JP6883839B2 - Component comparison biosensor chip and component comparison biosensor using this - Google Patents

Description

In the present invention, in order to obtain information on the amount of a specific substrate component in a liquid sample, an enzyme that selectively reacts with the enzyme is fixed to a reaction part in the flow path, and the substrate is brought into contact with the corresponding enzyme. by measuring the temperature rise change by contact heat reaction are those with component comparison biosensor chips to know the amount and ratio of the plurality of substrates, to a component comparison biosensor using the same.

One of the inventors of the present application catalytically activated information on the amount of a specific substrate component in a body fluid or the like as a liquid sample, for example, glucose (substrate), using a corresponding enzyme, for example, glucose oxidase. We have invented an "enzyme-fixed biosensor" that measures, acquires, and evaluates as a change in temperature rise within a uniform temperature due to the heat of reaction (Patent Document 1). There, a reaction part is formed in the flow path formed on the thin film of the crosslinked structure, and the enzyme is fixed therein, and a first temperature sensor is formed for detecting the temperature rise due to the enzyme reaction in the reaction part. In order to correct the temperature of the liquid sample injected into the flow path, a reference temperature sensor for differential amplification is formed on the thin film of the crosslinked structure near the first temperature sensor. However, in order to increase the sensitivity, a reaction portion and a first temperature sensor for measuring the temperature thereof are formed in the substantially center of the thin film having a crosslinked structure. However, when the liquid sample is a body fluid such as urine, it often has a warm temperature deviating from room temperature. According to the experiment, since the temperature sensitivity is highest near the center of the crosslinked thin film, the reference temperature sensor formed at a position deviated from the center of the crosslinked thin film is approximately in the center of the crosslinked thin film. It is difficult to completely correct the temperature dependence of the liquid sample by the differential operation of the reaction part formed in the above with the first temperature sensor, and the first temperature sensor and the reference temperature sensor are heat. It was necessary to make them almost equal.

When urinary as a liquid sample to be detected is used, it is necessary to measure the amount of each component such as urinary sugar and urinary protein. For example, the amount of protein (for example, albumin) and the amount of creatinine. There is a request for these measurements from the information that the evaluation of the ratio with and is suitable for grasping the progress of diseases such as kidney disease. For example, the ratio of the amount of protein in urine to the amount of creatinine is directly measured. There was a request to be able to do it. In the measurement of urinary components by the conventional electrochemical method, the amount of urinary sugar generated by the enzymatic reaction of hydrogen peroxide (H2O2) can be obtained from the measurement of the redox current of H2O2 generated, but proteins. Was extremely difficult to detect.

Japanese Patent Application No. 2016-97151

The present invention has been improved in order to solve the above-mentioned problems in the past, and is a contact-catalyzed thermal reaction between a minute substrate derived from a living body in a liquid sample and an enzyme corresponding thereto (hereinafter, this is referred to as a thermal reaction). ) Is designed to provide a temperature measuring means that can measure the temperature change with high sensitivity and high accuracy, and the temperature is measured using the calorific value generated by the contact catalytic reaction between the substrate and the corresponding enzyme. Therefore, it is not always necessary to generate H2O2, and it is compact and high for determining the amount of substrate in a liquid sample such as urine and the component ratio of multiple substrates from a simple measurement of the heat of reaction generated in the reaction process. An object of the present invention is to provide an accurate and inexpensive component comparison biosensor chip and a component comparison biosensor using the chip.

In order to achieve the above object, the component comparison biosensor chip according to claim 1 of the present invention corresponds a specific substrate component in a liquid sample flowing in from at least one inflow port to the substrate component. A biosensor chip that detects by the heat of reaction of catalytic action with an enzyme, in which at least one flow path through which a liquid sample passes is formed in a plurality of thin films thermally separated from the substrate. A reference portion is formed in at least one of the thin films, and a reaction portion that is thermally equivalent to the reference portion is formed in each of the remaining thin films in the same flow path. The members are connected in series via the flow path, and each of the plurality of reaction parts, such as the first reaction part and the second reaction part, are different from each other corresponding to the specific substrate component. Although the predetermined enzyme is fixed, the enzyme is not fixed in the reference portion, and further, the reaction heat of each of the first reaction portion, the second reaction portion, etc. and the reference portion is detected. A first temperature sensor, a second temperature sensor, and the like and a reference temperature sensor are provided, respectively, and the temperature is based on the heat of reaction at each of the reaction parts of the first temperature sensor, the second temperature sensor, and the like. The change is measured with reference to the reference temperature sensor, and is characterized in that it is configured so that information on the amount of each of the substrate components or comparable information on those amounts can be obtained. .. In the above description, the "first reaction unit, second reaction unit, etc." refers to a plurality of reaction units such as the first reaction unit, the second reaction unit, the third reaction unit, and so on. The same applies to "first temperature sensor, second temperature sensor, etc."

This component comparison biosensor chip uses a silicon (Si) single crystal SOI substrate, and uses the SOI layer to create minute dimensions using known MEMS technology, such as 1 mm in length and 0.2 mm in width. In order to provide an enzyme-immobilized biosensor chip using a crosslinked structure with a thickness of about 0.01 mm or, if necessary, a cantilever-shaped thin film, an integrated circuit that serves as a peripheral circuit is required. It is convenient because it can be formed on the same substrate, and the semiconductor thin film thermocouple can be used when the first temperature sensor, the second temperature sensor, the reference temperature sensor, etc. with high sensitivity and high accuracy are composed of thermocouples. Often.

The component comparison biosensor chip of the present invention may use a plastic or glass substrate other than Si, and a flow path, a reaction part, and a temperature sensor are formed in a thin film thermally separated from this substrate, and an enzyme is used in the reaction part. May be fixed and configured. Further, for example, a flow path, a reaction part or a reference part in the flow path, a first temperature sensor, a second temperature sensor, a reference temperature sensor, and the like are formed on the first substrate, and then they are formed. A thick film is formed by stacking layers on the above, and the thick film is formed so as to be used as a second substrate, and further, a thin film that is thermally separated from the second substrate is formed on the above. A first temperature sensor, a second temperature sensor, a reference temperature sensor, and the like may be formed. Then, if necessary, the first substrate may be removed, leaving the second substrate, and the second substrate may be treated as the above-mentioned substrate. Further, as the thin film, the layered thin film forming the flow path can also be used as the above-mentioned thin film. In this case, a flow path having a reaction part, a first temperature sensor, a second temperature sensor, a reference temperature sensor, etc., which is thermally separated from the substrate and fixed with an enzyme, floats in the air such as a crosslinked structure or a diaphragm structure. A biosensor chip for comparing this component of the state is provided.

For example, a plurality of equivalent thin films having a crosslinked structure are formed on a substrate, and one flow path is formed through the plurality of thin films, and heat is formed in each thin film in the flow path. There are one reaction part that is substantially equivalent to each other, and these plurality of reaction parts will be referred to as a first reaction part, a second reaction part, and the like. Different predetermined enzymes corresponding to each specific substrate component are immobilized on each reaction part. A first temperature sensor, a second temperature sensor, and the like for detecting the heat of reaction are provided in the first reaction unit, the second reaction unit, and the like (or in the vicinity thereof), respectively. When the crosslinked thin film is uniformly heated, the highest temperature is almost in the central part, so that a first reaction part and a second reaction part that are substantially equivalent in heat are formed in the flow path. It is most desirable to arrange each reaction part in the same shape in the substantially central part of the crosslinked thin film. This reference temperature sensor is mainly used to measure only the temperature change in measuring the minute temperature change in the first temperature sensor and the second temperature sensor, and is used for measuring only the temperature change, and the first temperature. It is necessary to be formed in a region where a thermal reaction does not occur even though the environment is equivalent to that of a sensor or a second temperature sensor. Mainly, the temperature of a liquid sample is measured, and the first temperature sensor is based on this temperature. And a second temperature sensor or the like for differential measurement of minute temperature changes. The above-mentioned "neighborhood" refers to a region or position within a range that can be regarded as having the same temperature.

A so-called highly selective enzyme that specifically reacts with the substrate is immobilized on the reaction section. For example, glucose oxidase, which is an enzyme corresponding to sugar (glue) as a substrate, trypsin corresponding to protein, creatinase corresponding to creatinine, bilirubin oxidase for bilirubin, and lactic acid oxidase for lactic acid, which are selectively and specific to specific substrates. The enzyme that reacts with the target is fixed. Further, one flow path is formed so as to pass through a plurality of thin films having a crosslinked structure, and the region between the first reaction section and the first temperature sensor, and the second reaction section and the second It passes through the area of the temperature sensor, reaches the substrate at the opposite position across the cavity forming the crosslinked structure from the front of the substrate, and then returns to the front through the flow path formed in the adjacent thin film of the crosslinked structure. Further, the specific substrate component is formed in one flow path formed in a plurality of thin films in a zigzag manner, such as returning to the substrate at the opposite position through the flow path formed in the adjacent crosslinked thin film. A liquid sample such as a body fluid containing is passed through. For example, when the two specific substrate components are protein and creatinine, which are substrates in urine as a liquid sample, it is preferable to use trypsin and creatinase, which are enzymes that selectively correspond to each other, as enzymes. Then, proteins and creatinine, which are substrates in the body fluid sample introduced through the flow path, and their corresponding enzymes trypsin (for example, fixed to the first reaction part) and creatinase (for example, to the second reaction part). The temperature is raised by the contact catalytic thermal reaction with (fixed), and the temperature rise is based on the reference temperature sensor by the first temperature sensor and the second temperature sensor formed in (or near) the reaction part. It can be detected and the respective amounts of the substrates protein and creatinase and their ratios can be calculated and obtained from the magnitude of the signal including the passage of time by using their differential amplification signals.

In the above description, as a liquid sample, a catalytic thermal reaction between a substrate as a specific substrate component in body fluids such as blood, urine, sweat, saliva, and tears and an enzyme thereof has been described, but as a liquid sample, for example, in fruits. Information such as the amount and ratio of at least two specific substrate components such as sugar and protein can be obtained in the same manner based on the catalytic thermal reaction with each enzyme.

The component comparison biosensor chip according to claim 2 of the present invention is a case where the reference unit is provided at a position substantially intermediate with the reaction unit such as the first reaction unit and the second reaction unit.

