JP5250385B2 - Concentration measurement system - Google Patents

Description

  The present invention relates to a concentration measurement system.

Conventionally, a concentration measurement system that measures the concentration of a detection target substance in a sample using a sensor is known.
Specifically, for example, using a biosensor equipped with an electrode, a concentration measurement system that measures the concentration of glucose or the like in jelly, or a concentration measurement system that measures the concentration of free amino acids, fatty acids, sugars, etc. in fish meat ( For example, refer to Patent Document 1), using a biosensor equipped with an electrode, a concentration measurement system (for example, refer to Patent Document 2) that measures the concentration of ammonia nitrogen or the like in wastewater, and using a biosensor equipped with an electrode The concentration of the human chorionic gonadotropin (hCG) in the liquid sample is measured using a concentration measurement system (see, for example, Patent Document 3) and a biosensor (enzyme sensor) equipped with electrodes. Using a concentration measurement system (for example, refer to Patent Document 4) for measuring concentrations of hydrogen oxide, hydrogen oxide, glucose, etc., and a biosensor (enzyme sensor) equipped with electrodes Concentration measuring system for measuring the concentration of dissolved oxygen and the like in a fluid sample (e.g., Patent Document 5 references) have been proposed.

  Here, Patent Document 3 proposes a method of determining the amount (concentration) of the detection target substance using the initial speed of the signal from the sensor (the initial slope of the signal value from the sensor). This method is preferable because the measurement time can be shortened because it is not necessary to wait until the signal value from the sensor reaches equilibrium.

By the way, when using the characteristics of biological materials such as enzymes and microorganisms as in the biosensors described in Patent Documents 1 to 5, the presence of water is necessary to make the biological materials function. Therefore, in the biosensor, the detection element (electrode or the like) or the detection unit containing the biological material is a liquid phase chamber filled with water (aqueous solution). For example, when a detection target substance in a gas sample is detected using such a sensor, the liquid phase chamber is controlled by a permeable membrane (gas permeable membrane) in order to prevent water from leaking or evaporating from the liquid phase chamber. Etc.).
Japanese Patent No. 2690053 Japanese Patent Publication No. 06-072858 Japanese Patent No. 2930809 Japanese Patent No. 3299211 JP 2001-208720 A

  However, when the liquid phase chamber is covered with the permeable membrane, the detection target substance moves from the gas sample to the liquid phase chamber through the permeable membrane, and is detected by the detection element in the liquid phase chamber. However, since the diffusion of the detection target substance in the liquid phase chamber depends on the distance between the permeable membrane and the detection element, the sensitivity of the sensor varies depending on the distance between the permeable membrane and the detection element. Therefore, when the gas sample is sprayed on the permeable membrane, the permeable membrane is deformed, and the distance between the permeable membrane and the detection element changes, the detection by the sensor cannot be stably performed, and the concentration of the detection target substance is It becomes difficult to determine with high accuracy using the initial inclination of the signal value from the sensor.

  An object of the present invention is to provide a concentration measurement system capable of measuring the concentration of a detection target substance in a gas sample with high reproducibility at high speed and high accuracy.

In order to solve the above-mentioned problem, the invention described in claim 1
In the concentration measurement system,
A sensor,
A measuring device for measuring the concentration of the detection target substance in the fluid sample based on the detection result by the sensor;
With
The sensor is
A supply unit to which the fluid sample is supplied;
A detection unit disposed adjacent to the supply unit;
A permeable membrane that is disposed so as to separate the supply unit and the detection unit, and through which at least the detection target substance passes;
A detection element disposed on the detection unit so as to face the permeable membrane;
A spacer disposed between the permeable membrane and the detection element and having a plurality of through holes penetrating in a direction substantially perpendicular to the detection element;
With
The measuring device is
A measuring means for measuring a signal value from the sensor;
Based on a time change curve representing a time change of the signal value measured by the measurement means, a calculation means for calculating an inclination of an approximate straight line obtained by approximating an initial portion of the time change curve as an initial inclination;
Storage means for storing in advance calibration curve data representing a relationship between the initial inclination and the concentration of the detection target substance;
An acquisition means for acquiring the concentration of the detection target substance corresponding to the initial slope calculated by the calculation means from the calibration curve data stored in the storage means;
Equipped with a,
Said initial portion of said time variation curve, from the first hour or more time of starting the supply of the fluid sample into the supply unit, Rukoto Oh the portion up to the second hour is larger than said first hour It is characterized by.

The invention described in claim 2
The concentration measurement system according to claim 1,
The measuring device is
A supply pump for supplying the fluid sample to the supply unit;
An introduction pump for introducing a predetermined fluid substance into the detection unit and cleaning the inside of the detection unit;
It is characterized by providing.

The invention according to claim 3
The concentration measurement system according to claim 1 or 2,
The detection element is a porous carbon electrode;
The detection unit contains ferrocenecarboxylic acid and / or ferrocenyltrimethylammonium bromide as an electron carrier.

The invention according to claim 4
The concentration measurement system according to claim 2 ,
It said sensor is an enzyme sensor comprising an enzyme that selectively reacts with the detection target substance,
It said fluid sample is a gaseous sample,
The supply unit is a gas phase chamber to which the gas sample is supplied,
The detection unit is a liquid phase chamber that is disposed adjacent to the gas phase chamber and into which a predetermined electrolytic solution is introduced,
The feed pump is a suction pump which supplies the gaseous sample to the gas phase chamber,
The introduction pump is characterized in that said predetermined electrolyte is a liquid feed pump to introduce to the liquid phase chamber.

According to the present invention, the sensor includes a spacer disposed between the permeable membrane and the detection element and having a plurality of through holes penetrating in a substantially vertical direction with respect to the detection element. Based on the time change curve representing the time change of the signal value of the initial value, the inclination of the approximate line obtained by approximating the initial part of the time change curve is calculated as the initial inclination, and the initial inclination and the detection target substance stored in advance are calculated. The concentration of the detection target substance corresponding to the calculated initial slope can be acquired from the calibration curve data representing the relationship with the concentration.
That is, since the spacer is provided, the distance between the permeable membrane and the detection element can be kept constant, and a spacer having a plurality of through holes penetrating in a substantially vertical direction with respect to the detection element is used as the spacer. Therefore, even if the spacer is provided, diffusion of the detection target substance in the detection unit is not easily limited.
Therefore, the detection target substance can be stably detected, and the detection target substance can be detected based on the slope of the approximate line obtained by approximating the initial portion of the time change curve (the initial slope of the signal value from the sensor). Since the concentration can be determined, the concentration of the detection target substance in the gas sample can be measured with high reproducibility at high speed and high accuracy.

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 the sensor.

<Concentration measurement system>
FIG. 1 is a diagram showing a configuration of a concentration measurement system 1 of the present embodiment.
The concentration measurement system 1 is a system that detects a detection target substance using the enzyme sensor 100 and measures the concentration of the detection target substance, for example.
Specifically, for example, as shown in FIG. 1, the concentration measurement system 1 includes an enzyme sensor 100 and a measurement device 200 that measures the concentration of a detection target substance in a gas sample based on a detection result by the enzyme sensor 100. , And so on.

<Measurement device>
FIG. 2 is a block diagram illustrating a functional configuration of the measurement apparatus 200 included in the concentration measurement system 1 of the present embodiment.
For example, as shown in FIGS. 1 and 2, the measurement device 200 includes a measurement circuit 211, a data processing device 212, a display device 213, an electrolyte tank 221, a waste liquid tank 222, a liquid feed pump 223, A sensor temperature adjusting device 231, an intake pump 241, a dust filter 242, a standard gas providing device 243, a valve switching device 250, a control device 260, and the like are configured.

