JPH06213856A - Detector for degradation of electrode activity of biosensor - Google Patents

Abstract

PURPOSE:To simply and highly accurately detect activity of an enzyme electrode by applying a predetermined voltage waveform between base electrodes and generating predetermined characteristic data based on an electric signal obtained according to the activity of the base electrodes. CONSTITUTION:In order to detect degradation in activity of base electrodes, a predetermined voltage waveform is applied between base electrodes from a triangular wave generating part. A characteristic data generating and holding part 32 generates predetermined characteristic data based on an electric signal generated between the base electrodes, while a good/defective electrode reference data holding part 33 holds characteristic data when the predetermined voltage waveform is applied when activity of the base electrodes is sufficiently maintained. A good/defective electrode determining part 34 determines degradation of the base electrode activity based on the characteristic data generated by the characteristic data generating and holding part 32 and the characteristic data at the time of being active held in the good/defective electrode reference data holding part 33. Thus replacing a reference immobilized enzyme film is not necessary, thereby simply determining the base electrode activity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for detecting a reduced electrode activity state of a biosensor, and more specifically, it automatically detects that the electrode activity of the biosensor is reduced at an arbitrary or predetermined timing. Regarding the device.

[0002]

2. Description of the Related Art Conventionally, various biosensors have been researched by paying attention to the characteristic that highly complex organic compounds and the like can be detected with high sensitivity and selectively. As a typical example of such a biosensor, for example, a working electrode made of Pt and a counter electrode made of Ag are formed, an enzyme is fixed on the electrode, and the biosensor is generated by a reaction between the fixed enzyme and a target substance. H ₂ O ₂ for guidance on the electrode surface through H ₂ O ₂ permeable film, presence or absence of an object to be measured is taken out electrical signals corresponding to the amount of the transmitted H ₂ O _2, it is intended to detect the abundance or the like proposed Has been done.

In order to improve the accuracy of measurement in such a biosensor, it is necessary to always maintain good activities of the enzyme electrode and the immobilized enzyme membrane provided on the enzyme electrode. In addition, whether the cause of the degradation when the output of the biosensor decreases is the degradation of the immobilized enzyme membrane,
Alternatively, it is necessary to judge whether it is due to the deterioration of the activity of the enzyme electrode.

[0004]

However, according to the conventional biosensor, although the deterioration of the immobilized enzyme membrane can be detected, the deterioration of the activity of the enzyme electrode itself cannot be detected. Therefore, conventionally, in order to determine the decrease in the activity of the enzyme electrode itself, a measurement process was performed using an immobilized enzyme membrane with a known activity (hereinafter referred to as a reference immobilized enzyme membrane) to obtain normal measurement data. If so, the method of assuming that the activity of the enzyme electrode used was normal was taken.

However, the above determination method has problems that the operation for obtaining the determination result as to whether or not the activity of the enzyme electrode is lowered is complicated and the measurement accuracy is poor. More specifically, the method of measuring the degree of activity of the enzyme electrode using the standard immobilized enzyme membrane requires that the standard immobilized enzyme membrane be prepared each time the activity of the enzyme electrode is examined. Therefore, the enzyme membrane currently being measured must be replaced with the reference immobilized enzyme membrane to perform the measurement, which significantly increases the work required for the measurement. Further, since the method of estimating the activity of the enzyme electrode by exchanging with the reference immobilized enzyme membrane is to estimate the activity of the enzyme electrode through the reference immobilized enzyme membrane, the measurement side that cancels the decrease in the activity of the enzyme electrode When a factor (for example, the degree of drying of the enzyme electrode changes, the degree of adhesion of the reference immobilized enzyme membrane changes, etc.), the accuracy of the electrode activity determination is significantly reduced.

[0006] Further, according to the conventional biosensor, it is not possible to identify the cause of lowering the activity of the enzyme electrode, so that the activity of the enzyme electrode is lowered in order to perform an appropriate treatment for recovering the activity of the enzyme electrode. It was desired to obtain information on the cause.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and provides an electrode activity lowering state detection device for a biosensor capable of detecting the active state of an enzyme electrode simply and accurately. It is an object. Another object of the present invention is to provide a biosensor electrode activity lowering state detection device capable of obtaining information on the cause of the enzyme electrode activity lowering.