The flow path provided with the reference temperature sensor is provided in the same flow path as the flow path flowing into the first reaction section and the second reaction section, and these flow paths are provided from the substrate. It is formed on different heat-separated thin films and is also thermally separated from each other. Moreover, since the first reaction section and the flow path flowing into the second reaction section are thermally equivalent and connected in series, a liquid sample having the same temperature can be used as an enzyme in the first reaction section, for example. It is heated by reacting with and cooled when flowing on the substrate, but if it flows into the second reaction part while being heated, the second reaction part does not generate heat due to the enzymatic reaction. The temperature of the reaction part is raised, and an error that reaction heat is generated is likely to occur. In the present invention, in order to reduce these errors, the reference portion is formed on a thin film at a position substantially intermediate with the reaction portions such as the first reaction portion and the second reaction portion, and one flow is formed. It measures and compensates for the average temperature of the liquid sample flowing in the path. In this way, when the signal related to the temperature with the first temperature sensor and the second temperature sensor is differentially amplified with reference to the reference temperature sensor, the liquid sample in the first temperature sensor and the second temperature sensor is used. It is possible to measure only the temperature change deviated from the temperature of the above with high accuracy.

The component comparison biosensor chip according to claim 3 of the present invention is a case where the first temperature sensor, the second temperature sensor, and the reference temperature sensor are all temperature difference sensors.

Thermocouples and thermopile are known as temperature difference sensors. The feature of the temperature difference sensor is that the output related only to the temperature difference between the reference point (for example, cold contact) and the measurement point (for example, warm contact) can be taken out as a voltage output, and moreover, the temperature difference is almost equal. The output voltage is proportional. Therefore, for example, a thermocouple is adopted as the first temperature sensor, the second temperature sensor, and the reference temperature sensor, and the first temperature sensor and the second temperature sensor are used with reference to the above reference temperature sensor. When measured as a measurement point (warm contact), the output of the first temperature sensor and the second temperature sensor is the temperature of the position of the reference temperature sensor and the position of the first temperature sensor and the second temperature sensor, respectively. Shows the difference output. In this case, if the output difference between the second temperature sensor and the first temperature sensor is measured by sharing the reference point between the second temperature sensor and the first temperature sensor, this is the second temperature sensor. It is the temperature difference output between the position of the temperature sensor and the position of the first temperature sensor. It is convenient to use an SOI layer (for example, an n-type silicon layer) that constitutes a common crosslinked structure as one thermoelectric material of each of the second temperature sensor and the first temperature sensor because the configuration is simple. With this configuration, the temperature is raised by the catalytic thermal reaction between the substrates protein and creatinine in the body fluid sample introduced through the flow path and the corresponding enzymes trypsin and creatinase. , Those temperature rises are detected by the first temperature sensor and the second temperature sensor formed in (or near) the reaction part, and the amount of substrate and their substrates are detected from the magnitude of the signal including the passage of time. You can get information about the ratio of the amount of.

In the component comparison biosensor chip of the present invention, when the first temperature sensor, the second temperature sensor, and the reference temperature sensor are thermocouples, which are temperature difference sensors, Si (silicon) is used as a thin film thermally separated from the substrate. ) By using the SOI layer of the substrate, it can be used as one of the thermocouples of this SOI layer as a thermoconducting material. Since the semiconductor, which is the SOI layer, has a larger Seebeck coefficient than that of metal, it is easy to manufacture a highly sensitive and highly accurate thermocouple. It is preferable to use a metal film as the thermocouple material for the other thermocouple because it can be easily formed. Since the Seebeck coefficient of a semiconductor is about an order of magnitude larger than that of a metal, the other metal film may be selected regardless of the sign of the Seebeck coefficient. Then, when a plurality of flow paths are formed on the same enzyme-fixed biosensor chip and the components in the liquid sample of multiple items are measured at the same time, the first flow path formed in each flow path. By using the temperature sensor, the second temperature sensor, and the reference temperature sensor as one thermoconducting material of the thermoelectric pair of the common SOI layer, the number of electrode pads can be reduced, and the number of electrode pads can be reduced, and the first in each flow path can be used. Since the output signal amplification circuit of the temperature sensor, the second temperature sensor, and the reference temperature sensor can also be used as a common ground, it can be amplified with low noise, and the first temperature sensor in the flow path is based on the reference temperature sensor. It is convenient because it is an electronic circuit that facilitates the selection of the second temperature sensor and the differential amplification of them.

Since the temperature near the inlet of the liquid sample on the substrate is generally not the optimum temperature for the reaction, the injected liquid sample has already reached the predetermined optimum environmental temperature for the reaction by the time it reaches the reaction part through the flow path. It is desirable that it is. However, if the ambient temperature does not deviate extremely from the optimum temperature even if it is not the optimum temperature, the specific component in the liquid sample can be calibrated by the ambient temperature correction. For example, in the case of urine, the liquid sample is close to the body temperature and often deviates from the ambient temperature, and in this component comparison biosensor chip that measures the temperature change of about 1 mK, the temperature of the substrate and the ambient temperature, etc. Furthermore, it is extremely sensitive to the temperature of the liquid sample. The liquid sample passing through the flow path can also be arranged so as to have a predetermined optimum temperature by an external heater or the like. In addition, even if there is no heater and the temperature is not the optimum environment, the reaction part and temperature sensing part are covered with a heat insulating material, and the temperature rise of the thermal reaction in a room temperature environment is measured to obtain the temperature characteristics of each enzyme in advance. The amount of the following substrates can be calibrated using the calibration data provided.

The component comparison biosensor chip of the present invention is a biosensor that detects a specific substrate component in a liquid sample flowing in from at least one inflow port by the heat of reaction of catalytic action with an enzyme corresponding to the substrate component. A first flow path, a second flow path, and the like, which are flow paths through which the liquid sample is diverted, are formed in a plurality of thin films that are thermally separated from the substrate and are connected in parallel. The first reaction section, the second reaction section, etc., which are one reaction section, and these reaction sections, respectively, in the same first flow path, second flow path, etc. The first reference portion, the second reference portion, and the like, which are substantially equivalent one reference portions, are connected in series at positions substantially symmetrical with respect to the central portion of each of the thin films. The first reaction section, the second reaction section, and the like are provided with corresponding first temperature sensors, second temperature sensors, and the like, respectively, and the reference section is provided with a reference temperature without fixing an enzyme. A sensor is provided, and predetermined different enzymes corresponding to the specific different substrate components are fixed to the first reaction part, the second reaction part, etc., which are the respective reaction parts. The temperature change based on the heat of reaction at each of the reaction sections of the first temperature sensor, the second temperature sensor, etc. is measured with reference to the reference temperature sensor, and information on the amount of each of the substrate components, Alternatively, there is one characterized in that it is configured so that comparable information of those amounts can be obtained.

Body fluids such as urine, blood, sweat and saliva as liquid samples contain many substrates such as glucose, protein, lactic acid, creatinine, bilirubin and uric acid, which are biological substances. Using these body fluids, we would like to identify as many types of substrate components as possible, their amounts, and their ratios at the same time. For that purpose, a plurality of thin films and channels on each of them are arranged on the same substrate, and one reaction part and an enzyme in which different enzymes are fixed in the channels through which a liquid sample passes are fixed in each of the thin films. A reference portion that is not formed is formed in series, and a first temperature sensor and a reference temperature sensor are formed at each portion. The liquid sample flowing from the inlet of the liquid sample provided on the substrate is formed to flow in each of the flow paths of the plurality of thin films so as to be divided into the flow paths on the plurality of thin films. When the differential output of the first temperature sensor and the reference temperature sensor is taken out in this way, one reaction part that causes a thermal reaction in the flow path on one thin film heat-separated from the substrate. Since there is only one and it can be measured thermally independently from other heat-generating reaction parts, there is no thermal crosstalk, and one piece rises from the temperature of the liquid sample passing through the flow path formed in each thin film. There is an advantage that only the temperature change generated in the reaction part can be measured.

In the reaction part of the flow path formed in each of the two thin films of at least the plurality of thin films, for example, the ratio of the amount of the urinary component of the protein (albumin) and creatinine in the urine as a liquid sample. Since different enzymes corresponding to each of the two types of substrates for which the ratio of the amount of a specific substrate component is to be obtained are fixed, the reaction heat is detected by each temperature sensor, and the reference liquid sample is used. By detecting the temperature with the reference temperature sensor and configuring each flow path so that the temperature sensor and the reference temperature sensor can be differentially detected, each reaction in each of at least two thin films. It is possible to detect a temperature change based on the reaction heat in the part. Then, information on the amounts of each of the two substrate components can be obtained, and the ratio of those amounts can be calculated from the information on the comparison of those amounts based on the calibration data prepared in advance. As described above, in the present invention, the reaction heat is detected by each temperature sensor in each flow path formed in the same thin film, and the temperature of each reference liquid sample is detected by the reference temperature sensor. Then, each flow path is configured so that the differential detection between the respective temperature sensor and the reference temperature sensor can be performed.

The component comparison biosensor chip of the present invention is a biosensor that detects a specific substrate component in a liquid sample flowing in from at least one inflow port by the heat of reaction of catalytic action with an enzyme corresponding to the substrate component. A first flow path, a second flow path, etc., which is a chip, which is a flow path through which the liquid sample is diverted and passed through a plurality of thin films that are thermally separated from the substrate and connected in parallel, and for reference. Each of the flow paths is formed, and in each of the same first flow path, second flow path, etc., the first reaction part and the first reaction part, which are substantially equivalent one reaction parts, respectively. The second reaction section and the like are formed near the center of each of the thin films, and the first reaction section, the second reaction section and the like are provided with a first temperature sensor, a second temperature sensor and the like, respectively. , The reference part formed in the reference flow path is provided with a reference temperature sensor without fixing the enzyme, and the first reaction part and the second reaction part, which are the respective reaction parts, respectively. Etc., a predetermined different enzyme corresponding to a specific different substrate component is immobilized, and a temperature based on the heat of reaction at each of the reaction parts such as the first temperature sensor and the second temperature sensor. some variations, the temperature sensor the reference measured as a reference, the amount of information of each of the matrix components, or by being configured so that it can be retrieved with comparable information for their amounts, also those characterized by ..