  The enzyme sensor 100 included in the concentration measurement system 1 includes an electrode 150 as a detection element, 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 includes a gas phase chamber R1 as a supply unit to which a gas sample (gas) containing a detection target substance is supplied, and a liquid phase chamber R2 as a detection unit to which an aqueous solution (predetermined electrolyte) is introduced. The electrode 150 is disposed in the liquid phase chamber R2, and the enzyme is contained in the liquid phase chamber R2.

For example, the measurement circuit 211 measures a signal value from the enzyme sensor 100 as a measurement unit, for example, according to a control signal input from the control device 260.
Specifically, for example, the measurement circuit 211 applies a voltage to the enzyme sensor 100 to measure a signal value (response current value) from the enzyme sensor 100 and outputs the measurement signal to the data processing device 212. .

For example, the data processing device 212 processes the measurement signal input from the measurement circuit 211 in accordance with the control signal input from the control device 260, creates numerical data based on the measurement signal, and outputs the numerical data to the display device 213. .
Here, the numerical data is arbitrary as long as it is data related to numerical values such as the response current from the enzyme sensor 100 and the concentration of the detection target substance, for example, data related to numerical values such as the response current and concentration. Alternatively, it may be data relating to a time change of the numerical value.

The display device 213 is composed of, for example, an LCD (Liquid Crystal Display) panel, and for example, numerical information based on numerical data input from the data processing device 212 or a concentration measurement system in accordance with a control signal input from the control device 260. 1 usage information is displayed.
Here, the displayed numerical information may be, for example, numerical values such as response current and concentration, may be numerically processed by the control device 260, or may indicate the time change of the numerical values. It may be a graph (such as a time change curve).
Note that the LCD panel or the like of the display device 213 may be integrated with a touch panel.

The electrolyte tank 221 is a tank for storing the electrolyte, and is connected to the enzyme sensor 100 (electrolyte introduction side of the liquid phase chamber R2) with a tube via a liquid feed pump 223 or a valve, for example. In addition, it is connected to the enzyme sensor 100 (electrolyte discharge side of the liquid phase chamber R2) through a valve through a tube.
Here, the electrolytic solution in the electrolytic solution tank 221 is, for example, an electrolytic solution containing an enzyme (a solution obtained by dissolving an enzyme in a buffer solution).

  Note that the measuring apparatus 200 may include, for example, an electrolyte temperature adjusting device (for example, a thermostatic bath) for adjusting the temperature of the electrolyte in the electrolyte tank 221. This is preferable because the temperature of the electrolyte introduced into the liquid phase chamber R2 can be adjusted and kept constant. Specifically, the electrolytic solution temperature adjusting device performs electrolysis so that the temperature of the electrolytic solution in the electrolytic solution tank 221 becomes a temperature designated by the control device 260 in accordance with a control signal input from the control device 260, for example. The temperature of the electrolytic solution in the liquid tank 221 is adjusted.

Moreover, in addition to the electrolyte tank 221, the measuring apparatus 200 can prevent, for example, the temperature of the electrolyte (electrolyte containing an enzyme) from being stored at the electrolyte storage temperature (deactivation of the enzyme contained in the electrolyte). You may provide the electrolyte storage tank which can be hold | maintained at temperature (for example, 4 degreeC). In this case, the enzyme contained in the electrolytic solution can be prevented from being deactivated, which is preferable. Specifically, for example, the measuring apparatus 200 includes an electrolyte storage tank and a temperature controller for the electrolyte storage tank, and the temperature of the electrolyte in the electrolyte storage tank is always set to the electrolyte storage temperature. And the electrolyte in the electrolyte storage tank is preferably transferred to the electrolyte tank 221 by using a pump or the like.
Here, when the control device 260 is provided with an electrolyte storage tank and a temperature control device for the electrolyte storage tank in addition to the electrolyte solution tank 221, the control device 260 stores the electrolyte so that the electrolyte storage temperature is set in advance. A control signal is input to the tank temperature 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 whether or not the concentration of the detection target substance using the enzyme sensor 100 is measured.

  The waste liquid tank 222 is a tank for storing the waste liquid discharged from the enzyme sensor 100. For example, the waste liquid tank 222 is connected to the enzyme sensor 100 (electrolyte discharge side of the liquid phase chamber R2) with a tube via a valve. Yes.

The liquid feed pump 223 is an introduction pump for introducing the electrolytic solution in the electrolytic solution tank 221 into the liquid phase chamber R2 of the enzyme sensor 100, and is connected to the electrolytic solution tank 221 with a tube through a valve, for example. In addition, it is connected to the enzyme sensor 100 (electrolyte introduction side of the liquid phase chamber R2) with a tube via a valve. By introducing the electrolytic solution into the liquid phase chamber R2 by the liquid feed pump 223, the liquid phase chamber R2 is filled with the electrolytic solution for detection by the enzyme sensor 100, and before and after the detection by the enzyme sensor 100 (detection of the detection). The inside of the liquid phase chamber R2 may be washed before or after the detection, or both before and after the detection.
Specifically, the liquid feed pump 223 introduces the electrolytic solution in the electrolytic solution tank 221 to the enzyme sensor 100 according to a control signal input from the control device 260, for example.
Here, the liquid feeding pump 223 is arbitrary as long as it can pump liquid using a tube. Specifically, for example, a diaphragm pump or the like, or a tube such as a peristaltic pump or a ring pump is used. It may be a type of pump or the like.

  The introduction pump is not limited to the liquid feed pump 223 connected to the previous stage (electrolyte introduction side) of the enzyme sensor 100, and the electrolyte in the electrolyte tank 221 is fed (introduced) to the enzyme sensor 100. ) Any pump can be used, and specifically, for example, it may be a discharge pump connected to the rear stage (electrolyte discharge side) of the enzyme sensor 100, or the rear stage (electrolyte discharge) of the enzyme sensor 100. The pump may be connected to both the front stage and the rear stage of the enzyme sensor 100. Here, when a liquid feed pump is connected to the subsequent stage of the enzyme sensor 100, the liquid feed pump is operated as a suction pump.

The sensor temperature adjusting device 231 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 231 determines that the temperature of the enzyme sensor 100 is a temperature specified by the control device 260 (for example, a temperature set by the user) according to a control signal input from the control device 260, for example. Then, the temperature of the enzyme sensor 100 is adjusted.

The intake pump 241 is a supply pump that sucks the external atmosphere (gas sample) and supplies it to the gas phase chamber R1 of the enzyme sensor 100. For example, the intake pump 241 is connected to the dust filter 242 with a tube. These are connected to the enzyme sensor 100 (the gas introduction side of the gas phase chamber R1) by a tube via a valve.
Specifically, for example, in accordance with a control signal input from the control device 260, the intake pump 241 intakes the external atmosphere via the dust filter 242, and introduces the intake external atmosphere into the enzyme sensor 100. .
The external air introduced into the enzyme sensor 100 is discharged from the enzyme sensor 100 (the gas discharge side of the gas phase chamber R1) to the outside.

The standard gas providing device 243 is, for example, a device for providing 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) is provided. R1 gas introduction side) and a tube through a valve.
Specifically, the standard gas providing device 243 provides the enzyme sensor 100 with a standard gas having a substrate concentration specified by the control device 260 according to a control signal input from the control device 260, for example.
The standard gas introduced into the enzyme sensor 100 is discharged from the enzyme sensor 100 (the gas discharge side of the gas phase chamber R1) to the outside.

  For example, the valve switching device 250 switches each valve included in the measurement device 200 in accordance with a control signal input from the control device 260.

