JP2007271287A - Detection sensor of oxidation stress substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection sensor of an oxidation stress substance having a simple structure, capable of miniaturizing the whole of a system, capable of being manufactured by utilizing a semiconductor manufacturing technique, excellent in mass productivity, usable repeatedly, excellent in resources saving properties, capable of precisely detecting the oxidation stress substance such as nitrogen monoxide or the like of an extremely small amount of about 1 ppm contained in a liquid to be inspected in an extremely short time and excellent in reliability and workability. <P>SOLUTION: The detection sensor of the oxidation stress substance includes a substrate, a pair of the electrodes arranged on the substrate at a predetermined interval and the insulating film applied to the surfaces of a pair of the electrodes and the surface of the substrate between a pair of the electrodes and coming into contact with the liquid to be inspected containing body fluids such as blood, lymph, cytoplasm substrate, etc. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an oxidative stress substance detection sensor capable of detecting the amount of an oxidative stress substance such as nitric oxide contained in a sample fluid including a body fluid such as blood, lymph, and cytoplasm based on a voltage change or a current change. About.

Conventionally, as a kind of micromachine technology, a liquid microsystem (fluid MEMS) used for analysis, reaction, and separation operation of a minute amount of liquid.
mechanical system) is known.
A liquid microsystem is a package of micropumps, mixers, valves, reactors, separators, sensors, etc. mounted on a substrate (including a chip) and packaged. Indispensable for the development of post-genome research and proteomic research. Not expected as a tool.
As a means for measuring the chemical change or physical change of the analyte liquid in such a liquid microsystem, for example, (Patent Document 1) includes “a substrate irradiated with light in a predetermined pattern and a predetermined interval on the substrate” And a pair of electrodes in contact with an analyte liquid such as blood, oral fluid, and DNA-containing liquid, and a liquid analyte detection sensor ”is disclosed.
JP 2005-90961 A

However, the above conventional techniques have the following problems.
(1) The analyte liquid characteristic detection sensor of (Patent Document 1) was filed by the applicants of the present application. When the analyte liquid is brought into contact with an electrode pair, the concentration of the analyte liquid or the intensity of light irradiated. Depending on the condition, resistance or voltage change can be detected by a detection unit such as a digital multimeter connected to the electrode pair, and the sample solution is an electrolyte solution or a non-electrolyte liquid containing blood, enzymes, proteins, cells, bacteria, etc. Quantitative and qualitative analysis is possible, and the substance concentration in the sample liquid can be detected in a very short time using a very small amount of the sample liquid, but the limit of the detectable substance concentration is about 100 ppm, and further detection Improvement of sensitivity was strongly desired.
(2) In addition, an inexpensive metal such as Cu is laminated on the substrate and patterned to produce a sensor electrode, and the sensor surface (electrode) is in direct contact with the analyte liquid to detect a voltage change. As a result, the electrode surface deteriorates at every measurement, which is not suitable for repeated use, and it is disposable and lacks resource saving.

  The present invention responds to the above-mentioned demands. The structure is simple, the entire system can be miniaturized, it can be manufactured using semiconductor manufacturing technology, is excellent in mass productivity, can be used repeatedly, and is resource-saving. Providing an oxidative stress substance detection sensor that excels in reliability, can detect oxidative stress substances such as nitric oxide, such as a very small amount of about 1 ppm contained in a sample liquid, in an extremely short time, and has excellent workability. Objective.

In order to solve the above problems, the oxidative stress substance detection sensor of the present invention has the following configuration.
The oxidative stress substance detection sensor according to claim 1 of the present invention includes a substrate, an electrode pair in which two electrodes are spaced apart on the substrate, a surface of the electrode, and a surface of the substrate between the electrodes. And an insulating film that comes into contact with an analyte fluid containing body fluid such as blood, lymph, and cytoplasmic substrate.
This configuration has the following effects.
(1) Since the surface of the electrode and the surface of the substrate between the electrodes are covered with an insulating film, it is contained in a sample liquid containing a small amount of blood, lymph, cytoplasmic substrate, etc. dripped onto the insulating film. It is possible to accurately detect the voltage value or current value between the electrodes that changes according to the amount of oxidative stress substance such as nitric oxide in a short time, and to easily determine the physiological state of the subject who provided the subject. Can do.
(2) Since the electrode surface is coated with an insulating film, the electrode surface can be chemically stabilized and mechanical strength can be enhanced, and it can be used repeatedly as a chemical sensor with excellent durability and reproducibility. Excellent resource saving.
(3) Electrode pairs with a simple structure can be integrated on a substrate with high density by a semiconductor fabrication technology, and can be easily miniaturized and excellent in mass productivity, and can be easily incorporated into electrical circuits and semiconductor integrated circuits. The detected chemical information can be processed as electrical signals in a short time, and a detection system capable of advanced and complicated analysis can be constructed.
(4) Since the electrode surface is covered with an insulating film, the separation of electric charges occurs at the interface between the insulating film and the sample liquid just by dropping or applying the sample liquid on the insulating film, and the sample liquid Can detect oxidative stress substances (radical active species) such as nitric oxide, which have a large electrolytic effect, so that it can be measured with a very small amount of analyte liquid of 1 to 10 μL. Since it is not necessary to perform any pre-treatment or the like, the sample liquid can be easily collected and handled, and the workability is excellent.