In the inventions of claims 1 and 2, in one flow path, a thermally equivalent first reaction part and a second reaction part in which predetermined enzymes different from each other are fixed are connected in series, and further referred to. Since the parts are also connected in series, some thermal crosstalk may occur due to the exothermic reaction between the first reaction part and the second reaction part. Therefore, it is easy to make an error in measuring the value of the amount of substrate corresponding to each enzyme, but since it is a simple configuration in the same flow path, it is necessary to obtain the ratio of the amounts of the two substrate components. Is suitable. Further, in the invention described in paragraph No. 18 , the same flow path has only one reaction section having heat of reaction, and the reaction section is thermally separated from the reaction sections of other flow paths. Since there is no crosstalk and there is a first temperature sensor and a reference temperature sensor that measure the temperature of the reaction part in each of the same flow paths that are divided, the temperature of the liquid sample in the same flow path is used. There is an advantage that the temperature rise in the reaction section can be measured with high accuracy, and the amount of the substrate component in each reaction section can be obtained with high accuracy. However, since the ratio of the amounts of the two substrate components is measured by measuring the heat generated by the enzyme reaction at the reaction sites formed in the flow paths on different thin films, there is a possibility that an error may occur in the highly accurate measurement. Of course, it is possible to create correction calibration data for which these errors have been obtained in advance and use them to reduce the errors. In addition, since the temperature sensor of each reaction unit and the reference temperature sensor are formed on a fine thin film that is thermally separated from the same substrate, it is necessary to consider that the wiring is congested at the time of designing.

On the other hand, in the component comparison biosensor chip of the present invention, only one reaction portion having a heat of reaction is formed in the same flow path, which is an advantage of the invention described in paragraph number 18. It is adopted that there is no thermal crosstalk with the reaction part of the other flow path, and further, in order to solve the difficulty in the invention described in paragraph No. 18 , the reaction part is thermally separated from the substrate. There is also one that is formed near the central part where the sensitivity is the highest, and further eliminates the point where the wiring is congested. By using the above-mentioned correction calibration data, it is possible to measure the amount of two or more substrates and measure the ratio of the amounts of those substrates with high sensitivity and high accuracy.

The component comparison biosensor chip according to claim 4 of the present invention is for placing beads for fixing surface enzymes in the first reaction part, the second reaction part, etc. in the flow path formed on the same thin film. The pillar is provided, and the injection port of the surface enzyme fixing beads is formed at a position different from the inlet of the liquid sample on the substrate of the same flow path through which the liquid sample flows.

Surface enzyme-fixing beads are generally prepared by using ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) on the surface carboxyl group of polystyrene beads (PS beads) having a diameter of 20 micrometer (μm). PS beads that are bound and further coated by binding a specific enzyme using the amino group of the enzyme that is a protein. If the surface enzyme fixing beads are placed in the reaction part in the flow path, the enzyme can be fixed in the reaction part. A pillar is provided in the reaction section, specific enzyme-fixing beads are poured from the injection port of the surface enzyme-fixing beads on the inlet side of the liquid sample, and the reaction is stopped by the pillar provided on the outlet side of the reaction section. If the enzyme-fixing beads are placed in the part, the specific enzyme is fixed in this reaction part. Here, in particular, when the first reaction unit, the second reaction unit, and the like, which are a plurality of reaction units, are connected in series or in parallel, the first reaction unit and the second reaction unit in the flow path When fixing different enzymes to, etc., with the same diameter enzyme fixing beads, even if the enzyme fixing beads are poured from one inlet side of the liquid sample, if they are in series, they will be in the first reaction part. In the case of staying in the provided pillars and in parallel, they are similarly placed in the first reaction part, the second reaction part and the like, and it becomes difficult to fix different enzymes in these reaction parts. Therefore, in the present invention, a plurality of injection ports for surface enzyme-fixing beads are provided at positions different from the inlet of the liquid sample for each of the first reaction section, the second reaction section, and the like in the flow path. , This is a case where predetermined different surface enzyme fixing beads are injected from these injection ports. After injecting the beads for fixing the surface enzyme and indwelling them in each reaction part, the injection port is covered to prevent liquid leakage.

Further, when a plurality of flow paths formed on the same substrate of the component comparison biosensor chip are arranged in parallel, the liquid sample flowing in from one inflow port is divided into each flow path. After splitting, it is better to merge and discharge from one outlet. Since it can be sucked from one outlet of the liquid sample, it is different from the inflow from one inlet of the liquid sample, the inflow of the washing liquid into the flow path, and the inlet of the beads for fixing the surface enzyme. The flow path system can be simplified by injecting from the injection port into the flow path and discharging the liquid sample or cleaning liquid to the outside, and the sensor chip can be formed compactly.

The component comparison biosensor chip according to claim 5 of the present invention is for placing beads for fixing surface enzymes in the first reaction part, the second reaction part, etc. in the flow path formed on the same thin film. Pillars having different intervals are provided, and the bead dimensions for fixing the surface enzyme of the predetermined different enzyme are used to form the first reaction part and the second, respectively, by utilizing the bead size of the bead for fixing the surface enzyme. This is a case where the pillars are arranged and formed so that they can be placed in the reaction part of the above.

There are a first reaction part and a second reaction part connected in series in the same flow path, and it is desired to fix different enzymes to the first reaction part and the second reaction part. Therefore, for example, when the first reaction section is provided in order from the inlet side of the liquid sample in one flow path and the second reaction section is formed after that, the first reaction section is used. The interval between the arranged pillars may be 18 μm, and the diameter of the enzyme-fixing beads to be placed in the first reaction section may be 20 μm. Enzyme-fixing beads having a diameter of 10 μm and pillars arranged in a second reaction section located behind the first reaction section with respect to the flow of the flow path are arranged so as to be, for example, 8 μm. Is used, it passes through the pillar provided in the first reaction part in front and is fastened at the pillar provided in the second reaction part, and is fixed to the second reaction part with an enzyme having a diameter of 10 μm. The beads are placed and a desired enzyme different from the enzyme fixed in the first reaction part is fixed in the second reaction part. The amount of the enzyme involved in the reaction can be adjusted by adjusting the amount of these enzyme-fixing beads.

As described above, when there are a first reaction part and a second reaction part connected in series in the same flow path, the amount of each substrate by each fixed enzyme in each reaction part and them. When measuring the ratio to the amount of, the heat generation temperature may be in the order of millikelvin (mK), and the reference part that corrects the temperature of the liquid sample and the reference temperature sensor provided there are mainly important. It becomes. The reference part has the same inflow port of the liquid sample as the flow path on the thin film that is thermally separated from the substrate forming the first reaction part and the second reaction part that are connected in series. It is formed in the flow path on the thin film connected in parallel so that the liquid sample injected from the inflow port is divided and flows. The flow path having this reference portion has substantially the same shape as the first reaction portion and the second reaction portion, but the enzyme is not fixed and neither of them is provided with a pillar for placing beads for fixing the enzyme. Then, the reference temperature sensor as the reference unit is formed at a position corresponding to either the first reaction unit or the second reaction unit. At this time, since there is no pillar in which the enzyme-fixing beads are placed in the flow path where the reference portion is located, the reference portion does not stay there, and there is no thermal reaction by the enzyme, so that the reference portion serves as a reference portion. The first reaction section and the second reaction section are differentially amplified by the first temperature sensor and the second temperature sensor provided in the first reaction section and the second reaction section, respectively. It is possible to obtain the amount of each substrate corresponding to a predetermined enzyme fixed to each of the above and the ratio of these amounts.

The first reaction section and the flow path having the second reaction section connected in series are connected in parallel to the first reaction section and the second reaction section formed in these flow paths. To place enzyme-fixing beads with different enzymes, a liquid sample is injected from one inlet with enzyme-fixing beads, the first reaction forming in each parallel-connected flow path. There is a problem that only the same two types of enzymes are fixed in the part and the second reaction part. To solve this, for example, a special injection port for injecting beads for fixing enzymes is provided in a region located on the substrate of each flow path that divides the liquid sample into one inflow port. Then, the enzyme-fixing beads of the predetermined two types of enzymes to be placed in the first reaction part and the second reaction part of each flow path are injected from each special injection port, and each first After the required amount of enzyme-fixing beads is placed and fixed in the reaction section and the second reaction section, it is preferable to cover the special injection port to prevent liquid leakage.

The component comparison biosensor chip of the present invention, the each reaction part, there is a case of a structure in which an electrode for enzyme immobilization by electroporation click method.

As a method for fixing an enzyme, a fixing material for fixing by a carrier binding method, a cross-linking method, a comprehensive method, etc. (for example, a gel-like substance such as porous silica gel, an electrodeposited polymer material, or a photocrosslinkable PVA) has been conventionally used. (Polymer material, etc.), fixed to a substance that is hydrophilic but insoluble in water, and undergoes a thermal reaction by the contact catalytic reaction between the substrate in the body fluid introduced through the flow path and the corresponding enzyme. It is also possible to raise the temperature, detect the temperature rise with a first temperature sensor formed in or near the reaction section, and measure the amount of the substrate from the magnitude of the signal including the passage of time. However, in the present invention, it is desirable to fix the enzyme to the reaction part in the flow path after the flow path is formed. Therefore, after electrodeposition of a substance such as chitosan on a metal film such as a gold electrode, the surface is alkylated. Then, a method of fixing the enzyme, which is a protein, is adopted by a so-called electroclick method (also referred to as an electroclick chemistry method) in which the azide-ized protein is fixed on the azide by electrodeposition. The contact (catalytic) thermal reaction between the substrate and the corresponding enzyme has an optimum temperature, and the reaction between the substrate and the corresponding enzyme in the body fluid is generally near the body temperature. Therefore, it is desirable to keep the optimum temperature of the reaction uniformly at least in the vicinity of the reaction part that contributes to the reaction.

The enzyme is electrodeposited and fixed to a metal film such as gold (Au) formed in the reaction part in the flow path of the enzyme-fixed biosensor chip by the electroclick method, for example, as follows. First, chitosan is dissolved in dilute hydrochloric acid, and the pH is adjusted with sodium hydroxide to a pH of about 5.5, which is then flowed from the inlet of the liquid sample in the flow path of the completed enzyme-immobilized biosensor chip excluding enzyme immobilization. Inject into the road. The metal film electrode formed on the reaction part such as gold (Au) for fixing the enzyme is conductive with the electrode pad formed on the substrate outside the flow path of the enzyme fixing biosensor chip. , The other counter electrode is inserted or formed on the inlet side of the flow path or the outlet side of the flow path, and since chitosan is positively charged, the electrode for immobilizing the enzyme is used as the negative electrode and the other counter electrode is used. Is wired so as to be a positive electrode, and is electrodeposited to a thickness of about 10 μm in about 5 minutes at a voltage of about 3 V and about 3-10 μA (microamp) (depending on the size of the electrode). After that, the electrodeposited chitosan film is semi-dried by flowing air through the flow path.