The control device 260 is, for example, a device for controlling each part constituting the measuring device 200.
Specifically, for example, as illustrated in FIG. 2, the control device 260 includes a CPU (Central Processing Unit) 261, a RAM (Random Access Memory) 262, a memory unit 263, a storage unit 264, and the like. ing.

  For example, the CPU 261 performs various control operations according to various processing programs for the measuring apparatus 200 stored in the storage unit 264.

  The RAM 262 includes, for example, a program storage area for expanding a processing program executed by the CPU 261, a data storage area for storing input data, a processing result generated when the processing program is executed, and the like.

For example, the memory unit 263 stores or deletes predetermined data according to a control signal input from the CPU 261.
Specifically, the memory unit 263 stores, for example, calibration curve data 253a representing the relationship between the initial slope of the signal value from the enzyme sensor 100 and the concentration of the detection target substance as a storage unit.

  The storage unit 264 includes, for example, a system program that can be executed by the measuring apparatus 200, 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 261. The data etc. are memorized. Note that the program is stored in the storage unit 264 in the form of a computer-readable program code.

  Specifically, the storage unit 264 stores, for example, a measurement preparation program 264a, a calibration curve creation program 264b, a measurement program 264c, and the like.

  For example, the measurement preparation program 264a causes the CPU 261 to realize a function of preparing for the concentration measurement of the detection target substance.

  For example, the calibration curve creation program 264b creates the calibration curve data 263a before measuring the concentration of the detection target substance, and causes the CPU 261 to realize a function of storing the created calibration curve data 263a in the memory unit 263.

For example, the measurement program 264c causes the CPU 261 to realize a function of measuring the concentration of the detection target substance using the enzyme sensor 100.
Specifically, for example, the CPU 261 causes the measurement circuit 211 to measure the signal value from the enzyme sensor 100, and the data processing device 212 uses the enzyme sensor based on the time change curve representing the time change of the measured signal value. The initial slope of the signal value from 100 is calculated, and the concentration of the detection target substance corresponding to the calculated initial slope of the signal value from the enzyme sensor 100 is obtained from the calibration curve data 263a stored in the memory unit 263.

Here, the initial slope of the signal value from the enzyme sensor 100 is the slope of the approximate line obtained by approximating the initial portion of the time change curve representing the time change of the measured signal value.
For example, assume that a time change curve as shown in FIG. 3 is obtained. The slope of the approximate line obtained by approximating the initial portion of the time change curve from the first time t1 to the second time t2 is the initial slope of the signal value from the enzyme sensor 100.
The first time t1 is arbitrary as long as it is greater than or equal to time 0 (when the supply of the gas sample to the enzyme sensor 100 is started), and the second time t2 is greater than the first time t1, and the third time t3 ( It is optional as long as the signal value from the enzyme sensor 100 is equal to or less than the time point when the equilibrium is reached. However, the first time period is sufficient to separate the initial portion (or approximate line) corresponding to each substrate concentration with a specified resolution. It is preferable to set t1 and the second time t2.
The first time t1 and the second time t2 are defined in advance, for example.
The CPU 261 functions as a calculation unit and an acquisition unit by executing the measurement program 264c.

<Enzyme sensor>
4 is a plan perspective view of the enzyme sensor 100 provided in the concentration measurement system 1 of the present embodiment, FIG. 5 is a diagram schematically showing a cross section taken along the line V-V in FIG. 4, and FIG. It is a figure which shows typically the cross section in the VI-VI line of FIG. FIG. 7A is a bottom view of the upper support 110 with the permeable membrane 130 attached thereto, and FIG. 7B is a plan view of the lower support 120 with the electrodes 150 formed thereon. is there. FIG. 8 is a schematic diagram for explaining the spacer 170.
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 on which the pad 160 is disposed is the front side, and the opposite side is the rear side. The direction orthogonal to both is the left-right direction.

The enzyme sensor 100 includes, for example, an upper support 110, a lower support 120, and a gas phase chamber R1 to which a gas sample (gas) containing a detection target substance is supplied, as shown in FIGS. The liquid phase chamber R2 that is disposed adjacent to the gas phase chamber R1 and into which the aqueous solution (predetermined electrolyte) is introduced is disposed so as to separate the gas phase chamber R1 and the liquid phase chamber R2, and at least the detection target A permeable membrane 130 through which a substance permeates, an O-ring 140 for fixing the permeable membrane 130 to the upper support 110, and three electrodes 150 (working electrode 151) disposed in the liquid phase chamber R1 facing the permeable membrane 130. The reference electrode 152, the counter electrode 153), the three pads 160 connected to the electrode 150 via the wiring 161, the spacer 170 disposed between the permeable membrane 130 and the electrode 150, and the upper support 110. Lower support 12 A plurality of screws 180 for securing to, and includes a like.
In the enzyme sensor 100, an enzyme that selectively reacts with the detection target substance is contained in the liquid phase chamber R2, and the enzyme sensor 100 has passed through the permeable membrane 130 and has moved from the gas phase chamber R1 to the liquid phase chamber R2. A substance to be detected 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 vertical direction at a position approximately at the center in the vertical 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.

For example, the upper support 110 is disposed at a substantially central position in the horizontal direction of the upper support 110, and has a substantially circular recess 112 in a plan view with an open lower surface, and is disposed around the recess 112, and the lower surface is open. An O-ring accommodating portion 113 having a substantially ring shape in plan view, a gas introduction port 114 for introducing a gas (gas sample) from the outside of the enzyme sensor 100 toward the gas phase chamber R1, a gas introduction port 114, and a gas phase chamber A gas introduction path 115 that communicates with R1, a gas exhaust port 116 for exhausting gas from the gas phase chamber R1 toward the outside of the enzyme sensor 100, and a gas that communicates the gas exhaust port 116 with the gas phase chamber R1. A discharge path 117 and the like are provided.
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 gas phase chamber R1, the liquid phase chamber R2, etc. It is preferably formed of a material having high corrosion resistance (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 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, and the lower side The region becomes the liquid phase chamber R2. Therefore, the distance between the 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 left-right direction on the upper surface of the lower support 120 and extending from the substantially central position in the horizontal direction to the exposed portion 121, and from the outside of the enzyme sensor 100. Electrolyte inlet 123 for introducing the electrolyte toward the liquid phase chamber R2, an electrolyte introduction path 124 that communicates the electrolyte inlet 123 and the liquid phase chamber R2, and the enzyme sensor 100 from the liquid phase chamber R2. There are provided an electrolyte outlet 125 for discharging the electrolyte toward the outside, an electrolyte outlet 126 for communicating the electrolyte outlet 125 and the liquid phase chamber R2, and the like.
Here, in particular, the surface of the lower support 120, the electrolyte solution introduction port 123, the electrolyte solution introduction channel 124, the electrolyte solution discharge port 125, the electrolyte solution discharge channel 126, the liquid phase chamber R2 (the gas phase chamber of the liquid phase chamber R2) The region constituting the surface opposite to R2) 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 preferable.

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 port 126 is, for example, a first portion formed in the vertical direction in a region from a position between the working electrode 151 and the counter electrode 153 to a position at a substantially central position in the vertical direction of the lower support 120. An electrolyte solution discharge path 126a and a second electrolyte solution discharge path 236b formed along the left-right direction on the right side of the first electrolyte solution discharge path 126a.

For example, the 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 permeable membrane 130, and moves upward with the O-ring 140 disposed on the lower surface side of the permeable membrane 130. By being accommodated in the O-ring accommodating portion 113, the upper support 110 is attached.
The detection target substance (detection target gas) introduced into the gas phase chamber R1 passes through the permeable membrane 130 and moves to the liquid phase chamber R2, and then reacts with the enzyme contained in the liquid phase chamber R2. ing. Therefore, the permeable membrane 130 is arbitrary as long as it is a gas permeable membrane 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 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. It 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.
In addition, the O-ring 140 is preferably formed of a material having high corrosion resistance against an electrolytic solution or a sample.
The size (diameter) of the liquid phase chamber R2 is defined by the inner diameter of the O-ring 140, but is not limited to this. For example, a portion other than the portion where the electrode 150 is disposed is resin. The size of the liquid phase chamber R2 may be defined by the size of the portion that is coated with an insulating film or the like and is not covered with the resin insulating film or the like.