[0008]

In order to achieve the above object, an electrode activity lowering state detecting device for a biosensor according to claim 1 applies a predetermined voltage waveform between base electrodes to detect electrode activity lowering. Voltage waveform applying means, characteristic data generating means for generating predetermined characteristic data based on an electric signal generated between the base electrodes, and the predetermined voltage waveform is applied in a state in which the activity of the base electrode is sufficiently maintained. Active characteristic data holding means for holding the characteristic data when the base electrode is activated, and based on the characteristic data generated by the characteristic data generating means and the characteristic data during activation held in the active characteristic data holding means And a determination means for determining.

In addition, the above-described biosensor electrode activity lowering state detection device further includes an inhibitor substance that retains characteristic data when the predetermined voltage waveform is applied in a state where a specific substance that inhibits the electrode activity is attached to the base electrode. A characteristic data holding means, and a failure cause specifying means for specifying the cause of the activity lowering of the base electrode based on the characteristic data generated by the characteristic data generating means and the characteristic data of the inhibitor held in the inhibitor characteristic data holding means. You can also add.

[0010]

According to the biosensor electrode activity lowering state detecting device of the present invention, when the voltage waveform applying means applies a predetermined voltage waveform between the base electrodes, an electric power corresponding to the activity of the base electrode is generated corresponding to the predetermined voltage waveform. The signal is obtained. Then, the characteristic data generating means generates predetermined characteristic data based on the obtained electric signal. On the other hand, the active characteristic data holding means holds the characteristic data when the predetermined voltage waveform is applied in advance in the state where the activity of the base electrode is sufficiently maintained, and the determining means generates it by the characteristic data generating means. The decrease in the activity of the base electrode is determined on the basis of the feature data corresponding to the current activity of the base electrode and the feature data of the active time stored in the active feature data holding means. Therefore, since the activity of the base electrode can be determined by applying a predetermined voltage waveform between the base electrodes, the work of replacing the reference-immobilized enzyme membrane on the base electrode becomes unnecessary, and the activity of the base electrode can be remarkably easier than the conventional one. Can be determined. In addition, the method of generating characteristic data by applying a predetermined voltage waveform to the base electrode uses so-called cyclic voltammetry, etc., and is a method with high accuracy and excellent reproducibility, so the immobilized enzyme is used. The accuracy of discrimination of the active state can be improved as compared with the method of replacing the membrane.

Further, the above-mentioned biosensor electrode activity lowering state detecting device further includes an inhibitor substance which retains characteristic data when the predetermined voltage waveform is applied in a state where a specific substance inhibiting the electrode activity is attached to the base electrode. Characteristic data holding means, defect cause specifying means for specifying the cause of the activity decrease of the base electrode based on the characteristic data generated by the characteristic data generating means and the characteristic data of the inhibitor held in the inhibitor characteristic data holding means, If you add
It is possible to specifically specify the cause when the activity of the base electrode is lowered, and it is possible to perform the activity recovery process of the base electrode corresponding to each inhibitor.

[0012]

Embodiments will be described in detail below with reference to the accompanying drawings showing embodiments. FIG. 9 is a vertical cross-sectional view showing an example of an enzyme electrode applied to the electrode state detection device for the biosensor of this embodiment. The enzyme electrode 1 has a convex surface on one side of the enzyme electrode body 2, and is provided with a working electrode 3 made of Pt and a ring-shaped counter electrode 4 made of Ag in a state where the convex surface is exposed to the outside. Then, an immobilized GOD film 5 on which glucose oxidase (hereinafter abbreviated as GOD) is fixed so as to cover the convex surface, a diffusion limiting film 6 made of cellophane, and a blood cell separation film 7 are provided in this order. Then, the immobilized GOD film 5 and the diffusion limiting film 6
Is integrally attached to the enzyme electrode main body 2 by a first folder cap 8, and the blood cell separation membrane 7 is attached to the enzyme electrode main body 2 with the second folder cap 9 surrounding the first folder cap 8. It is installed against. It should be noted that the working electrode 3 and the signal output terminals 10 and 11 connected to the counter electrode 4 are provided at predetermined positions on the other side of the enzyme electrode body 2.

