… Open circuit period 9 …… Constant potential measurement pulse 10 …… Potentiostat 11 …… Function generator 12 …… Digital memory scope 13 …… 20mM Glucose response 14 …… Blank solution response 15 …… 20mM Response to fructose solution 16 …… 5mM Response to glucose 17 …… Blank solution response 18 …… 5mM glucose response 19 …… Blank solution response 20 …… 10mM glucose response 21 …… 5mM glucose response 22 …… 2mM Response to glucose

Claims (1)

[Claims] 1. A potentiostatic measuring pulse in a system for detecting a substance to be measured or determining a concentration by an electrochemical unsteady method using a porous electrode such as platinum black and gold black on which a biofunctional substance is immobilized. Analytical method characterized in that an electrochemical pretreatment with a constant potential preliminary pulse prior to the above and an open circuit state for a certain period of time are maintained.