The electrode 150 is supported by the lower support 120, for example.
Specifically, for example, the electrode 150, the pad 160, and the wiring 161 that connects the electrode 150 and the pad 160 are formed in 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 permeable membrane 130 and the electrode 150 can be kept constant and uniform. Can do.
Here, the material for forming the electrode 150 is arbitrary, but from the viewpoint that the voltage applied to the enzyme sensor 100 can be lowered due to the relationship with the electron carrier, the electrode 150 is a porous carbon electrode. preferable.

The spacer 170 is disposed, for example, in the liquid phase chamber R2, and is used to keep the distance between the permeable membrane 130 and the electrode 150 constant. Therefore, as the spacer 170, for example, a spacer having a thickness substantially equal to the distance between the permeable membrane 130 and the electrode 150 is preferable.
The material of the spacer 170 is arbitrary as long as the distance between the permeable membrane 130 and the electrode 150 can be kept constant by the spacer 170 and the substrate (detection target substance) has good permeability. For example, as shown in FIG. 8, a material having a plurality of through-holes 170a penetrating in a substantially vertical direction with respect to the electrode 150 is preferable. Specifically, as the spacer 170, specifically, for example, a porous body such as an anodized film (alumina oxide film or the like) having a plurality of through holes 170a penetrating in a substantially vertical direction with respect to the electrode 150, Examples thereof include a hydrophilic film such as a hydrophilic Teflon film and a mesh body such as a nylon mesh.

The enzyme may be any enzyme as long as it 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 cross section of the enzyme molecule or the enzyme containing the active site may be, for example, extracted from animals or plants or microorganisms, or may be cleaved if desired, genetic engineering or chemical It may also be synthesized.

The enzyme contained in the liquid phase chamber R2 may be one type of enzyme or two or more types of enzymes.
Specifically, the enzyme contained in the liquid phase chamber R2 may be, for example, one type of enzyme, or two or more types of enzymes having substantially the same molecular weight and / or size (diameter). It may be two or more kinds of enzymes having different molecular weights and / or sizes. In addition, when there are two or more types of enzymes contained in the liquid phase chamber R2, the enzymes may be, for example, two or more types of enzymes that act on the same type of substrate (substance to be detected), or different types of substrates. Two or more types of enzymes acting on the substrate may be used, or two or more types of enzymes acting on the same and / or different substrates may be used.
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 substrates, the enzyme sensor 100 is configured to use the different types of substrates (two or more types of enzymes). Detection target substance) can be detected at the same time.

  In addition to the enzyme, the electrolyte introduced into the liquid phase chamber R2 promotes the transfer of electrons between the enzyme and the electrode 150 (working electrode 151) and a coenzyme for catalyzing the expression of the enzyme activity. An electron carrier for the purpose may be contained.

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.

  As the electron carrier, for example, potassium ferricyanide, ferrocene, ferrocene derivative, benzoquinone, quinone derivative, osmium complex and the like are used. From the viewpoint that the voltage applied to the enzyme sensor 100 can be lowered, the liquid phase The chamber R2 preferably contains ferrocenecarboxylic acid and / or ferrocenyltrimethylammonium bromide as an electron carrier.

<Density measurement process>
An example of processing related to the measurement of the concentration of the detection target substance using the enzyme sensor 100 by the concentration measurement system 1 will be described with reference to the flowchart of FIG.

  First, for example, when the user operates an operation button (not shown) or a touch panel (not shown) included in the concentration measurement stem 1 to instruct to start the concentration measurement of the detection target substance, the CPU 261 reads the measurement preparation program. 264a is executed to prepare for the concentration measurement of the detection target substance (step S1).

Specifically, for example, the CPU 261 inputs a control signal to the sensor temperature adjusting device 231 and adjusts the temperature of the enzyme sensor 100 so as to be a preset temperature such as an initial preset temperature.
In addition to (or instead of) adjusting the temperature of the enzyme sensor 100 by the sensor temperature adjusting device 231, the electrolyte temperature in the electrolyte tank 231 is adjusted to a predetermined set temperature such as an initial set temperature. Thus, the temperature of the enzyme sensor 100 may be adjusted.

  Next, the CPU 261 inputs a control signal to the measurement circuit 211, for example, causes the reference electrode 152 of the enzyme sensor 100 to apply a voltage of a predetermined magnitude to the working electrode 151, and the signal value from the enzyme sensor 100 Start measuring.

Next, for example, the CPU 261 inputs a control signal to the valve switching device 250, the electrolyte in the electrolyte tank 221 is introduced into the enzyme sensor 100, and the electrolyte discharged from the enzyme sensor 100 is input to the waste liquid tank 222. Switch the valve so that the liquid is delivered.
Next, the CPU 261 inputs a control signal to the liquid feeding pump 223, for example, and starts feeding the electrolytic solution in the electrolytic solution tank 221. Thereby, enzyme sensor 100 (liquid phase room R2) is washed.

Next, after a lapse of a certain time from the start of feeding of the electrolytic solution, the CPU 261 inputs a control signal to the valve switching device 250, for example, and the electrolytic solution discharged from the enzyme sensor 100 is sent to the electrolytic solution tank 221. That is, the valve is switched so that the electrolyte in the electrolyte tank 221 circulates. Thereby, the enzyme sensor 100 (liquid phase chamber R2) is washed (circular washing).
Next, when the signal value from the enzyme sensor 100 is stabilized, the CPU 261 inputs a control signal to the liquid feeding pump 223, for example, and stops the feeding of the electrolytic solution in the electrolytic solution tank 221.
Here, at the stage where the feeding of the electrolytic solution is stopped, the liquid phase chamber R2 of the enzyme sensor 100 is completely filled with the electrolytic solution (electrolytic solution containing the enzyme). From the viewpoint of completely filling the liquid phase chamber R2 with the electrolyte 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 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.

  Next, the CPU 261 executes a calibration curve creation program 264b to create calibration curve data 263a (step S2).

  Specifically, for example, the CPU 261 inputs a control signal to the valve switching device 250 and switches the valve so that the standard gas in the standard gas providing device 243 is introduced into the enzyme sensor 100.

  Next, the CPU 261, for example, inputs a control signal to the standard gas providing device 243, specifies the substrate concentration of the standard gas, generates the standard gas having the specified substrate concentration, and generates the generated standard gas. The enzyme sensor 100 is supplied for a certain time (until the elapsed time from the start of the standard gas supply becomes the second time t2 or more). Then, a time change curve representing a time change of the signal value from the enzyme sensor 100 is created in the data processing device 232 and stored in a storage area (may be the memory 263 or the like) in the data processing device 212. The CPU 261 repeats this series of processing by switching the type of the designated substrate concentration (the designated substrate concentration value). Note that the substrate concentration to be specified is three or more.

  Next, the CPU 261, for example, based on the time change curve corresponding to each substrate concentration stored in the storage area in the data processing device 212, the initial slope of the signal value from the enzyme sensor 100, the concentration of the detection target substance, and Calibration curve data 263a representing the relationship is created. That is, for example, the CPU 261 creates calibration curve data 263a in which the vertical axis is the initial slope of the signal value from the enzyme sensor 100 and the vertical axis is the substrate concentration of the standard gas.