Therefore, an enzyme reaction of glucose + O ₂ + H ₂ O → gluconic acid + H ₂ O ₂ is carried out in the immobilized GOD film 5, and a current corresponding to the amount of H ₂ O ₂ produced opposes the working electrode 3. It is generated between the electrode 4 and is taken out to the outside through the signal taking-out terminals 10 and 11.

FIG. 1 is a block diagram showing an electrode state detecting device of a biosensor. The electrode state detection device of this biosensor includes an enzyme electrode 1 as shown in FIG. 9, a triangular wave voltage generator 21 for generating a triangular wave voltage to determine whether or not the enzyme electrode 1 is good, and a normal target electrode. Measuring voltage generator 22 for generating a measuring voltage for measuring the concentration of the test substance
, And the connection between the current-voltage conversion unit 25 and the triangular wave voltage generation unit 21 or the measured voltage generation unit 22 is a terminal and b, respectively.
The applied voltage changeover switch unit 23 selected by switching to the terminal, and the current-voltage conversion unit 25 having one end connected to the signal extraction terminal 11 and the other end connected to the applied voltage changeover switch unit 23, and a current-voltage conversion unit. A / D conversion unit 26 that converts an analog signal of the voltage converted by unit 25 into a digital signal, a calculation control unit 27 that performs calculation and determination processing described later, and a display unit that displays information from calculation control unit 27. 28 and. The terminals of the triangular wave voltage generator 21 and the measurement voltage generator 22 on the side opposite to the applied voltage changeover switch 23 side are connected to the signal extraction terminal 10.

FIG. 2 is a diagram showing a detailed configuration of the arithmetic control unit 27. The arithmetic control unit 27 has a signal processing changeover switch 31 connected to the A / D conversion unit 26 and a c side of the signal processing changeover switch 31. A characteristic data generation / holding unit 32 which is connected to a terminal and generates characteristic data for determining the quality of an electrode and holds the characteristic data, and an electrode quality reference data holding unit 33 which holds reference data for determining the quality of the electrode. And an electrode pass / fail discriminating unit 34 for discriminating the pass / fail of an electrode based on the characteristic data of the characteristic data generation / holding unit 32 and the reference data of the electrode pass / fail reference data holding unit 33, a signal processing changeover switch unit 31, and an applied voltage changeover switch unit. 23, a control unit 35 that outputs a switching signal, a defect cause reference data storage unit 36 that stores reference data of electrode defect causes, and A defect cause specifying unit 37 that specifies a cause of a defect based on the reference data of the cause reference data holding unit 36 and the characteristic data of the characteristic data generation holding unit 32, and a d-side terminal of a signal processing changeover switch, and is connected to a substance to be inspected. The measurement result calculation unit 38 calculates the measurement data result such as the concentration of

In this electrode state detecting device of the biosensor, an electrode quality check signal is output at a predetermined time before and after measuring the concentration of the substance to be inspected or when an operator inputs to inspect the activity of the enzyme electrode 1. Is configured to. FIG. 3 is a diagram showing an example of the triangular wave voltage generated by the triangular wave voltage generator 21. In this way, the working electrode 3
By sweeping the triangular wave voltage from 0.75V to -0.6V between the counter electrode 4 and the counter electrode 4 and back to 0.75V,
An electrochemical measurement called cyclic voltammetry can be performed. The cyclic voltammetry is information about the reaction on the electrode (for example,
It is one of the electrochemical measurement methods for obtaining equilibrium potential, electrode area, diffusion coefficient).