Next, the surface of this electrodeposited chitosan film is alkylated as a pretreatment for enzyme electrodeposition fixation. For this, a mixed solution of sodium hydroxide and isopyl alcohol is injected into the flow path, brought into contact with the electrodeposited chitosan membrane, a small amount of propargyl bromide solution is added, and the mixture is left at about 60 ° C. for about 5 hours. Achieved by doing. In addition, the enzyme, which is a protein, is azidized and electrodeposited on the surface of the alkylated chitosan membrane by the electroclick method to fix the enzyme on the electrode of the reaction part in the flow path. There are various methods for azidating the enzyme, and it is achieved, for example, by immersing the enzyme in a solution of LVDS3 containing NHS-dPEG (4) -N3 for about 4 hours. Then, the unreactive component is removed by leaving it in a buffer solution at a low temperature (about 4 ° C.) for about 8 hours. Next, for enzyme fixation by the electroclick method, for example, copper sulfate, asconic acid and EDTA are dissolved in a phosphate buffer solution, further dissolved in the azide-ized buffer solution of this enzyme, and injected into the flow path. .. A counter electrode is inserted or formed on the inlet side, and a reference electrode is inserted or formed on the outlet side. By repeatedly applying a scanning voltage of about 250 mV / s between and -300 mV and electrodepositing the enzyme for about 10 minutes, enzyme immobilization of the reaction part in the flow path by the electroclick method is achieved. ..

The component comparison biosensor chip of the present invention, each reaction part of the thin film, Yes to form a heater, there is a case configured to allow heating the reaction section.

In each combination of the substrate of the biological substance and the corresponding enzyme, each contact-catalyzed thermal reaction has an optimum ambient temperature, often at around 35 ° C to 40 ° C near the human body temperature. Yes, it is a temperature higher than the general room temperature of 20 ° C. A heater is installed outside so that the contact-catalyzed thermal reaction occurs in such an optimum temperature environment or in a temperature environment in which a thermal reaction can be easily observed, and the thin film having a flow path has a predetermined uniform temperature. It is good to be able to set the temperature that will be the distribution. In the present invention, the temperature of the reaction part can be set to the optimum temperature for the enzyme, and at the same time, it can be used for calibration of the heat of reaction in the thermal reaction with the enzyme. In particular, the activity of the enzyme often decreases with time and environment, and the heater formed in this reaction part is useful for checking and calibrating the activity of the enzyme. For example, the temperature rise due to the addition of 10 μW (microwatt), which is a predetermined wattage, to the heater is measured in advance, and the temperature rise of the reaction heat of a specific enzyme due to the enzyme heat reaction by flowing a standard liquid sample. If the minutes are measured from time to time, the degree of enzyme activity can be checked by the change over time, and the amount of components of the standard liquid sample can be calibrated based on this.

The component comparison biosensor chip according to claim 6 of the present invention is a case where the shape of the flow path in each reaction section and the reference section is streamlined at least in the width direction.

According to the experiment, if the shape of the reaction part in the width direction in the flow path through which the liquid sample flows has a corner such as a rectangle, the liquid sample or the cleaning liquid stagnates near the four corners, and a new liquid sample or the cleaning liquid is used. There was a problem that they could not be replaced. This phenomenon was solved by making the shape of the flow path in each reaction section and the reference section at least in the width direction streamlined. In addition, it is desirable that the height direction of the flow path has a structure in which there is no step at the reaction part or the reference part, and if there is a step, it is necessary to make the step gentle slope so that the fluid does not stagnate. There is.

The biosensor module has an inlet and an outlet for a liquid sample or a cleaning liquid, and the component comparison biosensor chip according to any one of claims 1 to 6 is mounted between the inlet and the outlet. That is, the liquid sample and the cleaning liquid are integrated so as to flow out to the outside through the inlet and outlet of the component comparison biosensor chip, and further, for electrical connection to the component comparison biosensor chip. It is characterized by being provided with a connector of.

When used as a component comparison biosensor, which is a handy calorimetric (calorific value detection type) biosensor equipped with the component comparison biosensor chip of the present invention, it is necessary to replace the component comparison biosensor chip due to enzyme deactivation or the like. Come out. The component comparison biosensor chip has an extremely fine pattern shape and has a structure that is easily broken. In such a case, it is desirable to replace the component comparison biosensor chip without directly touching the component comparison biosensor chip, instead of handling the component comparison biosensor chip alone. The present invention has an inlet and an outlet for a liquid sample or a cleaning liquid and a component comparison so that a general person using a component comparison biosensor can easily replace the component comparison biosensor chip. It is modularized as a biosensor module equipped with a connector for electrical connection to the biosensor chip. By attaching this biosensor module to the component comparison biosensor like a cassette, the component comparison bio The sensor chip can be replaced.

In the component comparison biosensor according to claim 7 of the present invention, the ratio of the first reaction part and the second reaction part, which are at least two reaction parts, in the liquid sample to the component comparison biosensor chip. A component comparison biosensor chip or biosensor module in which a predetermined enzyme corresponding to a specific substrate component to be obtained is fixed is mounted, and each temperature change based on the reaction heat is formed on the component comparison biosensor chip. The first temperature sensor and the second temperature sensor are measured by differential detection with reference to the reference temperature sensor, and based on predetermined calibration data, at least the amount of the two substrate components, or It is characterized by the fact that the ratios thereof have been obtained.

As described in claims 1 and 2 above, for example, one flow path is formed in a plurality of thin films having a crosslinked structure, and the first reaction section is thermally equivalent in the flow path. Different enzymes are fixed to the second reaction part. Since the first reaction section and the second reaction section are arranged in the same shape substantially symmetrically on both sides of the central portion of the crosslinked thin film, the calorific value of the enzymatic reaction is increased. The temperature rises. Since the first temperature sensor and the second temperature sensor are formed in the first reaction section and the second reaction section, respectively, the reference temperature is provided with a separate temperature rise according to the calorific value of these sensors. Differential measurement is performed with reference to the sensor. The reference temperature sensor is provided in a separately provided flow path in which the first reaction section and the flow path passing through the second reaction section are connected in parallel to separate and flow, and is a region in which the enzyme is not fixed. Therefore, since the same liquid sample as the first reaction part and the second reaction part flows, as a reference part, mainly based on the temperature of the liquid sample, the first reaction part and the second reaction part, respectively. It is possible to measure the temperature rise of. In this way, the amount of the two substrate components generated by the respective enzymes in the first reaction section and the second reaction section, or their ratios are determined. The relationship between the calorific value and the temperature change of each of the first reaction section and the second reaction section is obtained in advance as calibration data, and based on this calibration data, the amount and ratio of the two substrate components are obtained. It is good to calibrate. In particular, when urine or blood is used as a liquid sample, the ratio of albumin and creatinine, which are proteins in the liquid sample, is important because it leads to early detection of kidney disease. It is preferable to fix an enzyme such as trypsin to albumin and an enzyme such as creatinase to creatinine as a substrate in the first reaction part and the second reaction part, respectively.

The component comparison biosensor chip described in paragraphs 18 and Numbering 21 described above, each of the thin film which is connected to a plurality of parallel that is thermally separated from the substrate, a flow path through which the liquid sample is formed, the they each flow path of, in some cases one reactive unit with a fixed predetermined enzyme is formed. In the component comparison biosensor chip described in paragraph No. 18, each flow path on the thin film has a reference portion, and a reference temperature sensor is provided therein. However, in the component comparison biosensor chip described in paragraph 21, in the flow path on the thin film which is connected in parallel with a plurality of flow paths having one reaction part and is thermally separated from each reaction part, A reference portion and a reference temperature sensor are formed at positions spatially and thermally equivalent to the reaction portion on the thin film. Since the first temperature sensor is formed in the reaction section, the temperature rise according to the calorific value is measured differentially with reference to a separately provided reference temperature sensor. The reference temperature sensor is provided in a separately provided flow path that is connected in parallel with the flow path passing through the reaction part and splits and flows, and since the enzyme is not fixed, the same liquid sample as the reaction part flows. As a reference unit, the temperature rise of the reaction unit can be measured mainly based on the temperature of the liquid sample. In this way, the amount or ratio of at least two substrate components that generate heat by different predetermined enzymes fixed to each reaction part of the parallel connection is determined.

Here, the above-mentioned component comparison biosensor chip is mounted, and a Joule heating heater is installed in the reaction section so that the contact catalytic thermal reaction can be performed in an optimum temperature environment or a temperature environment in which a thermal reaction can be easily observed. Therefore, this is a case where a component comparison biosensor is provided which can make the temperature of the reaction part the optimum temperature for the enzyme and can also be used for calibrating the reaction heat in the thermal reaction with the enzyme. In particular, the activity of the enzyme often decreases with time and environment, and the heater formed in this reaction part is useful for checking and calibrating the activity of the enzyme. As described above, for example, the amount of temperature increase due to the addition of 10 μW (microwatt), which is a predetermined wattage, to the heater is measured in advance, and the enzyme heat reaction by flowing a standard liquid sample is used to obtain a specific enzyme. If the temperature rise of the heat of reaction is measured from time to time, the degree of enzyme activity can be checked by the change over time, and based on this, the amount of a specific substrate component in the liquid sample or at least two specific substrate components can be checked. The ratio of can be calibrated.

The component comparison biosensor according to claim 8 of the present invention includes at least a power supply circuit, an amplifier circuit, an arithmetic circuit, and a control circuit, and the amount of a specific substrate component in the liquid sample or at least the amount of the two substrate components. This is the case when information about the ratio can be obtained.

When the component comparison biosensor chip is manufactured using an SOI substrate which is a Si single crystal, it is preferable that the MEMS technology can be easily applied. A power supply circuit, an amplifier circuit, an arithmetic circuit, and a control circuit can also be integrated on the sensor chip made of the SOI substrate by integrated circuit technology, or these can be integrated on another semiconductor substrate or the like to be modularized. By doing so, it is possible to provide an extremely compact, for example, handy type component comparison biosensor. Of course, these circuits can also be formed and placed on a compact printed circuit board separately from the component comparison biosensor chip, placed close to the component comparison biosensor chip, and electrically connected for use.

