JP2009270980A - Biosensor, enzyme sensor, and gas detecting system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor, an enzyme sensor, and a gas detecting system, easily detecting a detection object material in a gas sample with a simple constitution. <P>SOLUTION: An enzyme sensor 100 includes: a gas phase chamber R1 where a gas containing a detection object material is introduced; a liquid phase chamber R2, disposed to be adjacent to the gas phase chamber R1, where a predetermined electrolytic solution is introduced; a gas-permeable membrane 130, disposed so as to separate the gas phase chamber R1 from the liquid phase chamber R2, where at least the detection object material passes through; an electrode 150 disposed to face to the gas gas-permeable membrane 130 in the liquid phase chamber R2; and an enzyme, involved in the liquid phase chamber R2, as a receptor which selectively react with the detection object material. The sensor is constituted so as to detect the detection object material which passed through the gas-permeable membrane 130 and transferred from the gas phase chamber R1 to the liquid phase chamber R2. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a biosensor, an enzyme sensor, and a gas detection system.

Conventionally, research and development of biosensors for detecting a specific substance in a sample have been actively conducted.
Specifically, for example, a biosensor that detects glucose concentration in jelly, a biosensor that detects free amino acids, fatty acids, sugars, etc. in fish meat (see, for example, Patent Document 1), ammonia nitrogen concentration in wastewater Biosensor to detect (for example, see Patent Document 2), biosensor to detect amino acid concentration in the electrolyte (for example, see Patent Document 3), biosensor to detect lactic acid content in tomato juice (for example, Patent Document) 4)) has been proposed.
Japanese Patent No. 2690053 Japanese Patent Publication No. 06-072858 Japanese Patent Laid-Open No. 06-043131 Japanese Patent Application Laid-Open No. 08-327581

  However, when utilizing the characteristics of a receptor (such as an enzyme or a microorganism) as in the biosensors described in Patent Documents 1 to 4, the presence of water is necessary to make the receptor function. Therefore, in order to detect the detection target substance in the gas sample, it is necessary to perform a process such as dissolving the detection target substance in the gas sample in water, which is troublesome.

  An object of the present invention is to provide a biosensor, an enzyme sensor, and a gas detection system that can easily detect a detection target substance in a gas sample with a simple configuration.

In order to solve the above-mentioned problem, the invention described in claim 1
In biosensors,
A gas phase chamber into which a gas containing a detection target substance is introduced;
A liquid phase chamber disposed adjacent to the gas phase chamber and into which a predetermined electrolyte is introduced;
A gas permeable membrane that is disposed so as to separate the gas phase chamber and the liquid phase chamber and allows at least the detection target substance to pass through;
An electrode disposed opposite to the gas permeable membrane in the liquid phase chamber;
A receptor contained in the liquid phase chamber and selectively reacting with the detection target substance,
The detection target substance that has passed through the gas permeable membrane and has moved from the gas phase chamber to the liquid phase chamber is detected.

The invention described in claim 2
The biosensor according to claim 1, wherein
A spacer is provided between the gas permeable membrane and the electrode.

The invention according to claim 3
The biosensor according to claim 1 or 2,
Constituting a surface of the liquid phase chamber facing the gas phase chamber, and comprising a support for supporting the electrode;
The support has a groove,
The electrode is formed in the groove;
The thickness of the electrode is substantially the same as the depth of the groove.

The invention according to claim 4
In the biosensor according to any one of claims 1 to 3,
An electrode part having the electrode;
An attachment portion for attaching the electrode portion to the liquid phase chamber,
The electrode part is detachable from the attachment part.

The invention described in claim 5
In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber is provided.

The invention described in claim 6
In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
An electrode temperature adjusting means for adjusting the temperature of the electrode is provided.

The invention described in claim 7
The gas detection system according to claim 6.
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Determination means for determining the temperature of the electrode at the time of detection of the detection target substance by the biosensor based on the response data acquired by the acquisition means;
An adjustment control means for causing the electrode temperature adjustment means to adjust the temperature of the electrode so that the temperature is determined by the determination means;
It is characterized by providing.

The invention according to claim 8 provides:
In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
Electrolytic solution temperature adjusting means for adjusting the temperature of the electrolytic solution introduced into the liquid phase chamber is provided.

The invention according to claim 9 is:
The gas detection system according to claim 8.
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Based on the response data acquired by the acquisition unit, a determination unit that determines the temperature of the electrolytic solution at the time of detection of the detection target substance by the biosensor;
Adjustment control means for causing the electrolyte temperature adjusting means to adjust the temperature of the electrolyte so as to be the temperature determined by the determining means;
It is characterized by providing.

The invention according to claim 10 is:
A gas detection system comprising the biosensor according to claim 4.
The receptor is contained in the liquid phase chamber by being fixed to the electrode,
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Determining means for determining whether or not to replace the electrode unit based on the response data acquired by the acquiring means;
It is characterized by providing.

The invention according to claim 11
In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
The receptor is contained in the liquid phase chamber by being contained in the electrolytic solution introduced into the liquid phase chamber,
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Determining means for determining whether or not to replace the electrolyte based on the response data acquired by the acquiring means;
It is characterized by providing.

The invention according to claim 12
In enzyme sensor,
A gas phase chamber into which a gas containing a detection target substance is introduced;
A liquid phase chamber disposed adjacent to the gas phase chamber and into which a predetermined electrolyte is introduced;
A gas permeable membrane that is disposed so as to separate the gas phase chamber and the liquid phase chamber and allows at least the detection target substance to pass through;
An electrode disposed opposite to the gas permeable membrane in the liquid phase chamber;
An enzyme contained in the liquid phase chamber and selectively reacting with the detection target substance;
A spacer disposed between the gas permeable membrane and the electrode;
A surface of the liquid phase chamber that faces the gas phase chamber, and a support that supports the electrode; and
The support has a groove,
The electrode is formed in the groove;
The thickness of the electrode is substantially the same as the depth of the groove,
The detection target substance that has passed through the gas permeable membrane and has moved from the gas phase chamber to the liquid phase chamber is detected.

The invention according to claim 13
A gas detection system comprising the enzyme sensor according to claim 12,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
Electrode temperature adjusting means for adjusting the temperature of the electrode;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determination means for determining the temperature of the electrode at the time of detection of the detection target substance by the enzyme sensor based on the response data acquired by the acquisition means;
An adjustment control means for causing the electrode temperature adjustment means to adjust the temperature of the electrode so that the temperature is determined by the determination means;
It is characterized by providing.

The invention according to claim 14
A gas detection system comprising the enzyme sensor according to claim 12,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
An electrolyte temperature adjusting means for adjusting the temperature of the electrolyte introduced into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Based on the response data acquired by the acquisition unit, a determination unit that determines the temperature of the electrolytic solution at the time of detection of the detection target substance by the enzyme sensor;
Adjustment control means for causing the electrolyte temperature adjusting means to adjust the temperature of the electrolyte so as to be the temperature determined by the determining means;
It is characterized by providing.

The invention according to claim 15 is:
A gas detection system comprising the enzyme sensor according to claim 12,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
Electrode temperature adjusting means for adjusting the temperature of the electrode;
An electrolyte temperature adjusting means for adjusting the temperature of the electrolyte introduced into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determination means for determining the temperature of the electrode and the temperature of the electrolytic solution at the time of detection of the detection target substance by the enzyme sensor based on the response data acquired by the acquisition means;
An adjustment control means for causing the electrode temperature adjustment means to adjust the temperature of the electrode so that the temperature is determined by the determination means, and for causing the electrolyte temperature adjustment means to adjust the temperature of the electrolyte solution;
It is characterized by providing.

The invention described in claim 16
In enzyme sensor,
A gas phase chamber into which a gas containing a detection target substance is introduced;
A liquid phase chamber disposed adjacent to the gas phase chamber and into which a predetermined electrolyte is introduced;
A gas permeable membrane that is disposed so as to separate the gas phase chamber and the liquid phase chamber and allows at least the detection target substance to pass through;
An electrode disposed opposite to the gas permeable membrane in the liquid phase chamber;
An enzyme contained in the liquid phase chamber and selectively reacting with the detection target substance;
A spacer disposed between the gas permeable membrane and the electrode;
An electrode part having the electrode;
An attachment portion for attaching the electrode portion to the liquid phase chamber,
The electrode part is detachable from the attachment part,
The detection target substance that has passed through the gas permeable membrane and has moved from the gas phase chamber to the liquid phase chamber is detected.

The invention described in claim 17
A gas detection system comprising the enzyme sensor according to claim 16,
The enzyme is contained in the liquid phase chamber by being fixed to the electrode,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determining means for determining whether or not to replace the electrode unit based on the response data acquired by the acquiring means;
It is characterized by providing.

The invention described in claim 18
A gas detection system comprising the enzyme sensor according to claim 12 or 16,
The enzyme is contained in the liquid phase chamber by being contained in the electrolyte solution introduced into the liquid phase chamber,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determining means for determining whether or not to replace the electrolyte based on the response data acquired by the acquiring means;
It is characterized by providing.

According to the present invention, the biosensor includes a gas phase chamber into which a gas containing a detection target substance is introduced, a liquid phase chamber that is disposed adjacent to the gas phase chamber and into which a predetermined electrolyte is introduced, The gas phase chamber is separated from the liquid phase chamber, and is contained in the liquid phase chamber, at least a gas permeable membrane through which the detection target substance permeates, an electrode disposed in the liquid phase chamber facing the gas permeable membrane, and And a receptor that selectively reacts with the detection target substance, and detects the detection target substance that has passed through the gas permeable membrane and moved from the gas phase chamber to the liquid phase chamber.
That is, the biosensor has a simple configuration in which a gas phase chamber and a liquid phase chamber are disposed via a gas permeable membrane. In addition, when a detection target substance in a gas sample is detected using a biosensor, it is not necessary to perform a process such as dissolving the detection target substance in the gas sample in water, and the gas sample is directly introduced into the gas phase chamber. Therefore, the detection target substance in the gas sample can be easily detected.

Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings. The scope of the invention is not limited to the illustrated example.
In the present embodiment, an enzyme sensor will be described as an example of a biosensor.

[First Embodiment]
First, the gas detection system 1000 provided with the enzyme sensor 100 and the enzyme sensor 100 in 1st Embodiment is demonstrated.

<Configuration of gas detection system>
FIG. 1 is a diagram illustrating a configuration of the gas detection system 1000, and FIG. 2 is a diagram illustrating a functional configuration of the gas detection system 1000.

  As shown in FIGS. 1 and 2, for example, the gas detection system 1000 includes an enzyme sensor 100, a measurement circuit 210, a data processing device 220, a data display device 230, a notification lamp 240, an electrolyte tank 310, and the like. The standard liquid providing device 320, the waste liquid tank 330, the liquid feeding pump 340, the sensor temperature adjusting device 410, the electrolyte temperature adjusting device 420, the intake pump 510, the dust filter 520, and the standard gas providing device 530 The valve switching device 600, the control device 700, and the like are configured.

The enzyme sensor 100 included in the gas detection system 1000 includes an electrode 150, and is a sensor that detects a detection target substance in a gas sample by an electrochemical measurement method using characteristics of the enzyme. The enzyme sensor 100 has a gas phase chamber R1 into which a gas (gas sample) containing a substance to be detected is introduced and a liquid phase chamber R2 into which a predetermined electrolytic solution is introduced, and the electrode 150 is a liquid. Arranged in the phase chamber R2, the enzyme is contained in the liquid phase chamber R2.
In the enzyme sensor 100, the enzyme may be contained in the liquid phase chamber R2 in the state of an immobilized enzyme, or may be contained in the liquid phase chamber R2 in the state of a free enzyme. Hereinafter, the enzyme sensor 100 in which the enzyme is contained in the liquid phase chamber R2 in the state of the immobilized enzyme may be referred to as “fixed enzyme sensor”, and the enzyme is contained in the liquid phase chamber R2 in the state of the free enzyme. The enzyme sensor 100 may be referred to as a “free enzyme sensor”.

Specifically, in the “fixed enzyme sensor”, for example, the enzyme is fixed to the electrode 150, or is fixed to a predetermined carrier disposed in the liquid phase chamber R2, thereby causing the enzyme to flow into the liquid phase chamber R2. It is supposed to be contained in Hereinafter, the enzyme sensor 100 in which the enzyme is fixed to the electrode 150 may be referred to as “electrode-fixed enzyme sensor”, and the enzyme sensor 100 in which the enzyme is fixed to a predetermined carrier is referred to as “carrier-fixed enzyme sensor”. May be called.
In the “free enzyme sensor”, for example, by using an electrolyte containing an enzyme as an electrolyte introduced into the liquid phase chamber R2, the enzyme is contained in the liquid phase chamber R2.

  For example, the measurement circuit 210 applies a voltage to the enzyme sensor 100 according to a control signal input from the control device 700, measures a response current from the enzyme sensor 100, and outputs the measurement signal to the data processing device 220. To do.

For example, the data processing device 220 processes the measurement signal input from the measurement circuit 210 in accordance with the control signal input from the control device 700, creates numerical data based on the measurement signal, and outputs the numerical data to the data display device 230. To do.
Here, the numerical data is arbitrary as long as it is data relating to numerical values such as response current and concentration (concentration of the detection target substance determined from the response current and a calibration curve prepared in advance), for example, response current It may be data relating to numerical values themselves such as or density, or data relating to changes in numerical values.

For example, the data display device 230 displays numerical information based on numerical data input from the data processing device 220 in accordance with a control signal input from the control device 700.
Here, the displayed numerical information may be, for example, numerical values such as response current or concentration, or graphs of changes in numerical values (for example, a graph showing a calibration curve or a change with time) Graph).

  For example, the notification lamp 240 lights up or blinks in accordance with a control signal input from the control device 700.

The electrolyte solution tank 310 is a tank for storing the electrolyte solution. For example, the enzyme sensor 100 (electrolyte solution introduction side of the liquid phase chamber R2), the first valve 610, the liquid feed pump 340, and predetermined valves are provided. And is connected to the enzyme sensor 100 (electrolyte discharge side of the liquid phase chamber R2) via a second valve 620 and a tube.
Specifically, for example, when the enzyme sensor 100 is a “fixed enzyme sensor”, the electrolyte solution in the electrolyte tank 310 is an electrolyte solution (buffer solution) that does not contain an enzyme. In the case of a “type enzyme sensor”, it is an electrolytic solution containing an enzyme (a solution obtained by dissolving an enzyme in a buffer solution).