In the electrode state detecting device of this biosensor, cyclic voltammetry is used to determine whether or not the activity of the enzyme electrode 1 is at a normal level and, if necessary, to elucidate the cause of the activity decrease. I am trying. A schematic process flow of the electrode state detection device of the biosensor will be described with reference to a flowchart shown in FIG.

In FIG. 4, first, in step SP1, it is determined whether or not the electrode quality check signal is output. If it is determined that the electrode quality check signal is output, in step SP2 the triangular wave voltage is swept to cyclically. Voltammetric measurement is performed, and step S
At P3, feature data is generated based on the cyclic voltammetry measurement data. Then step S
In P4, whether or not the activity of the enzyme electrode 1 is normal is determined by comparing the generated characteristic data with reference data in advance when the activity of the enzyme electrode 1 is normal. If it is determined that, the enzyme electrode 1 is displayed as normal in step SP5, the concentration measurement process is performed by the measured voltage in step SP6, and the series of processes is ended.

On the other hand, in step SP4, the enzyme electrode 1
If it is determined that the activity of is not normal, step SP7
At the step S1, the operator is made to select whether or not to search for the cause of the defect of the enzyme electrode 1, and when it is determined that the worker should search for the cause of the defect of the enzyme electrode 1, the characteristic data generated in step SP8 and the characteristic data generated in advance are obtained. Based on the reference data of the cyclic voltammetry of the electrode activity inhibitor attached to the existing enzyme electrode 1, the electrode activity inhibitor is identified,
In step SP9, the specified inhibitor is displayed, and in step SP10, a treatment corresponding to each inhibitor (for example, if an electrode inhibitor is attached, the surface of the enzyme electrode 1 is washed with a solvent that removes the inhibitor). Is performed by the worker and the series of processes is completed. Further, when it is determined in step SP7 that the cause of the defect is not searched (for example, when the worker replaces the enzyme electrode 1), the electrode activity inhibiting substance is not specified, and the series of processes is ended.

The operation of the electrode state detecting device for the biosensor will be described in more detail. FIG. 5 is a diagram showing a voltage-current characteristic called cyclic voltamgram obtained by sweeping a triangular wave voltage when the enzyme electrode 1 is normal. In the figure, desorption of oxygen and adsorption of hydrogen occur in the process from + 0.75V to -0.6V (going process), and hydrogen desorption in the process from -0.6V to + 0.75V (return process). It is considered that the desorption and the adsorption of oxygen occur to obtain the voltage-current characteristic in which the current values flowing in the going and returning are different as shown in FIG.

FIG. 6 is a cyclic voltammogram when the activity of the enzyme electrode 1 is reduced due to the adhesion of Ag from the counter electrode 4 to the surface of the enzyme electrode 1, and FIG. 7 is the working electrode on the surface of the enzyme electrode 1. 3 is a diagram showing cyclic voltammgrams when the activity of the enzyme electrode 1 is reduced due to the adhesion of an epoxy resin component that fixes the counter electrode 3 and the counter electrode 4. FIG. Ag
In addition to the difference in current value due to the adsorption and desorption of hydrogen and oxygen described above, the peak 50a, which is considered to be caused by each reaction of Ag ⁺ + e ⁻ → Ag Ag → Ag ⁺ + e ⁻ , 50b
Occurs, and a unique voltage-current characteristic is exhibited.

When the epoxy resin is attached to the surface of the enzyme electrode 1, adsorption and desorption of hydrogen and oxygen are suppressed,
The difference between the going and returning current values is significantly reduced. Therefore, if the characteristic data of the voltage-current characteristic when the enzyme electrode 1 shown in FIG. 5 is normal is held as the electrode quality reference data in advance, the reference data is compared with the measured characteristic data of the voltage-current characteristic. By doing so, it is possible to determine how much the activity of the enzyme electrode has deteriorated compared to the normal state. Then, for example, it is possible to determine a normal or abnormal state of the enzyme electrode by setting a predetermined threshold value.