  Next, the CPU 261 stores the calibration curve data 263a created in step S2 in the memory unit 263 (step S3).

Next, the CPU 261 cleans the enzyme sensor 100 (step S4).
Specifically, for example, the CPU 261 inputs a control signal to the valve switching device 250, the electrolytic solution in the electrolytic solution tank 221 is introduced into the enzyme sensor 100, and the electrolytic solution discharged from the enzyme sensor 100 is the waste liquid. The valve is switched so that the liquid is fed to the tank 222.
Next, the CPU 261 inputs a control signal to the liquid feeding pump 223, for example, and starts feeding the electrolytic solution in the electrolytic solution tank 221.

Next, after a predetermined time has elapsed from the start of cleaning of the enzyme sensor 100 in step S4, the CPU 261 circulates and cleans the enzyme sensor 100 (step S5).
Specifically, the CPU 261 inputs a control signal to the valve switching device 250, for example, after a lapse of a certain time from the start of feeding of the electrolytic solution in step S4, and the electrolytic solution discharged from the enzyme sensor 100 is the electrolytic solution. The valve is switched so that the liquid is fed to the tank 221, that is, the electrolytic solution in the electrolytic solution tank 221 is circulated.
Next, when the signal value from the enzyme sensor 100 is stabilized, the CPU 261 inputs a control signal to the liquid feeding pump 223, for example, and stops the feeding of the electrolytic solution in the electrolytic solution tank 221.

  Next, the CPU 261 executes the measurement program 264c and measures the concentration of the detection target substance using the enzyme sensor 100 (step S6).

Specifically, for example, the CPU 261 inputs a control signal to the valve switching device 250 and switches the valve so that the external atmosphere (gas sample) sucked by the suction pump 241 is introduced into the enzyme sensor 100.
Next, the CPU 261, for example, inputs a control signal to the intake pump 241 and sucks the external air that has been inhaled for a certain period of time (until the elapsed time from the start of external air supply becomes the second time t2 or more). The enzyme sensor 100 is supplied.

  Next, when the time change of the signal value is measured by the measurement circuit 211, the CPU 261 creates a time change curve in the data processing device 212, and calculates the initial inclination of the enzyme sensor 100 based on the created time change curve. The concentration of the detection target substance corresponding to the calculated initial inclination is obtained from the calibration curve data 263a calculated and stored in the memory unit 263.

  Next, for example, the CPU 261 inputs a control signal to the display device 230 to display the acquired concentration of the detection target substance (step S7).

  Next, the CPU 261 cleans the enzyme sensor 100 (step S8).

  Next, after a predetermined time has elapsed from the start of cleaning of the enzyme sensor 100 in step S8, the CPU 261 circulates and cleans the enzyme sensor 100 (step S9).

Next, the CPU 261 determines whether or not to perform the next measurement (step S10).
Specifically, for example, when the user instructs to perform the next measurement by operating an operation button (not shown) or a touch panel (not shown) included in the concentration measurement stem 1, the CPU 261 Judge that measurement is to be performed. Alternatively, the CPU 261 determines whether or not to perform the next measurement based on the number of times or the measurement time set in advance by operating the operation unit by the user.

  If it is determined in step S10 that the next measurement is to be performed (step S10; Yes), the CPU 261 repeats the processes in and after step S6 after the signal value from the enzyme sensor 100 is stabilized by the process in step S9.

  On the other hand, when determining that the next measurement is not performed (step S10; No), the CPU 261 ends the process.

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

In the present example, an enzyme sensor 100 for detecting formaldehyde gas was created and evaluated, and calibration curve data 263a was created using the created enzyme sensor 100.
A porous carbon electrode is used as the electrode 150, an alumina oxide film having a plurality of through-holes 170a penetrating in a direction substantially perpendicular to the electrode 150 is used as the spacer 170, and a coenzyme (NAD ⁺ ) -dependent enzyme is used as the enzyme. Formaldehyde dehydrogenase (formaldehyde dehydrogenase) was used, NAD ⁺ was used as a coenzyme, and ferrocenecarboxylic acid was used as an electron carrier.

<Evaluation of combination of electrode and electron carrier>
By performing cyclic voltammetry measurement (CV measurement) of the electrode 150, the combination of the porous carbon electrode as the electrode 150 and the ferrocenecarboxylic acid as the electron carrier contained in the liquid phase chamber R2 was evaluated.

(Create electrode)
First, the lower support 120 was created using a lathe, a milling machine, etc. using the peak material which is an insulator. The depth of the groove part 122 was 10 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 working electrode 152 and sintered at 120 ° C. to prepare a reference electrode 152 which is a silver / silver chloride electrode.

(Creation of electrolyte for first evaluation)
A reduced coenzyme (NAD ⁺ ) (NADH) and an oxidized electron carrier (oxidized ferrocenecarboxylic acid) are prepared, and 20 mg of NADH and 5 mg of oxidized ferrocenecarboxylic acid are prepared in 20 mL. It dissolved in the phosphate buffer solution (pH 7.4), and the electrolyte solution for 1st evaluation (electrolyte solution which does not contain an enzyme) was created.

(Evaluation)
CV measurement was performed by dropping 60 μL of the prepared first electrolyte for evaluation on the prepared electrode 150. The result is shown in FIG.
Here, when NADH, which is a reduced coenzyme, coexists with an oxidized electron carrier, it is oxidized to NAD ⁺ , and the oxidized electron carrier is reduced to a reduced electron carrier. (NADH + oxidized electron mediator of → NAD ^{+ +} reduced electron mediator of). At this time, if a voltage is applied to the electrode 150, the reduced electron carrier gives electrons to the electrode 150 and returns to the oxidized electron carrier (reduced electron carrier → oxidized type). Electron carrier + H ⁺ + 2e ⁻ ). Therefore, the oxidation current of NADH can be examined by CV measurement of the electrode 150.

  In FIG. 10, the vertical axis represents the response current (μA), and the horizontal axis represents the applied voltage (V). Moreover, the result of this Example is shown by a solid line, and the result of Comparative Example 1 (the result when naphthoquinone is used in place of the ferrocenecarboxylic acid contained in the first evaluation electrolytic solution) is shown by a broken line. The results of Comparative Example 2 (results when a platinum electrode is used instead of the porous carbon electrode as the electrode 150 included in the lower support 120) are shown by chain lines.

From the results of FIG. 10, the NADH oxidation current peak was observed at +0.08 V in the example, the NADH oxidation current peak was observed at +0.35 V in Comparative Example 1, and the NADH oxidation peak was observed in Comparative Example 2. No oxidation current peak was observed.
In other words, it was found that the peak of the oxidation current of NADH was obtained in the example at a lower applied voltage than in Comparative Example 1 and Comparative Example 2.
Therefore, from the viewpoint of lowering the applied voltage when detecting the detection target substance, the combination of the porous carbon electrode and naphthoquinone (Comparative Example 1) and the combination of the platinum electrode and ferrocenecarboxylic acid (Comparative Example 2). The combination of the porous carbon electrode and the ferrocenecarboxylic acid (Example) was found to be preferable.

<Evaluation of spacer>
By measuring the response current value from the enzyme sensor 100, the spacer 170 having a plurality of through holes 170a penetrating in a substantially vertical direction with respect to the electrode 150 was evaluated.

(Creation of sensor head)
First, a sensor head was created.
Here, the sensor head is the enzyme sensor 100 before the electrolytic solution (electrolytic solution containing an enzyme) is introduced into the liquid phase chamber R2.

First, the upper support 110 and the lower support 120 were formed using a lathe, a milling machine, or the like using a peak material that is an insulator.
The depth of the groove 122 was 10 μm, and the volume of the liquid phase chamber R2 was 100 μL. Further, the height of the partition wall 118 was adjusted so that the distance between the permeable membrane 130 and the electrode 150 was 30 μm.