As described above, adopting a crosslinked structure as the thin film has an advantage that stable holding of the thin film is achieved, and as described later, in order to use the sensor chip many times, a flow path is used. It is necessary to clean the inside. In this case, for example, a liquid sample such as urine or blood needs to be flowed through the flow path, and after the thermal reaction, the liquid sample or the washing liquid needs to be discharged to the outside of the sensor chip. Therefore, a crosslinked structure is preferable. Of course, if the thin film is made into a cantilever shape, a smaller thin film is required compared to the crosslinked structure, but in order to use the sensor chip many times, the liquid sample or cleaning liquid is thermally separated from the substrate. In order to discharge the liquid sample while preventing evaporation on the existing thin film, it is necessary to return the flow path on the cantilever to the substrate support portion of the cantilever, which causes a problem that the structure becomes complicated.

As described above, in specifying the amount and type of substrate in body fluids such as urine and blood as a specific substrate component, an enzyme corresponding to the substrate which is a specific substrate component is used as a reaction part in the flow path on each of a plurality of thin films. By fixing to, the body fluid of the liquid sample injected from the inflow port is distributed, passes through each flow path formed in a plurality of thin films, and the catalytic heat with a specific enzyme in each reaction part. By generating heat due to the reaction and extracting the differential output of the first temperature sensor and the second temperature sensor, the combination of a specific enzyme and substrate can be determined almost at the same time. For example, a liquid sample injected from one inlet is split into each flow path formed in each thin film, and a thermal reaction with each enzyme corresponding to the substrate of each specific substrate component in each reaction part. Then, when the amount and ratio can be obtained from the information on the amount of each different specific substrate component and the comparable information by measuring the temperature rise with each of the first temperature sensor and the second temperature sensor. Is. The present invention has the advantage that various substrates in a liquid sample can be measured at the same time. Of course, conversely, it is also possible to fix the substrate in the reaction section as a substance corresponding to the specific substrate component and detect the enzyme as the specific substrate component.

The component comparison biosensor of the present invention is a temperature measurement system having a resolution of about 1 mK in which a fine flow path is formed in a thin film having an extremely small heat capacity thermally separated from a substrate, and is used for highly accurate detection of the amount of a specific substrate component. It is most important to prevent the influence of heat transfer and convection from the outside world other than the thermal reaction between the enzyme and the substrate. It is necessary to have a structure that is not affected by heat convection or changes in outside air temperature, and in order to prevent the effects of room temperature fluctuations and the flow of external air, etc., at least one cover should be attached to the substrate to enhance the heat insulation effect. At the same time, it is better to allow the liquid sample to flow in and out of the inlet and outlet of the enzyme-fixed biosensor chip of the liquid sample. Of course, it is necessary that the flow path region of the crosslinked structure floating in the air, which is a portion of the cover that is thermally separated from the substrate, has a recess such as a cavity. As a result, the temperature of the reaction part can be thermally separated from the cover. By attaching a cover to the back side of the board, the heat insulating effect is increased. Depending on how the component comparison biosensor chip is attached to the sensor, the back side of the substrate may be fixed to the plate instead of the cover so as to cover the cavity on the back side of the substrate.

When the component comparison biosensor of the present invention is used for measuring urine sugar, urine protein, etc., information on the amount and ratio of a specific substrate component from a certain individual component comparison biosensor is available not only at that time but also in daily life. It is important to know the daily changes and their trends in Japan. It is important to accumulate past data as well as the numerical values at the time of measurement to graph and predict diurnal changes, and it may be necessary to contact medical institutions. , It is convenient to enable information to be transmitted to an external computer wirelessly or by wire so that various processes can be performed.

In the component comparison biosensor chip of the present invention, the reference part and all the plurality of reaction parts are formed in series or in parallel in the flow path, and the reference part is thermally equivalent to the reaction part. There is an advantage that the temperature sensor output at the reaction unit based on the temperature sensor can be easily compensated thermally.

In the component comparison biosensor chip of the present invention, the first temperature sensor, the second temperature sensor, etc. and the reference temperature sensor are all temperature difference sensors such as thermocouples, and in particular, the reference temperature sensor formed equally is used. As a reference, the differential outputs of the first temperature sensor and the second temperature sensor enable highly accurate measurement of only the temperature rise based on the thermal reaction with their corresponding enzymes, and at least two reaction parts. There is an advantage that the amount of the substrate as a desired specific substrate component and the ratio thereof can be measured with high accuracy.

In the component comparison biosensor chip of the present invention, a heater is formed in the reaction part so that the reaction part can be heated. Therefore, this heater is driven by a predetermined amount of Joule heat to lose the enzyme. Since the degree of activity can be checked and used for calibration, there is an advantage that a highly accurate component comparison biosensor with a long life can be provided.

In the component comparison biosensor chip of the present invention, enzyme fixation can be selectively formed in the reaction portion in this flow path by the click chemistry method or the placement method of surface enzyme fixation beads. Therefore, since different enzymes can be immobilized on a plurality of thin films and flow paths formed on the same substrate, there is an advantage that, for example, multiple specific substrate components in urine of a liquid sample can be simultaneously measured.

The biosensor module of the present invention has an advantage that it can be easily replaced without directly touching the component comparison biosensor chip by attaching the component comparison biosensor to the component comparison biosensor in units of the biosensor module like a cassette. ..

In the component comparison biosensor of the present invention, a plurality of thin films having a temperature sensor in each of the flow path, the plurality of reaction parts and the reference part, and the plurality of reaction parts and the reference part are formed on the same substrate, and a predetermined substrate and the predetermined substrate are formed. By arranging them in each so that they can be measured in combination with the corresponding enzymes, the identification of multiple types of specific substrate components contained in body fluids such as urine and their amounts and their ratios can be measured almost simultaneously. There is an advantage that it can be done.

In the component comparison biosensor of the present invention, since the flow path has a closed structure, evaporation of the liquid sample can be prevented and the structure can be surrounded by a heat insulating material, so that minute temperature changes can be stably measured. There is an advantage that it can be done.

The component comparison biosensor of the present invention has a structure in which the flow path can be washed with a washing liquid, and the measurement is based on the thermal reaction between the substrate and the enzyme, but since the enzyme acts as a catalyst, it is not consumed and is essential. There is an advantage that it can be used many times if the substrate is washed or the like.

In the component comparison biosensor of the present invention, a sensor chip and a heater can be formed by MEMS technology, and a power supply circuit, an amplifier circuit, an arithmetic circuit and a control circuit can be easily integrated. It has the advantage that it can also be provided as a comparative biosensor.

A schematic plan view of an embodiment of the conventional biosensor chip 100'is shown. (Example 1) It is a cross-sectional schematic view along the X-ray line of FIG. (Example 1) It is a plan schematic diagram which shows one Example of the comparative biosensor chip 100 of this invention. (Example 1) It is a plan schematic diagram which shows another Example of the comparative biosensor chip 100 of this invention. (Example 2) It is a plan schematic diagram which shows another Example of the comparative biosensor chip 100 of this invention. (Example 3) It is a plan schematic diagram which shows another Example of the comparative biosensor chip 100 of this invention. (Example 4) It is a plan schematic diagram which shows another Example of the comparative biosensor chip 100 of this invention. (Example 5) It is a plan schematic diagram which shows another Example of the comparative biosensor chip 100 of this invention. (Example 6) It is a plan schematic diagram which shows another Example of the comparative biosensor chip 100 of this invention. (Example 7) A schematic cross-sectional view of an embodiment of the biosensor module 500 of the present invention of the present invention is shown (Example 8). The schematic diagram of one Example of the component comparison biosensor 1000 of this invention is shown. (Example 9) A block diagram of an embodiment of a schematic configuration diagram when driving the component comparison biosensor 1000 of the present invention is shown. (Example 10)

The component comparison biosensor chip of the present invention can be formed of a silicon (Si) substrate using MEMS technology. We will mainly explain some examples of manufacturing using this silicon (Si) substrate, especially the SOI substrate, and also implement one of the modularized biosensor modules on which the component comparison biosensor chip of the present invention is mounted. For example, an embodiment of the component comparison biosensor of the present invention using the component comparison biosensor chip of the present invention and a module on which the chip is mounted will be described with reference to the drawings. In the present specification and the drawings, components having substantially the same functional configuration are designated by the same reference numerals, so that duplicate description will be omitted.

FIG. 1 shows a schematic plan view of a conventional biosensor chip 100'in the case of having a crosslinked structure 12 of a plurality of thin films 10. Further, FIG. 2 is a schematic cross-sectional view taken along the line XX of FIG. Also in this embodiment, the first temperature sensor 20A and the reference temperature sensor 20R are formed by a thermocouple, but at that time, the SOI layer (n-type semiconductor layer) is shared as the thermoelectric conductor 120a. The thermoelectric conductor 120b is formed by using a sputtering thin film of nichrome (NiCr) formed through the insulating film 50. At the measurement points (warm contacts) 26 of these thermocouples, they are electrically connected by an ohm-like contact 60 through the through hole of the insulating film 50. Here, an example is shown in which five single thin films 10 are arranged in parallel, and each of them has a crosslinked structure 12-shaped thin film 10a, thin film 10b, thin film 10c, or the like. The urine of the liquid sample injected from one inflow port 16 provided on the substrate 1 is distributed, and for example, the respective flow paths 17 to be introduced into the thin film 10a, the thin film 10b and the thin film 10c are formed. Then, when a substrate as a specific substrate component in urine of a liquid sample, for example, glucose, lactic acid, protein, etc. is detected, glucose oxidase, lactate dehydrogenase, which are enzymes 4A, 4B, 4C, etc. corresponding to these substrates, respectively, A predetermined amount of trypsin or the like is fixed to the reaction portions 6A, 6B, 6C arranged at substantially the center of the thin film 10a, the thin film 10b, and the thin film 10c by the above-mentioned electrodeposition or the like. The flow paths 17 are rejoined near the end of the substrate 1 of the biosensor chip 100'to be guided to the outlet 18. Further, a pump or the like, which is a driving means 610, may be capable of sucking a liquid sample, washing water, or the like. The enzyme 4 is used as a catalyst and is not consumed in the reaction with the substrate. It is lost by immobilizing the enzyme 4 and removing the reaction product and the unreacted substrate with washing water. If it is not activated, it can be used repeatedly. Here, only five single thin films 10 are arranged in parallel, but of course, the number may be further increased.