  Here, when the sample liquid is brought into contact with the surface of the insulating film by dropping or applying it, charge separation occurs at the interface between the insulating film and the sample liquid, and the solution side and the insulating film side have the same number of polarities. Charged with different charges. As a result, the insulating film is inductively polarized and charges are generated inside the insulating film in contact with the electrodes. When a voltage is applied between the electrodes, this charge accelerates the flow of electrons, so that a current value or a voltage value between the electrodes changes. Oxidative stress substances (radical active species) such as nitric oxide contained in the sample liquid have a large inductive polarization effect. Therefore, it is possible to detect a change in current value or voltage value proportional to the concentration of the oxidative stress substance. it can.

As the material of the substrate, any material can be used as long as it can form an electrode pair and an insulating film on the substrate without being affected by the analyte liquid to be analyzed, and can electrically insulate the electrode pair. Glass, ceramics, etc. are preferably used. In particular, when a transparent material such as glass is used, the sample liquid can be observed with a microscope or the like, and the versatility is excellent. The substrate can be formed in various shapes such as a rectangular shape, a polygonal shape, and a disk shape.
As a material for the electrode, for example, a metal such as Pt, Au, Ag, Fe, Ni, Co, Cr, Cu, Al, Ti, Mn, Zn, an alloy such as stainless steel, tin plate, or the like can be used. The electrode may be formed by chemical vapor deposition on the substrate, or may be formed by patterning a metal thin film previously formed on the substrate by dry etching or wet etching. Moreover, the same kind of metal may be used for each electrode of the electrode pair, or different kinds of metals may be combined. In addition, by forming an Au electrode after depositing Cr on a glass substrate, Cr can be a binder to improve adhesion.

One or a plurality of electrode pairs can be formed on one substrate, and the arrangement can be arbitrarily selected. In addition, the shape of each electrode of the electrode pair is not limited, but can be formed in a triangular shape, a rectangular shape, a semicircular shape, or the like. The electrode pairs may be asymmetrical or different in size, and are arranged so that the sides face each other.
The interval between the sides of the two electrodes facing each other depends on the analyte liquid, the type of electrode, and the like, but is preferably in the range of 5 μm to 10 mm, preferably 10 μm to 5 mm. As the distance between the side portions becomes narrower than 10 μm, the correlation between the electrical characteristics such as the current value with respect to the concentration of the oxidative stress substance in the sample liquid decreases, and the response sensitivity tends to decrease, and the distance is wider than 5 mm. This is because the detection sensitivity tends to be lowered and the data reproducibility tends to be lacking. In particular, as the distance between the sides of the electrode becomes narrower than 5 μm or wider than 10 mm, the signal noise increases, and it tends to be difficult to accurately detect changes in the current value and voltage value. Absent.

The insulating film preferably covers the entire surface of the substrate in order to maintain the mechanical strength of the electrode. However, it is sufficient that the insulating film covers at least the surface of the electrode.
Although the thickness of the insulating film varies depending on the material, it is necessary to select it within a range in which the analyte liquid and the electrode pair can be reliably insulated and the responsiveness as a sensor can be maintained. As the film thickness of the insulating film decreases, the effect of the insulating film becomes insufficient and the sensitivity of the sensor tends to decrease.As the film thickness increases, the voltage value and current are changed even if the concentration of the oxidative stress substance in the sample liquid changes. There is a tendency that the change in the value is not observed and the responsiveness of the sensor is lost. For example, 0.2 μm to 0.8 μm is preferable for a fluoroolefin vinyl ether polymer (molecular weight distribution 100 to 1000), and 5 μm to 20 μm is preferable for a novolac phenol resin (molecular weight distribution 1000 to 10,000).

