Sensors using particles with molecularly imprinted polymers on their surface

Abstract

To resolve the problem of reduced sensor sensitivity caused by silicone oil in a molecularly imprinted polymer paste, in a sensor that includes an electrode immobilized with the paste. [Solution] A sensor that utilizes conductive carbon for wiring, wherein the sensor has (a) an working electrode, (b) a counter electrode, (c) a reference electrode, (d) a wiring section, and (e) a connection section to a measuring instrument, the working electrode is formed from a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and silicone oil, and a portion of the wiring section between the connection section to the measuring instrument and the working electrode is replaced with a conductive and non-lipophilic substance. A sensor comprising an electrode substrate coated or filled with a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and an amphiphilic substance.

Description

Technical Field

[0001] The present invention relates to a sensor using particles having a molecularly imprinted polymer on the surface, and a measuring method using the sensor.

Background Art

[0002] In order to effectively use a therapeutic drug with strong side effects, therapeutic drug monitoring (TDM) for confirming that its blood concentration is within the effective range is required. However, there is no appropriate sensing technology, and the spread of TDM in the medical field has not progressed.

[0003] A molecularly imprinted polymer (MIP) is a molecular recognition element obtained by copolymerizing a monomer (functional monomer) having an affinity for a recognition target substance (template) and a crosslinkable monomer in the presence of the recognition target substance. It is a molecular recognition element that can be tailor-made for any target substance by a simple and economical process. The present inventor has found that the redox current in an electrode grafted with MIP on the surface depends on the concentration of the template. This is considered to be because the change in the accessibility of the redox species to the base electrode (gate effect) occurs due to the specific interaction between the template and MIP. By measuring this current, the template can be sensed simply and quickly. Based on this finding, Patent Document 1 describes a sensor for measuring an anticoagulant drug composed of a substrate on which a molecularly imprinted polymer is immobilized. Further, Patent Document 2 describes a sensor composed of a substrate on which a molecularly imprinted polymer is directly immobilized on the surface, and a redox species is immobilized on the molecularly imprinted polymer and / or the substrate. Further, Patent Document 3 describes a sensor composed of conductive particles having a molecularly imprinted polymer graft-polymerized on the surface of conductive particles on which a polymerization initiator is immobilized, and a support.

Prior Art Documents

Patent Documents

[0004] [Patent Document 1] International Publication No. WO2012 / 124800 [Patent Document 2] International Publication No. WO2016 / 140337 [Patent Document 3] International Publication No. WO2018 / 117067 [Overview of the project] [Problems that the invention aims to solve]

[0005] In the technologies described in Patent Documents 1 and 2, a method is employed to graft MIP by irradiating ITO with a polymerization initiator immobilized in a solution containing each monomer and a template with ultraviolet light. During the polymerization process, attempts have been made to reduce variations between the produced electrodes by thinning the large liquid layer on the electrode or increasing the intensity of the light source, but it has been difficult to ensure reproducibility sufficient for single-use disposable applications. This is thought to be due to the difficulty in obtaining homogeneous products through radical polymerization. In the technology described in Patent Document 3, a molecular imprinted polymer carbon paste (MIP-CP) obtained by kneading graphite particles with molecular imprinted polymers (MIP) immobilized on their surface with silicone oil as a binder is used to produce an MIP-CP electrode filled into a tube. The measurement principle is thought to be that the MIP on the graphite surface swells due to interaction with the target substance and becomes significantly hydrophilic, pushing the oil on the electrode surface deeper inside, thereby increasing the effective area of ​​the electrode. The inventors have developed a sensor capable of measuring the concentration of a target substance by applying molecular imprinted polymer carbon paste (MIP-CP) electrodes to a substrate wired with carbon screen printing, since MIP-CP electrodes adhere stably to carbon wiring. This sensor has the advantage of being able to rapidly measure various water-soluble substances. However, the sensor with MIP-CP applied to screen-printed carbon wiring has low sensitivity, and the sensitivity decreased even further when measuring in samples containing a large amount of protein, such as serum. In other words, this sensor often could not ensure sufficient sensitivity when measuring the target substance in samples containing a large amount of interfering substances such as proteins, such as serum. This is thought to be because conductive carbon ink has an affinity for MIP-CP, allowing MIP-CP to adhere stably to the carbon wiring, but the conductive carbon ink absorbs the silicone oil, which is the binder in MIP-CP, and the surface oil responsible for sensitivity is lost, thus impairing the sensitivity to the target substance. Therefore, when silver ink was used instead of carbon ink for screen printing, MIP-CP could not be stably applied to the wiring, and the cost also increased.Furthermore, while sensors coated with MIP-CP on the circuit board have the advantage of being able to rapidly measure various water-soluble substances, they were unable to measure the concentration of substances in lipid-heavy dispersed solutions such as serum obtained by blood collection immediately after a meal or milk. In other words, it was found that sensors using MIP-CP exhibited significantly low sensitivity in samples containing a large amount of lipid colloids, such as milk or serum containing chyle. This is thought to be because the lipid colloids penetrate the silicone oil on the surface of the MIP-CP, making it hydrophilic and impairing the sensitivity to the target substance. There is a strong demand for sensors that can detect indicator substances of milk freshness (histamine). Also, since patient blood often contains a large amount of lipid colloids, it is important that the sensor is not interfered with by them.

[0006] The present invention aims to solve the problem of reduced sensor sensitivity caused by the silicone oil in a molecular imprint polymer paste (MIP-P) containing a molecular imprint polymer and silicone oil, in a sensor including an electrode with an immobilized MIP-P. Specifically, the present invention aims to provide a sensor including an electrode with an immobilized MIP-P that can measure the concentration of a target substance in a sample containing a large amount of protein, such as serum or meat drip, with high sensitivity. Furthermore, the present invention aims to provide a sensor including an electrode with an immobilized MIP-P that can measure a target substance in a sample containing a large amount of lipid colloid, such as milk or serum containing chyle, with high sensitivity. [Means for solving the problem]

[0007] The inventors diligently studied to solve the above problems. The inventors fabricated a sensor that utilizes conductive carbon in its wiring, wherein the sensor comprises (a) a working electrode, (b) a counter electrode, (c) a reference electrode, (d) a wiring section, and (e) a connection section to a measuring instrument, the working electrode being formed from a molecularly imprinted polymer paste containing conductive particles having molecularly imprinted polymers on its surface and silicone oil, and a portion of the wiring section between the connection section to the measuring instrument and the working electrode being replaced with a conductive and non-lipophilic substance. Differential pulse voltameometry was performed in human serum containing vancomycin to confirm the response to the vancomycin concentration in the serum. As a result, the inventors found that a sensor fabricated on a substrate in which a portion of the carbon wiring section connecting the working electrode coated with MIP-CP to the connector (connection section to the measuring instrument) was missing, and the missing portion was filled with silver ink, showed high sensitivity even in human serum, thus completing the present invention. Furthermore, a sensor was fabricated containing an electrode substrate coated or filled with a molecularly imprinted polymer paste, which includes conductive particles having molecularly imprinted polymers on their surface and an amphiphilic substance. Cyclic voltameometry was performed in milk containing histamine to confirm the response to the histamine concentration in the milk. As a result, it was found that sensitivity to the target substance could be obtained even in milk using a sensor containing an electrode substrate coated or filled with MIP-CP, obtained by kneading conductive particles having molecularly imprinted polymers on their surface with polyethylene glycol monoacrylate, an amphiphilic liquid, as a binder instead of silicone oil, thereby completing the present invention.