As a method for such determination, as shown in FIG. 8, a sweep voltage v and a function f (v, i) of a current i flowing between the electrodes are obtained by using a voltage-current characteristic curve when the activity of the enzyme electrode 1 is normal. ), It is determined whether or not the detected current i at the same voltage value on the way back and forth with respect to the function F (v, i) serving as the reference data is within the predetermined threshold Δd. There is a method of finally determining the active state by the number of voltage values within the threshold value Δd.

Further, as a method for determining whether Ag is attached to the enzyme electrode and the activity is reduced, the method shown in FIG.
Presence or absence of peaks 50a and 50b caused by adhesion of g, data of height H and width W of peaks 50a and 50b, voltage values Va and Vb generated by peaks 50a and 50b.
There is a method of preliminarily holding such as characteristic data. When it is determined that the enzyme electrode 1 is not normal, the electrode inhibitor can be identified based on the characteristic data of each electrode activity inhibitor. For example, as an example of distinguishing the state in which Ag is attached and the state in which the epoxy resin is attached, first, the threshold value described in FIG. 8 for the reference data of the state in which Ag is attached and the state in which the epoxy resin is attached Adhesion of Ag is determined by determining based on Δd and whether or not peaks 50a and 50b corresponding to Ag are present, and by confirming that peaks 50a and 50b are not present, determination of adhesion of epoxy resin. You can

As the electrode inhibiting substances other than the attachment of Ag and the attachment of epoxy resin, proteins in biological materials, extracts from enzyme membranes and the like are considered. It is easily understood that even when considering these electrode inhibitory substances, it is possible to similarly identify them by appropriately selecting appropriate characteristic data for distinguishing each electrode inhibitory substance. Next, the operation of the electrode state detection device for the biosensor shown in FIGS. 1 and 2 will be described.

First, when an electrode pass / fail check signal for determining whether the activity of the enzyme electrode 1 is lowered by a system or operator input at a predetermined time before and after measurement is input,
The control unit 35 of the arithmetic and control unit 27 causes the signal processing changeover switch unit 31 to switch so as to be connected to the c terminal side, and causes the applied voltage changeover switch unit 23 to be a.
Switching is performed so that it is connected to the terminal side. Then, when the sweep voltage as shown in FIG. 3 is applied between the working electrode 3 and the counter electrode 4 by the triangular wave voltage generation unit 21, the current i flowing between both electrodes is detected by the current-voltage conversion unit 25 and converted into a voltage. After the conversion, it is converted into a digital signal by the A / D conversion unit 26 and input to the arithmetic control unit 27. Then, the characteristic data generation / holding unit 32 in the arithmetic control unit 27 generates and holds the above-described characteristic data. Next, it is determined whether the activity of the enzyme electrode 1 is normal by comparing the reference data of the electrode quality reference data holding unit 33 with the generated feature data. And enzyme electrode 1
If it is determined that the activity of the enzyme electrode 1 is normal, the determination signal is output to the display unit 28 to display that the activity of the enzyme electrode 1 is normal. Further, the determination signal that the activity of the enzyme electrode 1 is normal is also output to the control unit 35, and the control unit 3
In response to the discrimination signal, the switch 5 switches so as to connect the signal processing changeover switch unit 31 to the d terminal side, and also connects the applied voltage changeover switch unit 23 to the b terminal side. Then, when the operator inputs an instruction to measure the concentration into the apparatus, the measurement voltage generation unit 22 generates the measurement voltage, obtains the conduction current by the measurement voltage, and the measurement result calculation unit 38 performs a predetermined calculation. As a result, the concentration of the substance to be inspected is calculated and displayed on the display unit 28.

On the other hand, when the electrode quality discriminating unit 34 discriminates that the activity of the enzyme electrode 1 is not normal, a discrimination signal indicating that the enzyme electrode 1 is not normal is output to the defect cause identifying unit 37, and the defect cause identifying unit 37 informs the operator. In response to the input of searching for the cause of electrode failure, the substance that inhibits the electrode activity is specified based on the defect cause reference data of the defect cause reference data holding unit 36 and the characteristic data of the characteristic data generation holding unit 32, and the inhibitor substance name and Predetermined data is displayed on the display unit 28 to prompt the operator to respond.