The oxidative stress substance detection sensor according to claim 2 has a configuration in which the insulating film is formed of an organic thin film layer.
With this configuration, in addition to the operation of the first aspect, the following operation is provided.
(1) Since the insulating film is formed of an organic thin film layer, when the analyte liquid is brought into contact by dropping or coating, charge separation occurs at the interface, and the solution side and the insulating film side are polar. It becomes charged with the same number of charges with different charges. As a result, the insulating film is inductively polarized and charges are generated inside the insulating film in contact with the electrodes. When a voltage is applied between the electrodes, this charge accelerates the flow of electrons between the electrodes, causing a change in the current value or voltage value between the electrodes. Since various functional groups derived from the structure (carboxyl group, ketone group, hydroxyl group, amino group, ether group, etc.) and polar groups (fluorine, chlorine, bromine, etc.) exist, this polarization phenomenon is large and improves detection sensitivity. be able to.
Here, as the insulating film, the above-mentioned fluoroolefin vinyl ether polymer, novolac phenol resin, or the like is preferably used.
Depending on the chemical species in the sample liquid, specific polarization occurs with various functional groups on the surface of the organic thin film. Therefore, it is possible to produce an oxidative stress substance detection sensor with high response sensitivity.

A third aspect of the present invention is the oxidative stress substance detection sensor according to the first or second aspect, comprising a detection unit that is connected to the electrode pair and detects a voltage change or a current change between the electrodes. have.
With this configuration, in addition to the operation of the first or second aspect, the following operation is provided.
(1) Since the detection unit connected to the electrode pair is provided, the voltage change or current change between the electrodes can be easily detected, and the concentration of the oxidative stress substance in the sample liquid is immediately determined based on the measured value. Can be requested.

  Here, as the detection unit, various measuring devices capable of measuring a minute current value or a voltage value generated between the electrodes can be used, for example, a tester having functions such as a potentiometer, an ammeter, a voltmeter, or the like. A measuring device such as a digital multimeter, a measuring device incorporating a Wheatstone bridge circuit, a data logger device having the above function, or the like can be used. Alternatively, it may be directly incorporated into an A / D converter or the like, and a current or voltage change may be converted from an analog signal to a digital signal and measured. A signal obtained from the electrode pair can be directly measured by a measuring device, but can be measured after being amplified by an amplifier circuit or the like, or can be processed through a noise filter, thereby improving data reliability.

A fourth aspect of the present invention is the oxidative stress substance detection sensor according to any one of the first to third aspects, wherein the oxidative stress substance detection sensor is included in the analyte liquid based on detection data detected by the detection unit. It has the structure described in that it comprises a determination unit for detecting the amount of the oxidative stress substance and determining the physiological state.
With this configuration, in addition to the operation of any one of claims 1 to 3, the following operation is provided.
(1) Since there is a determination unit that detects the amount of oxidative stress substance contained in the sample liquid based on the detection data detected by the detection unit and determines the physiological state, the sample liquid is immediately submitted in a short time It is possible to know the physiological state of the subject and is excellent in handling.

Here, the determination unit stores in advance the relationship between the concentration of the oxidative stress substance and the voltage value or current value, and compares the standard data with the detection data (measurement data), thereby making a reliable and quick determination. be able to.
Further, by providing a storage unit and storing the detection data continuously or periodically, it is possible to manage the change and progress of the physiological state of the subject, which is excellent in versatility.

As described above, according to the oxidative stress substance detection sensor of the present invention, the following advantageous effects can be obtained.
According to invention of Claim 1, it has the following effects.
(1) By measuring the voltage value or the current value between the electrodes, it is possible to accurately detect an extremely small amount of an oxidative stress substance such as nitric oxide contained in the sample liquid in a short time. Therefore, it is possible to provide an oxidative stress substance detection sensor excellent in reliability and workability that can easily determine the physiological state.

According to invention of Claim 2, in addition to the effect of Claim 1, it has the following effects.
(1) By having an insulating film formed of an organic thin film layer, the polarization phenomenon generated at the interface between the insulating film and the sample liquid increases, and the amount of charge increases. As a result, inductive polarization occurs and charges are also generated inside the insulating film in contact with the electrodes. By applying a voltage between the electrodes, the charge accelerates the flow of electrons and changes in the current value and voltage value between the electrodes. In addition, depending on the chemical species in the analyte liquid, specific polarization occurs with various functional groups on the surface of the organic thin film, which makes it possible to detect oxidative stress substances with high response sensitivity and excellent reliability. A sensor can be provided.

According to invention of Claim 3, in addition to the effect of Claim 1 or 2, it has the following effects.
(1) The change in voltage or current between the electrodes can be easily detected by the detection unit connected to the electrode pair, and the concentration of the oxidative stress substance in the sample liquid can be immediately obtained based on the measured value. It is possible to provide an oxidative stress substance detection sensor excellent in workability.