[0008] In other words, aspects of the present invention relate to the following. <1> A sensor that uses conductive carbon in its wiring, The sensor has (a) an working electrode, (b) a counter electrode, (c) a reference electrode, (d) a wiring section, and (e) a connection section for a measuring instrument. The working electrode is formed from a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and silicone oil. A portion of the wiring between the connection point to the measuring instrument and the working electrode is replaced with a conductive and non-lipophilic substance. Sensor. <2> The conductive and non-lipophilic substance is silver and / or a silver salt. <1> The sensor described above. <3> The conductive particles are graphite particles. <1> The sensor described above. <4> Conductive particles having molecularly imprinted polymers on their surface are obtained by polymerizing particles immobilized with an initiator by contacting them with a functional monomer, a crosslinkable monomer, and a substance to be measured. <1> The sensor described above. <5> The substance being measured is a hormone, antibacterial agent, anticoagulant, or a toxic substance or freshness indicator substance in food. <1> The sensor described above. <6> The substance being measured is heparin, warfarin, serotonin, vancomycin, phenobarbital, theophylline, edoxaban, or histamine. <1> The sensor described above. <7> <1> from <6> A method for measuring a substance, comprising contacting a sample containing the substance to be measured with a sensor described in any one of the items described above, and detecting a change in the signal. <8> This includes detecting changes in current as changes in signal, <7> The method for measuring the substance described. <9> The aforementioned sample is whole blood or blood components, or food. <7> The measurement method described above. <10> A sensor comprising an electrode substrate coated or filled with a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and an amphiphilic substance. <11> The sensor according to claim 10, wherein the molecular imprinted polymer paste functions as a working electrode. <12> The aforementioned amphiphilic substance is polyethylene glycol monoacrylate. <10> The sensor described above. <13> The conductive particles are graphite particles. <10> The sensor described above. <14> The sensor according to <10>, wherein the conductive particles having a molecularly imprinted polymer on the surface are conductive particles obtained by bringing a functional monomer, a crosslinkable monomer, and a measurement substance into contact with particles immobilizing an initiator and polymerizing them. <15> The sensor according to <10>, wherein the measurement substance is a hormone, an antibacterial agent, an anticoagulant, or a toxic substance or freshness index substance in food. <16> The sensor according to <10>, wherein the measurement substance is heparins, warfarin, serotonin, vancomycin, phenobarbital, theophylline, edoxaban, or histamine. <17> A method for measuring a measurement substance, comprising bringing a sample containing the measurement substance into contact with the sensor according to any one of <10> to <16> and detecting a change in a signal. <18> The method for measuring a measurement substance according to <17>, comprising detecting a change in current as the change in the signal. <19> The measurement method according to <17>, wherein the sample is whole blood or a blood component, or food.

Advantages of the Invention

[0009] According to the sensor of the present invention, even for a target substance in a sample containing a large amount of protein such as serum or meat drip liquid, the concentration can be measured with higher sensitivity compared to the conventional method. Further, according to the sensor of the present invention, even for a target substance in a sample containing a large amount of lipid colloid such as milk or serum containing chyle, the concentration can be measured with higher sensitivity compared to the conventional method. Further, according to the present invention, a sensor capable of sensitively detecting vancomycin in serum can be provided. According to the present invention, a sensor capable of sensitively detecting histamine in milk can be provided. Furthermore, even when the patient's blood contains a large amount of lipid colloid, highly sensitive monitoring of therapeutic drugs in blood and the like can be enabled.

Brief Description of the Drawings

[0010] [Figure 1] Figure 1 shows the principle of molecular imprinting. [Figure 2] Figure 2 shows an overview of the manufacturing procedure of the sensor chip substrate. Figure 2(a) shows the manufacturing procedure of a standard sensor chip substrate, and (b) shows the manufacturing procedure of a sensor chip substrate in which part of the carbon ink is replaced with Ag / AgCl ink. [Figure 3] Figure 3 shows the wiring of a standard sensor chip substrate. (a) shows an overall view of the screen made of carbon ink, and (b) shows the structure of one unit of the sensor substrate. [Figure 4] Figure 4 shows a standard sensor chip substrate (a) after insulation treatment and (b) after formation of the reference electrode. [Figure 5] Figure 5 shows the ink replacement procedure for manufacturing a sensor chip substrate in which part of the carbon ink is replaced with Ag / AgCl ink. [Figure 6] Figure 6 shows a sensor completed using a standard sensor chip substrate. [Figure 7] Figure 7 shows the relationship between the vancomycin concentration in human serum and the response current. The circular plots show the results when using a substrate wired only with carbon ink, and the square plots show the results when using a substrate with combined use of Ag / AgCl. [Figure 8] Figure 8 shows the relationship between the histamine concentration in milk and the reduction current. (a) shows the results when using a histamine MIP-CP control containing silicone oil as the binder, and (b) shows the results when using a histamine MIP-CP containing polyethylene glycol monomethacrylate as the binder.

Embodiments for Carrying Out the Invention

[0011] Hereinafter, embodiments of the present invention will be described. The sensor of the present invention is a sensor that uses conductive carbon for wiring, <* the sensor has (a) a working electrode, (b) a counter electrode, (c) a reference electrode, (d) a wiring part, and (e) a connection part to a measuring instrument. The working electrode is formed from a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and silicone oil. A portion of the wiring between the connection point to the measuring instrument and the working electrode is replaced with a conductive and non-lipophilic substance. It is a sensor. Furthermore, the sensor of the present invention is The sensor includes an electrode substrate coated or filled with a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and an amphiphilic substance.