  Next, three electrodes of the working electrode 151, the reference electrode 152, and the counter electrode 153 are formed on the upper surface of the lower support 120 in the same manner as the above-described “<Evaluation of combination of electrode and electron carrier>”. A structural pattern was created.

Next, the O-ring 140 is used to fix the permeable membrane 130 (Gore-Tex Teflon membrane) to the upper support 110, and the spacer has substantially the same thickness as the distance (30 μm) between the permeable membrane 130 and the electrode 150. 170 (thickness: 30 μm, pore diameter: 200 nm, alumina oxide film) was placed on the electrode 150.
Next, the upper support body 110 was placed on the lower support body 120, and the upper support body 110 was fixed to the lower support body 120 using screws 180, thereby creating a sensor head.

(Creation of electrolyte for second evaluation)
4 mg of formaldehyde dehydrogenase was dissolved in 2 mL of phosphate buffer (pH 7.41) containing 1 mM of NAD ⁺ and 1 mM of ferrocenecarboxylic acid to prepare a second evaluation electrolyte.

(Evaluation)
First, the prepared electrolyte for second evaluation was placed in the electrolyte tank 221.
Next, the created sensor head was assembled at the position of the enzyme sensor 100 in the concentration measurement system 1. Specifically, the electrolyte inlet 123 of the sensor head is connected to the liquid feeding pump 223 of the measuring device 200, and the electrolyte outlet 125 of the sensor head is connected to the waste liquid tank 222 and the liquid feeding pump 223 of the measuring device 200. The gas inlet 114 of the sensor head was connected to the intake pump 241 and the standard gas providing device 243 of the measuring device 200, and the pad 160 of the sensor head was connected to the measuring circuit 211 of the measuring device 200.

Next, preparation for measurement was performed. Specifically, the prepared sensor head is cleaned and circulated and cleaned with the second evaluation electrolytic solution, thereby introducing an electrolytic solution (electrolyte containing an enzyme) into the liquid phase chamber R2 to obtain the enzyme sensor 100. . Washing and circulation washing were performed at a liquid feed rate of 0.1 mL / min, and circulation washing was performed for 100 seconds.
Next, a standard gas (formaldehyde gas) was generated by the standard gas providing device 243, supplied to the enzyme sensor 100, and a signal value (response current value) from the enzyme sensor 100 was measured. The standard gas substrate concentration was 100 ppb, and the standard gas introduction rate was 300 mL. The result is shown in FIG.

  FIG. 11 is a time change curve, in which the vertical axis represents response current (nA) and the horizontal axis represents elapsed time (min) from the start of gas supply. Although two time change curves are shown in FIG. 11, the upper time change curve is the result of this example, and the lower time change curve is the result of Comparative Example 3 (as the spacer 170 included in the enzyme sensor 100). This is a result of using a Teflon film (thickness: 30 μm) having random holes instead of the alumina oxide film having a plurality of through holes 170 a penetrating in a substantially vertical direction with respect to the electrode 150.

From the results of FIG. 11, it was found that Comparative Example 3 has a slower rise in time change curve and lower sensitivity than the Example. In Comparative Example 3, it was found that it is difficult to determine the concentration of the detection target substance using the initial slope of the signal value from the enzyme sensor 100 because the rise of the time change curve is slow.
Therefore, from the viewpoint of determining the concentration of the detection target substance by using the initial slope of the signal value from the enzyme sensor 100, the electrode 150 is compared with the case where a spacer with a random hole is used (Comparative Example 3). Thus, it was found that the case where the spacer 170 having the through hole 170a penetrating in a substantially vertical direction is used (Example) is preferable.

<Creation of calibration curve data>
Calibration curve data 263a was created by sequentially changing the substrate concentration of the standard gas introduced into the enzyme sensor 100 and measuring the response current from the enzyme sensor 100.

(Creation of sensor head)
A sensor head was produced in the same manner as the production method in “<Spacer evaluation>” described above.

(Creation of electrolyte for second evaluation)
A second evaluation electrolytic solution was prepared in the same manner as the preparation method in “<Spacer evaluation>” described above.

(Creation of time change curve)
First, the prepared electrolyte for second evaluation was placed in the electrolyte tank 221.
Next, the created sensor head was assembled at the position of the enzyme sensor 100 in the concentration measurement system 1.

Next, preparation for measurement was performed. Specifically, the prepared sensor head is cleaned and circulated and cleaned with the second evaluation electrolytic solution, thereby introducing an electrolytic solution (electrolyte containing an enzyme) into the liquid phase chamber R2 to obtain the enzyme sensor 100. . Washing and circulation washing were performed at a liquid feed rate of 0.1 mL / min, and circulation washing was performed for 100 seconds.
Next, a standard gas (formaldehyde gas) was generated by the standard gas providing device 243, introduced into the enzyme sensor 100, and a signal value (response current value) from the enzyme sensor 100 was measured. The substrate concentration of the standard gas was 4 ppb, 40 ppb, 100 ppb, 200 ppb, 300 ppb, 400 ppb and 1200 ppb, and the introduction rate of the standard gas was 300 mL. The result is shown in FIG.

  FIG. 12 is a time change curve, in which the vertical axis represents response current (nA) and the horizontal axis represents elapsed time (min) from the start of gas supply. FIG. 12 shows seven time change curves, which are time change curves obtained by introducing standard gases having substrate concentrations of 4 ppb, 40 ppb, 100 ppb, 200 ppb, 300 ppb, 400 ppb, and 1200 ppb in order from the bottom. .

  From the results of FIG. 12, it was found that the rise of the time change curve becomes sharper as the substrate concentration increases.

(Get approximate line)
Next, with the first time t1 as 1 minute and the second time t2 as 6 minutes, the initial part was extracted from the seven time change curves shown in FIG. 12, and the initial part was approximated to obtain an approximate line. The result is shown in FIG.

  FIG. 13 shows an initial portion of the time change curve, in which the vertical axis represents response current (nA) and the horizontal axis represents elapsed time (min) from the start of gas supply. FIG. 13 shows seven time-varying curves, which are time-varying curves obtained by introducing standard gases having substrate concentrations of 4 ppb, 40 ppb, 100 ppb, 200 ppb, 300 ppb, 400 ppb, and 1200 ppb in order from the bottom. . Moreover, although the straight line is shown with the broken line along the initial part of each time change curve in FIG. 13, these are the approximate straight lines obtained.

  From the results of FIG. 13, it was found that the initial part of the time change curve coincided with the straight line with high accuracy.

(Creation of calibration curve data)
Next, calibration curve data 263a (first calibration curve data 263a) was created by obtaining the slope of each approximate straight line shown in FIG.
Subsequently, the above-described “(time-change curve creation)” and “(approximate straight line acquisition)” processes are performed again, the slope of each approximate straight line is obtained, and calibration curve data 263a (second calibration curve data) is obtained. 263a) was made.
Subsequently, the above-described “(time-change curve creation)” and “(approximate straight line acquisition)” processes are performed again, the slope of each approximate straight line is obtained, and calibration curve data 263a (third calibration curve data) is obtained. 263a) was made.
The result is shown in FIG.

  In FIG. 14, the vertical axis represents the initial slope of the signal value (response current value) from the enzyme sensor 100, and the horizontal axis represents the substrate concentration (ppb) of the standard gas introduced into the enzyme sensor 100. Further, a square plot (□) indicates the first calibration curve data 263a, a triangle plot (Δ) indicates the second calibration curve data 263a, and a rhombus plot (◇) indicates the third calibration curve data 263a.