According to invention of Claim 4, in addition to the effect of any one of Claims 1 thru | or 3, it has the following effects.
(1) The determination unit determines the physiological state based on the detection data detected by the detection unit, so that the physiological state of the subject can be immediately known in a short time without performing complicated work. It is possible to provide an oxidative stress substance detection sensor excellent in properties.

(Embodiment 1)
An oxidative stress substance detection sensor according to Embodiment 1 of the present invention will be described below with reference to the drawings.
FIG. 1A is a plan view showing an oxidative stress substance detection sensor according to Embodiment 1, and FIG. 1B is a schematic cross-sectional view taken along line AA in FIG.
In FIG. 1, reference numeral 1 denotes an oxidative stress substance detection sensor according to Embodiment 1 of the present invention, 2 denotes a substrate of the oxidative stress substance detection sensor 1 formed of various synthetic resins, glass, ceramics, etc., 3 denotes an upper surface of the substrate 2. The electrode pair of the oxidative stress substance detection sensor 1, which is arranged so that two substantially semicircular electrodes 4 are opposed to each other at the side portions 4 a, 4 b extend to the arc-shaped side portions of the respective electrodes 4. The terminal portion 5 of the electrode pair 3 is an insulating film such as a fluoroolefin vinyl ether polymer or a novolac resin covering the entire surface of the substrate 2 including the electrode pair 3, and 6 is an electrode pair 3 by the terminal portion 4 b. A detection unit such as a digital multimeter that is electrically connected to detect a voltage value or a current value between the electrodes 4, and 7 is connected to the detection unit 6, and is applied to the sample liquid based on detection data detected by the detection unit 6. Contained oxidative stress A determination unit for determining physiological condition by detecting the amount of quality.

In FIG. 1, the material of the substrate 2 is not limited to the present embodiment, and the electrode pair 3 and the insulating film 5 can be formed on the substrate 2 without being affected by the analyte liquid to be analyzed. Any electrode that can electrically insulate the electrode pair 3 may be used. In particular, when a transparent material such as glass is used, the sample liquid can be observed with a microscope or the like, and the versatility is excellent. Moreover, the shape of the board | substrate 2 can be formed in various shapes, such as polygonal shape and disk shape other than rectangular shape.
In the present embodiment, the adhesion of the electrode 4 and the substrate 2 is improved by using Cr as a binder by forming the Au electrode 4 after depositing Cr on the substrate 2.

The interval between the side portions 4a of the two electrodes 4 facing each other was formed in a range of 1 μm to 10 mm, depending on the sample liquid and the type of the electrode 4. As the distance between the side portions 4a becomes narrower than 1 μm, the correlation between the electrical characteristics such as the current value with respect to the concentration of the oxidative stress substance in the sample liquid decreases, and the response sensitivity tends to decrease, and the distance is 10 mm. This is because it has been found that the detection sensitivity tends to decrease as the length increases, and the reproducibility of data tends to be lacking.
For convenience of explanation, only a pair of electrode pairs 3 is shown, but one or a plurality of electrode pairs 3 can be formed on the substrate 2, and the arrangement thereof can be arbitrarily selected. In addition, the shape of each electrode of the electrode pair is not limited, but can be formed in a triangular shape, a rectangular shape, a semicircular shape, or the like. The electrode pairs may be asymmetrical or different in size, and are arranged so that the sides face each other. Further, in the present embodiment, the two terminal portions 4b extending from the side portions of the respective electrodes 4 are formed in a substantially L shape and taken out from one end portion of the substrate 2. However, the present invention is not limited to this. Instead, the take-out position and take-out direction of the terminal portion 4b can be arbitrarily selected.

In the present embodiment, the entire surface of the substrate 2 is covered with the insulating film 5, but the insulating film 5 only needs to cover at least the surface of the electrode 4.
Although the thickness of the insulating film 5 varies depending on the material, it is necessary to select it within a range in which the analyte liquid and the electrode pair 3 can be reliably insulated and the responsiveness as a sensor can be maintained. As the film thickness of the insulating film 5 decreases, the effect of the insulating film 5 becomes insufficient and the sensitivity of the sensor tends to decrease. As the film thickness increases, the voltage value changes even if the concentration of the oxidative stress substance in the sample liquid changes. This is because it has been found that there is a tendency that the change in the current value is not observed and the response of the sensor is lowered. For example, 0.2 μm to 0.8 μm is preferable for a fluoroolefin vinyl ether polymer (molecular weight distribution 100 to 1000), and 5 μm to 20 μm is preferable for a novolac phenol resin (molecular weight distribution 1000 to 10,000).