[0012] By copolymerizing a functional monomer with a crosslinkable monomer in a self-assembled state consisting of a specific substance (template) and a functional monomer that reversibly binds to it, a molecularly imprinted polymer can be synthesized that memorizes the molecular structure of the template and specifically re-bonds to it (Figure 1). Compared to biopolymers, this molecularly imprinted polymer is highly chemically and physically stable and can be prepared at low cost and in a short time. To use molecularly imprinted polymers as sensor elements, it is necessary to generate signals such as electrical signals corresponding to the specific bonding of the template. However, because this method had not been established, the application of molecularly imprinted polymers to biosensors had not progressed. The inventors of this invention have discovered that the size of voids inside the thin film of the molecularly imprinted polymer changes due to a specific reaction with the template, and furthermore, the rate at which solutes pass through the molecularly imprinted polymer thin film changes significantly (J.Chem.Eng.Jpn., 34, 1466-1469, 2001), and have named this phenomenon the gate effect.

[0013] In this invention, conductive particles having a molecularly imprinted polymer on their surface are used. The type of conductive particles is not particularly limited, but graphite particles, carbon black particles, titanium oxide particles, tin oxide particles, etc., can be used. The particle size of the conductive particles is not particularly limited, but is generally 1 μm to 100 μm, preferably 3 μm to 50 μm, more preferably 3 μm to 20 μm, and particularly preferably 3 μm to 10 μm.

[0014] Conductive particles having molecularly imprinted polymers on their surface can be produced by polymerizing particles immobilized with an initiator by contacting them with a functional monomer, a crosslinkable monomer, and a substance to be measured. Alternatively, during polymerization, in addition to the functional monomer and the crosslinkable monomer, a degree of crosslinking adjustment monomer and / or an electron transfer monomer (redox monomer) may also be used to produce conductive particles having molecularly imprinted polymers on their surface. In one example, conductive particles having molecularly imprinted polymers on their surface can be produced by immobilizing an initiator (such as a photopolymerization initiator) on conductive particles, dispersing the initiator-immobilized conductive particles in a polymerization solution containing a functional monomer, a degree of crosslinking adjustment monomer, a crosslinkable monomer, and a substance to be measured (template), and performing photopolymerization.

[0015] The functional monomers used in this invention are not particularly limited, and can include acrylic acid, methacrylic acid, acrylamide, itaconic acid, vinylphenylboronic acid, acrylamideboronic acid, 2-acrylamido-2-methylpropanesulfonic acid, 2-(trifluoromethyl)acrylic acid, etc. Furthermore, it is preferable to use cationic monomers to manufacture a sensor for measuring heparin. Since heparin contains many sulfonic acid groups, using cationic functional monomers makes it possible to synthesize molecularly imprinted polymers that specifically bind to heparin. Cationic monomers include those that have cationic groups in their molecules, such as primary to tertiary amino group-containing (meth)acrylamide, primary to tertiary amino group-containing (meth)acrylate, quaternary ammonium base-containing (meth)acrylamide, quaternary ammonium base-containing (meth)acrylate, diallyldialkylammonium halide, etc. Examples of tertiary amino group-containing (meth)acrylamides include dimethylaminoethyl (meth)acrylamide, dimethylaminopropyl (meth)acrylamide, diethylaminoethyl (meth)acrylamide, diethylaminopropyl (meth)acrylamide, and dialkylaminoalkyl (meth)acrylamide. Examples of tertiary amino group-containing (meth)acrylates include dimethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, diethylaminoethyl (meth)acrylate, diethylaminopropyl (meth)acrylate, and dialkylaminoalkyl (meth)acrylate. Examples of primary and secondary amino group-containing (meth)acrylamides include primary amino group-containing (meth)acrylamides such as aminoethyl (meth)acrylamide, or secondary amino group-containing (meth)acrylamides such as methylaminoethyl (meth)acrylamide, ethylaminoethyl (meth)acrylamide, and t-butylaminoethyl (meth)acrylamide.Examples of primary and secondary amino group-containing (meth)acrylates include primary amino group-containing (meth)acrylates such as aminoethyl (meth)acrylate, or secondary amino group-containing (meth)acrylates such as methylaminoethyl (meth)acrylate, ethylaminoethyl (meth)acrylate, and t-butylaminoethyl (meth)acrylate. Examples of quaternary ammonium base-containing (meth)acrylamides and quaternary ammonium base-containing (meth)acrylates include monoquaternary base-containing monomers obtained by quaternizing tertiary amino group-containing (meth)acrylamide or tertiary amino group-containing (meth)acrylate with quaternizing agents such as methyl chloride, benzyl chloride, methyl sulfate, and epichlorohydrin. Specifically, examples include acrylamidopropyltrimethylammonium chloride, acrylamidopropylbenzyldimethylammonium chloride, methacryloyloxyethyldimethylbenzylammonium chloride, acryloyloxyethyldimethylbenzylammonium chloride, (meth)acryloylaminoethyltrimethylammonium chloride, (meth)acryloylaminoethyltriethylammonium chloride, (meth)acryloyloxyethyltrimethylammonium chloride, and (meth)acryloyloxyethyltriethylammonium chloride. Among the above, specific examples of cationic monomers include ethyltrimethylammonium methacrylate, vinylpyridine, and diethylaminoethyl methacrylate. These may be used individually or in combination of two or more. Furthermore, it is preferable to use acrylamide to manufacture a sensor for measuring vancomycin. Furthermore, it is preferable to use methacrylic acid to manufacture a sensor for measuring histamine.

[0016] Examples of crosslinkable monomers used in the present invention include methylenebisacrylamide, 1,4-butyl diacrylate, 1,6-hexanediol dimethacrylate, polyethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, nonaethylene glycol dimethacrylate, divinylbenzene, polypropylene glycol dimethacrylate, neopentyl glycol dimethacrylate, pentaerythritol dimethacrylate, trimethylolplupane trimethacrylate, pentaerythritol trimethacrylate, pentaerythritol tetramethacrylate, dipentaerythritol hexamethacrylate, epoxy acrylate, polyester acrylate, and urethane acrylate. Among these, particularly preferred are, for example, ethylenebisacrylamide, methylenebisacrylamide, polyethylene glycol dimethacrylate, ethylene glycol dimethacrylate, and triethylene glycol dimethacrylate. Among the crosslinkable monomers used in the present invention, ethylene glycol dimethacrylate is the most preferred. These may be used individually or in combination of two or more types.

[0017] During polymerization, monomers for adjusting the degree of crosslinking can be used. Examples of monomers for adjusting the degree of crosslinking include acrylamide. These may be used individually or in combination of two or more.

[0018] During polymerization, electron transfer monomers (redox monomers) can be used. Suitable redox monomers include compounds having a redox ferrocenyl group (e.g., ferrocene carboxylic acid, aminoferrocene, allylamine carboxypropionate-3-ferrocene, vinylferrocene, mercaptoalkylferrocene), compounds having a benzoquinone group (alkyl-introduced benzoquinone, etc.), compounds with introduced ruthenium complexes (tetrachlororuthenium), osmium complexes, hemin compounds, cytochrome C, etc. These may be used individually or in combination of two or more. If a mediator (such as a redox marker) is not added to the sample, it is preferable to use the above-mentioned redox monomers during polymerization to produce conductive particles having a molecularly imprinted polymer with the redox monomer immobilized on its surface. For manufacturing a sensor to measure vancomycin, it is preferable to use allylamine carboxypropionate-3-ferrocene. For manufacturing a sensor to measure histamine, it is preferable to use vinylferrocene.