  Here, when the enzyme sensor 100 is a “free enzyme sensor”, in addition to the electrolytic solution tank 310, the temperature of the electrolytic solution (electrolytic solution containing an enzyme) is set so that the enzyme contained in the electrolytic solution is deactivated. It is preferable to provide an electrolyte storage tank that can be maintained at a temperature that can be prevented (eg, 4 ° C.). Specifically, for example, an electrolyte storage tank and a temperature controller for the electrolyte storage tank are provided, and the temperature of the electrolyte in the electrolyte storage tank is always set to the electrolyte storage temperature (contained in the electrolyte). It is preferable that the electrolyte in the electrolyte storage tank be transferred to the electrolyte tank 310 using a pump or the like as much as it is used.

  Further, when the enzyme sensor 100 is a “fixed enzyme sensor”, the temperature of the enzyme sensor 100 or the temperature of the electrolyte solution in the enzyme sensor 100 and the electrolyte solution tank 310 is set to the immobilized enzyme when the enzyme sensor 100 is stored. It is preferable that the temperature is kept at a temperature (for example, 4 ° C.) that can prevent the deactivation, and the enzyme sensor 100 is adjusted to have a measurement temperature (for example, 30 ° C.) when used. Thereby, the lifetime of the “fixed enzyme sensor” can be extended.

The standard solution providing device 320 is a device for providing, for example, a standard solution (electrolyte solution containing a substrate (detection target substance)) used when creating a calibration curve. For example, the enzyme solution 100 (liquid phase) The electrolytic solution introduction side of the chamber R2 is connected to the tube through a first valve 610, a liquid feed pump 340, and a predetermined valve.
Specifically, the standard solution providing device 320 provides the enzyme sensor 100 with a standard solution having a substrate concentration designated by the control device 700 according to a control signal input from the control device 700, for example.

  The waste liquid tank 330 is a tank for storing the waste liquid discharged from the enzyme sensor 100. For example, the waste liquid tank 330 is a tube through the enzyme sensor 100 (electrolyte discharge side of the liquid phase chamber R2) and the second valve 620. It is connected.

The liquid feeding pump 340 is a pump for feeding the electrolytic solution in the electrolytic solution tank 310 and the standard solution in the standard solution providing device 320 to the enzyme sensor 100. For example, the electrolytic solution tank 310 and the standard solution providing device 320 are used. Are connected by a tube via the first valve 610, and are connected by a tube via a predetermined valve to the enzyme sensor 100 (electrolyte introduction side of the liquid phase chamber R2).
Specifically, the liquid feed pump 340 sends the electrolytic solution in the electrolytic solution tank 310 or the standard solution in the standard solution providing device 320 to the enzyme sensor 100 according to a control signal input from the control device 700, for example. To do.
The liquid feed pump 340 may be any pump as long as it can send liquid using a tube. Specifically, for example, it may be a diaphragm pump or the like, or a tube type such as a peristaltic pump or a ring pump. A pump or the like may be used.
Further, although the liquid feed pump 340 is configured to be connected to the front stage (electrolyte introduction side) of the enzyme sensor 100, the electrolyte solution in the electrolyte tank 310 and the standard solution in the standard solution providing device 320 are sent to the enzyme sensor 100. For example, instead of the liquid feed pump 340, a discharge pump may be connected to the downstream of the enzyme sensor 100 (electrolyte discharge side) and operated. A liquid feed pump 340 may be connected to the rear stage (electrolyte discharge side) of the sensor 100 and the liquid feed pump 340 may be operated as a suction pump, or the front stage (electrolyte introduction side) and the rear stage (electrolyte) of the enzyme sensor 100. A pump may be connected to both the liquid discharge side and the liquid discharge side) to operate.

The sensor temperature adjustment device 410 is, for example, a device for keeping the temperature of the enzyme sensor 100 constant, and specifically, for example, a thermostat.
Specifically, the sensor temperature adjustment device 410 is, for example, as an electrode temperature adjustment means for adjusting the temperature of the electrode 150, and the temperature of the enzyme sensor 100 is controlled according to a control signal input from the control device 700, for example. The temperature of the enzyme sensor 100 is adjusted so that the temperature specified by

The electrolyte temperature adjusting device 420 is, for example, a device for keeping the temperature of the electrolyte in the electrolyte tank 310 constant, and specifically, for example, a thermostat.
Specifically, the electrolyte temperature adjusting device 420 is, for example, as an electrolyte temperature adjusting means for adjusting the temperature of the electrolyte introduced into the liquid phase chamber R2, according to a control signal input from the control device 700, for example. The temperature of the electrolytic solution in the electrolytic solution tank 310 is adjusted so that the temperature of the electrolytic solution in the electrolytic solution tank 310 becomes a temperature designated by the control device 700.

The intake pump 510 is a pump for sucking the external atmosphere (gas sample), and is connected to, for example, the enzyme sensor 100 (the gas introduction side of the gas phase chamber R1) via a third valve 630 with a tube. .
Specifically, the intake pump 510, for example, inhales the external atmosphere through the dust filter 520 in accordance with a control signal input from the control device 700, and introduces the intake external atmosphere into the enzyme sensor 100.
The external air introduced into the enzyme sensor 100 is discharged to the outside from the enzyme sensor 100 (the gas discharge side of the gas phase chamber R1).

The standard gas providing device 530 is a device for providing, for example, a standard gas (a gas containing a substrate (detection target substance)) used when preparing a calibration curve. For example, the enzyme sensor 100 (gas phase chamber) R1 gas introduction side) and a third valve 630 through a tube.
For example, the standard gas providing device 530 provides the enzyme sensor 100 with a standard gas having a substrate concentration specified by the control device 700 in accordance with a control signal input from the control device 700.
The standard gas introduced into the enzyme sensor 100 is discharged to the outside from the enzyme sensor 100 (the gas discharge side of the gas phase chamber R1).

  For example, the valve switching device 600 switches each valve (the first valve 610, the second valve 620, the third valve 630, etc.) included in the gas detection system 1000 in accordance with a control signal input from the control device 700.

The control apparatus 700 is an apparatus for controlling each apparatus which comprises the gas detection system 1000, for example.
Specifically, for example, as illustrated in FIG. 2, the control device 700 includes a CPU (Central Processing Unit) 710, a RAM (Random Access Memory) 720, a storage unit 730, and the like.

  For example, the CPU 710 performs various control operations according to various processing programs for the control device 700 stored in the storage unit 730.

  The RAM 720 includes, for example, a program storage area for expanding a processing program executed by the CPU 710, a data storage area for storing input data, a processing result generated when the processing program is executed, and the like.

  The storage unit 730 includes, for example, a system program that can be executed by the control device 700, various processing programs that can be executed by the system program, data that is used when these various processing programs are executed, and processing results that are calculated by the CPU 710. The data etc. are memorized. The program is stored in the storage unit 730 in the form of a computer readable program code.

  Specifically, the storage unit 730 stores, for example, a measurement preparation program 731, a calibration program 732, a circulation cleaning program 733, a calibration curve creation program 734, a measurement program 735, a cleaning program 736, and the like. is doing.

  For example, the measurement preparation program 731 causes the CPU 710 to realize a function of preparing for measurement (detection of a detection target substance).

  Specifically, the CPU 710 inputs a control signal to the sensor temperature adjustment device 410, for example, and sets the temperature of the enzyme sensor 100 and / or the electrolyte tank 310 so that a preset temperature such as an initial temperature is set. Adjust the temperature of the electrolyte inside.

  Next, for example, the CPU 710 inputs a control signal to the measurement circuit 210 and applies a voltage of a predetermined magnitude (about 0.01 to 1 V) to the working electrode 151 with respect to the reference electrode 152 of the enzyme sensor 100. Then, measurement of the response current from the enzyme sensor 100 is started.

  Next, for example, the CPU 710 inputs a control signal to the valve switching device 600 to switch the first valve 610 so that the electrolyte in the electrolyte tank 310 is sent to the enzyme sensor 100, and from the enzyme sensor 100. The second valve 620 is switched so that the discharged electrolyte solution is sent to the waste solution tank 330, and a control signal is input to the solution feed pump 340 to feed the electrolyte solution in the electrolyte solution tank 310 for a certain period of time. . Thereafter, a control signal is input to the valve switching device 600 to switch the second valve 620 so that the electrolyte discharged from the enzyme sensor 100 is sent (circulated) to the electrolyte tank 310, and the response of the enzyme sensor 100. The liquid supply is stopped when the output (response current from the enzyme sensor) is stabilized. At this time, the liquid phase chamber R2 of the enzyme sensor 100 is completely filled with the electrolytic solution. From the viewpoint of completely filling the liquid phase chamber R2 with the electrolytic solution and removing impurities and bubbles in the liquid phase chamber R2, for example, when the volume of the liquid phase chamber R2 is 100 μL, a preferable liquid feeding speed is 0.05 to 3 0.0 mL / min, a more preferable liquid feeding speed is 0.1 mL / min, a preferable liquid feeding time is 1 to 5000 seconds, and a more preferable liquid feeding time is 100 seconds.

  For example, the calibration program 732 causes the CPU 710 to realize a function of performing calibration (calibration) of the enzyme sensor 100 before measurement (detection of a detection target substance).

Specifically, when the enzyme sensor 100 is a “fixed enzyme sensor”, the CPU 710 inputs a control signal to the valve switching device 600, for example, and the standard solution in the standard solution providing device 320 is supplied to the enzyme sensor 100. The first valve 610 is switched so that the liquid is fed, and the second valve 620 is switched so that the standard liquid discharged from the enzyme sensor 100 is fed to the waste liquid tank 330, so that the standard liquid providing device 320 and the liquid feeding pump are switched. A control signal is input to 340 to sequentially generate and introduce standard solutions having different substrate concentrations, and a control signal is input to the data processing device 220 to generate a calibration curve (response data).
When the enzyme sensor 100 is a “fixed enzyme sensor” or a “free enzyme sensor”, the CPU 710 inputs a control signal to the valve switching device 600, for example, and the standard gas in the standard gas providing device 530 is The third valve 630 is switched to be introduced into the enzyme sensor 100, a control signal is input to the standard gas providing device 530, standard gases having different substrate concentrations are sequentially generated and introduced, and the control signal is sent to the data processing device 220. To create a calibration curve (response data).

  Next, the CPU 710 obtains, for example, the slope of the created calibration curve, and determines the temperature of the enzyme sensor 100 and / or the temperature of the electrolyte introduced into the enzyme sensor 100 at the time of measurement based on the slope.

Here, when a biological material such as an enzyme is used, it is generally necessary to make the temperature and pH conditions constant so that the biological material functions stably. Therefore, in the gas detection system 1000, in order to make the temperature of the enzyme sensor 100 (electrode 150) constant, a sensor temperature adjusting device 410 such as a thermostatic bath is provided, and the temperature of the electrolyte introduced into the enzyme sensor 100 is also provided. In order to make the temperature constant, an electrolyte temperature adjusting device 420 such as a thermostatic bath is provided. In order to make the pH conditions constant, a buffer solution (such as a buffer solution or an enzyme dissolved in the buffer solution) is used as the electrolytic solution.
In particular, with regard to temperature, biological substances such as enzymes have low activity at low temperatures and are damaged by heat at high temperatures, so there is an optimum temperature.
Therefore, in the gas detection system 1000, the enzyme fixed to the electrode 150 (when the enzyme sensor 100 is an “electrode-fixed enzyme sensor”) from response data (calibration curve) related to the response output of the enzyme sensor 100 before measurement. , Contained in an enzyme (when the enzyme sensor 100 is a “carrier-fixed enzyme sensor”) fixed to a predetermined carrier disposed in the liquid phase chamber R2, or in an electrolyte introduced into the liquid phase chamber R2. By determining the degree of deterioration of the enzyme (when the enzyme sensor 100 is a “free enzyme sensor”) and adjusting the temperature below the optimum temperature of the enzyme, the response output of the enzyme sensor 100 becomes substantially constant. Thus, the enzyme sensor 100 is calibrated.

Specifically, for example, when the slope of the calibration curve is equal to or less than a predetermined adjustment threshold, the CPU 710 sets a temperature that is equal to or higher than the initial set temperature and equal to or lower than the optimal temperature (measurement of the enzyme sensor 100 at the time of measurement). Temperature and / or temperature of the electrolyte introduced into the enzyme sensor 100 at the time of measurement), and when the slope of the calibration curve exceeds a predetermined adjustment threshold, a temperature lower than the initial set temperature is determined as the set temperature.
More specifically, for example, when the optimum temperature is 40 ° C. and the initial set temperature is 25 ° C., the temperature of 25 ° C. or more and 40 ° C. or less is set when the slope of the calibration curve is below the adjustment threshold. When the slope of the calibration curve exceeds the adjustment threshold, a temperature lower than 25 ° C. (for example, a temperature of 10 ° C. or higher and lower than 25 ° C.) is determined as the set temperature. Further, for example, when the adjustment threshold is 5, when “5 <calibration curve slope ≦ 6”, 20 ° C., and when “6 <calibration curve slope”, 15 ° C. is set as the set temperature. When “4 <calibration curve slope ≦ 5”, the calibration curve slope is 30 ° C., and when “calibration curve slope ≦ 4”, 35 ° C. is set as the set temperature. It is preferable to determine the set temperature step by step according to the slope of the calibration curve so that the set temperature at the time of measurement increases as the value decreases.

  Next, the CPU 710, for example, inputs a control signal to the sensor temperature adjustment device 410 to adjust the temperature of the enzyme sensor 100 and / or the temperature of the electrolyte in the electrolyte tank 310 so that the determined set temperature is reached. .

Here, the acquisition means for acquiring the response data regarding the response output of the enzyme sensor 100 before the detection of the detection target substance by the enzyme sensor 100 is the measurement circuit 210, the data processing device 220, and the CPU 710 that has executed the calibration program 732. , Etc.
Further, the CPU 710 executes the calibration program 732 to determine the temperature of the electrode 150 when the detection target substance is detected by the enzyme sensor 100 based on the response data acquired by the acquisition unit. Electrolytic solution at the time of detection of the detection target substance by the enzyme sensor 100 based on the response data acquired by the adjustment control means and the acquisition means for adjusting the temperature of the electrode 150 by the sensor temperature adjustment device 410 so that the determined temperature is obtained. It functions as an adjustment control means for causing the electrolyte temperature adjusting device 420 to adjust the temperature of the electrolytic solution so as to be the temperature determined by the determining means and the determining means.

  The circulation cleaning program 733 stabilizes the response output of the enzyme sensor 100, for example, by removing the impurities and bubbles in the liquid phase chamber R2 by circulating the electrolyte with the liquid feed pump 340 (cleaning the enzyme sensor 100). The CPU 710 realizes the function to be performed.