Hereinafter, the operation principle of the oxidative stress substance detection sensor will be described.
FIG. 2 is a schematic cross-sectional view showing a usage state of the oxidative stress substance detection sensor in the first embodiment.
In FIG. 2, reference numeral 10 denotes a sample liquid containing body fluid such as blood, lymph, cytoplasmic substrate, etc. dropped on the insulating film 5 on the electrode pair 3.
When the analyte liquid 10 is brought into contact with the surface of the insulating film 5 by dropping or applying it or the like, charge separation occurs at the interface between the insulating film 5 and the analyte liquid 10, and the solution side and the insulating film side have the same number of polarities. Charged with different charges. As a result, the insulating film is inductively polarized and charges are generated inside the insulating film in contact with the electrodes. When a voltage is applied between the electrodes, this charge accelerates the flow of electrons, so that a current value or a voltage value between the electrodes changes. Since the oxidative stress substance (radical active species) such as nitric oxide contained in the sample liquid 10 has a large induced polarization effect, a change in the current value or voltage value proportional to the concentration of the oxidative stress substance is detected by the detection unit 6. Can be detected.
The determination unit 7 stores the relationship between the concentration of the oxidative stress substance and the voltage value or current value in advance, and compares the standard data with the detection data (measurement data), thereby making a reliable and quick determination of the physiological state. It can be carried out.
In addition, by providing a storage unit in the determination unit 7 and storing the detection data continuously or periodically, it is possible to manage changes and progress of the physiological state of the subject, which is excellent in versatility.

Since the oxidative stress substance detection sensor of Embodiment 1 is configured as described above, it has the following actions.
(1) Since the surface of the electrode 4 and the surface of the substrate 2 between the electrodes 4 are covered with an insulating film, the subject liquid 10 contains a small amount of lymph fluid dropped on the insulating film 5 and body fluid such as cytoplasmic substrate. The voltage value or current value between the electrodes 4 that changes in accordance with the amount of oxidative stress substance such as nitric oxide contained in can be detected with high accuracy in a short time, and the physiological state of the subject who provided the subject can be easily Can be determined.
(2) The electrode pair 3 having a simple structure can be integrated on the substrate 2 with a high density by a semiconductor manufacturing technique, and can be easily miniaturized and excellent in mass productivity, and can be easily incorporated into an electric circuit or a semiconductor integrated circuit. Therefore, it is possible to construct a detection system capable of processing the detected chemical information as an electrical signal in a short time and capable of advanced and complex analysis.
(3) Since the surface of the electrode 4 is covered with the insulating film 5, charges can be separated at the interface between the insulating film 5 and the analyte liquid 10 simply by dropping or applying the analyte liquid 10 on the insulating film 5. Occurs, and the amount of charge increases to induce polarization. Since charges are also generated inside the insulating film 5 in contact with the electrodes 4, by applying a voltage between the electrodes 4, the charges accelerate the flow of electrons and have a large polarization effect contained in the analyte liquid 10. Since oxidative stress substances (radical active species) such as nitric oxide can be detected, measurement can be performed with a very small amount of the sample liquid 10 of 1 to 10 μL, and special pretreatment or the like is required before measurement. Therefore, the sample liquid 10 can be easily collected and handled and has excellent workability.
(4) When the analyte liquid 10 is brought into contact with the insulating film 5 by dropping or applying it, charges are separated at the interface between the insulating film 5 and the analyte liquid 10, but the organic that forms the insulating film 5 Various functional groups (carboxyl group, ketone group, hydroxyl group, amino group, ether group, etc.) and polar groups (fluorine, chlorine, bromine, etc.) derived from the chemical structure of the polymer terminal or branch terminal exist on the surface of the thin film layer Therefore, this polarization phenomenon is large, and the responsiveness of the oxidative stress substance detection sensor 1 can be improved. In addition, depending on the chemical species in the analyte liquid 10, specific polarization occurs with various functional groups on the surface of the organic thin film, so that the oxidative stress substance detection sensor 1 with high response sensitivity can be produced.
(5) Since the detection unit 6 connected to the electrode pair 3 is provided, a voltage change or a current change between the electrodes 4 can be easily detected, and immediately in the sample liquid 10 based on the measured value. An oxidative stress substance concentration can be obtained.
(6) Since the determination unit 7 that determines the physiological state by detecting the amount of the oxidative stress substance contained in the sample liquid 10 based on the detection data detected by the detection unit 6 is provided, the sample liquid is instantly obtained in a short time. It is possible to know the physiological state of the subject who submitted 10 and is excellent in handleability.