From the results of FIG. 14, it was found that good calibration curve data 263a can be obtained. In particular, it was found that the linearity at 0 to 400 ppb was good.
In addition, it was found that the calibration curve data 263a for the first to the third time coincide with each other with high accuracy, the variation is within 5%, and the reproducibility is high.
That is, it was found that the concentration of the detection target substance at the sub ppb level can be measured only by measuring the signal value from the enzyme sensor 100 for 6 minutes from the start of gas supply to the enzyme sensor 100.
From the above results, it was found that the enzyme sensor 100 of the present example can measure the concentration of the detection target substance in the gas sample at high speed (for example, in a measurement time of 10 minutes or less) with high accuracy and good reproducibility.

According to the concentration measurement system 1 of the present invention described above, the enzyme sensor 100 and the measurement device 200 that measures the concentration of the detection target substance in the gas sample based on the detection result by the enzyme sensor 100 are provided. The enzyme sensor 100 is disposed adjacent to the gas phase chamber R1 to which a gas sample is supplied, the gas phase chamber R1, and a liquid phase chamber R2 into which a predetermined electrolytic solution is introduced, and the gas phase chamber R1. A permeable membrane 130 that is disposed so as to be separated from the liquid phase chamber R2, and at least allows the detection target substance to pass therethrough, an electrode 150 that is disposed in the liquid phase chamber R2 so as to face the permeable membrane 130, a permeable membrane 130, and an electrode 150 And a spacer 170 having a plurality of through holes 170 a penetrating in a substantially vertical direction with respect to the electrode 150. In addition, the measuring apparatus 200 generates calibration curve data 263a representing the relationship between the measurement circuit 221 that measures the signal value from the enzyme sensor 100, the initial slope of the signal value from the enzyme sensor 100, and the concentration of the detection target substance. A memory unit 263 for storing in advance, and based on a time change curve representing a time change of the signal value measured by the measurement circuit 221, an inclination of an approximate line obtained by approximating an initial portion of the time change curve is obtained. The concentration of the detection target substance corresponding to the calculated initial inclination can be acquired from the calibration curve data 263a calculated as the initial inclination and stored in the memory unit 263.
That is, since the spacer 170 is provided, the distance between the permeable membrane 130 and the electrode 150 can be kept constant. In addition, when a spacer is provided between the permeable membrane 130 and the electrode 150, diffusion of the detection target substance in the liquid phase chamber R2 is limited, the sensor response becomes slow, and the concentration of the detection target substance is determined from the sensor. However, it is difficult to determine with high accuracy using the initial slope of the signal value, but since the spacer 170 has a plurality of through-holes 170a penetrating in a direction substantially perpendicular to the electrode 150, the spacer 170 However, the diffusion of the detection target substance in the liquid phase chamber R2 is not easily restricted.
Therefore, the detection target substance can be detected stably and detected based on the slope of the approximate line (initial slope of the signal value from the enzyme sensor 100) obtained by approximating the initial part of the time change curve. Since the concentration of the target substance can be determined, the concentration of the detection target substance in the gas sample can be measured at high speed and with high reproducibility.

Further, according to the concentration measuring system 1 of the present invention described above, the measuring device 200 includes the intake pump 241 that supplies a gas sample to the gas phase chamber R1, and a predetermined electrolyte to the liquid phase chamber R2. And a liquid feed pump 223 for introducing and cleaning the inside of the liquid phase chamber R2.
Therefore, since a gas sample can be forcibly supplied to the gas phase chamber R1, measurement can be performed at a higher speed. Moreover, since the inside of the liquid phase chamber R2 can be cleaned by forcibly introducing the electrolytic solution into the liquid phase chamber R2, the measurement can be started at a higher speed and the process can be shifted to the next measurement at a higher speed. be able to.

Further, according to the concentration measuring system 1 of the present invention described above, the electrode 150 is a porous carbon electrode, and the liquid phase chamber R2 has ferrocenecarboxylic acid and / or ferrocenyltrimethylammonium bromide as an electron carrier. Is contained.
Therefore, the voltage applied to the enzyme sensor 100 can be lowered as compared with the conventional case, and the concentration of the detection target substance in the gas sample can be measured more selectively.
That is, in the conventional sensor, a voltage of 300 mV or higher is applied even when an electron carrier is used, but in this case, even if the selectivity of the enzyme itself is high, other voltage contained in the liquid phase chamber R2 There is a problem in that the selectivity is lowered due to oxidation or reduction of the substance at the electrode 150. However, in the present invention, the applied voltage can be set to 100 mV or less, and the applied voltage to the enzyme sensor 100 can be lowered as compared with the conventional case. Therefore, the concentration of the detection target substance in the gas sample can be selected more selectively. Can be measured.

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

  In the said embodiment, the sensor temperature adjustment apparatus 231 is not limited to a thermostat, It is arbitrary if the temperature of the enzyme sensor 100 (electrode 150) can be adjusted, for example, the enzyme sensor 100 A built-in small heater or the like may be used.

  The structure of the sensor head (enzyme sensor 100) is not limited to that of the above embodiment, and includes a gas phase chamber R1, a liquid phase chamber R2, a permeable membrane 130, an electrode 150, and a spacer 170. If it is, it is arbitrary. For example, the electrolyte flow path (electrolyte introduction path 124 and electrolyte discharge path 126) is formed so as to be substantially parallel to the gas flow path (gas introduction path 115 and gas discharge path 116). For example, the electrolyte solution flow path may be formed so as to be substantially orthogonal to the gas flow path.

In the above embodiment, the enzyme is contained in the liquid phase chamber R2 by using the electrolytic solution containing the enzyme as the electrolytic solution introduced into the liquid phase chamber R2. However, the enzyme is contained in the liquid phase chamber R2. However, the method of fixing is not limited to this, and may be a method of fixing on the electrode 150 (working electrode 151) by a known fixing method, or a method of fixing to the spacer 170 by a known fixing method. It may be. In this case, an electrolytic solution that does not contain an enzyme may be used as the electrolytic solution introduced into the liquid phase chamber R2.
The same applies to electron carriers and coenzymes.

In the above embodiment, the concentration measuring system 1 (measuring device 200) includes the standard gas providing device 243, and the calibration curve data 263a is created using the standard gas. However, the present invention is not limited to this. For example, The concentration measurement system 1 may not include the standard gas providing device 243. The standard gas providing device 243 is too large to carry, but if the standard gas providing device 243 is not provided, the enzyme sensor 100 and the measuring device 200 are placed in a housing (size: for example, 20 cm × 10 cm × 15 cm). ), The concentration measuring system 1 can be carried.
In this case, for example, calibration curve data created in advance using the standard gas providing device 243 that is an external device is acquired and stored in the memory unit 263 or the like, and is stored in the memory unit 263 or the like in advance every time concentration measurement is performed. The calibration curve data stored in the memory unit 263 or the like is corrected based on the stored correction data (for example, data on deterioration with time or temperature dependence of the enzyme sensor 100), and the corrected It is preferable to determine the concentration using calibration curve data (calibration curve data 263a). In this case, in FIG. 9, the process of step S2 and step S3 as operation is omitted.