Specifically, for example, the CPU 710 inputs a control signal to the valve switching device 600 to switch the first valve 610 so that the electrolytic solution in the electrolytic solution tank 310 is sent to the enzyme sensor 100, and the enzyme The second valve 620 is switched so that the electrolyte discharged from the sensor 100 is sent (circulated) to the electrolyte tank 310, and a control signal is input to the liquid feed pump 340, so that the electrolyte in the electrolyte tank 310 is Liquid supply is started, and the liquid supply is stopped when the response output of the enzyme sensor 100 (response current from the enzyme sensor) is stabilized.
Here, the circulation means for discharging the electrolyte introduced into the liquid phase chamber R2 from the liquid phase chamber R2 and introducing it again into the liquid phase chamber R2 includes an electrolyte tank 310, a liquid feed pump 340, and a circulation cleaning program. CPU 710 that executed 733, and the like.

  For example, the calibration curve creation program 734 causes the CPU 710 to realize a function of creating a calibration curve before measurement (detection of a detection target substance).

Specifically, when the enzyme sensor 100 is a “fixed enzyme sensor”, the CPU 710 inputs a control signal to the valve switching device 600, for example, and the standard solution in the standard solution providing device 320 is supplied to the enzyme sensor 100. The first valve 610 is switched so that the liquid is fed, and the second valve 620 is switched so that the standard solution discharged from the enzyme sensor 100 is sent to the waste liquid tank 330, so that the standard solution providing device 320 and the liquid feeding pump are switched. A control signal is input to 340 to sequentially generate and introduce standard solutions having different substrate concentrations, and the control signal is input to the measurement circuit 210 and the data processing device 220 to create a calibration curve.
When the enzyme sensor 100 is a “fixed enzyme sensor” or a “free enzyme sensor”, the CPU 710 inputs a control signal to the valve switching device 600, for example, so that the standard gas in the standard gas providing device 530 is The third valve 630 is switched so as to be introduced into the enzyme sensor 100, a control signal is input to the standard gas providing device 530, standard gases having different substrate concentrations are sequentially generated and introduced, and the control signal is sent to the data processing device 220. To create a calibration curve.
The created calibration curve is stored in, for example, the RAM 172 or the storage unit 173, and is used, for example, when obtaining the concentration of the detection target substance detected by the enzyme sensor 100.

  For example, the measurement program 735 causes the CPU 710 to realize a function of performing measurement (detection of a detection target substance) using the enzyme sensor 100.

  Specifically, for example, the CPU 710 inputs a control signal to the valve switching device 600 and switches the third valve 630 so that the external atmosphere (gas sample) sucked by the suction pump 510 is introduced into the enzyme sensor 100. The control signal is input to the intake pump 510 to introduce the external atmosphere, the control signal is input to the data processing device 220 and the data display device 230, and the measurement result (numerical information or the like) measured by the measurement circuit 210 is input. Is displayed.

  The cleaning program 736 stabilizes the response output of the enzyme sensor 100, for example, by removing the impurities and bubbles in the liquid phase chamber R2 by feeding the electrolyte with the liquid feed pump 340 (cleaning the enzyme sensor 100). The CPU 710 realizes the function to be performed.

Specifically, for example, the CPU 710 inputs a control signal to the valve switching device 600 to switch the first valve 610 so that the electrolytic solution in the electrolytic solution tank 310 is sent to the enzyme sensor 100, and the enzyme The second valve 620 is switched so that the electrolyte discharged from the sensor 100 is sent to the waste liquid tank 330, and a control signal is input to the liquid feed pump 340 to feed the electrolyte in the electrolyte tank 310. The liquid feeding is stopped when the response output of the enzyme sensor 100 (response current from the enzyme sensor) is stabilized.
In the case where the circulation cleaning is performed after the cleaning, for example, the cleaning is performed until the electrolyte solution in the electrolyte flow path (in the tube or the enzyme sensor 100) is completely replaced, and then the circulation cleaning is performed.

  When the control device 700 is provided with an electrolyte storage tank and an electrolyte storage tank temperature adjusting device in addition to the electrolyte tank 310, the temperature of the electrolyte storage tank is set to a preset electrolyte storage temperature. A control signal is input to the adjusting device to adjust the temperature of the electrolytic solution in the electrolytic solution storage tank. In addition, it is preferable that the temperature of the electrolyte solution in the electrolyte solution storage tank is always maintained at the electrolyte solution storage temperature regardless of the presence or absence of measurement using the enzyme sensor 100.

<Configuration of enzyme sensor>
3 is a plan perspective view of the enzyme sensor 100, FIG. 4 is a diagram schematically showing a cross section taken along line IV-IV in FIG. 3, and FIG. 5 is a cross section taken along line VV in FIG. It is a figure shown typically. 6A is a bottom view of the upper support 110 with the gas permeable membrane 130 attached thereto, and FIG. 6B is a plan view of the lower support 120 with the electrode 150 formed thereon. It is.
Here, the gas phase chamber R1 side of the enzyme sensor 100 is the upper side, the liquid phase chamber R2 side is the lower side, the side where the pad 160 is disposed is the front side, and the side facing it is the rear side. The direction orthogonal to both is the left-right direction.

For example, as shown in FIGS. 3 to 6, the enzyme sensor 100 includes an upper support 110, a lower support 120, a gas phase chamber R <b> 1 into which a gas containing a detection target substance is introduced, and a gas phase chamber A gas permeable membrane that is disposed adjacent to R1 and is disposed so as to separate the liquid phase chamber R2 into which a predetermined electrolytic solution is introduced and the gas phase chamber R1 and the liquid phase chamber R2, and allows at least the detection target substance to pass therethrough. 130, an O-ring 140 for fixing the gas permeable membrane 130 to the upper support 110, and three electrodes 150 (the working electrode 151 and the reference electrode 152) disposed in the liquid phase chamber R1 so as to face the gas permeable membrane 130. , The counter electrode 153), the three pads 160 connected to the electrode 150 through the wiring 161, the spacer 170 disposed between the gas permeable membrane 130 and the electrode 150, and the lower support for the upper support 110. Solid on body 120 A plurality of screws 180 to configured to include the like.
In the enzyme sensor 100, an enzyme as a receptor that selectively reacts with a substance to be detected is contained in the liquid phase chamber R2, and the enzyme sensor 100 permeates the gas permeable membrane 130 from the gas phase chamber R1 to the liquid phase chamber R2. The substance to be detected that has shifted to is detected by an electrochemical measurement method.

The upper support body 110 and the lower support body 120 are formed, for example, by dividing a substantially cylindrical body in a direction (horizontal direction) perpendicular to the up-down direction at a substantially central position in the up-down direction.
The upper support body 110 is formed, for example, so as to have a substantially D shape in plan view, that is, to form a flat surface portion 111 having a part of a side surface cut out in the vertical direction. Therefore, a part of the upper surface of the lower support 120 is, for example, an exposed portion 121 exposed to the outside.
The material constituting the upper support 110 and the lower support 120 is a material having high corrosion resistance to an electrolyte solution or a sample (for example, a hydrophobic insulating material, an insulating material whose surface is subjected to hydrophobic treatment, etc.). Specifically, for example, ceramics, glass, plastic, Teflon (registered trademark), peak material, and the like can be used.

The upper support 110 is, for example, disposed at a substantially central position in the horizontal direction of the upper support 110, and is disposed around the concave portion 112 having a substantially circular shape in plan view with the lower surface opened, and the lower surface opened. The O-ring accommodating portion 113 having a substantially ring shape in plan view, a gas introduction port 114 for introducing gas from the outside of the enzyme sensor 100 toward the gas phase chamber R1, and the gas introduction port 114 and the gas phase chamber R1 communicate with each other. A gas introduction passage 115 for discharging gas from the gas phase chamber R1 toward the outside of the enzyme sensor 100, and a gas exhaust passage 117 for communicating the gas discharge port 116 with the gas phase chamber R1. , Etc. are provided.
Here, in particular, the surface of the upper support 110, the gas inlet 114, the gas inlet 115, the gas outlet 116, the gas outlet 117, the recess 112 (gas phase chamber R1, liquid phase chamber R2), etc. are electrolyzed. It is preferably formed of a material having high corrosion resistance to a liquid, a sample or the like (for example, a hydrophobic insulating material, an insulating material whose surface is subjected to hydrophobic treatment, etc.).

The recess 112 and the O-ring housing portion 113 are separated by a partition wall 118.
The recess 112 is separated in the vertical direction when the gas permeable membrane 130 is attached to the upper support 110, and the region above the recess 112 in the separated state becomes the gas phase chamber R1, The region on the side becomes the liquid phase chamber R2. Therefore, the distance between the gas permeable membrane 130 and the electrode 150 (working electrode 151) is defined by the height (length in the vertical direction) of the partition wall 118.

  The gas introduction path 115 is formed along the left-right direction at a position on the left side of the recess 112, for example, and the gas discharge path 117 is formed along the left-right direction on the right side of the recess 112, for example.

The lower support 120 includes, for example, three grooves 122 arranged in the horizontal direction on the upper surface of the lower support 120 from the position in the horizontal center to the exposed portion 121, and the outside of the enzyme sensor 100. From the liquid phase chamber R2 to the liquid phase chamber R2, an electrolytic solution introduction port 123 for introducing the electrolytic solution toward the liquid phase chamber R2, an electrolytic solution introduction path 124 communicating the electrolytic solution inlet 123 and the liquid phase chamber R2, and an enzyme sensor from the liquid phase chamber R2 An electrolyte solution discharge port 125 for discharging the electrolyte solution toward the outside of 100, an electrolyte solution discharge passage 126 that connects the electrolyte solution discharge port 125 and the liquid phase chamber R2, and the like are provided.
Here, in particular, the surface of the lower support 120, the electrolytic solution introduction port 123, the electrolytic solution introduction passage 124, the electrolytic solution discharge port 125, the electrolytic solution discharge passage 126, the liquid phase chamber R2 (the gas phase of the liquid phase chamber R2). The region constituting the surface facing the chamber R1) is formed of a material having high corrosion resistance to the electrolyte or sample (for example, a hydrophobic insulating material, an insulating material whose surface is subjected to hydrophobic treatment, etc.). It is preferred that

The electrolyte solution introduction path 124 is, for example, a first electrolyte solution formed along the vertical direction in a region from a position between the working electrode 151 and the reference electrode 152 to a position at a substantially central position in the vertical direction of the lower support 120. An introduction path 124a and a second electrolyte introduction path 124b formed along the left-right direction on the left side of the first electrolyte introduction path 124a.
In addition, the electrolyte discharge path 126 is, for example, a first line formed in the vertical direction in a region from a position between the working electrode 151 and the counter electrode 153 to a position approximately in the vertical direction of the lower support 120. An electrolyte solution discharge path 126a and a second electrolyte solution discharge path 126b formed along the left-right direction on the right side of the first electrolyte solution discharge path 126a.

For example, the gas permeable membrane 130 covers the recess 112 and the O-ring housing portion 113 from the lower surface side of the upper support 110 with the gas permeable membrane 130, and the O-ring 140 is arranged on the lower surface side of the gas permeable membrane 130. It is attached to the upper support 110 by being moved in the direction and housed in the O-ring housing part 113.
The detection target substance (detection target gas) introduced into the gas phase chamber R1 passes through the gas permeable membrane 130 and moves to the liquid phase chamber R2, and reacts with the enzyme contained in the liquid phase chamber R2. It has become. Therefore, the gas permeable film 130 is arbitrary as long as it is a gas permeable film through which at least the detection target substance permeates, and can be appropriately changed depending on the type of the detection target substance.

The O-ring 140 is, for example, for attaching the gas permeable membrane 130 to the upper support 110, and for sealing the liquid phase chamber R2 to prevent the electrolyte introduced into the liquid phase chamber R2 from leaking. Is. Therefore, as the O-ring 140, for example, one having a thickness equal to or greater than the height (length in the vertical direction) of the O-ring housing portion 113 is preferable.
As a material constituting the O-ring 140, a material having high corrosion resistance against an electrolytic solution or a sample is preferable.

The electrode 150 is supported by the lower support body 120 as a support body, for example.
Specifically, the electrode 150, the pad 160, and the wiring 161 that connects the electrode 150 and the pad 160 are formed in, for example, the groove 122 included in the lower support 120, and the electrode 150 and the wiring 161 are formed. Are processed so that the thickness of the electrode 150 and the wiring 161 is substantially the same as the depth of the groove 122. Accordingly, since the thickness (height) of the electrode 150 and the wiring 161 can be defined uniformly and precisely without troublesome control, the distance between the gas permeable film 130 and the electrode 150 is kept constant and uniform. be able to.

The spacer 170 is disposed, for example, in the liquid phase chamber R2, and is used to keep the distance between the gas permeable membrane 130 and the electrode 150 constant. Therefore, as the spacer 170, for example, a spacer having a thickness equivalent to the distance between the gas permeable membrane 130 and the electrode 150 is preferable.
The material of the spacer 170 is arbitrary as long as the distance between the gas permeable membrane 130 and the electrode 150 can be kept constant by the spacer 170 and the substrate (detection target substance) has good permeability. Specifically, for example, a porous body such as an anodized film having a plurality of through holes penetrating in a substantially vertical direction with respect to the electrode 150, a hydrophilic film such as a hydrophilic Teflon film, a mesh body such as a nylon mesh, etc. Can be mentioned. Further, for example, when the enzyme sensor 100 is a “carrier-fixed enzyme sensor”, the spacer 170 may be a carrier on which an enzyme is immobilized, or the carrier on which the enzyme is immobilized and the carrier are interposed between the spacer 170 and the carrier. It may be composed of a sandwiched hydrophilic film or the like.

The enzyme is arbitrary as long as it is an enzyme that selectively reacts with the detection target substance, and can be appropriately changed depending on the type of the detection target substance.
Specifically, the enzyme is an enzyme (enzyme protein) such as an oxidoreductase, a hydrolase, a transferase, and an isomerase.
The enzyme may be, for example, a natural enzyme molecule or an enzyme fragment containing an active site. The enzyme molecule or the fragment of the enzyme containing the active site is synthesized, for example, genetically or chemically, whether it is extracted from animals or plants or microorganisms, or is cleaved if desired. It may be a thing.