Hereinafter, the present invention will be specifically described by way of examples.
First, with respect to the oxidative stress substance detection sensor 1 described in the first embodiment, an experiment was conducted on the ability to detect an oxidative stress substance.
(Experimental example 1)
The substrate 2 of the oxidative stress substance detection sensor 1 is a glass substrate having a thickness of 1 mm, and the diameter of the substantially circular electrode pair 3 in which the semicircular electrodes 4 are arranged to face each other with a separation of 10 μm is 150 μm. The electrode 4 was formed by laminating a Cr layer of 0.1 μm and an Au layer of 1 μm by sputtering and patterning by photolithography. Further, the entire surface of the substrate 2 was covered with an insulating film 5 of a novolac phenol resin (molecular weight distribution 1000 to 10000) having a film thickness of 5 μm.
1 μHydroxy-2-oxo-3- (N-methyl-3-aminopropyl) -3-methyl-1-triazene (hereinafter abbreviated as NOC) + NaOH solution 1 μm each as the analyte liquid 10 on the surface of the insulating film 5 After dropping, a phosphate buffer solution (PBS) was added to generate NO as an oxidative stress substance.
The voltage value between the electrodes 4 detected by the detection unit 6 was measured as the NO generation voltage with respect to the NOC concentration at this time.
FIG. 3 is a diagram showing the relationship between the NOC concentration and the NO generation voltage.
As shown in FIG. 3, it was found that the NO generation voltage increases as the NOC concentration increases. Thereby, it was confirmed that the concentration of the oxidative stress substance can be detected by the oxidative stress substance detection sensor 1.

Next, with respect to the oxidative stress substance detection sensor 1, an experiment was conducted on the film thickness of the insulating film 5 and the detection sensitivity of the oxidative stress substance.
(Experimental example 2)
An oxidative stress substance detection sensor 1 similar to that in Experimental Example 1 was used except that the thickness of the insulating film 5 was 0.5 μm. NaNO ₃ having different concentrations was dropped as the analyte liquid 10, and the voltage value between the electrodes 4 was measured by the detection unit 6 as a sensor voltage.

(Experimental example 3)
The voltage value between the electrodes 4 was measured as a sensor voltage in the same manner as in Experimental Example 2 except that the thickness of the insulating film 5 was 2.5 μm.

(Experimental example 4)
The voltage value between the electrodes 4 was measured as a sensor voltage in the same manner as in Experimental Example 2 except that the thickness of the insulating film 5 was 5 μm.

4 to 6 are diagrams showing the relationship between the NO _x concentration and the sensor voltage in Experimental Examples 2 to 4, respectively.
According to FIGS. 4-6, but linearity is not observed, the thickness 5μm of Example 4 between the Experimental Examples 2 and 3 having a thickness of 0.5μm and NO _x concentration and the sensor voltage at 2.5μm Further, linearity was observed between the NO _x concentration and the sensor voltage, and it was confirmed that a very small amount of NO _x (a chemically stable species of the oxidative stress substance NO in an aqueous solution) could be detected.

Next, an experiment was conducted on the difference in detection sensitivity of the oxidative stress substance detection sensor 1 due to the difference in the analyte liquid 10.
(Experimental example 5)
Except that the test solution 10 was NaNO ₂ were measured voltage value between the to electrodes 4 in the same manner as in Experimental Example 4 as a sensor voltage.
FIG. 7 is a diagram showing the relationship between the NO _x concentration and the sensor voltage in Experimental Example 5.
According to FIG. 7, even when the analyte liquid 10 is NaNO ₂ , relatively good linearity is recognized between the NO _x concentration and the sensor voltage, and NO _x ( It was confirmed that a chemically stable species of the oxidative stress substance NO in an aqueous solution can be detected.

Next, an experiment was conducted on the difference in the type of the insulating film 5 and the difference in the film thickness of the insulating film 5 and the difference between the coating location of the insulating film 5 and the oxidative stress substance detection sensitivity.
(Experimental example 6)
The substrate 2 of the oxidative stress substance detection sensor 1 is made of glass / epoxy resin having a thickness of 1 mm, and the diameter of the substantially circular electrode pair 3 in which the semicircular electrodes 4 are arranged facing each other with a separation of 200 μm is 4 mm. The electrode was formed by laminating a Cu layer by electroplating and patterning by photolithography. Further, the entire surface of the substrate 2 was covered with an insulating film 5 of a fluoroolefin vinyl ether polymer (molecular weight distribution 100 to 1000) having a film thickness of 0.2 μm. 10 μL of NaNO ₂ (chemically stable species of oxidative stress substance NO in an aqueous solution) having a different concentration was dropped as the analyte liquid 10, and the voltage value between the electrodes 4 was measured by the detection unit 6 as a sensor voltage.