In the above embodiment, the concentration measuring system 1 (measuring device 200) includes the standard gas providing device 243, and the calibration curve data 263a is created using the standard gas. However, the present invention is not limited to this. For example, In addition to (or instead of) the standard gas providing device 243, a standard solution providing device may be provided, and calibration curve data may be created using the standard solution. In this case, the standard solution providing device may be connected to, for example, a front stage of the liquid feed pump 223 (that is, a valve between the electrolyte solution tank 221 and the liquid feed pump 223).
When a standard liquid providing device is provided instead of the standard gas providing device 243, the standard liquid providing device is smaller in size than the standard gas providing device 243. Therefore, the enzyme sensor 100 and the measuring device 200 are arranged in a casing (size: for example, The concentration measurement system 1 can be carried by being housed in a 20 cm × 10 cm × 15 cm).
When the concentration measurement system 1 measures the concentration of the detection target substance in the gas sample, it is preferable to determine the concentration using the calibration curve data created using the standard gas, so the calibration curve created using the standard solution is used. When determining the concentration of the detection target substance using the data, the memory unit 263 obtains the correlation between the calibration curve data created using the standard gas and the calibration curve data created using the standard solution in advance. The calibration curve data created based on the correlation (calibration curve data created using the standard solution) each time calibration curve data is created using the standard solution when the concentration is measured. It is preferable to determine the concentration using the corrected calibration curve data (calibration curve data 263a). When preparing calibration curve data using a standard solution, the enzyme is immobilized on the electrode 150 by a known immobilization method, or the enzyme is immobilized on the spacer 170 by a known immobilization method. Is preferred.

In the above embodiment, the gas sample is supplied to the gas phase chamber R1 which is the supply unit. However, the sample supplied to the supply unit is arbitrary as long as it is a fluid sample. For example, a liquid sample is supplied. Also good.
In the above embodiment, the predetermined electrolytic solution is introduced into the liquid phase chamber R2 that is the detection unit. However, the substance that fills the detection unit (the substance that cleans the detection unit) may be any fluid substance. For example, a predetermined gas may be used.

In the above embodiment, the enzyme is contained in the liquid phase chamber R2. However, the present invention is not limited to this, and the liquid phase chamber R2 includes a biological material that selectively reacts with the detection target substance (biological origin). Molecular identification element), specifically, for example, a biocatalyst such as an enzyme, an antigen, an antibody, a lipid, a cell, a fungus, DNA, a sugar chain, or the like may be contained.
Furthermore, in the above embodiment, the liquid phase chamber R2 contains an enzyme (biological substance), but the present invention is not limited to this, and the liquid phase chamber R2 selectively reacts with the detection target substance. The reaction material, specifically, for example, the biological material, metal catalyst, organic catalyst, inorganic catalyst, various polymers, polymer complex, polyion complex, light-absorbing material, fluorescent material and the like may be contained. In addition, the kind of the reactive material contained in the liquid phase chamber R2 may be one kind or plural kinds.

The electrode 150 is not limited to that of the above-described embodiment, and may be any as long as it is formed from platinum, gold, silver, carbon, or the like by a screen printing method, a vapor deposition method, a sputtering method, or the like. These are determined by compatibility with the electron carrier used (difficulty of oxidation on the electrode 150 differs depending on the electrode material).
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, or the like, and then immersed in a second silver chloride aqueous solution. 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 above embodiment, the detection element is not limited to the electrode 150, but a predetermined change (for example, substance change, reaction) between the detection target substance and a reaction substance that selectively reacts with the detection target substance. Any change is possible as long as color change, endothermic change, exothermic change, mass change, resistance change, capacitance change, etc.) can be detected and converted into an electrical signal. Specifically, as the detection element, for example, an electrode (hydrogen peroxide electrode, dissolved oxygen electrode, conductivity electrode, ion electrode, redox potential electrode, comb electrode, parallel plate electrode, etc.), semiconductor, light receiving element A thermal element, a piezoelectric element, a thermistor, a cantilever, an ion-sensitive field effect transistor (ISFET), a crystal resonator (QCM), a surface acoustic wave (SAW) device, or the like can be used.

It is a figure which shows the structure of the density | concentration measuring system of this embodiment. It is a block diagram which shows the functional structure of the measuring apparatus with which the density | concentration measuring system of this embodiment is provided. It is a figure for demonstrating an initial stage inclination. It is a top perspective view of enzyme sensor 100 with which concentration measuring system 1 of this embodiment is provided. It is a figure which shows typically the cross section in the VV line | wire of FIG. It is a figure which shows typically the cross section in the VI-VI line of FIG. It is a bottom view (a) of the upper side support body in the state where the permeable membrane was attached, and is a top view (b) of the lower side support body in the state where the electrode was formed. It is a schematic diagram for demonstrating a spacer. It is a flowchart for demonstrating an example of the process regarding the measurement of the density | concentration of the detection target substance using an enzyme sensor by the concentration measurement system of this embodiment. It is the result of the CV measurement performed for evaluation of the combination of an electrode and an electron carrier in Example 1. 2 is a time change curve obtained for evaluating a spacer in Example 1. FIG. 2 is a time change curve obtained for creating calibration curve data in Example 1. FIG. FIG. 13 shows an initial portion of the time change curve shown in FIG. 12 and an approximate straight line obtained by approximating the initial portion. It is the calibration curve data created in Example 1.

Explanation of symbols

1 Concentration measurement system 100 Enzyme sensor (sensor)
130 Permeation membrane 150 Electrode (detection element)
170 Spacer 170a Through-hole 200 Measuring device 211 Measuring circuit (measuring means)
223 Liquid feed pump (introduction pump)
241 Intake pump (supply pump)
261 CPU (calculation means, acquisition means)
263 Memory unit (storage means)
263a Calibration curve data 264c Measurement program (calculation means, acquisition means)
R1 Gas phase chamber (supply section)
R2 Liquid phase chamber (detector)

Claims (4)

A sensor,
A measuring device for measuring the concentration of the detection target substance in the fluid sample based on the detection result by the sensor;
With
The sensor is
A supply unit to which the fluid sample is supplied;
A detection unit disposed adjacent to the supply unit;
A permeable membrane that is disposed so as to separate the supply unit and the detection unit, and through which at least the detection target substance passes;
A detection element disposed on the detection unit so as to face the permeable membrane;
A spacer disposed between the permeable membrane and the detection element and having a plurality of through holes penetrating in a direction substantially perpendicular to the detection element;
With
The measuring device is
A measuring means for measuring a signal value from the sensor;
Based on a time change curve representing a time change of the signal value measured by the measurement means, a calculation means for calculating an inclination of an approximate straight line obtained by approximating an initial portion of the time change curve as an initial inclination;
Storage means for storing in advance calibration curve data representing a relationship between the initial inclination and the concentration of the detection target substance;
An acquisition means for acquiring the concentration of the detection target substance corresponding to the initial slope calculated by the calculation means from the calibration curve data stored in the storage means;
Equipped with a,
Said initial portion of said time variation curve, from the first hour or more time of starting the supply of the fluid sample into the supply unit, Rukoto Oh the portion up to the second hour is larger than said first hour Concentration measurement system characterized by The concentration measurement system according to claim 1,
The measuring device is
A supply pump for supplying the fluid sample to the supply unit;
An introduction pump for introducing a predetermined fluid substance into the detection unit and cleaning the inside of the detection unit;
A concentration measurement system comprising: The concentration measurement system according to claim 1 or 2,
The detection element is a porous carbon electrode;
The concentration measurement system, wherein the detection unit contains ferrocenecarboxylic acid and / or ferrocenyltrimethylammonium bromide as an electron carrier. The concentration measurement system according to claim 2,
It said sensor is an enzyme sensor comprising an enzyme that selectively reacts with the detection target substance,
It said fluid sample is a gaseous sample,
The supply unit is a gas phase chamber to which the gas sample is supplied,
The detection unit is a liquid phase chamber that is disposed adjacent to the gas phase chamber and into which a predetermined electrolytic solution is introduced,
The feed pump is a suction pump which supplies the gaseous sample to the gas phase chamber,
The introduction pump, the concentration measurement system, which is a liquid feed pump for introducing a predetermined electrolyte to the liquid phase chamber.

Images