[0019] Particles formed by graft polymerization of molecularly imprinted polymers (MIP) onto the surface of conductive particles can be further mixed with a binder in a specific weight ratio and kneaded together to form a paste. The binder can be an oil or an amphiphilic substance. For example, silicone oil or liquid paraffin can be used as the oil, with silicone oil being preferred. The amphiphilic substance is preferably a liquid, such as polyethylene glycol monomethacrylate, with CAS number 25736-86-1 and molecular weight 360 being the most preferred. The binder can be mixed with the total MIP-P at a weight ratio of 10-50%, more preferably at 20-30%. For example, the weight ratio of silicone oil or liquid paraffin to the total MIP-P is preferably 30%, and the weight ratio of polyethylene glycol monomethacrylate to the total MIP-P is preferably 20%. In one example, MIP-P can be prepared by mixing particles in which a molecularly imprinted polymer has been grafted onto the surface of conductive particles with the above-mentioned binder in the above-mentioned weight ratio, and kneading them in an alumina mortar. In another example, for a sensor that measures vancomycin, an MIP-CP paste can be used, which is made by kneading graphite particles with MIP for vancomycin fixed to the surface with silicone oil. For a sensor that measures histamine, an MIP-CP paste can be used, which is made by kneading graphite particles with MIP for histamine fixed to the surface with polyethylene glycol monomethacrylate.

[0020] Sensors using MIP-P can be either electrochemical or non-electrochemical. Electrochemical sensors can be constructed by using electrodes coated or filled with MIP-P. Non-electrochemical sensors can include surface plasmon resonance (SPR) sensors (e.g., BIACORE) and quartz crystal microbalance (QCM) sensors.

[0021] Conductive particles having molecularly imprinted polymers on their surface can be made into a paste, and therefore the type and form of the support are not particularly limited, allowing them to be applied to a wide range of supports. For example, by coating the surface of an insulating support with the MIP-P fixed electrode having a homogeneous surface using printing technology, a homogeneous surface can be manufactured. Alternatively, conductive particles having molecularly imprinted polymers on their surface can be used as MIP-P fixed electrodes by filling them into tubes or PET films. For example, an MIP-P fixed electrode can be manufactured by filling the tip of a polyether ether ketone tube, hematocrit capillary tube, or glass tube with lead wires attached with MIP-P, and then polishing the filled area with weighing paper while applying pressure. Alternatively, for example, a PET film (100 μm / sheet) with 4 x 4 mm holes can be placed on top of a cleaned ITO glass, and the depressions can be filled with MIP-P (using liquid paraffin as a binder), and the surface can be polished with weighing paper to manufacture an MIP-P fixed plate electrode. These MIP-P fixed electrodes can be used to fabricate sensors.

[0022] In one example, the sensor of the present invention is a sensor comprising an electrode substrate coated or filled with a molecularly imprinted polymer paste containing conductive particles having a molecularly imprinted polymer on its surface and an amphiphilic substance. That is, the MIP-P containing conductive particles having a molecularly imprinted polymer on its surface and an amphiphilic substance is coated or filled into the working electrode of the sensor. The working electrode coated or filled with the paste is physically connected to wiring made of carbon paste. In addition to the working electrode containing the MIP-P containing conductive particles having a molecularly imprinted polymer on its surface and an amphiphilic substance, the sensor may also be used as a counter electrode and / or reference electrode. In one example, the amphiphilic substance of the sensor is polyethylene glycol monoacrylate.

[0023] Furthermore, MIP-P can be applied to the surface of the working electrode portion of screen-printed wiring to create a sensor that includes an electrode with MIP-P immobilized on it. Screen-printed electrodes are electrodes made by printing ink onto a substrate using printing technology, and include a working electrode, a counter electrode, and / or a reference electrode, a connector for connecting to a measuring instrument, and wiring that connects each of the working electrode, counter electrode, and / or reference electrode to the connector. Using screen-printed electrodes makes it easy to create low-cost, single-use disposable sensors. By changing the material of the ink used to print the working electrode, various electrodes such as carbon electrodes, gold electrodes, platinum electrodes, silver electrodes, bismuth electrodes, ruthenium oxide electrodes, and palladium electrodes can be made. In addition, the substrate material (e.g., PET film, glass, alumina, plastic, etc.) can be changed depending on the purpose. Furthermore, the sensor configuration can be appropriately modified by changing the material of the ink used for printing (e.g., carbon, gold, platinum, silver, silver / silver salt, bismuth, ruthenium oxide, palladium, etc.) for the counter electrode, reference electrode, connection to the measuring instrument, and wiring, similar to the working electrode. It is also possible to create only the wiring on a substrate using a screen printing method with one or more types of conductive ink, and then appropriately form the desired working electrode, counter electrode, and / or reference electrode, or change the configuration of the wiring. For example, by applying a desired ink or paste to the surface of the fabricated wiring, it is possible to form an working electrode and / or reference electrode with the desired function. Alternatively, a portion of the wiring on the substrate can be partially missing, and ink of a different material can be added to the missing portion to change the configuration, function, and / or properties of the wiring. In this way, a sensor with the desired function can be manufactured.

[0024] In one example, the substrate used in the sensor of the present invention is a PET film. In one example, conductive carbon ink is first screen printed onto this substrate to create wiring, and then the carbon ink wiring is processed as appropriate to form an working electrode, a counter electrode, a reference electrode, a connector for a measuring instrument, and wiring that connects the working electrode, counter electrode, and reference electrode to the connector, thereby creating a sensor. Specifically, the working electrode can be formed by applying MIP-P to a part of the wiring (wiring for the working electrode). The reference electrode can be formed by applying Ag / AgCl ink or the like to a part of the wiring (wiring for the reference electrode). A part of the wiring (wiring for the counter electrode) can be left as carbon ink wiring without applying paste or ink, and used as the counter electrode. The wiring sections for forming the working electrode, counter electrode, and reference electrode are each connected to the wiring sections for forming the connectors for the measuring instrument. By applying insulating ink to the wiring sections between the working electrode, counter electrode, and reference electrode and the wiring sections for forming the connectors, the connectors are formed, and wiring sections connecting the working electrode, counter electrode, and reference electrode to the connectors are also formed. The connectors may remain as carbon ink wiring without the application of paste or ink. Furthermore, a portion of the wiring section connecting the working electrode and the connector may be replaced from conductive carbon ink to an ink that is conductive and non-lipophilic. In this specification, the wiring section for forming the working electrode may be referred to as "wiring for the working electrode" or "working electrode section," and these terms may be used interchangeably. Similarly, the wiring section for forming the counter electrode may be referred to as "wiring for the counter electrode" or "counter electrode section," and these terms may be used interchangeably. The wiring section for forming the reference electrode may be referred to as "wiring for the reference electrode" or "reference electrode section," and these terms may be used interchangeably.