(Experimental example 7)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 6 except that the thickness of the insulating film 5 was 0.4 μm.

(Experimental example 8)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 6 except that the thickness of the insulating film 5 was 0.6 μm.

(Experimental example 9)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 6 except that the insulating film 5 was formed thicker than 0.8 μm.

(Experimental example 10)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 7 except that only the electrode 4 on one side of the electrode pair 3 was covered with the insulating film 5.

(Experimental example 11)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 7 except that only the gap between the electrodes 4 of the electrode pair 3 was covered with the insulating film 5.

(Comparative Example 1)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 6 except that the insulating film 5 was not formed.

The measurement results of the sensor voltage in Experimental Examples 6 to 11 and Comparative Example 1 are shown in (Table 1).

According to Experimental Examples 6 to 8, when the insulating film 5 is a fluoroolefin vinyl ether polymer, the sensor voltage between the electrodes 4 is NO _x (aqueous solution) when the film thickness is in the range of 0.2 μm to 0.6 μm. It was found to be proportional to the concentration of the chemically stable species of the oxidative stress substance NO.
According to Experimental Example 9, when the insulating film 5 is a fluoroolefin vinyl ether polymer, when the film thickness becomes thicker than 0.8 μm, NO _x (oxidative stress substance NO in aqueous solution) It was found that the voltage value no longer changes even when the concentration changes, and the oxidative stress substance detection sensor 1 does not respond.
According to Experimental Example 10, when only one of the electrodes 4 of the electrode pairs 3 and covered with an insulating film 5, the voltage value irrespective of the concentration of NO _x becomes unstable, was found to be impossible to detect the voltage.
According to Experiment 11, if only between 4 electrode pairs 3 of the electrode covered with the insulating film 5, the sensor voltage decreases with increasing concentration of NO _x, the trend reversed and the Experimental Example 6-8 observed I understood it. This tendency is the same as in the case where the insulating film 5 of Comparative Example 1 is not formed, and is because the electrode 4 is in contact with the sample liquid 10 without being insulated. When the electrode 4 is covered with the insulating film 5, the voltage generated by the induced polarization of the insulating film induced by the voltage value (E) of the detection unit 6, the resistance value (R) of the insulating film 5, and the polarization of the interface is the electrode. The relation of the acceleration current (I) flowing between the four is in accordance with Ohm's law E = R × I (Equation 1)
The relationship is established. Here, when the insulating film 5 does not change due to deterioration or the like, R is a constant value. I in (Equation 1) changes according to the concentration of the oxidative stress substance contained in the sample liquid 10. Therefore, E responds in proportion to I.
On the other hand, when the electrode 4 is not covered with the insulating film 5 and is in direct contact with the analyte liquid 10, R is replaced with the conductivity (≈resistance value) of the analyte liquid 10. For this reason, when the concentration of the analyte liquid 10 is low, R increases, and as a result, the detection voltage E increases. Conversely, when the concentration of the analyte liquid 10 is high, R decreases, and as a result, the detection voltage E also decreases. Due to such a difference in mechanism, the responsiveness opposite to that when the insulating film 5 is present is exhibited.

Next, the relationship between the NO _x (chemically stable species of oxidative stress substance NO in aqueous solution) concentration of the analyte liquid 10 and the sensor voltage (voltage value) between the electrodes 4 detected by the detection unit 6 will be described in detail. It was measured.
FIG. 8 is a diagram showing the relationship between the NO _x concentration and the sensor voltage.

(Experimental example 12)
The same oxidative stress substance detection sensor 1 as that of Experimental Example 6 was used except that the insulating film 5 was formed of a fluoroolefin vinyl ether polymer having a film thickness of 0.2 μm. 10 μL of NaNO ₂ (chemically stable species of oxidative stress substance NO in an aqueous solution) having a different concentration was dropped as the analyte liquid 10, and the voltage value between the electrodes 4 was measured by the detection unit 6 as a sensor voltage.

(Experimental example 13)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 12 except that the thickness of the insulating film 5 was 0.4 μm.

(Experimental example 14)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 12 except that the thickness of the insulating film 5 was 0.6 μm.

As shown in FIG. 8, the sensor voltage is proportional to the NO _x concentration. Particularly when the thickness of the insulating film 5 is 0.6 μm, the sensor voltage 10 has a good linear relationship and is very small from the analyte liquid 10. It was found that NO _x (chemically stable species of the oxidative stress substance NO in an aqueous solution) can be detected with high accuracy.