This conventional biosensor chip 100'can be formed by a known MEMS manufacturing technique. Here, the thin film 10 having a crosslinked structure 12 is formed by using the SOI layer 11 of the SOI substrate, and here, the temperature of the first temperature sensor 20A, the second temperature sensor 20B, and the reference temperature sensor 20R is set. This is a case where it is realized as a thin film thermocouple which is a difference sensor. In these thin film thermocouples, an SOI layer 11 (for example, an n-type silicon single crystal film having a thickness of about 10 μm) is used as a first thermoconductor 120a, and a silicon oxide film formed by thermally oxidizing the SOI layer 11 on the first thermoconductor 120a. A second thermal conductor 120b (for example, a nickel thin film, an aluminum thin film, or a silicon thin film) is formed through the insulating film 50, and an ohm-like contact 60 as a measurement point (warm contact) 26 is formed. To. A common reference point (cold contact) 27 for the thermocouples of these thin films is formed on the substrate 1. The length of the crosslinked structure 12-shaped thin film 10 may be about 800 μm. Further, here, in order to measure the temperature of the substrate 1, a pn junction diode (semiconductor diode) is formed on the substrate 1, and it is used as an absolute temperature sensor 23 for measuring the absolute temperature of the substrate 1. .. In the method of using the semiconductor diode as the absolute temperature sensor 23, in the measurement of room temperature which is a relatively low temperature of 150 ° C. or less, a constant forward voltage is applied to the semiconductor diode, and the temperature dependence of the diode current at that time is used. There is a method of obtaining it, and a method of obtaining it from the temperature dependence of the diode forward voltage at that time by passing a constant current. Of course, a resistance temperature detector may be used as the absolute temperature sensor 23.

For example, urine of the liquid sample injected from the inflow port 16 is distributed and installed in the reaction portions 6A, 6B, 6C, etc., which are arranged in the substantially central portions of the thin film 10a, the thin film 10b, the thin film 10c, and the like. When contacted with glucose oxidase, lactate dehydrogenase, trypsin, etc., which are the enzymes 4A, 4B, 4C, etc. of the fixed enzymes under the optimum environmental temperature, they are installed in the vicinity of the reaction parts 6A, 6B, 6C, etc. The first temperature sensor 20A, the second temperature sensor 20B, the third temperature sensor 20C, and the like for measuring the temperature are similarly formed in the middle of the thin film 10a, the thin film 10b, and the thin film 10c by the reference temperature sensor 20R. When the temperature difference ΔT for the temperature rise is recognized by the temperature difference measurement with each of the reference temperature sensors 20RA, 20RB, 20RC, etc., the temperature difference ΔT among the reaction parts 6A, 6B, 6C is recognized. It can be said that different specific substrate components (sugar, lactic acid, protein, etc.) corresponding to each location are present in the urine. Then, the amount of the specific substrate component which is the substrate can be obtained from the calibration data prepared in advance from the magnitude of the temperature difference ΔT corresponding to the temperature rise. For example, the reaction section 6A formed on the thin film 10a (here, an enzyme called glucose oxidase is fixed as the enzyme 4A) generates heat due to a catalytic thermal reaction, and the first temperature sensor 20A and the reference temperature sensor When the temperature difference ΔT for the temperature rise is measured to a predetermined size or more by the temperature difference measurement with 20RA, the value of urinary sugar is obtained by using the calibration data according to the size. .. Of course, when the temperature difference ΔT for the temperature rise is measured by the differential output of the second temperature sensor 20B formed on the thin film 10b and the reference temperature sensor 20RB to a predetermined size or more, it depends on the size. Therefore, the lactate level in urine can be obtained by using the calibration data. Since the manufacturing method and other actions of the biosensor chip 100'and the like are the same as those described in a known document, for example, document 1, detailed description thereof will be omitted here.

However, in the prior art such as Document 1 described above, the first temperature sensor 20A, the second temperature sensor 20B, the third temperature sensor 20C and the like are substantially central to the thin film 10a, the thin film 10b, the thin film 10c and the like, respectively. The reference temperature sensors 20RA, 20RB, 20RC, etc., which are installed in the respective reaction units 6A, 6B, 6C, etc. arranged in the above, are located at the center of each of the thin film 10a, thin film 10b, thin film 10c, etc. Since the reference part is deviated from the part, the reference part is different from the reaction part 6, and the central part of the thin film 10 has the highest sensitivity, the liquid sample having a temperature deviated from room temperature can flow through each flow path. For example, even though the first temperature sensor 20A and the reference temperature sensor 20RA do not have the heat of enzyme reaction, a slight temperature difference occurs and the actual measurement accuracy of the heat of enzyme reaction deteriorates. There was a problem.

The component comparison biosensor chip 100 of the present invention has the same basic principle of detecting a temperature change due to a thermal reaction between a conventional biosensor chip 100'and a corresponding substrate by enzyme fixation, but the above-mentioned measurement of enzyme reaction heat The accuracy is improved, and the ratio of the amount of the substrate, which is two or more predetermined components in the liquid sample, can be compared. An example of the component comparison biosensor chip of the present invention shown in FIG. 3 will be described. FIG. 3 is a plan view of the comparative biosensor chip of the present invention. For example, a silicon chip (Si chip) substrate 1 is crosslinked with an SOI layer 11 as described in FIGS. 1 and 2 above. This is an example of a comparative biosensor chip in a case where a plurality of thin films 10 thermally separated from the substrate 1 of the structure 12 by a cavity 40 are connected in series by one flow path 17. The flow path 17 is, for example, a photoresist film such as SU-8, which can be easily formed on the thin film 10 of the SOI layer 11 by photolithography using a known technique. When a thermal reaction occurs in the first reaction section 6A on the side close to the inflow port 16 of the liquid sample, the heat reaches the second reaction section 6B downstream of the thermal reaction along the flow of the liquid sample. It is expected that the temperature of the reaction section 6B will also increase. As a countermeasure, the flow path 17 includes a first reaction unit 6A and a second reaction unit 6B, which are two reaction units 6 which are the inflow port 16 of the liquid sample and the reaction unit 6 on the crosslinked structure 12. A reference portion 7 is provided in the thin film 10 between the reaction portions 6A and 6B, and the liquid sample is discharged to the outside through the outlet 18 of the liquid sample to improve the measurement accuracy. This is the case.

Then, for example, when the liquid sample is urine, which is a body fluid, glucose oxidase, the substrate of which is the corresponding enzyme 4A for detecting glucose (urine sugar, etc.), is immobilized on the reaction unit 6A. In the reaction section 6B, trypsin, which is an enzyme 4B corresponding to the protein as a substrate, which is different from the reaction section 6A (it is better to add an enzyme that further decomposes the protein into a peptide having a small molecular weight). I try to keep it fixed. A first temperature sensor 20A and a second temperature sensor 20B are mounted on the reaction unit 6A and the reaction unit 6B of the two different thin films 10, respectively, and a reference unit provided on the different thin film 10 is provided. Reference numeral 7 is equipped with a reference temperature sensor 20R, and all the first temperature sensors 20A, the second temperature sensor 20B and the reference temperature sensor 20R on the bridge structure 12 are composed of a thin film thermocouple. If you are. In these thin-film thermocouples, the measurement point 26, which is a warm contact, is arranged at the reaction portion 6 and the reference portion 7 on the crosslinked structure 12, and the reference point (cold contact) 27 is arranged in common with the substrate 1. .. Then, as shown in FIGS. 1 and 2 of the above-mentioned prior art, the thermoelectric conductor 120b is formed through the insulating film 50 by sharing the SOI layer (n-type semiconductor layer) as the thermoelectric conductor 120a. This is the case where the (NiCr) film forms a thermocouple. Further, in this embodiment, an enzyme fixing electrode 72 is formed in the reaction part 6 and the reference part 7 for fixing the enzyme to the reaction part 6 by the electroclick method. Further, the enzyme is not fixed to the reference unit 7 due to the heat balance with the reaction unit 6, but it is better to form a chitosan film or the like on the enzyme fixing electrode 72 before that. Further, in the insulation separation groove 110 for the SOI layer provided at the support portion of the substrate 1 of each thin film 10, since the SOI layer 11 covers the entire surface of the substrate 1, each of the first temperature sensors 20A and the first This is a groove provided in the SOI layer 11 for insulation separation so that the temperature sensor 20B and the reference temperature sensor 20R of No. 2 are not electrically independent.

In this embodiment, each reaction unit 6 and reference unit 7 are provided with a heater 25, and calibrates the amount of substrate based on the enzymatic thermal reaction, calibrates the change over time, and further adjusts the optimum temperature of the enzymatic reaction. It can be used for correction and so on. One wiring of each heater 25 is connected to each enzyme fixing electrode 72 via an ohm-like contact 60, and the heater electrode pad 71P and the enzyme fixing electrode pad 72P arranged around the substrate 1 are shared. And saves the number of pads. Further, a counter electrode 73 used for the electroclick method is formed at the inflow port 16, and a reference electrode 74 is formed at the outflow port 18. In the electroclick method, for example, an aqueous solution of chitosan whose pH is adjusted to the acidic side is passed through the flow path of the electrode 72 for fixing the enzyme with respect to the counter electrode 73, and a negative (-) voltage is applied to form a chitosan film. Electrode electrodeposition. After that, the surface of the chitosan film is alkylated, and a separately prepared azide-ized predetermined enzyme is used as a catalyst for copper (Cu) ions to form a chitosan film whose surface is alkylated with respect to the reference electrode 74. By applying alternating positive and negative voltages between the working electrode of the enzyme fixing electrode 72 and the counter electrode 73, the predetermined enzyme is selectively fixed to the alkylated chitosan film by electroclicking. is there. Since such an electroclick method is described in detail in Reference 1, it will be omitted here.

For example, when measuring the amount of a substrate such as glucose as a specific substrate component in urine as a liquid sample, a small amount of urine injected into the flow path 17 from the inflow port 16 provided on the substrate 1 is applied onto the thin film 10. In order to guide urine, which is mostly water, to the reaction section 6 by a capillary phenomenon or a driving means, at least the wall surface of the flow path 17 that comes into contact with urine is hydrophilic. It is better to form with the material of. For that purpose, a thick film can be formed in order to maintain the strength, and it is preferable to form the thick film with a hydrophilic photosensitive resist. Further, it is rather preferable to use only the pump as the driving means 610 and forcibly extrude or suck out the liquid sample by applying pressure without depending on the capillary phenomenon, because stable operation can be obtained. is there.