[0025] An example of a specific method for manufacturing the sensor of the present invention is shown in Figure 2(b) and below. First, conductive carbon ink is screen printed onto a PET film sheet, which serves as the substrate. Then, a portion of the wiring connecting the working electrode and the connector is cut out, and the carbon ink is cured on the substrate. The area to be cut out is not particularly limited as long as it is a portion of the wiring connecting the working electrode and the connector, but in one example, it is preferably near the working electrode or in contact with the working electrode. The length of the wiring to be cut out is not particularly limited as long as it can stop the penetration of the silicone oil of the MIP-CP, but in one example, it is 1 mm to 5 mm, preferably 2 mm. Next, the cut-out wiring is filled with an ink that is a conductive and non-lipophilic substance. The conductive and non-lipophilic ink is not particularly limited as long as it can transmit electrical signals to the measuring instrument while stopping the penetration of the silicone oil of the MIP-CP, but for example, inks made of gold, platinum, silver, silver / silver salt, bismuth, ruthenium oxide, palladium, etc. can be used. In one example, silver / silver salt ink is preferred, and Ag / AgCl ink is most preferred. After filling in any missing wiring sections and ensuring conductivity, insulating ink is applied to and covering the wiring sections connecting the working electrode, counter electrode, and reference electrode to the connector. In other words, only the working electrode, counter electrode, reference electrode, and connector are exposed on the surface of the sensor. Subsequently, silver / silver chloride ink is applied to the reference electrode section and MIP-CP is applied to the working electrode section to form the reference electrode and working electrode. After that, the sensor of the present invention can be manufactured by separating each unit. In this application, a sensor chip substrate in which a portion of the wiring section connecting the working electrode and the connector is replaced from carbon to a conductive and non-lipophilic substance is sometimes referred to as a replacement-type sensor chip substrate, and a sensor manufactured using such a replacement-type sensor chip substrate is sometimes referred to as a replacement-type sensor.

[0026] In other words, the sensor of the present invention is a sensor that utilizes conductive carbon in its wiring, and the sensor has (a) an working electrode, (b) a counter electrode, (c) a reference electrode, (d) a wiring section, and (e) a connection section to a measuring instrument, the working electrode is formed from a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and silicone oil, and a portion of the wiring section between the connection section to the measuring instrument and the working electrode is replaced with a conductive and non-lipophilic substance.

[0027] In one example, the sensor of the present invention has a portion of the wiring between the connection to the measuring instrument and the working electrode replaced from carbon with a conductive and non-lipophilic substance. The conductive and non-lipophilic substance may be, for example, gold, platinum, silver, silver and / or silver salts, bismuth, ruthenium oxide, or palladium. In one example, the conductive and non-lipophilic substance is silver and / or silver salts.

[0028] The sensor of the present invention is not particularly limited in terms of the substances to be measured, and can measure any substance, such as hormones (serotonin, dopamine, adrenaline, acetylcholine, γ-aminobutyric acid, etc.), antibacterial agents (vancomycin, teicoplanin, etc.), central nervous system drugs (phenobarbital, etc.), bronchodilators (theophylline, etc.), anticoagulants (heparin, warfarin, edoxaban, etc.), anesthetics, pesticides, anticancer drugs (gefitinib, fluorouracil, methotrexate, etc.), toxic substances in food (histamine, etc.), and freshness indicator substances in food (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosinic acid, inosine, hypoxanthine, etc.). In the sensor of the present invention, the substance to be measured may be contained in a sample that contains a large amount of protein and / or lipid colloids.

[0029] Examples of anticoagulants include heparin, heparin-like substances (including low molecular weight heparin), warfarin, asenocumarol, phenindione, and edoxaban, but are not limited to these. The heparin used in the examples of this specification is unfractionated heparin with a molecular weight range of 7,000 to 25,000 (mostly 10,000 to 20,000), but in this invention, not only unfractionated heparin but also low molecular weight heparin (molecular weight 4,000 to 8,000) can be measured.

[0030] Mechanical prosthetic valves are highly durable and, once implanted in the body, can be used semi-permanently. Similarly, ventricular assist devices (VADs) have reached a point where they demonstrate therapeutic effects comparable to transplantation. However, because both involve prolonged contact with blood, the blood is prone to clotting. If blood clotting occurs, the resulting clots can block capillaries in the brain and other organs, endangering life. To prevent this, patients using mechanical prosthetic valves or VADs require anticoagulant medication. However, since most of these patients live outside the hospital, administering anticoagulants requiring injections, such as heparin, is difficult, and oral anticoagulants are the primary treatment. The most commonly used oral anticoagulant is warfarin, a vitamin K antagonist. Excessive administration of anticoagulants can cause bleeding (especially intracerebral hemorrhage). Furthermore, the anticoagulant effect of warfarin is easily affected by external factors, such as the intake of foods rich in vitamin K (natto, parsley, etc.), requiring medication design based on monitoring of anticoagulant function. Monitoring the anticoagulant effect of warfarin involves adding calcium and thromboplastin to plasma, measuring the clotting time (prothrombin time), and calculating a relative value (international normalized ratio) by comparing it to the normal value, or measuring the functional impairment of vitamin K-dependent clotting factors (thrombotest). Both are excellent methods for evaluating blood clotting ability. However, as mentioned earlier, the anticoagulant effect of warfarin is strongly influenced by external factors, so the results of prothrombin time and thrombotest do not necessarily directly indicate whether the warfarin concentration is excessive or insufficient. The intestinal absorption rate and metabolic rate of warfarin vary greatly from person to person and are greatly affected by internal and external factors such as health status. Therefore, therapeutic drug monitoring (TDM), which involves monitoring changes in blood warfarin concentration while formulating medication regimens, is ideally required. However, currently, methods for selectively measuring warfarin in the blood are limited to those requiring large-scale equipment such as liquid chromatography (LC-MS) using mass spectrometry as the detection method. It is not practical to frequently measure blood warfarin concentration using such analytical methods. There is a need to develop a low-cost, easy-to-operate sensor for warfarin.

[0031] Vancomycin is a first-line treatment for Gram-positive bacterial infections. While excessive administration of vancomycin can cause side effects such as hearing loss and kidney damage, under-administering it not only fails to produce therapeutic effects but also promotes the development of drug-resistant bacteria. Therefore, vancomycin is considered a prime example of a drug requiring monitoring of its blood concentration (drug monitoring). However, currently, outside of large hospitals, the quantitative analysis of blood vancomycin is outsourced to testing laboratories, resulting in the inability to adjust dosages in real time. A simple vancomycin sensor that can be used at the bedside is needed.

[0032] Histamine is a causative agent of histamine poisoning and is also used as an indicator of food freshness. Histamine is produced from free histidine, an amino acid found in food, by histamine-producing bacteria that possess histidine decarboxylase. When food contaminated with histamine-producing bacteria is stored at inappropriate temperatures or for extended periods, the bacteria in the food multiply, leading to a large accumulation of histamine. Histamine poisoning occurs when such food with a high histamine accumulation is consumed. Histamine is not easily broken down by heat, making it difficult to remove histamine produced and accumulated in food. Furthermore, even if histamine accumulates in food, it does not cause changes in the appearance or smell of the food, making it difficult to detect histamine accumulation before consumption. Therefore, there is a need for a simple histamine sensor that can easily detect or quantify histamine in food. There is also a strong demand for sensors that can detect histamine as an indicator of milk freshness.

[0033] According to one example of the present invention, a vancomycin sensor for electrochemically measuring vancomycin in serum is provided. Vancomycin hydrochloride, which is a template, acrylamide, which is a functional monomer, and ethylene glycol dimethacrylate, which is a crosslinkable monomer, are dissolved in a mixed solvent of the organic solvent dimethylformamide and water to prepare a metastable solution. At this time, allylamine carboxypropionate-3-ferrocene (ACPF), which is an electron transfer monomer (redox monomer), may also be dissolved in the mixed solvent. Conductive particles having a molecular imprinted polymer on their surface can be produced by first immobilizing a radical polymerization agent onto conductive particles by covalent bonds, dispersing the conductive particles in the above metastable solution, and graft polymerization by light irradiation.

[0034] Conductive particles having molecularly imprinted polymers on their surface, manufactured as described above, can be kneaded with silicone oil in a specific weight ratio (for example, a weight ratio of 7:3) to form a paste. By applying this paste containing conductive particles having molecularly imprinted polymers on their surface to the working electrode portion of a screen-printed carbon wiring, a working electrode can be formed, and a sensor containing a screen-printed carbon electrode with fixed MIP can be manufactured. The sensor of the present invention is a substitution type sensor in which, in order to sensitively measure substances in samples containing a large amount of protein, such as serum, a part of the material of the wiring portion connecting the working electrode of the screen-printed carbon electrode to the connection portion of the measuring instrument is replaced from carbon to a conductive and non-lipophilic substance. That is, the sensor of the present invention can be manufactured using a substitution type sensor chip substrate made by coating or filling a part of the wiring portion between the connection portion of the screen-printed carbon wiring and the working electrode portion with a conductive and non-lipophilic ink (for example, silver / silver chloride ink) instead of conductive carbon ink. In replacement-type sensor chip substrates, a portion of the wiring between the screen-printed carbon wiring connection to the measuring instrument and the working electrode portion is replaced from conductive carbon to a conductive and non-lipophilic substance (e.g., silver / silver salt).

[0035] According to the present invention, the substance to be measured can be measured by bringing a sample containing the substance to be measured into contact with the working electrode, counter electrode, and reference electrode of the sensor described above, and detecting a change in signal (preferably a change in current). For example, a method can be employed in which a potential is applied and the resulting oxidation-reduction current is measured. In one example, the sensitivity of a sensor including an MIP-CP fixed electrode to vancomycin can also be measured and evaluated by performing differential pulse voltammetry in human serum containing vancomycin. If electron transfer monomers (redox monomers) were not mixed when preparing the molecularly imprinted polymer, mediators (redox markers, etc.) such as potassium phalicyanide, potassium ferrocyanide, benzoquinone, and hydroquinone should be added to the sample. Furthermore, mediators (such as redox markers) that are present in the body include uric acid and ascorbic acid, as well as glucose, lactic acid, bilirubin, and cholesterol. Oxidoreductases (e.g., glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, etc.) may also be used. As samples, whole blood or blood components (e.g., plasma or serum), or food (e.g., meat drip) can be used. According to the present invention, even if the sample contains a large amount of protein, the substance to be measured can be measured with high sensitivity.

[0036] According to one example of the present invention, a histamine sensor for electrochemically measuring histamine in milk is provided. A metastable solution was prepared by dissolving histamine dihydrochloride, a template, methacrylic acid, a functional monomer, and ethylene glycol dimethacrylate, a crosslinkable monomer, in the organic solvent dimethyl sulfoxide. At this time, vinylferrocene, an electron transfer monomer (redox monomer), may also be dissolved in the solvent. Conductive particles having a molecular imprint polymer corresponding to histamine on their surface can be produced by pre-immobilizing a radical polymerization agent by covalent bonds onto conductive particles, dispersing the conductive particles in the above metastable solution, and graft polymerization by light irradiation.

[0037] Conductive particles having the molecularly imprinted polymer on their surface, manufactured as described above, can be mixed with an amphiphilic substance (e.g., polyethylene glycol monomethacrylate) as a binder in a specific weight ratio (e.g., 8:2) to form a paste. By applying this paste containing conductive particles with molecularly imprinted polymers on their surface (MIP-CP) to the surface of a support (e.g., an insulating substrate) using printing technology, a MIP-CP fixed electrode with a homogeneous surface can be manufactured. Alternatively, the conductive particles with molecularly imprinted polymers on their surface may be filled into a support (e.g., a tube) to form an MIP-P fixed electrode. Sensors can be manufactured using these MIP-P fixed electrodes. Furthermore, sensors can also be manufactured using MIP-P fixed screen-printed carbon electrodes, where the wiring for the working electrode of screen-printed carbon is used as a support, and the MIP-P paste is applied to the working electrode portion.

[0038] According to the present invention, a substance to be measured can be measured by bringing a sample containing the substance to be measured into contact with the sensor described above and detecting a change in signal (preferably a change in current). As the sample, whole blood or blood components (e.g., plasma or serum) or food (e.g., milk) can be used. According to the present invention, even if the sample contains a large amount of lipid colloid, the substance to be measured can be measured with high sensitivity.

[0039] When using the MIP-P fixed electrode of the present invention as a sensor, a method can be employed in which the electrode, together with the counter electrode and reference electrode, is immersed in the sample, a potential is applied, and the resulting oxidation-reduction current is measured. For example, the sensitivity of the MIP-P fixed electrode to histamine can also be measured and evaluated by performing cyclic voltameometry in milk containing histamine. Furthermore, if electron transfer monomers (redox monomers) were not mixed during the preparation of the molecularly imprinted polymer, a method can be employed in which the electrode, together with the counter electrode and reference electrode, is immersed in a sample to which mediators (redox markers, etc.) such as potassium phalicyanide, potassium ferrocyanide, benzoquinone, and hydroquinone have been added, a potential is applied, and the resulting oxidation-reduction current is measured. Furthermore, mediators (such as redox markers) that are present in the body include uric acid and ascorbic acid, as well as glucose, lactate, bilirubin, and cholesterol. Oxidoreductases (e.g., glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, etc.) may also be used. If uric acid or ascorbic acid itself is used as a mediator, measurement can be performed directly from perfusion blood, enabling minimally invasive measurement without causing blood loss. The sensor of the present invention can be attached to an extracorporeal circulation device. For example, by bringing perfusion blood into contact with the sensor of the present invention and detecting the change, the substance to be measured in the blood can also be measured.

[0040] The present invention will be described in more detail by the following examples, but the present invention is not particularly limited by the following examples. [Examples]

[0041] Example 1: (1) Initiator immobilization onto graphite particles A diethyldithiocarbamylmethylene group, a photoradical polymerization initiator, was introduced onto the surface of graphite particles. First, chloromethyl groups were introduced to the graphite surface using hydrogen chloride and formaldehyde. 5 g of spherical graphite particles (SG-BH8, Ito Graphite Industry Co., Ltd.) with a particle diameter of 8 μm were placed in a three-necked flask along with a mixture of 65 g of concentrated hydrochloric acid (containing 0.25 g of zinc chloride) and 65 g of acetic acid. Argon gas was bubbled through the flask for 1 hour while vigorously stirring with a magnetic stirrer and cooling with ice. Next, 19.0 g of a 37.0% aqueous formaldehyde solution was added to the flask, and stirring was continued for 4 hours under ice cooling while hydrogen chloride gas was bubbled through the flask. Then, the supply of hydrogen chloride was stopped, and the flask was stirred at room temperature for 6 hours. The flask was washed with 700 mL of distilled water and 200 mL of methanol in that order using a suction filter, and then vacuum dried.

[0042] Chloromethylated graphite particles were dispersed in 50 mL of a 0.3 M ethanol solution of sodium diethyldithiocarbamate and stirred at room temperature for 24 hours to introduce diethyldithiocarbamyl methylene groups with initiator function by desodium chloride condensation. The mixture was washed with 700 mL of distilled water and 200 mL of methanol in sequence using a suction filter and then vacuum dried. The initiator-introduced graphite obtained from these operations was stored in a refrigerator, protected from light.

[0043] (2) Graphite graft polymerization 0.0899 g of vancomycin hydrochloride was used as the template material, 0.5002 g of acrylamide (Wako Pure Chemical Industries, Osaka) as the functional monomer, 0.5002 g of allylamine carboxypropionate-3-ferrocene (ACPF) as the electron transfer monomer (redox monomer), and 2.9781 g of ethylene glycol dimethacrylate as the crosslinking monomer, all dissolved in a mixed solvent of 24 mL of dimethylformamide (DMF) (Wako Pure Chemical Industries, Osaka) and 4 mL of water. This solution was used as the polymerization solution. 0.6018 g of initiator-introduced graphite was dispersed in the polymerization solvent in a quartz tube, and dissolved oxygen was removed by bubbling with nitrogen gas saturated with a mixture of DMF and water (same composition as the polymerization solvent) for 30 minutes while stirring with a magnetic stirrer. Then, while continuing bubbling and stirring, the mixture was irradiated with xenon lamp light for 3 hours. Subsequently, the graphite particles were washed by suction filtration using DMF, 1 M NaCl aqueous solution, and distilled water in that order, and then vacuum dried.

[0044] (3) Vancomycin MIP-CP Graphite with MIP (Mass Inhibitor Protein) fixed to its surface against vancomycin was mixed with silicone oil (KF-96-300CS: Shin-Etsu Silicone Co., Ltd.) in a weight ratio of 7:3, and kneaded in an alumina mortar to form a paste.

[0045] (4) Fabrication of sensor chip substrate As shown in Figure 2, a sensor chip substrate was fabricated. (4-1) Fabrication of a standard sensor chip substrate A conductive carbon ink (RAFS090S, Toyo Ink) was printed onto a 100 μm thick PET film using a screen printing method with a 400-mesh nylon screen to create the wiring shown in Figure 3. Next, as shown in Figure 4, insulating ink was applied to the substrate, and silver / silver chloride (ALS, Tokyo) was applied to the reference electrode area to create the reference electrode. This standard sensor chip substrate was used as a comparison.

[0046] (4-2) Fabrication of a sensor chip substrate in which part of the carbon ink is replaced with Ag / AgCl ink In (4-1), after fabricating the wiring by screen printing, as shown in Figure 5, a portion of the wiring connecting the working electrode and the connection to the measuring instrument was rubbed with a cotton swab containing acetone to create a defect of about 2 mm. After filling this defect with the same silver / silver chloride ink as the reference electrode, insulation treatment was applied in the same manner as a standard sensor chip substrate to form the reference electrode and fabricate a replacement type sensor chip substrate.

[0047] (5) Fabrication of sensors As shown in Figure 6, the vancomycin MIP-CP prepared in (3) was applied to the working electrode area of ​​the sensor chip substrates prepared in (4-1) and (4-2), respectively, to form the working electrode. The sensors were then separated into individual units to complete each sensor. The completed replacement type sensors are the same as standard sensors, except that a portion of the wiring section is missing and filled with silver / silver chloride ink.

[0048] (6) Vancomycin sensing using electrochemical methods Vancomycin hydrochloride was dissolved in human serum (residual serum from biochemical samples) to a concentration of 0-40 μg / mL. The sensors prepared in (5) were each connected to an electrochemical analyzer (Ivium Pocketstat). The vancomycin-dissolved serum sample was dropped onto the area around the working electrode of the sensor and spread to cover the working electrode, reference electrode, and counter electrode. Differential pulse voltammetry was performed while manipulating the working electrode potential in the range of 0 to 0.9 V (pulse width 10 ms, pulse height 60 mV, potential step 10 mV, scanning speed 20 mV / s). The relationship between the current at 0.8 V and the serum vancomycin concentration was investigated.

[0049] Figure 7 shows the results of a comparison of the sensitivity of different substrates to vancomycin in serum. The results showed that when measured with a substitution-type sensor in which a portion of the carbon ink was replaced with Ag / AgCl ink, the change in response current to concentration changes was approximately twice as large as when measured with a standard sensor (Figure 7). In other words, it was revealed that the substitution-type sensor of the present invention exhibits approximately twice the sensitivity of conventional sensors. Furthermore, even one month after MIP-CP application, the substitution-type sensor showed higher sensitivity to vancomycin in serum compared to conventional sensors, similar to the case immediately after MIP-CP application. The low sensitivity of conventional sensors to vancomycin in serum is thought to be due to the fact that while carbon ink has affinity for MIP-CP, it absorbs the silicone oil, which is the binder of MIP-CP. However, by replacing a portion of the wiring with Ag / AgCl ink, the penetration of silicone oil in the Ag / AgCl portion is stopped, resulting in improved sensor sensitivity. Therefore, by using a substitution type sensor in which a portion of the wiring is replaced with a non-lipophilic conductive material such as Ag / AgCl, it becomes possible to measure the target substance in protein-rich samples such as serum with higher sensitivity compared to conventional sensors.

[0050] Example 2: (1) Graphite graft polymerization 0.37 g of histamine dihydrochloride was dissolved in 19 mL of dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, Osaka) as the template material, 0.86 g of methacrylic acid as the functional monomer, 0.15 g of vinylferrocene as the electron transfer monomer (redox monomer), and 0.31 g of ethylene glycol dimethacrylate as the crosslinking monomer. This solution was used as the polymerization solution. 0.25 g of the initiator-introduced graphite prepared in Example 1(1) was dispersed in the polymerization solvent in a quartz tube, and dissolved oxygen was removed by bubbling nitrogen gas saturated with DMSO for 20 minutes while stirring with a magnetic stirrer. Then, while continuing bubbling and stirring, the mixture was irradiated with xenon lamp light for 1 hour. After that, the graphite particles were washed by suction filtration in DMSO, acetic acid, and distilled water in that order, and then vacuum dried.

[0051] (2) Histamine MIP-CP Graphite with histamine-targeting MIP immobilized on its surface was mixed with polyethylene glycol monomethacrylate (CAS number: 25736-86-1, molecular weight 360) in a weight ratio of 8:2, and kneaded in an alumina mortar to form a paste. This histamine MIP-CP was designated as histamine MIP-CP. For comparison, MIP-CP was similarly prepared by mixing it with conventionally used silicone oil in a weight ratio of 8:2 instead of polyethylene glycol monomethacrylate. This histamine MIP-immobilized carbon paste using silicone oil as a binder was designated as the histamine MIP-CP control.

[0052] (3) Fabrication of the sensor As shown in Figure 6, the histamine MIP-CP or histamine MIP-CP control prepared in (2) was applied to the working electrode area of ​​the standard sensor chip substrate prepared in Example 1 (4-1), and the working electrode was formed. The sensors were then completed by separating them into individual units.

[0053] (4) Histamine sensing using electrochemical methods Histamine dihydrochloride was dissolved in fresh, commercially available milk to a concentration of 0-100 ppm. The sensor prepared in (3) was connected to an electrochemical analyzer (Ivium Pocketstat). The milk sample was dropped around the working electrode of the sensor and spread to cover the working electrode, reference electrode, and counter electrode. The potential was scanned in the order of 0.0 V → 1.0 V → -1.2 V → 0.0 V (speed 100 mV / s), and cyclic voltameometry was performed to record the current. The correlation between the peak value of the reduction current and the histamine concentration in the milk was examined.

[0054] As a result, when using the histamine MIP-CP control, the reduction current did not correlate with the histamine concentration in milk and showed no sensitivity to histamine (Figure 8(a)). On the other hand, when using histamine MIP-CP, the reduction current correlated with the histamine concentration in milk and showed sensitivity to histamine in milk (Figure 8(b)). The decrease in sensitivity when using the histamine MIP-CP control is thought to be due to the infiltration of lipid colloids, which are abundant in milk, into the silicone oil in the histamine MIP-CP control. On the other hand, although polyethylene glycol monomethacrylate in histamine MIP-CP is amphiphilic, its hydrophobic regions are aggregated, so it is thought that the lipid colloids in milk did not infiltrate into the histamine MIP-CP. As a result, it is thought that histamine in milk, which contains a large amount of lipid colloids, could be measured with good sensitivity. Therefore, by using polyethylene glycol monomethacrylate as the binder for MIP-CP, it becomes possible to measure the concentration of target substances in samples containing large amounts of lipid colloids, which could not be measured with conventional sensors.

Claims

1. A sensor that uses conductive carbon in its wiring, The sensor has (a) an working electrode, (b) a counter electrode, (c) a reference electrode, (d) a wiring section, and (e) a connection section for a measuring instrument. The working electrode is formed from a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and silicone oil. A portion of the wiring between the connection point to the measuring instrument and the working electrode is replaced with a conductive and non-lipophilic substance. Sensor.

2. The sensor according to claim 1, wherein the conductive and non-lipophilic substance is silver and / or a silver salt.

3. The sensor according to claim 1, wherein the conductive particles are graphite particles.

4. The sensor according to claim 1, wherein the conductive particles having a molecularly imprinted polymer on their surface are conductive particles obtained by polymerizing particles immobilized with an initiator by contacting a functional monomer, a crosslinkable monomer, and a measurement substance.

5. The sensor according to claim 1, wherein the substance to be measured is a hormone, an antibacterial agent, an anticoagulant, or a toxic substance or freshness indicator substance in food.

6. The sensor according to claim 1, wherein the substance to be measured is heparin, warfarin, serotonin, vancomycin, phenobarbital, theophylline, edoxaban, or histamine.

7. A method for measuring a substance, comprising contacting a sample containing the substance to be measured with a sensor according to any one of claims 1 to 6, and detecting a change in the signal.

8. A method for measuring a substance according to claim 7, comprising detecting a change in current as a change in signal.

9. The measurement method according to claim 7, wherein the sample is whole blood or blood components, or food.

10. A sensor comprising an electrode substrate coated or filled with a molecular imprint polymer paste containing conductive particles having molecular imprint polymers on their surface and an amphiphilic substance.

11. The sensor according to claim 10, wherein the molecular imprinted polymer paste functions as the working electrode.

12. The sensor according to claim 10, wherein the amphiphilic substance is polyethylene glycol monoacrylate.

13. The sensor according to claim 10, wherein the conductive particles are graphite particles.

14. The sensor according to claim 10, wherein the conductive particles having a molecularly imprinted polymer on their surface are conductive particles obtained by polymerizing particles immobilized with an initiator by contacting a functional monomer, a crosslinkable monomer, and a measurement substance.

15. The sensor according to claim 10, wherein the substance to be measured is a hormone, an antibacterial agent, an anticoagulant, or a toxic substance or freshness indicator substance in food.

16. The sensor according to claim 10, wherein the substance to be measured is heparin, warfarin, serotonin, vancomycin, phenobarbital, theophylline, edoxaban, or histamine.

17. A method for measuring a substance, comprising contacting a sample containing the substance to be measured with a sensor according to any one of claims 10 to 16 and detecting a change in the signal.

18. A method for measuring a substance according to claim 17, comprising detecting a change in current as a change in signal.

19. The measurement method according to claim 17, wherein the sample is whole blood or blood components, or food.

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