Next, an experiment was conducted on the relationship between the distance between the side portions 4a of the two electrodes 4 facing each other in the electrode pair 3 and the detection sensitivity of the oxidative stress substance.
(Experimental example 15)
The substrate 2 of the oxidative stress substance detection sensor 1 is a glass substrate having a thickness of 1 mm, and the diameter of the substantially circular electrode pair 3 in which the semicircular electrodes 4 are arranged to face each other with a separation of 1 μm is 150 μm. The electrode 4 was formed by laminating a Cr layer of 0.1 μm and an Au layer of 1 μm by sputtering and patterning by photolithography. Further, the entire surface of the substrate 2 was covered with an insulating film 5 of a novolac phenol resin (molecular weight distribution 1000 to 10000) having a film thickness of 5 μm. NaNO ₃ was dropped as the analyte liquid 10, and the voltage value between the electrodes 4 was measured by the detection unit 6 as a sensor voltage.

(Experimental example 16)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 15 except that the distance between the side portions 4a of the electrodes 4 was 5 μm.

(Experimental example 17)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 15 except that the interval between the side portions 4a of the electrodes 4 was 10 μm.

(Experiment 18)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 15 except that the distance between the side portions 4a of the electrodes 4 was 200 μm.

(Experimental example 19)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 15 except that the distance between the side portions 4a of the electrodes 4 was 5 mm.

(Experiment 20)
The voltage value between the electrodes 4 was measured in the same manner as in Experimental Example 15 except that the distance between the side portions 4a of the electrodes 4 was 10 mm.

The measurement results of the sensor voltage in Experimental Examples 15 to 20 are shown in (Table 2).

According to Experimental Example 15, when the distance between the side portions 4a of the electrode 4 is 1 μm, the voltage is maintained even if the concentration of NO _x (chemically stable species of the oxidative stress substance NO in the aqueous solution) in the sample liquid 10 changes. It was found that the value was not changed and the oxidative stress substance detection sensor 1 stopped responding.
According to Experimental Example 16, it was found that when the distance between the side portions 4a of the electrode 4 is 5 μm, the signal noise increases and it is difficult to accurately detect the change in the voltage value.
According to Experimental Examples 17 to 19, when the distance between the side portions 4a of the electrode 4 is in the range of 10 μm to 5 mm, the sensor voltage proportional to the concentration of NO _x (chemically stable species of the oxidative stress substance NO in the aqueous solution) is increased. It turns out that it can be detected.
According to Experimental Example 20, it was found that when the distance between the side portions 4a of the electrode 4 is 10 mm, the signal noise increases and it is difficult to accurately detect the change in the voltage value.

  The present invention is simple in structure, can downsize the entire system, can be manufactured using semiconductor manufacturing technology, has excellent mass productivity, can be used repeatedly, has excellent resource savings, Providing an oxidative stress substance detection sensor with excellent reliability and workability that can accurately detect oxidative stress substances such as nitric oxide, such as trace amounts of 1ppm contained in the liquid, in a very short time. Thus, it is possible to easily determine the physiological state of the subject.

(A) Plan view showing the oxidative stress substance detection sensor in Embodiment 1 (b) Schematic cross-sectional view taken along line AA in FIG. Sectional schematic diagram which shows the use condition of the oxidative stress substance detection sensor in Embodiment 1 Diagram showing the relationship between NOC concentration and NO generation voltage The figure which shows the relationship between the NOx density _| concentration and sensor voltage in Experimental example 2. The figure which shows the relationship between the NOx density _| concentration and sensor voltage in Experimental example 3. The figure which shows the relationship between the NOx density _| concentration and sensor voltage in Experimental example 4. The figure which shows the relationship between the NOx density _| concentration and sensor voltage in Experimental example 5. Diagram showing the relationship between the concentration of NO _x and the sensor voltage

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Oxidative stress substance detection sensor 2 Board | substrate 3 Electrode pair 4 Electrode 4a Side part 4b Terminal part 5 Insulating film 6 Detection part 7 Determination part 10 Analyte liquid

Claims (4)

A subject including a substrate, electrode pairs arranged on the substrate at a predetermined interval, and body fluids such as blood, lymph, and cytoplasmic substrate covering the surfaces of the electrode pairs and the surface of the substrate between the electrode pairs. An oxidative stress substance detection sensor comprising an insulating film in contact with a liquid. The oxidative stress substance detection sensor according to claim 1, wherein the insulating film is formed of an organic thin film layer. 3. The oxidative stress substance detection sensor according to claim 1, further comprising a detection unit that is connected to the electrode pair and detects a voltage change or a current change between the electrode pair. 4. The apparatus according to claim 1, further comprising a determination unit that detects a physiological state by detecting an amount of an oxidative stress substance contained in the sample liquid based on detection data detected by the detection unit. The oxidative stress substance detection sensor of any one of Claims.

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