FIG. 4 is a schematic plan view of an embodiment of the component comparison biosensor chip 100 of the present invention. Similar to FIG. 3 of Example 1, a thin film 10 having a plurality of crosslinked structures 12 is provided, and these thin films 10 are formed. This is an example of a schematic plan view of a comparative biosensor chip when one flow path 17 is connected in series. Each thin film 10 is provided with a reaction unit 6 and a reference unit 7, and in this embodiment, each of the thin films 10 has three reaction units 6A, 6B, 6C and three reference units 7A, 7B, 7C, respectively. Different enzymes 4A, 4B, and 4C are fixed in the reaction parts 6A, 6B, and 6C, and the substrates of the urine components corresponding to each are fixed to the reaction parts 6A, 6B, and 6C by selective contact thermal reaction with the enzyme. The first temperature sensor 20A, the second temperature sensor 20B, the third temperature sensor 20C provided in each of the 6Cs, and the reference temperature sensors 20RA of the three reference portions 7A, 7B, and 7C paired with each other. , 20RBA, 20RC, respectively, by detecting the heat of contact reaction between the substrate and the enzyme, the amount of the substrate component of urine, which is a liquid sample, and if necessary, comparing the amount and their ratio The component comparison biosensor chip 100 is configured so that it can be calculated.

For example, the reaction portion 6A and the reference portion 7A paired with the reaction portion 6A formed on each thin film 10 have an equivalent shape so as to be thermally balanced, and pass through the central portion of each thin film 10. It is formed symmetrically with respect to the YY line. Further, each reaction unit 6 and reference unit 7 are streamlined so that the flow of the liquid sample in the flow path does not stagnate. Therefore, it is possible to accurately compare the amounts of substrates and their amounts. The reference units 7A, 7B, and 7C, which are paired with the reaction units 6A, 6B, and 6C, respectively, have a first temperature sensor 20A, a second temperature sensor 20B, and a third temperature sensor 20C, respectively. The reference temperature sensors 20RA, 20RB, and 20RC are formed, and a differential output between the first temperature sensor 20A and the paired reference temperature sensor 20RA in each of the thin films 10 can be obtained. It is configured as follows. The temperature sensors formed on each of these thin films 10 are all composed of thin film thermocouples as described in FIGS. 1 and 2 of the conventional biosensor chip, and are provided on the substrate 1. A differential output can be obtained by using one common reference point (cold contact) 27 (the electrode pad is the common electrode pad 75P for the SOI layer). The component comparison biosensor chip 100 of the present invention undergoes a selective thermal reaction with different enzymes 4A, 4B, and 4C fixed to the respective reaction portions 6A, 6B, and 6C among the substrate components in the liquid sample. , The amount of each substrate and the amount of them can be compared. Further, in this embodiment, the enzyme fixing electrodes 72 are provided in the reaction portions 6A, 6B, 6C so that the enzymes 4A, 4B, and 4C can be fixed by the electroclick method, and the heater 25 is also the same. Since the operations thereof are the same as those described with respect to FIG. 3 of the first embodiment, they are omitted here.

FIG. 5 is a schematic plan view of another embodiment of the component comparison biosensor chip 100 of the present invention, which has a plurality of thin films 10 having a crosslinked structure 12 as in FIG. 4 of the second embodiment. In the example of FIG. 4 of Example 2, these thin films 10 were connected in series by one flow path 17, but in this example, the liquid sample flowing in from one inflow port 16 is An embodiment is characterized in that the flow is divided into the respective flow paths 17 formed in the thin films 10 of the plurality of crosslinked structures 12 connected in parallel, and is integrated so as to be discharged to the outside from one outlet 18. This is a difference from the case of FIG. 4 of 2. That is, each thin film 10 of the crosslinked structure 12 is provided with a reaction unit 6 and a reference unit 7 as in the case of FIG. 4 of the second embodiment, and in this embodiment, the same as in FIG. 4 of the second embodiment. , Each of which has three reaction parts 6A, 6B, 6C and three reference parts 7A, 7B, 7C, and different enzymes 4A, 4B, 4C are fixed to the three reaction parts 6A, 6B, 6C, respectively. The first temperature sensor 20A, the second temperature sensor 20B, and the third temperature sensor 20B, which are provided in the respective reaction units 6A, 6B, and 6C by the selective contact thermal reaction with the substrate of the urine component corresponding to each. By the operational amplification of the temperature sensor 20C and the three reference parts 7A, 7B, and 7C paired with each other, the amount of the substrate component of urine, which is a liquid sample, is detected by detecting the heat of contact reaction between the substrate and the enzyme. , It is configured so that the amounts can be compared as needed. Therefore, the detection method and operation of predetermined substrate components in those liquid samples are the same as those in FIG. 4 of Example 2, and thus are omitted here.

FIG. 6 is a schematic plan view of another embodiment of the component comparison biosensor chip 100 of the present invention, which has a thin film 10 having a plurality of crosslinked structures 12 and one stream, as in FIG. 5 of the third embodiment. The liquid sample flowing in from the inlet 16 diverges and flows into each of the flow paths 17 formed in the thin films 10 of the plurality of crosslinked structures 12 connected in parallel, and is integrated and discharged to the outside from one outlet 18. The points are the same, but in this embodiment, one reaction unit 6A is located in the substantially central portion of the thin film 10 of each crosslinked structure 12, and one is located in the approximately central portion of another thin film 10. This is a case where one reference portion 7 is formed at substantially the center of the reaction portion 6B, which is one reaction portion 6, and the thin film 10 of another crosslinked structure 12, and this point is the same as that of FIG. 5 of Example 3. However, the fact that one reaction portion 6A, one reaction portion 6B, and one reference portion 7 are formed at substantially the center of the thin film 10 of the crosslinked structure 12 is shown in FIG. 3 of Example 1. Same as the case. Then, in the case of FIG. 3 of the first embodiment, the reaction units 6A and 6B and the reference unit 7 are formed in series in one flow path 17, but in this embodiment, since they are connected in parallel, the embodiment This is different from the case of FIG. 3 of 1. However, as in the case of FIG. 3 of Example 1, enzymes 4A and 4B, which are different enzymes, can be fixed to the reaction portions 6A and 6B by the electroclick method, and the reference portion 7 can be attached to the reference unit 7. Enzymes 4 are not fixed, and enzymes 4A and 4B having different substrate components in the liquid sample are used by using the first temperature sensor 20A and the second temperature sensor 20B, which are thermocouple temperature sensors formed therein. The method and operating principle of measuring the temperature change due to the contact thermal reaction with the reference temperature sensor 20R by the differential output is the same as that of FIG. 3 of the first embodiment. Therefore, the details of the operating principle will be omitted here.

FIG. 7 is a schematic plan view of another embodiment of the component comparison biosensor chip 100 of the present invention. Similar to FIG. 3 of Example 1, a plurality of thin films 10 are serialized in one flow path 17 in series. It is an example of the comparative biosensor chip in the case of being connected, and the difference from the case of FIG. 3 of Example 1 is simply the case of changing the enzyme immobilization method, and the case of FIG. 3 of Example 1. Is provided with an enzyme fixing electrode 72 for heat balance in each of the reaction parts 6A and 6B, and also in the reference part 7, and one of the heater electrode pads 71P for the heater 25 is used for the enzyme fixing electrode 72. Although it was shared with the enzyme fixing electrode pad 72P, in this example, different enzymes 4 were fixed on the surface of the surface enzyme fixing beads 90 such as polystyrene (PS), and each reaction part 6 was used. Since the surface enzyme fixing beads 90 are used for fixing the enzyme 4, the enzyme fixing electrode 72 is no longer necessary, and therefore the role as the enzyme fixing electrode pad 72P is no longer necessary. The point is different.

The method of placing the surface enzyme fixing beads 90 in the respective reaction parts 6 is to inject the surface enzyme fixing beads 90 into the reaction parts 6A and 6B from the inflow port 16 which is also used as the injection port 160 of the surface enzyme fixing beads 90. Pillars 81 acting as stoppers for indwelling the beads 90 in the first reaction section 6A and the second reaction section 6B are provided on the downstream side in the first reaction section 6A and the second reaction section 6B. For example, showing an example in which the surface enzyme fixing beads 90 of polystyrene beads are used, various diameters of the surface enzyme fixing beads 90 are sold, for example, 1 micrometer (μm), 5 μm, 10 μm, and so on. There are 15 μm, 20 μm, etc., and the desired enzyme 4 can be used for the surface enzyme fixing beads 90 by utilizing the carboxyl group on the surface thereof, the reaction with the drug EDAC, and the binding with the amino group of the enzyme which is a protein. Can be chemically bound to fix the enzyme 4 on the surface of the surface enzyme fixing beads 90. The fixed thickness of the enzyme depends on the molecular weight of the enzyme, but is about several μm. The surface enzyme fixing beads 90 on which the desired enzyme 4 is fixed are used together with the buffer solution in the flow path 17 as the injection port 160 (here, the inflow port 16 of the liquid sample) of the surface enzyme fixing beads 90. ) To inject. In this embodiment, the distance between the pillars 81 formed in the first reaction section 6A, which is the reaction section near the injection port 160 side of the flow path 17, is made larger than that of the second reaction section 6B, and the diameter is large. Only the surface enzyme fixing beads 90 are placed in the first reaction part 6A so that the enzyme 4A is fixed there, and the distance between the pillars 81 formed in the second reaction part 6B on the downstream side is set. This is a case where the surface enzyme fixing beads 90 having a small diameter smaller than the first reaction part 6A are placed in the second reaction part 6B so that the enzyme 4B is fixed there. Since the reference portion 7 provided in the thin film 10 between the first reaction portion 6A and the second reaction portion 6B does not form the pillar 81 for placing the surface enzyme fixing beads 90, the pillar 81 for placement is not formed. The surface enzyme fixing beads 90 pass through and arrive at the second reaction unit 6B and are placed and fixed.