The enzyme contained in the liquid phase chamber R2 may be one type of enzyme or two or more types of enzymes.
Specifically, the molecular weight of the enzyme contained in the liquid phase chamber R2 may be, for example, one kind of enzyme or two or more kinds of enzymes having substantially the same molecular weight and / or size (diameter). And / or two or more enzymes having different sizes. Further, when there are two or more types of enzymes contained in the liquid phase chamber R2, even if the enzymes are two or more types of enzymes that act on the same type of detection target substance (substrate), for example, different types of detection target substances It may be two or more types of enzymes that act on two or more types of enzymes that act on the same type and / or different types of detection target substances.
Here, in particular, when there are two or more types of enzymes contained in the liquid phase chamber R2 and the two or more types of enzymes act on different types of detection target substances, the enzyme sensor 100 is configured to detect the different types of detection target substances. (Two or more types of detection target substances) can be detected simultaneously.

In the case of a “fixed enzyme sensor”, a coenzyme and an electron carrier may be fixed together with the enzyme.
For example, in the case of a “fixed enzyme sensor” in which a coenzyme and an electron carrier are not fixed, an electrolyte solution that does not contain an enzyme or an electrolyte solution that does not contain an enzyme and a coenzyme (an electron carrier may be contained) .) Is introduced into the liquid phase chamber R2.
For example, in the case of a “fixed enzyme sensor” in which a coenzyme is fixed and an electron carrier is not fixed, an electrolyte solution containing no enzyme or an electrolyte solution containing no enzyme and a coenzyme (containing an electron carrier) May be introduced into the liquid phase chamber R2.
For example, in the case of a “fixed enzyme sensor” in which a coenzyme and an electron carrier are fixed, an electrolyte solution containing no enzyme or an electrolyte solution containing no enzyme and a coenzyme is introduced into the liquid phase chamber R2. I will do it.

In the case of the “free enzyme sensor”, a coenzyme and an electron carrier may be contained in the electrolytic solution introduced into the liquid phase chamber R2 together with the enzyme.
That is, for example, in the case of a “free enzyme sensor”, an electrolytic solution containing an enzyme (a coenzyme or an electron carrier may be contained together with the enzyme) is introduced into the liquid phase chamber R2. .
In the case of a “free enzyme sensor”, for example, a coenzyme or an electron carrier is fixed to a predetermined carrier or the like disposed in the electrode 150 or the liquid phase chamber R2, so that the coenzyme or the electron carrier is liquid. It may be contained in the phase chamber R2.

The coenzyme can be appropriately selected according to the type of enzyme (coenzyme-dependent enzyme). Specifically, examples of the coenzyme include nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), coenzyme I, coenzyme II, flavin mononucleotide (FMN), and flavin. Examples thereof include one or a combination of two or more of adenine dinucleotide (FAD), lipoic acid, coenzyme Q and the like. Among these, NAD coenzymes such as nicotinamide adenine dinucleotide (NAD ⁺ ) and nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) are used.
Examples of the electron carrier include potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, and the like.

  As a method for fixing the enzyme, coenzyme, and electron carrier to the electrode 150 or the carrier, for example, a known method such as a method using a predetermined cross-linking agent such as glutaraldehyde or a photocrosslinkable resin is appropriately used. Can do.

<Measurement process>
An example of processing related to measurement (detection of a detection target substance) using the enzyme sensor 100 by the gas detection system 1000 will be described with reference to a flowchart of FIG.

  First, the CPU 710 executes the measurement preparation program 731 to prepare for measurement (step S1).

  Next, the temperature of the enzyme sensor 100 (sensor temperature adjusting device 410) and / or the temperature of the electrolyte solution (electrolyte temperature adjusting device 420) introduced into the enzyme sensor 100 becomes the set temperature by the process of step S1, and the enzyme When the response output of the sensor 100 is stabilized, the CPU 710 executes the calibration program 732 to calibrate the enzyme sensor 100 (step S2).

  Next, the CPU 710 executes the cleaning program 736, supplies the electrolytic solution with the liquid supply pump 340, cleans the enzyme sensor 100 (step S3), executes the circulation cleaning program 733, and executes the circulation cleaning program 733 with the liquid supply pump 340. The enzyme sensor 100 is circulated and washed by circulating the electrolyte (step S4).

  Next, the temperature of the enzyme sensor 100 (sensor temperature adjustment device 410) and / or the temperature of the electrolyte solution (electrolyte temperature adjustment device 420) introduced into the enzyme sensor 100 becomes the determined set temperature by the process of step S2. When the response output of the enzyme sensor 100 is stabilized by the process of step S4, the CPU 710 executes the calibration curve creation program 734 to create a calibration curve (step S5).

  Next, the CPU 710 executes the cleaning program 736, supplies the electrolytic solution with the liquid supply pump 340 to clean the enzyme sensor 100 (step S6), executes the circulation cleaning program 733, and executes the circulation cleaning program 733 with the liquid supply pump 340. The enzyme sensor 100 is circulated and washed by circulating the electrolytic solution (step S7).

  Next, when the response output of the enzyme sensor 100 is stabilized by the process of step S7, the CPU 710 executes the measurement program 735 and performs measurement (detection of a detection target substance) using the enzyme sensor 100 (step 8). .

  Next, the CPU 710 executes the cleaning program 736, supplies the electrolytic solution with the liquid supply pump 340 to clean the enzyme sensor 100 (step S9), executes the circulation cleaning program 733, and executes the circulation cleaning program 733 with the liquid supply pump 340. The enzyme sensor 100 is circulated and washed by circulating the electrolyte (step S10).

Next, the CPU 710 determines whether or not to perform the next measurement (step S11).
Specifically, for example, when the user operates the operation unit (not shown) included in the gas detection system 1000 to instruct to perform the next measurement, or according to a preset number of measurements or side hand time. CPU 710 determines to perform the next measurement.

  If it is determined in step S11 that the next measurement is to be performed (step S11; Yes), the CPU 710 repeats the processes in and after step S8 after the response output of the enzyme sensor 100 is stabilized by the process in step S10.

  On the other hand, when the CPU 710 determines that the next measurement is not performed (step S8; No), the CPU 710 ends the process.

  Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited thereto.

<Example 1-1>
The enzyme sensor 100 was created and the enzyme sensor 100 and the gas detection system 1000 were evaluated.
In this example, “carrier-fixed enzyme sensor” and “free enzyme sensor” were prepared as the enzyme sensor 100.

<1> Production of enzyme sensor 100 In this example, an enzyme sensor 100 for detecting formaldehyde gas was produced. As the enzyme, formaldehyde dehydrogenase (formaldehyde dehydrogenase), which is a coenzyme (NAD ⁺ ) -dependent enzyme, was used.

<1-1> “Carrier immobilized enzyme sensor”
First, the upper support 110 and the lower support 120 were formed using a lathe or a milling machine using a peak material which is an insulator.
The depth of the groove 122 was 10 μm, and the volume of the liquid phase chamber R2 was 10 μL. Further, the height of the partition wall 118 was adjusted so that the distance between the gas permeable membrane 130 and the electrode 150 was 30 μm.

Next, a tripolar structure pattern of the working electrode 151, the reference electrode 152, and the counter electrode 153 was formed on the upper surface of the lower support 120.
Specifically, carbon is applied to the groove portion 122 by screen printing, post-baked at 120 ° C. for 2 hours using a hot plate, and then dried overnight in a dark room. The thickness of the electrode 150 is equal to the depth of the groove portion 122. The surface of the electrode 150 was polished by a polishing machine so as to be substantially the same. Thereafter, a silver / silver chloride ink was applied on the pattern of the reference electrode 152 and sintered at 120 ° C., thereby creating a reference electrode 152 which was a silver / silver chloride electrode.

Next, an enzyme-immobilized carrier is prepared by immobilizing formaldehyde dehydrogenase on a predetermined carrier (silica-based mesoporous material (FSM)), and the enzyme-immobilized carrier is made of a hydrophilic membrane (pore size 0.1 μm, hydrophilic manufactured by Millipore). A spacer 170 having a thickness substantially the same as the distance (30 μm) between the gas permeable membrane 130 and the electrode 150 was formed by sandwiching between the Teflon membrane.
Next, the gas permeable membrane 130 was fixed to the upper support 110 using the O-ring 140, and the created spacer 170 was disposed on the electrode 150.
Next, the upper support 110 was placed on the lower support 120, and the upper support 110 was fixed to the lower support 120 using the screws 180, thereby creating a "carrier-fixed enzyme sensor".

Next, an electrolytic solution for “carrier-fixed enzyme sensor” was prepared.
Specifically, a phosphate buffer solution (pH 7.41) containing 1 mM NAD ⁺ and 1 mM quinone was prepared to prepare an electrolyte for a “carrier-fixed enzyme sensor”. Then, when using the “carrier-fixed enzyme sensor”, this electrolyte solution was put in the electrolyte solution tank 310 and the enzyme sensor 100 and the gas detection system 1000 were evaluated.

<1-2> “Free enzyme sensor”
In the same manner as in the case of the “carrier-fixed enzyme sensor”, the upper support 110 and the lower support 120 are prepared, and the working electrode 151, the reference electrode 152, and the counter electrode 153 are formed on the upper surface of the lower support 120. A tripolar pattern was created.

Next, the gas permeable membrane 130 is fixed to the upper support 110 using an O-ring 140, and a spacer 170 (thickness: 30 μm) having a thickness substantially the same as the distance (30 μm) between the gas permeable membrane 130 and the electrode 150. , Pore diameter: 0.1 μm, Millipore hydrophilic Teflon membrane) was disposed on the electrode 150.
Next, the upper support 110 was placed on the lower support 120, and the upper support 110 was fixed to the lower support 120 using screws 180, thereby creating a “free enzyme sensor”.

Next, an electrolytic solution for “free enzyme sensor” was prepared.
Specifically, a phosphate buffer solution (pH 7.41) containing 1 mM NAD ⁺ and 1 mM quinone is prepared, and 20 mg of formaldehyde dehydrogenase is dissolved in the phosphate buffer (pH 7.41). An electrolyte was prepared. Then, when using the “free enzyme sensor”, the electrolyte solution was put in the electrolyte solution tank 310 and the enzyme sensor 100 and the gas detection system 1000 were evaluated.

<2> Evaluation of Enzyme Sensor 100 and Gas Detection System 1000 In this embodiment, the calibration of the enzyme sensor 100 is not performed, and the temperature of the enzyme sensor 100 and the temperature of the electrolyte in the electrolyte tank 310 are set to 30 ° C. The experiment was conducted.
The gas introduction rate was 300 mL / min, and the electrolyte introduction rate was 0.1 mL / min.

<2-1> Selection of Gas Permeation Membrane 130 Using the created “free enzyme sensor”, an experiment for selecting a preferred gas permeable membrane 130 from among a plurality of types of gas permeable membranes 130 was conducted.
Specifically, first, as the gas permeable membrane 130, a “free enzyme sensor” that employs neoflon (Daikin Industries' fluororesin membrane) and a “free enzyme sensor” that employs monotran (Nac nanoporous membrane). ”And a“ free enzyme sensor ”employing a zytex membrane (a porous PTFE membrane made by Saint-Gobain) and a“ free enzyme sensor ”employing a Gore-tex membrane (a porous Teflon membrane made by Gore-Tech). .
Next, for each “free enzyme sensor”, after preparing for measurement, a standard gas having a substrate concentration (formaldehyde concentration) of 100 ppb is generated and introduced, and the response current is measured, followed by washing and circulation washing. went. The result is shown in FIG.

In FIG. 8, the horizontal axis represents time, the vertical axis represents the response current, the broken line represents the result of “free enzyme sensor” employing neoflon, and the two-dot chain line represents “free enzyme sensor” employing monotran. The results are shown, and the results of the “free enzyme sensor” employing the zytex membrane are shown by the one-dot chain line, and the results of the “free enzyme sensor” employing the Gore-tex membrane are shown by the dotted line.
From the results of FIG. 8, it was found that the “free enzyme sensor” employing a Gore-Tex membrane, which is a porous material having a high porosity and excellent gas permeability, as the gas permeable membrane 130 has the highest sensitivity.

<2-2> Creation of calibration curve An experiment for creating a calibration curve was performed using the created "free enzyme sensor".
Specifically, for a “free enzyme sensor” employing a Gore-Tex membrane, after preparing for measurement, a standard gas with different formaldehyde concentrations is sequentially generated and introduced, and a calibration curve is obtained by measuring the response current. After preparation, washing and circulation washing were performed. The formaldehyde concentration of the standard gas was 1 bp, 10 ppb, 100 ppb, 400 ppb, 1200 ppb. The measurement results are shown in FIG.

In FIG. 9 (a), the horizontal axis represents time, the vertical axis represents the response current, the broken line represents the result when the formaldehyde concentration is 1 ppb, and the two-dot chain line represents the result when the formaldehyde concentration is 10 ppb. A chain line indicates the result when the formaldehyde concentration is 100 ppb, a dotted line indicates the result when the formaldehyde concentration is 400 ppb, and a solid line indicates the result when the formaldehyde concentration is 1200 ppb.
In addition, FIG. 9B shows a calibration curve created using the result of FIG.
From the result of FIG. 9 (a), it was found that the prepared “free enzyme sensor” had high sensitivity and could be sufficiently measured to the sub ppb level.
Further, from the result of FIG. 9B, it was found that the prepared “free enzyme sensor” has good linearity in a concentration range of 1 to 400 ppb.
Although calibration curves were created with five types of formaldehyde concentrations, it is sufficient in practice to create calibration curves with three types of formaldehyde concentrations.

<2-3> Detection of formaldehyde in the external atmosphere An experiment for detecting formaldehyde in the external atmosphere was performed using the prepared "free enzyme sensor".
Specifically, for `` free enzyme sensor '' employing Gore-Tex membrane, after preparing measurement, creating calibration curve, washing and circulating washing, introducing external air and measuring response current, Washing and circulation washing were performed. The result is shown in FIG.

In FIG. 10, the horizontal axis represents time, and the vertical axis represents response current.
From the results shown in FIG. 10, it was found that formaldehyde in the external atmosphere can be detected using the prepared “free enzyme sensor”.
Moreover, from the result of FIG. 10, it was found that the concentration of formaldehyde in the external air used in this example was 70 ppb.

<2-4> Cleaning of enzyme sensor 100 Using the created "free enzyme sensor", the effect of cleaning the enzyme sensor 100 by feeding the electrolytic solution with the liquid feed pump 340 (washing and circulation washing) Experiments were performed to verify.
Specifically, for a “free enzyme sensor” employing a Gore-Tex membrane, after preparing for measurement, a standard gas having a formaldehyde concentration of 160 ppb was generated and introduced, and then cleaning and circulation cleaning were performed. And the process of this gas introduction-cleaning (cleaning and circulation cleaning) was repeated, and the response current during that time was measured. The result is shown in FIG.