As described above, according to the electrode state detecting device for the biosensor, the triangular wave voltage generated by the triangular wave voltage generator 21 is applied between the working electrode 3 and the counter electrode 4, and the applied voltage is changed to the applied voltage. The activity of the enzyme electrode 1 is determined by detecting the accompanying conduction current, generating characteristic data, and comparing with the reference data when the activity of the enzyme electrode 1 is normal, so that the enzyme electrode 1 can be easily and accurately measured. It is possible to determine the active state of. Furthermore, since a substance that inhibits the activity of the enzyme electrode 1 can be specified as necessary, there is an advantage that the activity recovery process of the enzyme electrode 1 can be performed accurately.

The present invention is not limited to the above embodiment, and various design changes can be made without departing from the scope of the present invention. For example,
Of the triangular wave voltage applied between the electrodes, the going process (0.
It is also possible to determine whether the active state of the enzyme electrode 1 is normal or specify the electrode activity inhibitor by using only 75 V to −0.6 V).

[0030]

As described above, according to the first aspect of the invention, the discriminating means retains the characteristic data corresponding to the current activity of the base electrode generated by the characteristic data generating means and the activated characteristic data retaining means. Since the lowering of the activity of the base electrode is determined based on the characteristic data at the time of activation, it is not necessary to replace the reference immobilized enzyme membrane with the base electrode, and the activity of the base electrode can be determined significantly more easily than before. In addition to the above, there is a unique effect that the accuracy of discrimination of the active state can be improved.

[Brief description of drawings]

FIG. 1 is a block diagram showing an embodiment of an electrode activity lowering state detection device for a biosensor of the present invention.

FIG. 2 is a block diagram showing a detailed configuration of a calculation control unit of the electrode activity lowering state detection device of the biosensor of the present invention.

FIG. 3 is a diagram showing a triangular wave voltage waveform applied between a working electrode and a counter electrode.

FIG. 4 is a flow chart as an example of processing in the electrode activity lowering state detection device of the biosensor of the present invention.

FIG. 5 is a diagram showing a cyclic voltammgram when the activity of the enzyme electrode is sufficiently maintained.

FIG. 6 is a diagram showing a cyclic voltammgram when Ag is attached to the enzyme electrode surface and the activity of the electrode is inhibited.

FIG. 7 is a diagram showing a cyclic voltammgram in the case where the resin is attached to the enzyme electrode surface and the activity of the electrode is inhibited.

FIG. 8 is a diagram for explaining an example of a method of comparing detection data with reference data of cyclic voltamgram.

FIG. 9 is a sectional view of an enzyme electrode.

[Explanation of symbols]

 3 Working Electrode 4 Counter Electrode 21 Triangular Wave Voltage Generating Section 32 Characteristic Data Generation / Holding Section 33 Electrode Quality Reference Data Holding Section 34 Electrode Quality Determining Section

Claims (1)

[Claims] 1. An immobilizing enzyme membrane (4) having a physiologically active substance immobilized thereon is provided on the surface of the underlying electrode (3) (4), whereby the underlying electrode (3) (4) is obtained based on the result of the enzyme reaction. )
In order to detect a decrease in activity of the base electrodes (3) and (4) in a biosensor that generates an electric signal by using the biosensor and measures the target substance based on the generated electric signal, the base electrode (3)
Voltage waveform applying means (21) for applying a predetermined voltage waveform between (4) and characteristic data generating means (32) for generating predetermined characteristic data based on an electric signal generated between the base electrodes (3) and (4). An active characteristic data holding means (33) for holding characteristic data when the predetermined voltage waveform is applied in a state where the base electrodes (3) and (4) are sufficiently activated, and characteristic data generating means. (32) Feature data generated by (32) and feature data holding means for activation (3
Discriminating means (3) for discriminating a decrease in the activity of the base electrodes (3) and (4) based on the active characteristic data held in 3).
4) The device for detecting a decreased electrode activity state of a biosensor, comprising: