JP5073629B2 - Measurement display device to which biosensor is attached and measurement method - Google Patents

Description

  The present invention relates to a measurement display device and a measurement method for measuring a component amount (concentration) of a substrate of a specimen with a biosensor attached.

  Conventionally, various biosensors and measurement display devices including the following patent documents have been developed and disclosed. FIG. 3 shows an example of the biosensor 14. A measuring working electrode 18 and a measuring counter electrode 20 are formed on a PET resin substrate 16. These electrodes 18 and 20 are made of platinum. A reaction part 26 containing an enzyme is formed on the electrodes 18 and 20. When an enzyme and a sample such as blood react, electrons are exchanged. When a voltage is applied to the measurement working electrode 18 by the measurement display device, a current flows between the measurement working electrode 18 and the measurement counter electrode 20. The value of the current that flows depends on the amount of the substrate component of the specimen. The measurement display device measures the current and determines the amount of the substrate component of the specimen from the current value.

  The biosensor 14 is provided with a cover 48 that is opposed to the reaction unit 26 at a predetermined interval by a spacer 46. The sample is sucked from the supply port 50 at the end of the biosensor 14 and gradually spreads by capillary action. Therefore, before actually measuring the amount of the substrate component of the specimen, it is checked whether the specimen has spread sufficiently on the reaction part. For this check, a detection working electrode 22 and detection counter electrodes 24 a and 24 b are provided in the vicinity of the measurement working electrode 18 and the measurement counter electrode 20. In the example of FIG. 3, there are two detection counter electrodes 24a and 24b, the end side of the biosensor 14 is the first detection counter electrode 24a, and the other is the second detection counter electrode 24b. The detection working electrode 22 and the first detection counter electrode 24a detect that the specimen is spotted. The detection working electrode 22 and the second detection counter electrode 24b detect that the specimen has spread over the reaction unit 26. The spread of the specimen on the reaction unit 26 is called inhalation.

  The checking method is performed by the following method. (1) A constant voltage V3 is applied to the detection working electrode 22 (FIG. 4). (2) While the constant voltage V3 is applied, the current value is measured at regular intervals from the detection counter electrodes 24a and 24b. In FIG. 4, the measurement is performed at a black circle. (3) A threshold value is provided in advance, and it is determined that the sample has been sufficiently inhaled onto the reaction unit 26 when the current value exceeds the threshold value, and the substrate component amount is measured.

  However, when the current value is measured at regular intervals, the peak Ip may be missed. For example, as shown in FIG. 4, even if the peak Ip exceeds the threshold, measurement is not performed when the peak Ip is higher than the threshold. It cannot be detected that the sample has been sufficiently inhaled on the reaction unit 26, and the process cannot proceed to measurement of the amount of the substrate component. Since the reaction between the enzyme and the sample is only once, it is necessary to start over from the beginning using a new biosensor.

  Note that all of the following patent documents are methods of applying a constant voltage, and there is a possibility that detection cannot be performed as described above.

Japanese Patent No. 2901678 Patent No. 2800981 Japanese Patent Laid-Open No. 4-357252

  An object of the present invention is to provide a measurement display device and a measurement method that can reliably check that a specimen is sufficiently inhaled onto an enzyme.

  The biosensor attached to the measurement display device includes an insulating substrate, a measurement working electrode provided on the substrate, and a measurement counter electrode provided at a predetermined interval with respect to the measurement working electrode. And on the substrate, the detection working electrode provided on the periphery of the measurement working electrode and the measurement counter electrode, and on the substrate, provided on the periphery of the measurement working electrode and the measurement counter electrode, A detection counter electrode paired with the detection working electrode; and the measurement working electrode, the measurement counter electrode, the detection working electrode, and a reaction part including an enzyme provided on the detection counter electrode. Prepare.

  The measurement display device measures a detection current (non-Faraday current) that flows when the pulse voltage is applied, and means for applying a pulse voltage at regular intervals between the detection working electrode and the detection counter electrode. Means.

  Further, the measurement display device is configured to apply a voltage between the measurement working electrode and the measurement counter electrode to generate a potential difference, and the measurement that has flowed between the measurement working electrode and the measurement counter electrode. Means for measuring an electric current (electrolytic current), and means for determining the amount of a substrate component of the sample that has reacted with the enzyme from the current value measured by the means for measuring the measuring current, wherein the detection current is less than a threshold value. Is higher, a voltage is applied between the measuring working electrode and the measuring counter electrode.

  The method for measuring the amount of a substrate component of a specimen using the measurement display device includes a step of applying a pulse voltage at a constant interval between the detection working electrode and the detection counter electrode, and when applying the pulse voltage Measuring the detection current flowing in the circuit.

  A step of applying a voltage between the measuring working electrode and the measuring counter electrode to generate a potential difference; and measuring a measuring current flowing between the measuring working electrode and the measuring counter electrode. And a step of obtaining the amount of a substrate component of the sample that has reacted with the enzyme from the current value measured by the means for measuring the measurement current, and when the detection current is higher than a threshold value, the working electrode for measurement And a voltage is applied between the counter electrode for measurement.

  Since the present invention detects a non-Faraday current generated in response to a pulse voltage, it cannot be overlooked that the specimen is sufficiently inhaled on the enzyme. Since the inhalation of the sample can be reliably checked, it is possible to reliably shift to the measurement of the amount of the substrate component of the sample thereafter.

  The present invention will be described with reference to the drawings. Examples of the sample to be measured include liquid samples such as blood and urine. As shown in FIG. 1, the measurement display device 12 is a part of the sample measurement device 10, and the sample measurement device 10 includes a biosensor 14 in addition to the measurement display device 12. The biosensor 14 is replaceable because the enzyme and the sample cause an irreversible reaction when measuring the amount of the substrate component of the sample. First, the biosensor 14 will be described.

  The biosensor 14 shown in FIG. 1 shows electrodes, and is actually the biosensor 14 shown in FIG. The biosensor 14 includes a substrate 16 made of an insulator, a plurality of electrodes provided on the substrate 16, and a reaction unit 26 provided on the electrodes.

  The insulator constituting the substrate 16 is, for example, a polyester resin sheet such as polyethylene terephthalate (PET), polyethylene naphthalate, a biodegradable polyester resin composed of an aliphatic unit and an aromatic unit, heat resistance, chemical resistance, and strength. Such as a polyamide sheet, a plastic sheet such as a polyimide film sheet, and an inorganic substrate such as a ceramic.

  The electrodes include the measurement working electrode 18, the measurement counter electrode 20, the detection working electrode 22, and the detection counter electrodes 24a and 24b. Each electrode is formed of a conductor such as platinum, gold, palladium, or indium-tin oxide. Examples of the forming method include hot stamping and the like, but a method by vacuum evaporation or sputtering is preferable because a fine electrode pattern can be formed with high accuracy. In the case of sputtering, it can be formed all at once by masking the outside of electrode formation.

  The measuring working electrode 18 and the measuring counter electrode 20 are opposed to each other with a predetermined interval. Both the electrodes 18 and 20 are substantially semicircular, and the both electrodes 18 and 20 have a shape close to a circle. These electrodes 18 and 20 are used for measuring the amount of the substrate component of the specimen.

  The detection working electrode 22 and the detection counter electrodes 24 a and 24 b are provided in a non-contact manner on the periphery of the measurement working electrode 18 and the measurement counter electrode 20. The detection working electrode 22 and the detection counter electrodes 24 a and 24 b are used to check whether or not the sample has been inhaled onto the reaction unit 26.

  There are two detection counter electrodes 24a and 24b. For convenience of description, the first detection counter electrode 24a and the second detection counter electrode 24b are used. Since the detection working electrode 22 and the first detection counter electrode 24a are at the end of the biosensor 14, it can be checked that the specimen has been spotted. Since the detection working electrode 22 and the second detection counter electrode 24 b are provided so as to sandwich the measurement working electrode 18 and the measurement counter electrode 20, it is possible to check whether the specimen has been sufficiently inhaled onto the reaction unit 26. The first detection counter electrode 24a and the second detection counter electrode 24b operate alternately and are paired with the detection working electrode 22 alternately.

  The reaction part 26 is a film containing an oxidoreductase and an electron acceptor. The reaction unit 26 is formed by dropping a liquid material at a desired position with a dispenser and drying it.

  When the sample is inhaled on the reaction unit 26, the reaction unit 26 and the sample react, and when a potential difference is generated between the electrodes, electrons are transferred. At this time, transfer of electrons proportional to the amount of the substrate component of the specimen can be caused by the material of the reaction unit 26. For example, in the case of blood, the amount of electrons exchanged depends on the difference in blood sugar level.

  In addition, a transparent cover 48 that faces the reaction unit 26 at a predetermined interval is provided as necessary. When a sample is spotted on the supply port 50 at the tip of the biosensor 14, the sample penetrates into the inside by capillary action.

  A terminal 28 extended from each electrode of the biosensor 14 and a terminal 30 of the measurement display device 12 are connected. The measurement display device 12 performs voltage application and current measurement by the control means 32. Specifically, the control means 32 includes a circuit that operates as follows, software, or both.

  The control means 32 includes a power supply unit 34 and a measurement unit 36. The power supply 34 applies pulse voltages V1 and V2 to the detection working electrode 22 at regular intervals (FIG. 2). The measuring unit 36 measures the non-Faraday current Io that flows between the detection working electrode 22 and the detection counter electrodes 24a and 24b when the pulse voltages V1 and V2 are applied.

  Conventionally, the constant voltage V3 is applied as shown in FIG. 4, but the reason why the present invention applies the pulse voltages V1 and V2 will be described. When the specimen is inhaled from the detection working electrode 22 to the detection counter electrodes 24a and 24b, the configuration is the same as that of the electric double layer capacitor. That is, the electrolytic solution of the electric double layer capacitor is replaced with the specimen. When a voltage is applied at this time, a non-Faraday current flows in the same manner as when the electric double layer capacitor is charged. Conventionally, since the constant voltage V3 is applied, the non-Faraday current Io flows at the moment when the specimen is sufficiently inhaled on the enzyme (FIG. 4). If no voltage is applied when the sample is inhaled, a non-Faraday current Io flows when a voltage is applied thereafter. If the pulse voltages are V1 and V2 as in the present invention, the non-Faraday current Io flows by the pulse voltages V1 and V2 after the sample is inhaled. Since the non-Faraday current Io flows in synchronization with the pulse voltages V1 and V2, if the current is measured in synchronization with the pulse voltages V1 and V2, it is not overlooked that the specimen has been sufficiently inhaled. Since the non-Faraday current Io flows for a very short time, the non-Faraday current Io can hardly be measured if the current is detected at an integral interval as in the prior art.

  The pulse voltages V1 and V2 generated by the power supply unit 34 include a first pulse voltage V1 for detection by the first detection counter electrode 24a and a second pulse voltage V2 for detection by the second detection counter electrode 24b ( Figure 2). The power supply unit 34 alternately outputs the respective pulse voltages V1 and V2, and switches the detection counter electrodes 24a and 24b operating in accordance with the pulse voltages V1 and V2.

  The pulse voltages V1 and V2 are appropriately designed according to the type of enzyme and the substrate to be measured. As an example of each of the pulse voltages V1 and V2, the cycle is 50 ms, the pulse width is 1 to 2 ms, and the pulse height is 0.2V. The second pulse voltage V2 is output between the first pulse voltages V1.

  A digital / analog conversion circuit 38 and a switch S1 are provided between the power supply unit 34 and the detection working electrode 22. Since the control means 32 is controlled by digital data, the voltage is applied after being converted into an analog value when a voltage is applied to the electrode 22. By turning on the switch S1, the power supply unit 34 and the detection working electrode 22 are connected.

  The measuring unit 36 measures the current flowing between the electrodes when the power supply unit 34 outputs the pulse voltages V1 and V2. The measurement timing may be any time as long as the pulse voltages V1 and V2 are being applied. For example, the measurement is performed when the pulse voltages V1 and V2 fall. In FIG. 2, the measurement is performed at a black circle. The measuring unit 36 stores a threshold value for the non-Faraday current Io. When the measured current value exceeds the threshold, the power supply unit 34 and the measurement unit 36 are switched so that the substrate component amount of the specimen can be measured. The threshold value is appropriately changed according to the type of specimen and the substrate to be measured.

  Between the detection counter electrodes 24a and 24b and the measurement unit 36, switches S2 and S3, a current-voltage conversion circuit 40, and an analog / digital conversion circuit 42 are provided. The current / voltage conversion circuit 40 converts the current into a voltage. The analog / digital conversion circuit 42 converts it into digital data so that processing can be performed in the control means 32. The first detection counter electrode 24a and the second detection counter electrode 24b are switched by switches S2 and S3 and connected to the measurement unit 36.

  After the non-Faraday current Io flows, the reaction between the enzyme and the specimen proceeds, and when a voltage is applied, an electrolytic current flows. By measuring this electrolytic current, the amount of the substrate component of the specimen is measured. Therefore, the operation of the power supply unit 34 and the measurement unit 36 is switched as follows. The power supply unit 34 applies a predetermined voltage to the measuring working electrode 18. The measurement unit 36 measures the measurement current flowing between the measurement working electrode 18 and the measurement counter electrode 20. At this time, the electrodes to be used are switched by turning on the switches S4 and S5 and turning off the other switches S1, S2 and S3.

  Furthermore, a calculation unit 44 that obtains the amount of the substrate component of the specimen from the current value measured by the measurement unit 36 is provided. Since the current value varies depending on the amount of the substrate component, the amount of the substrate component is obtained by performing a predetermined calculation or table conversion.

  Switches S1 to 5 use elements such as MOSFETs that can be turned on / off electrically. A signal is input to the gate from the control means 32 to switch on / off.

  In addition, a memory used when calculating the amount of the substrate component, a display for displaying the amount of the substrate component, a button for turning the apparatus 12 on and off, and the like are provided.

  Next, a method for measuring the amount of the substrate component using the measurement display device 12 will be described. (1) The biosensor 14 is connected to the measurement display device 12. (2) The pulse voltages V1 and V2 are applied from the power supply unit 34 to the detection working electrode 22 at regular intervals. (3) The measuring unit 36 measures the current flowing between the measuring working electrode 22 and the measuring counter electrodes 24a and 24b by applying the pulse voltages V1 and V2. (4) When the current value exceeds the threshold value, the power supply unit 34 and the measurement unit 36 switch so as to measure the amount of the substrate component of the specimen. (5) The power supply unit 34 applies a predetermined voltage to the working electrode 18 for measurement. (6) The measuring unit 36 measures the current flowing between the measuring working electrode 18 and the measuring counter electrode 20. (7) The calculation unit 44 obtains the substrate component amount from the current value measured by the measurement unit 36.

  As described above, the present invention utilizes the electric double layer phenomenon that occurs when a DC voltage is applied to the specimen, and a non-Faraday current is passed between the working electrode and the counter electrode to confirm whether the specimen is inhaled. Since a non-Faraday current always flows according to the pulse voltage, the inhalation of the specimen is not missed.

  As mentioned above, although embodiment of this invention was described, this invention is not limited to said embodiment. For example, although there is one detection working electrode 22 and two detection counter electrodes 24a and 24b, the number may be reversed. The power supply unit 34 alternately applies a pulse voltage to the two detection working electrodes, and the measurement unit 36 connected to the detection counter electrode measures the current.

  The numbers of the detection working electrode 22 and the detection counter electrodes 24a and 24b are appropriately changed according to the structure of the biosensor 14 to detect that the specimen has been inhaled onto the reaction unit 26. For example, only the detection working electrode 22 and the second detection counter electrode 24b of FIG. 3 may be used. Without confirming that the specimen has been spotted, it is confirmed only that the specimen has been sufficiently inhaled onto the reaction unit 26.

  Although the switches S1 to S5 are shown in FIG. 1, the switches S1 to S5 may be incorporated in the power supply unit 34 or the measurement unit 36. Means for checking whether or not the terminals extending from the electrodes are connected to the measurement display device may be provided.

  Note that when the amount of the sample is smaller than the predetermined amount, the sample does not spread sufficiently on the reaction part. Therefore, a timer may be provided so that an electric current flows through only one detection electrode, and if no electric current flows through the other detection electrode for a certain period of time, it is determined that an error has occurred and that fact is displayed on the display. The user replaces the biosensor, causes the new biosensor to aspirate the sample, and measures again.

  In FIG. 3, the reaction portion 26 covers the detection working electrode 22 and the detection counter electrodes 24 a and 24 b, but the non-Faraday current Io flows even without the reaction portion 26. Therefore, the reaction portion 26 may not be provided on the detection working electrode 22 and the detection counter electrodes 24a and 24b. In addition, since the electrolysis current after the non-Faraday current Io flows without the reaction part 26, the reaction part 26 is always provided on the measurement working electrode 18 and the measurement counter electrode 20.

  In addition, the present invention can be carried out in a mode in which various improvements, modifications, and changes are added based on the knowledge of those skilled in the art without departing from the gist thereof.

It is a figure which shows the structure of the measurement display apparatus of this invention. It is a figure which shows the relationship between the pulse voltage which the measurement display apparatus of this invention outputs, the inhalation amount of a test substance, and a non-Faraday current. It is a figure which shows a biosensor, (a) is a front view, (b) is an AA sectional view. It is a figure which shows the relationship between the voltage which the conventional measurement display apparatus outputs, the inhalation amount of a test substance, and electrolysis current.

Explanation of symbols

10: Specimen measuring device 12: Measurement display device 14: Biosensor 16: Substrate 18: Measurement working electrode 20: Measurement counter electrode 22: Detection working electrode 24a, 24b: Detection counter electrode 26: Reaction unit 28, 30 : Terminal 32: control means 34: power supply unit 36: measuring unit 38: digital / analog conversion circuit 40: current / voltage conversion circuit 42: analog / digital conversion circuit 44: calculation unit 46: spacer 48: cover 50: supply port

Claims (4)

An insulating substrate;
A working electrode for measurement provided on the substrate;
A counter electrode for measurement provided at a constant interval with respect to the working electrode for measurement;
A detection working electrode provided on the substrate;
A counter electrode for detection provided on the substrate and operating when a voltage is applied to the working electrode for detection;
A reaction part containing an enzyme covering at least the working electrode for measurement and the counter electrode for measurement;
A biosensor comprising
Means for applying a pulse voltage at regular intervals between the working electrode for detection and the counter electrode for detection;
Means for measuring a detection current that flows when the pulse voltage is applied;
A measurement display device comprising: Means for applying a voltage between the working electrode for measurement and the counter electrode for measurement to generate a potential difference;
Means for measuring a measuring current flowing between the measuring working electrode and the measuring counter electrode;
Means for determining the amount of the substrate component of the specimen that has reacted with the enzyme from the current value measured by the means for measuring the measurement current;
With
The measurement display device according to claim 1, wherein when the detection current is higher than a threshold value, a voltage is applied between the measurement working electrode and the measurement counter electrode. An insulating substrate;
A working electrode for measurement provided on the substrate;
A counter electrode for measurement provided at a constant interval with respect to the working electrode for measurement;
A detection working electrode provided on the substrate;
A counter electrode for detection provided on the substrate and operating when a voltage is applied to the working electrode for detection;
A reaction part containing an enzyme covering at least the working electrode for measurement and the counter electrode for measurement;
A biosensor comprising
Applying a pulse voltage at regular intervals between the working electrode for detection and the counter electrode for detection;
Measuring a detection current that flows when the pulse voltage is applied;
A method for measuring a substrate component amount of a specimen comprising Applying a voltage between the measuring working electrode and the measuring counter electrode to generate a potential difference;
Measuring a measurement current flowing between the measurement working electrode and the measurement counter electrode;
Determining the amount of a substrate component of a sample that has reacted with an enzyme from the current value measured by the means for measuring the measurement current;
With
The measurement method according to claim 3, wherein a voltage is applied between the measurement working electrode and the measurement counter electrode when the detection current is higher than a threshold value.

Images