In FIG. 11, time is plotted on the horizontal axis, response current is plotted on the vertical axis, and the results when no cleaning (cleaning and circulation cleaning) is performed are shown by broken lines, and the results are shown by solid lines (cleaning and circulation cleaning). The result of performing is shown.
From the results shown in FIG. 11, when cleaning is performed after gas introduction (after measurement), the response output of the enzyme sensor 100 becomes stable about 600 seconds after the start of cleaning, and the next measurement can be performed. I understood.
It was also found that when the enzyme sensor 100 was cleaned, the response current of the enzyme sensor 100 recovered to an equilibrium state at a higher speed than when the enzyme sensor 100 was not cleaned. Thereby, it turned out that a high-speed continuous measurement is attained by wash | cleaning the enzyme sensor 100. FIG.

<2-5> Repetitive reproducibility of enzyme sensor 100 Using the created "free enzyme sensor", an experiment for evaluating repetitive reproducibility was performed.
Specifically, for a “free enzyme sensor” employing a Gore-Tex membrane, after preparing for measurement, a standard gas with a formaldehyde concentration of 40 ppb was generated and introduced, and the response current was measured, followed by washing and circulation. Washing was performed. Then, this measurement-washing (washing and circulation washing) process was repeated. The result is shown in FIG.

In FIG. 12, the horizontal axis represents the relative number of repetitions, and the vertical axis represents the relative response with the response current at the first time being 100%.
From the results of FIG. 12, it was found that the variation was within 5% after 20 measurements, and that the reproducible measurement could be performed by using the prepared “free enzyme sensor”.

<2-6> Cleaning of enzyme sensor 100 Using the created “free enzyme sensor”, the effect of cleaning the enzyme sensor 100 (cleaning and circulating cleaning) by circulating the electrolyte with the liquid feed pump 340 is demonstrated. An experiment was conducted.
Specifically, for a “free enzyme sensor” employing a Gore-Tex membrane, after preparing for measurement, cleaning and circulating cleaning, a standard gas with a formaldehyde concentration of 100 ppb is generated and introduced, and the response current is measured. It was measured. Then, this washing (washing and circulation washing) -measurement process was repeated. It should be noted that the electrolytic solution in the electrolytic solution tank 310 was not replaced while the cleaning (cleaning and circulation cleaning) -measurement process was repeatedly performed. Further, the temperature of the electrolytic solution in the electrolytic solution storage tank was maintained at 30 ° C. The result is shown in FIG.

In FIG. 13, the horizontal axis represents time and the vertical axis represents the relative response with the response current at 0 hour being 100%, and the square (□) plot does not perform cleaning (cleaning and circulation cleaning) (Comparative Example). ) And shows the results when washing (washing and circulation washing) is performed with a diamond (♦) plot.
From the result of FIG. 13, when the enzyme sensor 100 is not washed, the relative response becomes 10% in about 60 hours, whereas when the enzyme sensor 100 is washed, the relative response is 50% even after 300 hours or more. It turns out that it is a grade. Thereby, it turned out that the lifetime of the enzyme sensor 100 extends by washing | cleaning the enzyme sensor 100. FIG.
In this experiment, the temperature of the electrolytic solution in the electrolytic solution storage tank was maintained at 30 ° C., but the temperature of the electrolytic solution in the electrolytic solution storage tank was set at, for example, the deactivation of the enzyme contained in the electrolytic solution. It is considered that the lifetime of the enzyme sensor 100 is further extended if the temperature is maintained at a temperature (for example, 4 ° C.) that can prevent the above.

<2-7> Selectivity of enzyme sensor 100 Using the created "free enzyme sensor", an experiment for demonstrating the selectivity of the enzyme sensor 100 was performed.
Specifically, first, acetaldehyde gas, ethanol gas, methanol gas, benzene gas, and acetone gas were prepared as comparative gases.
Next, after preparing for measurement of the “free enzyme sensor” employing a Gore-Tex membrane, a standard gas having a formaldehyde concentration of 100 ppb was generated and introduced, and the response current was measured, followed by washing and circulation washing. It was. And this measurement preparation-measurement-cleaning (cleaning and circulation cleaning) processing was performed by using each of the comparative gases in place of the standard gas. The concentration of the comparison gas was the same as the standard gas concentration (100 ppb). The result is shown in FIG.

In FIG. 14, the horizontal axis shows the relative response with the response current when formaldehyde gas is introduced being 100%.
From the results of FIG. 14, it was found that even when a gas other than formaldehyde gas was introduced, no response was observed, and the prepared “free enzyme sensor” had high selectivity. This is due to the substrate specificity of the enzyme contained in the liquid phase chamber R2 of the produced “free enzyme sensor”.

<Example 1-2>
The enzyme sensor 100 was created and the enzyme sensor 100 and the gas detection system 1000 were evaluated.
In this example, an “electrode-fixed enzyme sensor” was prepared as the enzyme sensor 100.

<1> Production of enzyme sensor 100 In this example, an enzyme sensor 100 for detecting formaldehyde gas was produced. As the enzyme, formaldehyde dehydrogenase (formaldehyde dehydrogenase), which is a coenzyme (NAD ⁺ ) -dependent enzyme, was used.

  In the same manner as in Example 1, the upper support body 110 and the lower support body 120 are formed, and the upper electrode of the lower support body 120 has a tripolar structure of the working electrode 151, the reference electrode 152, and the counter electrode 153. Created a pattern.

Next, formaldehyde dehydrogenase was immobilized on the working electrode 151 using a photocrosslinking reagent (Tokyo Kasei).
Next, the O-ring 140 is used to fix the gas permeable membrane 130 (Goretech porous Teflon membrane) to the upper support 110, and the distance between the gas permeable membrane 130 and the electrode 150 (30 μm) is substantially the same. A spacer 170 (thickness: 30 μm, pore diameter 0.1 μm, Millipore hydrophilic Teflon membrane) was placed on the electrode 150.
Next, the upper support 110 was placed on the lower support 120, and the upper support 110 was fixed to the lower support 120 using screws 180, thereby creating an “electrode-fixed enzyme sensor”.

Next, an electrolytic solution for “electrode-fixed enzyme sensor” was prepared.
Specifically, an electrolyte solution for “electrode-fixed enzyme sensor” was prepared by preparing a phosphate buffer solution (pH 7.41) containing 1 mM NAD ⁺ and 1 mM quinone. And this electrolyte solution was put in the electrolyte solution tank 310, and the enzyme sensor 100 and the gas detection system 1000 were evaluated.

<2> Evaluation of Enzyme Sensor 100 and Gas Detection System 1000 In this example, an experiment related to calibration of the enzyme sensor 100 was performed.

<2-1> Temperature dependence of activity of formaldehyde dehydrogenase An experiment for observing the temperature dependence of the activity of formaldehyde dehydrogenase immobilized on electrode 150 using the created “electrode-fixed enzyme sensor” went.
Specifically, for the “electrode-fixed enzyme sensor”, after preparing the measurement by adjusting the temperature of the enzyme sensor 100 and the temperature of the electrolyte in the electrolyte tank 310 to a constant temperature, the substrate concentration ( A standard solution having a formaldehyde concentration (6840 μM) was produced and introduced, and the response current was measured, followed by washing and circulation washing. And this measurement preparation-measurement-washing (washing and circulation washing) was performed for each constant temperature (23 ° C., 30 ° C., 37 ° C., 40 ° C., 44 ° C., 52 ° C.). As a standard solution, a solution in which formaldehyde was diluted in a phosphate buffer solution (pH 7.41) containing 1 mM NAD ⁺ and 1 mM quinone was used. Assuming vapor-liquid equilibrium, the formaldehyde concentration of 6840 μM in the standard solution corresponds to the formaldehyde concentration of 100 ppb in the standard gas. The standard solution introduction rate was 0.1 mL / min. The result is shown in FIG.

In FIG. 15, the horizontal axis represents temperature and the vertical axis represents the relative response with the highest response current being 100%.
From the results of FIG. 15, it was found that the optimum temperature of formaldehyde dehydrogenase immobilized on the electrode 150 of the created “electrode-fixed enzyme sensor” was around 40 ° C.
In addition, since enzyme activity falls rapidly on the high temperature side from the optimum temperature, the temperature lower than the optimum temperature is adopted as the initial set temperature and the set temperature at the time of measurement.

<2-2> Creation of Calibration Curve An experiment for creating a calibration curve was performed using the created “electrode-fixed enzyme sensor”.
Specifically, the “electrode-fixed enzyme sensor” was prepared for measurement by adjusting the temperature of the enzyme sensor 100 and the temperature of the electrolyte in the electrolyte tank 310 to the initial set temperature (30 ° C.). Thereafter, standard solutions having different formaldehyde concentrations were sequentially generated and introduced, and a calibration curve was created by measuring the response current, followed by washing and circulation washing. As a standard solution, a solution in which formaldehyde was diluted in a phosphate buffer solution (pH 7.41) containing 1 mM NAD ⁺ and 1 mM quinone was used. The formaldehyde concentration of the standard solution was 68.4 μM, 684 μM, and 6840 μM, and the standard solution introduction rate was 0.1 mL / min. Assuming vapor-liquid equilibrium, the formaldehyde concentration of the standard solution of 68.4 μM corresponds to the formaldehyde concentration of 1 ppb of the standard gas, the formaldehyde concentration of the standard solution of 684 μM corresponds to the formaldehyde concentration of the standard gas of 10 ppb, and the formaldehyde concentration of the standard solution 6840 μM corresponds to a formaldehyde concentration of 100 ppb in the standard gas. The result is shown in FIG.

In FIG. 16 (a), the horizontal axis represents time, the vertical axis represents the response current, the one-dot chain line shows the result when the formaldehyde concentration is equivalent to 1 ppb (68.4 μM formaldehyde standard solution), and the dotted line represents the formaldehyde concentration. The result in the case of 10 ppb equivalent (684 μM formaldehyde standard solution) is shown, and the solid line shows the result in the case where the formaldehyde concentration is equivalent to 100 ppb (6840 μM formaldehyde standard solution).
FIG. 16B shows a calibration curve created using the result of FIG.
From the result of FIG. 16A, compared with the case of using the standard gas, when the standard solution is used, the dissolution time and the diffusion time of the gas in the electrolytic solution can be omitted. It was found that an equilibrium state was reached. Thus, it was found that the calibration of the enzyme sensor 100 can be performed at a higher speed by creating a calibration curve using the standard gas.
Further, from the result of FIG. 16B, it was found that the created “electrode-fixed enzyme sensor” has a good linearity in a concentration range of 1 to 100 ppb.

<2-3> Temperature calibration of enzyme sensor 100 Using the created “electrode-fixed enzyme sensor”, an experiment for verifying the effect of temperature calibration of the enzyme sensor 100 was performed.
Specifically, for the “electrode-fixed enzyme sensor”, the temperature of the enzyme sensor 100 and the temperature of the electrolytic solution in the electrolytic solution tank 310 were adjusted to the initial set temperature (30 ° C.) to prepare for measurement. Thereafter, standard gases having different formaldehyde concentrations were sequentially generated and introduced, and a calibration curve was created by measuring the response current, followed by washing and circulation washing. Then, the slope of the created calibration curve was obtained and used as the adjustment threshold value.
Subsequently, for the same “electrode-fixed enzyme sensor” as the “electrode-fixed enzyme sensor” used for the preparation of the calibration curve, the temperature of the enzyme sensor 100 and the temperature of the electrolyte in the electrolyte tank 310 are subsequently used. Are adjusted to the initial set temperature (30 ° C.), and the measurement preparation is performed. Then, calibration, washing and circulation washing of the enzyme sensor 100 are performed, and a standard gas having a formaldehyde concentration of 100 ppb is introduced to measure the response current. Thereafter, washing and circulation washing were performed. The standard gas introduction rate was 300 mL / min.

  In the calibration process of the enzyme sensor 100, the adjustment threshold (the slope of the calibration curve created on the first day (0.3)) and the temperature dependence of the activity of formaldehyde dehydrogenase shown in FIG. 15 are taken into consideration. When “0.3 <calibration curve slope ≦ 0.35”, 28 ° C., and when “0.35 <calibration curve slope ≦ 0.4”, 26 ° C., “0.4 < In the case of “the slope of the calibration curve”, 23 ° C. is determined as the set temperature (enzyme sensor 100 at the time of measurement), and in the case of “0.25 <the slope of the calibration curve ≦ 0.3”, 31 ° C. When “0.2 <calibration curve slope ≦ 0.25”, 33 ° C., and when “0.15 <calibration curve slope ≦ 0.2”, set the temperature at 37 ° C. The enzyme sensor 100) was determined. Further, when “the slope of the calibration curve ≦ 0.15”, an error report is made by turning on the notification lamp 240 or the like. In addition, the temperature of the electrolyte solution introduced into the enzyme sensor 100 at the time of measurement was not adjusted and was kept constant (30 ° C.). Further, the temperature of the electrolytic solution in the electrolytic solution storage tank was maintained at 30 ° C. The result is shown in FIG.

In FIG. 17, the horizontal axis shows the relative response with the number of days and the vertical axis the response current on the first day as 100%, and shows the result when the calibration is not performed with a square (□) plot (comparative example). ◆) Shows the result of calibration with plot.
From the results of FIG. 17, when the enzyme sensor 100 is not calibrated, the relative response is less than 60% on the 10th day, whereas when the enzyme sensor 100 is calibrated, the relative response is almost constant even after 10 days or more. It was found to be 100%. Thus, it was found that the response output of the enzyme sensor 100 can be stabilized by calibrating the enzyme sensor 100.
In this experiment, the temperature of the electrolytic solution in the electrolytic solution storage tank was maintained at 30 ° C., but the temperature of the electrolytic solution in the electrolytic solution storage tank was set at, for example, the deactivation of the enzyme contained in the electrolytic solution. If the temperature is maintained at a temperature (for example, 4 ° C.) that can prevent the enzyme sensor 100, the life of the enzyme sensor 100 is further extended, and the frequency of calibration can be suppressed.

According to the enzyme sensor 100 in the first embodiment described above, the gas phase chamber R1 into which the gas containing the detection target substance is introduced and the gas phase chamber R1 are arranged adjacent to each other, and a predetermined electrolytic solution Is disposed so as to separate the gas phase chamber R1 from the gas phase chamber R1 and the liquid phase chamber R2, and at least the gas permeable membrane 130 through which the detection target substance permeates, and the gas permeable membrane 130 in the liquid phase chamber R2. The electrode 150 disposed oppositely and an enzyme as a receptor that is contained in the liquid phase chamber R2 and selectively reacts with the substance to be detected, and passes through the gas permeable membrane 140 to be liquid from the gas phase chamber R1. The detection target substance that has moved to the phase chamber R2 is detected.
That is, the enzyme sensor 100 has a simple configuration in which the gas phase chamber R1 and the liquid phase chamber R2 are disposed via the gas permeable membrane 130. Further, when the detection target substance in the gas sample is detected using the enzyme sensor 100, it is not necessary to perform a process such as dissolving the detection target substance in the gas sample in water, and the gas sample is used as it is in the gas phase chamber R1. Therefore, the detection target substance in the gas sample can be easily detected.

Moreover, according to the enzyme sensor 100 in the first embodiment described above, the spacer 170 disposed between the gas permeable membrane 130 and the electrode 150 is provided.
Therefore, since the distance between the electrode 150 and the permeable membrane 130 can be kept constant by the spacer 170, the detection target substance can be stably detected.

In addition, according to the enzyme sensor 100 in the first embodiment described above, the lower support 120A that supports the electrode 150 by configuring the surface of the liquid phase chamber R2 that faces the gas phase chamber R1 is provided. The lower support 120 </ b> A has a groove 122, the electrode 150 is formed in the groove 122, and the thickness of the electrode 150 is substantially the same as the depth of the groove 122.
Therefore, since the electrode thickness can be easily defined without finely adjusting the electrode material, the distance between the electrode 150 and the permeable membrane 130 can be more reliably maintained constant, and the detection target Substance detection can be performed stably.

Further, according to the gas detection system 1000 including the enzyme sensor 100 in the first embodiment described above, the electrolyte introduced into the liquid phase chamber R2 is discharged from the liquid phase chamber R2, and again the liquid phase chamber R2. And an electrolyte tank 310 to be introduced into the apparatus, a liquid feed pump 340, and a CPU 710 that executes a circulation cleaning program 733.
Therefore, evaporation of the electrolytic solution in the liquid phase chamber R2 can be prevented, so that the life of the enzyme sensor 100 can be extended. In addition, since the response current of the enzyme sensor 100 is restored to the equilibrium state at high speed, high speed measurement (continuous measurement) is possible.

Further, according to the gas detection system 1000 including the enzyme sensor 100 in the first embodiment described above, the sensor temperature adjustment device 410 that adjusts the temperature of the electrode 150 is provided, and before the detection of the detection target substance by the enzyme sensor 100 is performed. In addition, response data relating to the response output of the enzyme sensor 100 is acquired, and based on the acquired response data, the temperature of the electrode 150 when the detection target substance is detected by the enzyme sensor 100 is determined so as to be the determined temperature. The sensor temperature adjusting device 410 adjusts the temperature of the electrode 150 (enzyme sensor 100).
That is, the enzyme sensor 100 can be calibrated by adjusting the temperature of the electrode 150 (enzyme sensor 100) so that a stable sensor output can be obtained.

In addition, according to the gas detection system 1000 including the enzyme sensor 100 in the first embodiment described above, the electrolyte temperature adjusting device 420 that adjusts the temperature of the electrolyte introduced into the liquid phase chamber R2 is provided, and the enzyme Before detecting the detection target substance by the sensor 100, the response data regarding the response output of the enzyme sensor 100 is acquired, and the temperature of the electrolytic solution when the detection target substance is detected by the enzyme sensor 100 is determined based on the acquired response data. Then, the temperature of the electrolytic solution is adjusted by the electrolytic solution temperature adjusting device 420 so that the determined temperature is reached.
That is, the enzyme sensor 100 can be calibrated by adjusting the temperature of the electrolyte so that a stable sensor output can be obtained.

  Moreover, according to the gas detection system 1000 provided with the enzyme sensor 100 in the first embodiment described above, when the enzyme sensor 100 is a “free enzyme sensor”, as a preferred form, an electrolyte storage tank, an electrolysis Since the liquid storage tank temperature adjusting device is provided, the activity of the enzyme contained in the electrolytic solution can be prevented from being lowered, and the electrolytic solution containing the enzyme can be used stably for a long period of time.

[Second Embodiment]
Next, an enzyme sensor 100A and a gas detection system 1000A including the enzyme sensor 100A in the second embodiment will be described.
The enzyme sensor 100A in the second embodiment is different from the enzyme sensor 100 in the first embodiment in that the electrode 150 is detachable. The gas detection system 1000A in the second embodiment is different from the gas detection system 1000A in the first embodiment in the way of calibrating the enzyme sensor 100A. Therefore, only different parts will be described, and other common parts will be denoted by the same reference numerals and detailed description thereof will be omitted.

<Configuration of gas detection system>
FIG. 18 is a diagram showing a configuration of the gas detection system 1000A, and FIG. 19 is a diagram showing a functional configuration of the gas detection system 1000A.

  As shown in FIGS. 18 and 19, for example, the gas detection system 1000A includes an enzyme sensor 100A, a measurement circuit 210, a data processing device 220, a data display device 230, a notification lamp 240, an electrolyte tank 310, and the like. The standard liquid providing device 320, the waste liquid tank 330, the liquid feeding pump 340, the sensor temperature adjusting device 410, the electrolyte temperature adjusting device 420, the intake pump 510, the dust filter 520, and the standard gas providing device 530 The valve switching device 600, the control device 700A, and the like are configured.

The enzyme sensor 100A included in the gas detection system 1000A includes an electrode substrate 190 having an electrode 150 in a detachable manner, and detects a substance to be detected in a gas sample by an electrochemical measurement method using the characteristics of the enzyme. It is a sensor. The enzyme sensor 100A has a gas phase chamber R1 into which a gas (gas sample) containing a detection target substance is introduced and a liquid phase chamber R2 into which a predetermined electrolytic solution is introduced. Arranged in the phase chamber R2, the enzyme is contained in the liquid phase chamber R2.
The enzyme sensor 100A may be an “electrode-fixed enzyme sensor” that is contained in the liquid phase chamber R2 in the state of an immobilized enzyme in which the enzyme is fixed to the electrode 150. It may be a “free enzyme sensor” contained in the chamber R2.

For example, the control device 700A is a device for controlling each device constituting the gas detection system 1000A.
Specifically, for example, as illustrated in FIG. 19, the control device 700A includes a CPU 710, a RAM 720, a storage unit 730A, and the like.

  Specifically, the storage unit 730A stores, for example, a measurement preparation program 731, a calibration program 732A, a circulation cleaning program 733, a calibration curve creation program 734, a measurement program 735, a cleaning program 736, and the like. is doing.

  For example, the calibration program 732A causes the CPU 710 to realize a function of performing calibration (calibration) of the enzyme sensor 100A before measurement (detection of a detection target substance).

Specifically, when the enzyme sensor 100A is an “electrode-fixed enzyme sensor”, the CPU 710 inputs a control signal to the valve switching device 600, for example, so that the standard solution in the standard solution providing device 320 is the enzyme sensor 100A. The first valve 610 is switched so that the liquid is fed to the waste liquid tank, and the second valve 620 is switched so that the standard liquid discharged from the enzyme sensor 100 is fed to the waste liquid tank 330. A control signal is input to the pump 340 to generate and introduce a standard solution having a substrate concentration set in advance, and a control signal is input to the data processing device 220 to change the response current from the enzyme sensor 100A over time. Data (response data) is created.
Further, when the enzyme sensor 100A is an “electrode-fixed enzyme sensor” or a “free enzyme sensor”, the CPU 710 inputs a control signal to the valve switching device 600, for example, and the standard gas in the standard gas providing device 530 is displayed. The third valve 630 is switched so as to be introduced into the enzyme sensor 100A, a control signal is input to the standard gas providing device 530, a standard gas having a preset substrate concentration is generated and introduced, and data processing is performed. A control signal is input to the device 220 to generate data (response data) related to the temporal change in the response current of the enzyme sensor 100A.

Next, when the enzyme sensor 100A is an “electrode-fixed enzyme sensor”, the CPU 710 obtains, for example, a time required for the response current to exceed a predetermined value from the created data, and based on the time required. Then, it is determined whether or not the electrode substrate unit 190 should be replaced. More specifically, when the required time is equal to or greater than a predetermined replacement threshold, it is determined that the enzyme immobilized on the electrode 150 of the electrode substrate unit 190 has deteriorated, and it is determined that the electrode substrate unit 190 should be replaced. To do.
On the other hand, when the enzyme sensor 100A is a “free enzyme sensor”, the CPU 710 obtains the time required for the response current to exceed a predetermined value from the created data, for example, and based on the time required, It is determined whether or not the electrolytic solution in the electrolytic solution tank 310 should be replaced. More specifically, when the required time is equal to or greater than a predetermined replacement threshold, it is determined that the enzyme contained in the electrolytic solution in the electrolytic solution tank 310 has deteriorated, and it is determined that the electrolytic solution should be replaced.

  Next, when it is determined that the battery should be replaced, the CPU 710 inputs a control signal to the notification lamp 240 and blinks the notification lamp 240 to notify the user to replace the electrode substrate unit 190 or the electrolytic solution.

Here, the acquisition means for acquiring response data regarding the response output of the enzyme sensor 100A before the detection of the detection target substance by the enzyme sensor 100A includes the measurement circuit 210, the data processing device 220, and the CPU 710 that executes the calibration program 732A. , Etc.
Further, the CPU 710 executes the calibration program 732A, and based on the response data acquired by the acquisition unit, the determination unit that determines whether or not the electrode substrate unit 190 should be replaced and the response data acquired by the acquisition unit. Based on the above, it functions as a judging means for judging whether or not the electrolytic solution should be replaced.

<Configuration of enzyme sensor>
20 is a plan perspective view of the enzyme sensor 100A, FIG. 21 is a diagram schematically showing a cross section taken along line XXI-XXI in FIG. 20, and FIG. 22 is a cross section taken along line XXII-XXII in FIG. It is a figure shown typically. FIG. 23A is a bottom view of the upper support 110A in a state where the gas permeable membrane 130 is attached, and FIG. 23B is a view of the lower support 120A in a state where the electrode substrate 190 is attached. It is a top view.

The enzyme sensor 100A includes, for example, as shown in FIGS. 20 to 23, an upper support 110A, a lower support 120A, a gas phase chamber R1 into which a gas containing a detection target substance is introduced, and a gas phase chamber A gas permeable membrane that is disposed adjacent to R1 and is disposed so as to separate the liquid phase chamber R2 into which a predetermined electrolytic solution is introduced and the gas phase chamber R1 and the liquid phase chamber R2, and allows at least the detection target substance to pass therethrough. 130, a first O-ring 141 for fixing the gas permeable membrane 130 to the upper support 110A, a second O-ring 142 for defining the size (diameter) of the liquid phase chamber R1, a gas permeable membrane 130 and an electrode 150, a plurality of screws 180 for fixing the upper support 110A to the lower support 120A, and 3 disposed in the liquid phase chamber R1 facing the gas permeable membrane 130. 150 electrodes Configured to include an electrode substrate 190 having a working electrode 151, reference electrode 152, counter electrode 153) and the electrode 150 connected through a wire 161 and has been three pads 160, and the like.
In the enzyme sensor 100A, an enzyme as a receptor that selectively reacts with a substance to be detected is contained in the liquid phase chamber R2, and the enzyme sensor 100A passes through the gas permeable membrane 130 and passes from the gas phase chamber R1 to the liquid phase chamber R2. The substance to be detected that has shifted to is detected by an electrochemical measurement method.

The upper support body 110A and the lower support body 120A are, for example, a substantially cylindrical body that is cut in a part of the side surface in the vertical direction so as to be substantially D-shaped in plan view, and at a substantially central position in the vertical direction. It consists of what was divided | segmented into the direction (horizontal direction) orthogonal to an up-down direction.
The upper support 110A is formed to be, for example, a dome shape that bulges upward.

The upper support 110A has, for example, a substantially ring shape in a plan view, which is disposed at a substantially central position in the horizontal direction of the upper support 110A. An O-ring housing part 113; a gas introduction port 114 for introducing gas from the outside of the enzyme sensor 100A toward the gas phase chamber R1; and a gas introduction path 115 communicating the gas introduction port 114 and the gas phase chamber R1. A gas discharge port 116 for discharging gas from the gas phase chamber R1 toward the outside of the enzyme sensor 100A, a gas discharge path 117 that connects the gas discharge port 116 and the gas phase chamber R1, and the outside of the enzyme sensor 100A From the liquid phase chamber R2 to the liquid phase chamber R2, an electrolytic solution introduction port 123 for introducing the electrolytic solution toward the liquid phase chamber R2, an electrolytic solution introduction path 124 communicating the electrolytic solution inlet 123 and the liquid phase chamber R2, and And an electrolytic solution outlet port 125 for discharging the electrolytic solution toward the 100A outside of the electrolyte discharge channel 126, and the like are provided for communicating the electrolyte outlet 125 and the liquid phase chamber R2.
Here, in particular, the surface of the upper support 110 </ b> A, the gas inlet 114, the gas inlet 115, the gas outlet 116, the gas outlet 117, the electrolyte inlet 123, the electrolyte inlet 124, and the electrolyte outlet 125. The electrolyte discharge passage 126, the recess 112 (the gas phase chamber R1, the liquid phase chamber R2) and the like are materials having high corrosion resistance to the electrolyte solution and the sample (for example, a hydrophobic insulating material, and the surface is subjected to a hydrophobic treatment) It is preferably formed of an insulating material or the like.

The recess 112 is, for example, a first recess 112a having a substantially circular shape in plan view, the position of the bottom surface (the surface facing the opening surface) separated by the partition wall 118 being above the approximate center in the vertical direction of the upper support 110A. And a second concave portion 112b having a substantially ring shape in plan view, the position of the bottom surface (the surface facing the opening surface) being below the substantially vertical center of the upper support 110A.
The first recess 112a is separated in the vertical direction when the gas permeable membrane 130 is attached to the upper support 110A, and the region above the first recess 112a in the separated state is a gas phase chamber. R1 and the lower region of the first recess 112a and the second recess 112b become the liquid phase chamber R2. Therefore, the distance between the gas permeable membrane 130 and the electrode 150 (working electrode 151) is defined by the height (length in the vertical direction) of the partition wall 118.

The gas introduction path 115 is formed, for example, on the left side of the first recess 112a and upward in the left direction, and the gas discharge path 117 is positioned on the right side of the first recess 112a in the right direction, for example. It is formed toward the upper side.
The angle of the gas introduction path 115 with respect to the gas permeable membrane 130 is arbitrary as long as it is 0 degree or more and 90 degrees or less, but the pressure applied to the gas permeable film 130 is increased to facilitate the permeation of the detection target substance. From the viewpoint, 45 degrees or more is preferable.

The electrolyte solution introduction path 124 is, for example, a first electrolysis formed in the vertical direction in a region from the upper surface of the second concave portion 112b behind the first concave portion 112a to a position approximately at the center in the vertical direction of the upper support 110A. It consists of a liquid introduction path 124a and a second electrolyte introduction path 124b formed rearward and upward at a position on the rear side of the first electrolyte introduction path 124a.
In addition, the electrolyte discharge path 126 is, for example, formed in a vertical direction in a region from the upper surface of the second recess 112b in front of the first recess 112a to a position approximately at the center of the upper support 110A in the vertical direction. The first electrolyte solution discharge path 126a and the second electrolyte solution introduction path 124b formed in the front direction and upward at a position on the front side of the first electrolyte solution discharge path 126a.

The lower support 120A includes, for example, an attachment portion 127 for detachably attaching the electrode substrate portion 190 disposed on the upper surface of the lower support 120A from the substantially horizontal center position toward the front side. Is provided.
Here, in particular, the surface of the lower support 120A, the attachment portion 127, and the like are made of a material having high corrosion resistance to an electrolyte solution or a sample (for example, a hydrophobic insulating material, an insulating material whose surface is subjected to hydrophobic treatment, etc.) ).

  The attachment portion 127 is formed in, for example, a concave shape having an open upper surface, and the depth of the attachment portion 127 is set to be substantially the same as the thickness of the electrode substrate portion 190, for example.

  For example, the gas permeable film 130 covers the first recess 112a from the lower surface side of the upper support 110A with the gas permeable film 130, and the first O-ring 141 is arranged on the lower surface side of the gas permeable film 130 and moves upward. By attaching to the periphery of the partition wall 118, it is attached to the upper support 110A.

The first O-ring 141 is, for example, for attaching the gas permeable membrane 130 to the upper support 110A.
The second O-ring 142 is, for example, for defining the horizontal size (diameter) of the liquid phase chamber R1, and the electrolysis introduced into the liquid phase chamber R2 by sealing the liquid phase chamber R2. This is for preventing the liquid from leaking, and is accommodated in the O-ring accommodating portion 113. Accordingly, the second O-ring 142 is preferably, for example, one having a thickness equal to or greater than the height (length in the vertical direction) of the O-ring housing portion 113.
As a material constituting the first O-ring 141 and the second O-ring 142, a material having high corrosion resistance against an electrolytic solution or a sample is preferable.

The electrode substrate 190 includes, for example, an electrode 150, a pad 160, a wiring 161 that connects the electrode 150 and the pad 160, a predetermined substrate 191 such as a glass substrate that supports these, and the like. .
The electrode substrate 190 is attached to the attachment part 127 by inserting it into the attachment part 127 from the front of the enzyme sensor 100A, for example.

<Measurement process>
An example of processing related to measurement (detection of a detection target substance) using the enzyme sensor 100A by the gas detection system 1000A will be described.
Since the measurement process by the gas detection system 1000A of the second embodiment is substantially the same as the flow of the measurement process (FIG. 7) by the gas detection system 1000 of the first embodiment, only different points will be described. Detailed description of other common parts is omitted.

Preparation for measurement is performed (step S1), and the enzyme sensor 100A is calibrated (step S2).
In step S2, when it is determined that the electrode substrate unit 190 or the electrolytic solution should be replaced, the notification lamp 240 is blinked to allow the user to replace the electrode substrate unit 190 or the electrolytic solution.

  Next, when it is determined in step S2 that the electrode substrate unit 190 or the electrolytic solution should be replaced, after confirming that the electrode substrate unit 190 or the electrolytic solution has been replaced, or in step S2, the electrode substrate unit 190 or the electrolytic solution is replaced. If it is determined that the liquid does not need to be replaced, after the determination, the enzyme sensor 100A is washed (step S3) and circulated and washed (step S4), and then the response output of the enzyme sensor 100A is stabilized. Then, a calibration curve is created (step S5), and the processes after step S6 are performed.

  Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited thereto.

The enzyme sensor 100A was created and the enzyme sensor 100A and the gas detection system 1000A were evaluated.
In this example, an “electrode-fixed enzyme sensor” was created as the enzyme sensor 100A.

<1> Production of enzyme sensor 100A In this example, an enzyme sensor 100A for detecting formaldehyde gas was produced. As the enzyme, formaldehyde dehydrogenase (formaldehyde dehydrogenase), which is a coenzyme (NAD ⁺ ) -dependent enzyme, was used.

First, an electrode substrate 190 having a tripolar pattern of the working electrode 151, the reference electrode 152, and the counter electrode 153 was created.
Specifically, carbon was applied to the substrate 191 by screen printing, and post-baked at 120 ° C. for 15 minutes using a hot plate. Further, an ultraviolet curable insulating film is applied to portions other than the analysis window (a region having a substantially circular shape in a plan view where the working electrode 151, the reference electrode 152, and the counter electrode 153 are disposed) and the pad 160 by screen printing, and ultraviolet exposure. The ultraviolet curable insulation film was cured using an apparatus. Here, the analysis window is formed in a region corresponding to the liquid phase chamber R2 when the electrode substrate portion 190 is attached to the attachment portion 127.
Thereafter, the film was dried overnight in a dark room, and a silver / silver chloride ink was applied onto the pattern of the reference electrode 152 and sintered at 120 ° C. to prepare a reference electrode 152 that is a silver / silver chloride electrode.

  Next, 10 μL of an electrolytic solution (enzyme solution) prepared by dissolving 2 mg of formaldehyde dehydrogenase in 1 mM phosphate buffer (pH 7.41) was dropped onto the analysis window. Then, it dried in glutaraldehyde gas overnight, the enzyme was fixed to the electrode 150, and the electrode substrate part 190 was created.

Next, the upper support 110A and the lower support 120A were formed using a lathe, a milling machine, or the like using a peak material that is an insulator.
The depth of the attachment portion 127 was made substantially the same as the thickness (0.5 mm) of the electrode substrate portion 190. Further, the height of the partition wall 118 was adjusted so that the distance between the gas permeable membrane 130 and the electrode 150 was 30 μm.

Next, the first O-ring 141 is used to fix the gas permeable membrane 130 (Goretech porous Teflon membrane) to the upper support 110A, and the second O-ring 142 is accommodated in the O-ring accommodating portion 113. A spacer 170 (thickness: 30 μm, pore diameter 0.1 μm, Millipore hydrophilic Teflon membrane) having a thickness substantially the same as the distance (30 μm) from the electrode 150 was disposed in the liquid phase chamber R2.
Next, the upper support body 110A is disposed on the lower support body 120A, the upper support body 110A is fixed to the lower support body 120A using screws 180, and the electrode substrate part 190 created as described above is attached to the attachment part 127. To attach an electrode-fixed enzyme sensor.

Next, an electrolytic solution for “electrode-fixed enzyme sensor” was prepared.
Specifically, an electrolyte solution for “electrode-fixed enzyme sensor” was prepared by preparing a phosphate buffer solution (pH 7.41) containing 1 mM NAD ⁺ and 1 mM quinone. And this electrolyte solution was put in the electrolyte solution tank 310, and the enzyme sensor 100A and the gas detection system 1000A were evaluated.

<2> Evaluation of Enzyme Sensor 100A and Gas Detection System 1000A In this example, an experiment relating to the removal of the electrode substrate 190 was performed by measuring the temperature of the enzyme sensor 100A and the temperature of the electrolyte in the electrolyte tank 310 at 30 ° C. I went there.

<2-1> Creation of Calibration Curve An experiment for creating a calibration curve was performed using the created “electrode-fixed enzyme sensor”.
Specifically, after preparing for measurement of the “electrode-fixed enzyme sensor”, a standard solution with different formaldehyde concentrations is sequentially generated and introduced, and a calibration curve is created by measuring the response current, followed by washing. And circulating cleaning was performed. As a standard solution, a solution in which formaldehyde was diluted in a phosphate buffer solution (pH 7.41) containing 1 mM NAD ⁺ and 1 mM quinone was used. The formaldehyde concentration of the standard solution was 68.4 μM, 684 μM, and 6840 μM, and the standard solution introduction rate was 0.1 mL / min. Assuming vapor-liquid equilibrium, the formaldehyde concentration of the standard solution of 68.4 μM corresponds to the formaldehyde concentration of 1 ppb of the standard gas, the formaldehyde concentration of the standard solution of 684 μM corresponds to the formaldehyde concentration of the standard gas of 10 ppb, and the formaldehyde concentration of the standard solution 6840 μM corresponds to a formaldehyde concentration of 100 ppb in the standard gas. The result is shown in FIG.

In FIG. 24 (a), the horizontal axis represents time, the vertical axis represents the response current, the one-dot chain line shows the result when the formaldehyde concentration is equivalent to 1 ppb (68.4 μM formaldehyde standard solution), and the dotted line represents the formaldehyde concentration. The result in the case of 10 ppb equivalent (684 μM formaldehyde standard solution) is shown, and the solid line shows the result in the case where the formaldehyde concentration is equivalent to 100 ppb (6840 μM formaldehyde standard solution).
FIG. 24B shows a calibration curve created using the results of FIG.
From the result of FIG. 24A, the “electrode-fixed enzyme sensor” created in this example can be detached from the electrode 150, but the “electrode-fixed type sensor” created in Example 2 of the first embodiment. Since the distance between the “enzyme sensor” (an “electrode-fixed enzyme sensor” in which the electrode 150 is not removable) and the gas permeable membrane 130 and the electrode 150 is the same (30 μm), there was no significant difference in sensitivity.
Further, from the result of FIG. 24B, it was found that the “electrode-fixed enzyme sensor” prepared in this example has a good linearity in a concentration range of 1 to 100 ppb.

<2-2> Degradation of enzyme immobilized on electrode 150 Using the created “electrode-immobilized enzyme sensor”, an experiment for observing degradation of the enzyme immobilized on the electrode 150 was performed.
Specifically, for the “electrode-fixed enzyme sensor”, after preparing for measurement, a standard solution having a formaldehyde concentration of 6840 μM was generated and introduced, and the response current was measured, followed by washing and circulation washing. And it preserve | saved at room temperature without replacing | exchanging the electrode board | substrate part 190, and the process of this measurement preparation-measurement-washing (washing | cleaning and circulation washing | cleaning) was performed again 10 days later. The standard solution introduction rate was 0.1 mL / min. The result is shown in FIG.

In FIG. 25, the horizontal axis indicates time, the vertical axis indicates response current, the dotted line indicates the result after 0 days (first day), and the solid line indicates the result after 10 days.
From the results shown in FIG. 25, it was found that when stored at room temperature for 10 days, the enzyme immobilized on the electrode 150 deteriorates and the response output of the enzyme sensor 100A decreases.

<2-3> Calibration of enzyme sensor 100A Using the created “electrode-fixed enzyme sensor”, it was determined that the effect of calibrating enzyme sensor 100A, that is, the enzyme fixed to electrode 150 was deteriorated. In some cases, an experiment was conducted to verify the effect of replacing the electrode substrate unit 190.
Specifically, for the “electrode-fixed enzyme sensor”, after preparing for measurement, the enzyme sensor 100A is calibrated, washed, and circulated, and a standard solution with a formaldehyde concentration of 6840 μM is generated and introduced. After measuring, washing and circulation washing were performed. And this measurement preparation-calibration-washing (washing and circulation washing) -measurement-washing (washing and circulation washing) was performed every day. The standard solution introduction rate was 0.1 mL / min.

  In the calibration process of the enzyme sensor 100A, the temperature of the enzyme sensor 100A and the temperature of the electrolytic solution in the electrolytic solution tank 310 are 30 ° C. (preset temperature), and the concentration of the introduced standard solution is 6840 μM ( When the time required for the response current to exceed 10 nA (predetermined value) is 20 sec (predetermined replacement threshold) when the concentration is a preset concentration), the electrode included in the electrode substrate unit 190 It is determined that the enzyme fixed to 150 has deteriorated, it is determined that the electrode substrate 190 should be replaced, the notification lamp 240 is blinked, and the user is allowed to replace the electrode substrate 190. The result is shown in FIG.

In FIG. 26, the horizontal axis represents the relative response with the number of days and the vertical axis the response current on the first day as 100%.
From the results shown in FIG. 26, it was found that the variation was within 5% after 20 measurements, and that measurement with good reproducibility was possible when the electrode substrate 190 was replaced.

According to the enzyme sensor 100A in the second embodiment described above, the electrode substrate 190 includes the electrode substrate 190 having the electrode 150 and the attachment 127 for attaching the electrode 150 to the liquid phase chamber R2. 190 is detachably attached to the mounting portion 120.
Therefore, since the electrode substrate 190 can be exchanged, the enzyme sensor 100A is particularly effective when the enzyme sensor 100A is an “electrode-fixed enzyme sensor”, and an inexpensive enzyme sensor 100A can be provided.

Moreover, according to the gas detection system 1000A in the second embodiment described above, when the enzyme sensor 100A is an “electrode-fixed enzyme sensor”, the enzyme sensor 100A detects the detection target substance before the enzyme sensor 100A. Response data regarding the response output of 100A is acquired, and it is determined whether or not the electrode substrate unit 190 should be replaced based on the acquired response data.
That is, when the enzyme sensor 100A is an “electrode-fixed enzyme sensor”, the enzyme sensor 100A can be calibrated by replacing the electrode substrate 190 so that a stable sensor output can be obtained.

Further, according to the gas detection system 1000A in the second embodiment described above, when the enzyme sensor 100A is a “free enzyme sensor”, the enzyme sensor 100A is detected before the detection of the detection target substance by the enzyme sensor 100A. The response data regarding the response output is acquired, and based on the acquired response data, it is determined whether or not the electrolytic solution should be replaced.
That is, when the enzyme sensor 100A is a “free enzyme sensor”, the enzyme sensor 100A can be calibrated by changing the electrolyte so that a stable sensor output can be obtained.

  Further, according to the gas detection system 1000A in the second embodiment described above, the response data is data related to the temporal change of the response current from the enzyme sensor 100A. In this case, the concentration of the standard solution or standard gas is Since only one type is required, the enzyme sensor 100A can be calibrated in a shorter time than when the concentration of the standard solution or standard gas needs to be sequentially changed (when the response data is a calibration curve). .

  The present invention is not limited to the above-described embodiment, and can be appropriately changed without departing from the gist thereof.

  In the first embodiment and the second embodiment, the receptor is not limited to an enzyme, and any receptor may be used as long as it is a biological substance (a molecular identification element derived from a living body) that selectively reacts with a detection target substance. Specifically, for example, an antibody or a microorganism may be used.

In the first embodiment and the second embodiment, the electrode 150 is not limited to that of the embodiment, but may be platinum, gold, silver, carbon, etc. by screen printing, vapor deposition, sputtering, or the like. As long as it is formed from, etc., it is arbitrary.
The reference electrode 152, which is a silver / silver chloride electrode, is formed by once forming a silver electrode by a screen printing method, a vapor deposition method, a sputtering method, etc., and then immersed in a second silver chloride aqueous solution by a method of electrolyzing a constant current. Any method may be used as long as it is formed by a method such as a method of applying and laminating silver chloride by a screen printing method.
In addition, the electrode 150 has a tripolar structure of the working electrode 151, the reference electrode 152, and the counter electrode 153, but the electrode 150 has a bipolar structure in which the reference electrode 152 is not provided (a bipolar structure of the working electrode 151 and the counter electrode 153). ).

  In the first embodiment and the second embodiment, when notifying the user, it may be displayed on the data display device 230 or the like instead of the notification lamp 240 and notified.

In the first embodiment, a calibration curve is used as response data and used to calibrate the enzyme sensor 100. However, the response data may be data relating to a temporal change in response current from the enzyme sensor 100. In this case, the set temperature is determined based on the time required for the response current to exceed a predetermined value.
Further, in the second embodiment, the data regarding the time change of the response current from the enzyme sensor 100A is used as the response data and used for the calibration of the enzyme sensor 100A. However, the response data may be a calibration curve. In this case, it is determined whether or not to exchange based on the slope of the calibration curve.

  In the first embodiment and the second embodiment, the electrode temperature adjusting means is not limited to the sensor temperature adjusting device 410 (a constant temperature bath), and may be arbitrary as long as the temperature of the electrode 150 can be adjusted. For example, a small heater or the like built in the enzyme sensors 100 and 100A may be used.

  In the second embodiment, the second O-ring 142 prevents the electrolyte introduced into the liquid-phase chamber R2 from leaking. However, the liquid-phase chamber R2 may be sealed using something other than the O-ring. The second O-ring has a configuration that can prevent the electrolyte introduced into the liquid phase chamber R2 from leaking, such as bonding and fixing the upper support 110A and the lower support 120A with an adhesive or the like. 142 may not be provided.

In the first embodiment, the electrolyte flow paths (electrolyte introduction path 124 and electrolyte discharge path 126) are formed so as to be substantially parallel to the gas flow paths (gas introduction path 115 and gas discharge path 116). However, the present invention is not limited to this. For example, the electrolyte flow path may be formed so as to be substantially orthogonal to the gas flow path as in the second embodiment.
In the second embodiment, the electrolyte flow paths (electrolyte introduction path 124 and electrolyte discharge path 126) are formed so as to be substantially orthogonal to the gas flow paths (gas introduction path 115 and gas discharge path 116). However, the present invention is not limited to this. For example, the electrolyte flow path may be formed so as to be substantially parallel to the gas flow path as in the first embodiment.

  In the first embodiment, when the enzyme sensor 100 is a “free enzyme sensor”, it is determined whether or not the electrolytic solution in the electrolytic solution tank 310 should be replaced as in the second embodiment. The enzyme sensor 100 may be calibrated by replacing the electrolytic solution when it is determined that it should be replaced.

  In the second embodiment, the groove portion 122 may be provided on the substrate 191 included in the electrode substrate portion 190. In this case, the substrate 191 becomes a support (a support that configures a surface of the liquid phase chamber R2 facing the gas phase chamber R1 and supports the electrode 150).

It is a figure which shows the structure of the gas detection system of 1st Embodiment. It is a figure which shows the functional structure of the gas detection system of 1st Embodiment. It is a top perspective view of the enzyme sensor of a 1st embodiment. It is a figure which shows typically the cross section in the IV-IV line of FIG. It is a figure which shows typically the cross section in the VV line | wire of FIG. It is a bottom view (a) of the upper side support body in the state where the gas permeable membrane was attached, and a top view (b) of the lower side support body in the state where the electrode was formed. It is a flowchart which shows an example of the process regarding the measurement (detection of a detection target substance) using an enzyme sensor by a gas detection system. It is a figure which shows the evaluation result (selection of a gas permeable film) of the enzyme sensor and gas detection system of the Example (Example 1-1) of 1st Embodiment. It is a figure which shows the evaluation result (creation of a calibration curve) of the enzyme sensor and gas detection system of the Example (Example 1-1) of 1st Embodiment. It is a figure which shows the evaluation result (detection of formaldehyde in external atmosphere) of the enzyme sensor and gas detection system of the Example (Example 1-1) of 1st Embodiment. It is a figure which shows the evaluation result (washing | cleaning (washing | cleaning and circulation washing | cleaning) of an enzyme sensor) of the enzyme sensor and gas detection system of the Example (Example 1-1) of 1st Embodiment. It is a figure which shows the evaluation result (repetitive reproducibility of an enzyme sensor) of the enzyme sensor and gas detection system of the Example (Example 1-1) of 1st Embodiment. It is a figure which shows the evaluation result (washing | cleaning (washing | cleaning and circulation washing | cleaning) of an enzyme sensor) of the enzyme sensor and gas detection system of the Example (Example 1-1) of 1st Embodiment. It is a figure which shows the evaluation result (selectivity of an enzyme sensor) of the enzyme sensor and gas detection system of the Example (Example 1-1) of 1st Embodiment. It is a figure which shows the evaluation result (temperature dependence of the activity of formaldehyde dehydrogenase) of the enzyme sensor and gas detection system of the Example (Example 1-2) of 1st Embodiment. It is a figure which shows the evaluation result (creation of a calibration curve) of the enzyme sensor and gas detection system of the Example (Example 1-2) of 1st Embodiment. It is a figure which shows the evaluation result (temperature calibration of an enzyme sensor) of the enzyme sensor and gas detection system of the Example (Example 1-2) of 1st Embodiment. It is a figure which shows the structure of the gas detection system of 2nd Embodiment. It is a figure which shows the functional structure of the gas detection system of 2nd Embodiment. It is a top perspective view of the enzyme sensor of a 2nd embodiment. It is a figure which shows typically the cross section in the XXI-XXI line | wire of FIG. It is a figure which shows typically the cross section in the XXII-XXII line | wire of FIG. It is the bottom view (a) of the upper side support body in the state where the gas permeable membrane was attached, and the top view (b) of the lower side support body in the state where the electrode part was attached. It is a figure which shows the evaluation result (creation of a calibration curve) of the enzyme sensor and gas detection system of the Example (Example 2) of 2nd Embodiment. It is a figure which shows the evaluation result (deterioration of the enzyme fixed to the electrode) of the enzyme sensor and gas detection system of the Example (Example 2) of 2nd Embodiment. It is a figure which shows the evaluation result (calibration of an enzyme sensor) of the enzyme sensor and gas detection system of the Example (Example 2) of 2nd Embodiment.

Explanation of symbols

100,100A Enzyme sensor (biosensor)
120 Lower support (support)
122 Groove 127 Attachment portion 130 Gas permeable membrane 150 Electrode 170 Spacer 190 Electrode portion 210 Measuring circuit (acquisition means)
220 Data processing device (acquisition means)
310 Electrolyte tank (circulation means)
340 Liquid feed pump (circulation means)
410 Sensor temperature adjusting device (electrode temperature adjusting means)
420 Electrolyte temperature adjusting device (electrolyte temperature adjusting means)
710 CPU (acquisition means, determination means, adjustment control means, circulation means, determination means)
732 Calibration program (acquisition means, determination means, adjustment control means)
732A Calibration program (acquisition means, determination means)
733 Circulation cleaning program (circulation means)
1000,1000A Gas detection system R1 Gas phase chamber R2 Liquid phase chamber

Claims (18)

A gas phase chamber into which a gas containing a detection target substance is introduced;
A liquid phase chamber disposed adjacent to the gas phase chamber and into which a predetermined electrolyte is introduced;
A gas permeable membrane that is disposed so as to separate the gas phase chamber and the liquid phase chamber and allows at least the detection target substance to pass through;
An electrode disposed opposite to the gas permeable membrane in the liquid phase chamber;
A receptor contained in the liquid phase chamber and selectively reacting with the detection target substance,
A biosensor that detects the detection target substance that has passed through the gas permeable membrane and has migrated from the gas phase chamber to the liquid phase chamber. The biosensor according to claim 1, wherein
A biosensor comprising a spacer disposed between the gas permeable membrane and the electrode. The biosensor according to claim 1 or 2,
Constituting a surface of the liquid phase chamber facing the gas phase chamber, and comprising a support for supporting the electrode;
The support has a groove,
The electrode is formed in the groove;
The biosensor is characterized in that the thickness of the electrode is substantially the same as the depth of the groove. In the biosensor according to any one of claims 1 to 3,
An electrode part having the electrode;
An attachment portion for attaching the electrode portion to the liquid phase chamber,
The biosensor according to claim 1, wherein the electrode part is detachable from the attachment part. In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
A gas detection system comprising circulation means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber. In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
A gas detection system comprising electrode temperature adjusting means for adjusting the temperature of the electrode. The gas detection system according to claim 6.
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Determination means for determining the temperature of the electrode at the time of detection of the detection target substance by the biosensor based on the response data acquired by the acquisition means;
An adjustment control means for causing the electrode temperature adjustment means to adjust the temperature of the electrode so that the temperature is determined by the determination means;
A gas detection system comprising: In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
A gas detection system comprising an electrolyte temperature adjusting means for adjusting the temperature of the electrolyte introduced into the liquid phase chamber. The gas detection system according to claim 8.
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Based on the response data acquired by the acquisition unit, a determination unit that determines the temperature of the electrolytic solution at the time of detection of the detection target substance by the biosensor;
Adjustment control means for causing the electrolyte temperature adjusting means to adjust the temperature of the electrolyte so as to be the temperature determined by the determining means;
A gas detection system comprising: A gas detection system comprising the biosensor according to claim 4.
The receptor is contained in the liquid phase chamber by being fixed to the electrode,
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Determining means for determining whether or not to replace the electrode unit based on the response data acquired by the acquiring means;
A gas detection system comprising: In a gas detection system comprising the biosensor according to any one of claims 1 to 4,
The receptor is contained in the liquid phase chamber by being contained in the electrolytic solution introduced into the liquid phase chamber,
An acquisition means for acquiring response data related to a response output of the biosensor before the detection of the detection target substance by the biosensor;
Determining means for determining whether or not to replace the electrolyte based on the response data acquired by the acquiring means;
A gas detection system comprising: A gas phase chamber into which a gas containing a detection target substance is introduced;
A liquid phase chamber disposed adjacent to the gas phase chamber and into which a predetermined electrolyte is introduced;
A gas permeable membrane that is disposed so as to separate the gas phase chamber and the liquid phase chamber and allows at least the detection target substance to pass through;
An electrode disposed opposite to the gas permeable membrane in the liquid phase chamber;
An enzyme contained in the liquid phase chamber and selectively reacting with the detection target substance;
A spacer disposed between the gas permeable membrane and the electrode;
A surface of the liquid phase chamber that faces the gas phase chamber, and a support that supports the electrode; and
The support has a groove,
The electrode is formed in the groove;
The thickness of the electrode is substantially the same as the depth of the groove,
An enzyme sensor, wherein the detection target substance that has passed through the gas permeable membrane and has migrated from the gas phase chamber to the liquid phase chamber is detected. A gas detection system comprising the enzyme sensor according to claim 12,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
Electrode temperature adjusting means for adjusting the temperature of the electrode;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determination means for determining the temperature of the electrode at the time of detection of the detection target substance by the enzyme sensor based on the response data acquired by the acquisition means;
An adjustment control means for causing the electrode temperature adjustment means to adjust the temperature of the electrode so that the temperature is determined by the determination means;
A gas detection system comprising: A gas detection system comprising the enzyme sensor according to claim 12,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
An electrolyte temperature adjusting means for adjusting the temperature of the electrolyte introduced into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Based on the response data acquired by the acquisition unit, a determination unit that determines the temperature of the electrolytic solution at the time of detection of the detection target substance by the enzyme sensor;
Adjustment control means for causing the electrolyte temperature adjusting means to adjust the temperature of the electrolyte so as to be the temperature determined by the determining means;
A gas detection system comprising: A gas detection system comprising the enzyme sensor according to claim 12,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
Electrode temperature adjusting means for adjusting the temperature of the electrode;
An electrolyte temperature adjusting means for adjusting the temperature of the electrolyte introduced into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determination means for determining the temperature of the electrode and the temperature of the electrolytic solution at the time of detection of the detection target substance by the enzyme sensor based on the response data acquired by the acquisition means;
An adjustment control means for causing the electrode temperature adjustment means to adjust the temperature of the electrode so that the temperature is determined by the determination means, and for causing the electrolyte temperature adjustment means to adjust the temperature of the electrolyte solution;
A gas detection system comprising: A gas phase chamber into which a gas containing a detection target substance is introduced;
A liquid phase chamber disposed adjacent to the gas phase chamber and into which a predetermined electrolyte is introduced;
A gas permeable membrane that is disposed so as to separate the gas phase chamber and the liquid phase chamber and allows at least the detection target substance to pass through;
An electrode disposed opposite to the gas permeable membrane in the liquid phase chamber;
An enzyme contained in the liquid phase chamber and selectively reacting with the detection target substance;
A spacer disposed between the gas permeable membrane and the electrode;
An electrode part having the electrode;
An attachment portion for attaching the electrode portion to the liquid phase chamber,
The electrode part is detachable from the attachment part,
An enzyme sensor, wherein the detection target substance that has passed through the gas permeable membrane and has migrated from the gas phase chamber to the liquid phase chamber is detected. A gas detection system comprising the enzyme sensor according to claim 16,
The enzyme is contained in the liquid phase chamber by being fixed to the electrode,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determining means for determining whether or not to replace the electrode unit based on the response data acquired by the acquiring means;
A gas detection system comprising: A gas detection system comprising the enzyme sensor according to claim 12 or 16,
The enzyme is contained in the liquid phase chamber by being contained in the electrolyte solution introduced into the liquid phase chamber,
Circulating means for discharging the electrolyte introduced into the liquid phase chamber from the liquid phase chamber and introducing it again into the liquid phase chamber;
An acquisition means for acquiring response data relating to a response output of the enzyme sensor before the detection of the detection target substance by the enzyme sensor;
Determining means for determining whether or not to replace the electrolyte based on the response data acquired by the acquiring means;
A gas detection system comprising:

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