In this embodiment, the distance between the pillars 81 formed in the first reaction section 6A, which is the reaction section near the injection port 160 side of the flow path 17, is widened to about 18 μm, and the first reaction section 6A is used. As a predetermined enzyme 4A fixed to the larger surface enzyme fixing bead 90 having a diameter of 20 μm to be indwelled, for example, trypsin is fixed, and a second reaction part which is a reaction part far from the injection port 160. The distance between the pillars 81 formed in 6B is narrowed to about 8 μm, and the smaller surface enzyme fixing beads 90 having a diameter of 10 μm to be placed in the second reaction section 6B, for example, the enzyme 4B that reacts with creatinine. An example of fixing creatinase is shown. In this way, the smaller surface enzyme-fixing beads 90 having a diameter of 10 μm on which creatinase as the enzyme 4B was immobilized passed through the pillars 81 at large intervals of the pillars 81 of the first reaction unit 6A near the injection port 160 side. , The reaction unit 6B, which is a reaction unit far from the injection port 160, stops at a small interval pillar 81 and is placed here. In this way, in the first reaction section 6A, the amount of urinary protein, which is a liquid sample, for example, a substrate in urine, is adjusted to the enzyme 4A, trypsin (it is better to use other degrading enzymes together). In the contact thermal reaction with, the first reaction unit 6A thermally detects differentially between the first temperature sensor 20A and the reference temperature sensor 20R, and the amount of creatinine, which is a substrate in urine, is detected in the second reaction. Part 6B thermally differentially detects the temperature of the second temperature sensor 20B and the reference temperature sensor 20R, and measures the amount of urinary protein and the amount of creatinine. Furthermore, these can be used to calculate the ratio of the amount of urinary protein to the amount of creatinine. For example, the ratio of albumin and creatinine, which are urinary proteins, is used as an index of renal diseases such as dialysis, and it is said that the value of the ratio of the amount of urinary protein to the amount of creatinine is important. The enzyme 4 may be any, but here, for example, trypsin is used as the enzyme 4A that reacts with the protein, but since trypsin decomposes only a part of albumin as a protein, another enzyme that further decomposes the protein. When used in combination with, it generates a large amount of heat and increases the sensitivity, which is preferable. In addition, trypsin itself, which is an enzyme, is also a protein, so it is a target for self-attack, but self-attack can be mitigated by reduction methylation or the like. Using the catalytic thermal reaction between the substrate component in a liquid sample such as urine and its corresponding highly selective enzyme and the temperature measurement and calibration data based on it, the amount of the substrate component and the comparison of the two component amounts can be compared. Since it is the same as the case shown in FIG. 3 of the above-described first embodiment, the detailed description thereof will be omitted here.

FIG. 8 is a schematic plan view of another embodiment of the component comparison biosensor chip 100 of the present invention. Similar to FIG. 4 of Example 2, a plurality of thin films 10 are serialized in one flow path 17 in series. It is an example of the comparative biosensor chip in the case of being connected, and the difference from the case of FIG. 4 of Example 2 is simply the case of changing the enzyme immobilization method, and the case of FIG. 4 of Example 2. Is provided with an enzyme fixing electrode 72 for heat balance in each of the first reaction section 6A, the second reaction section 6B, and the reference section 7, and one of the heater electrode pads 71P for the heater 25 is provided. It was shared with the enzyme fixing electrode pad 72P for the enzyme fixing electrode 72, but in this example, a different enzyme 4 is fixed on the surface using a surface enzyme fixing bead 90 such as polystyrene (PS). Then, the point that it was placed in each reaction part 6 and the surface enzyme fixing beads 90 were used for fixing the enzyme 4, so that the enzyme fixing electrode 72 became unnecessary, and therefore, the enzyme fixing electrode pad 72P The difference is that the role as is no longer necessary. Using the catalytic thermal reaction between the substrate component in a liquid sample such as urine and the corresponding highly selective enzyme and the temperature measurement and calibration data based on it, the amount of the substrate component and the comparison of the two component amounts can be compared. It is the same as the case shown in FIG. 4 of the above-mentioned Example 2, and the fixing by the placement of the surface enzyme fixing beads 90 is the same as the case of the above-mentioned Example 5, so the detailed description thereof is omitted here. ..

FIG. 9 is a schematic plan view of an embodiment of the component comparison biosensor chip 100 of the present invention, which has a plurality of thin films 10 having a crosslinked structure 12 and one inflow port 16 as in FIG. 6 of the fourth embodiment. The liquid sample flowing in from the above is divided and flowed into each of the flow paths 17 formed in the thin films 10 of the plurality of crosslinked structures 12 connected in parallel, and is integrated and discharged to the outside from one outlet 18. In addition to the above points, the first reaction section 6A, which is one reaction section 6 in the substantially central portion of the thin film 10 of each crosslinked structure 12, and the second reaction section 6, which is one reaction section 6 in the substantially central portion of another thin film 10. It is the same in that one reference portion 7 is formed in a certain reaction portion 6B and further in a substantially central portion of the thin film 10 of another crosslinked structure 12, but the difference from the case of FIG. 6 of Example 4 is. In the case of FIG. 6 of Example 4, the case of performing the enzyme fixation method by the electroclick method was shown, but the enzyme fixation method of this Example 7 is described in FIG. 7 and Examples of Example 5 described above. Similar to FIG. 8 of 6, the enzyme 4 is fixed to each reaction unit 6 by using the surface enzyme fixing beads 90. Further, in FIG. 9 of the present embodiment 7, as described above, the liquid sample flowing in from one inflow port 16 enters each of the flow paths 17 formed in the thin films 10 of the plurality of crosslinked structures 12 connected in parallel. In order to divide the flow and allow the flow to be integrated and discharged from one outlet 18 to the outside, an injection port 160 of the surface enzyme fixing beads 90 is provided separately for each of the different reaction sections 6. The difference is that the pillars 81 of the respective reaction portions 6 are spaced at the same distance so that the surface enzyme-fixing beads 90 having the same diameter may be used. This is a difference from FIG. Although not drawn in FIG. 9, the injection port 160 of the surface enzyme fixing beads 90 is covered with a cover prepared separately after injecting each of the predetermined enzymes 4. After fixing each of the predetermined enzymes 4A and 4B, the operating principle is essentially the same as in the case of FIG. 6 of Example 4 and FIG. 7 of Example 5 described above, and thus the detailed description thereof is omitted here. To do.

In one embodiment of the component comparison biosensor chip 100 of the present invention described above, two or three cases are shown in which the reaction unit 6 and the reference unit 7 formed on the thin films 10 of each of the plurality of crosslinked structures 12 are provided. However, the number of them may be further increased to the same substrate 1, and the substrate size is increased by that amount, but at the same time, it is possible to analyze the substrate components in a multi-item liquid sample, which is preferable. In addition, the measurement concentration range of each substrate can be adjusted by adjusting the amount of enzyme 4. For example, in the thermal reaction between the substrate glucose and the corresponding enzyme glucose oxidase, the dissolved oxygen in the liquid sample is involved in the reaction, the thermal reaction is suppressed due to the lack of dissolved oxygen, and the glucose concentration and the thermal reaction output are linear. It deviates from the above and shows a tendency to saturate. Therefore, in order to measure a high-concentration substrate, it is better to adjust the amount of enzyme 4 to a predetermined amount and fix it. Further, as a result of the reaction of the substrate with the enzyme, a reaction product is generated, which needs to be promptly removed from the reaction unit 6 or the like. For this purpose, the measuring device may be provided with a flow rate adjusting means for allowing the liquid sample to flow at a predetermined flow rate. Further, in the above description, the fixation of the enzyme 4 to the reaction portion 6 is described only in the case of using the electroclick method or the surface enzyme fixation beads 90, but of course, the fixation is not limited to these methods, and for example, A method of immobilizing an enzyme adsorbed on thin-film silica gel is also permitted. In addition, although urine was included in the example as a liquid sample, blood, sweat, tears, saliva, etc. can also be targeted. Further, when the surface enzyme fixing beads 90 are used, the example in which the surface enzyme fixing beads 90 are not placed in the reference portion 7 is described in the above-described embodiment, but the thermal contact with the reaction portion 6 is further increased. In order to obtain a balance, it is better to provide a pillar 81 also in the reference portion 7 so that the beads in which the enzyme is not fixed by the surface enzyme fixing beads 90 are placed in the reference portion 7. At this time, if necessary, it is preferable to provide the substrate 1 with a bead injection port 160 in which the enzyme is not fixed to the reference portion 7.

FIG. 10 shows a schematic cross-sectional view of an embodiment of the biosensor module 500 of the present invention. The component comparison biosensor chip 100 is provided with a fine flow path 17 of the crosslinked structure 12, and careless touching with a finger or tweezers is dangerous because there is a risk of destruction. Further, when used in a component comparison biosensor such as a urine sugar meter or a blood glucose level sensor, the enzyme 4 is a protein, and therefore, depending on its storage state, inactivation and deterioration of catalytic action over time are problems. Become. Therefore, although the enzyme 4 can be used repeatedly by washing or the like, it becomes necessary to replace the component comparison biosensor chip 100 due to deterioration. In this case, it is difficult for a general user to replace only the component comparison biosensor chip 100. Therefore, there is a need for modularization in which a component comparison biosensor chip 100 is attached and which can be easily replaced as a cassette. The present invention provides a biosensor module 500 that includes a component comparison biosensor chip 100 and can be easily replaced in a cassette manner.

Since the flow path 17, the inflow portion 160, the discharge portion 180, and the like of the module member 510 of the biosensor module 500 are fine and need to ensure accuracy, they may be formed by a plastic 3D printer. Further, the wiring from the various electrode terminals of the component comparison biosensor chip 100 is, for example, crimp-contacted with the bump electrode 310, provided with the terminal pin 350, and inserted into an external connector for electrical wiring. The component comparison biosensor chip 100 is fixed by, for example, a sensor chip fixing plate 520 so that it can be fixed with one touch. Further, the liquid sample flows in from the inflow section 160, passes through the flow path 17 from the inflow port 16 of the component comparison biosensor chip 100, and further passes through the discharge section 180 from the outflow port 18 so as to be discharged to the outside. ing. Component comparison The biosensor chip 100 and the flow path 17 of the module member 510 are prevented from leaking by packing 530. The biosensor module 500 may be inserted and fixed to the connector fixing plate 580 at the attachment location of the component comparison biosensor 1000 shown in FIG. 12, which will be described later, with one touch.

FIG. 11 shows a schematic view of an embodiment of the component comparison biosensor 1000 of the present invention. In this embodiment, the biosensor module 500 shown in the above-mentioned Example 8 is provided in a plastic case 570 with the connector 550 provided on the connector fixing plate 580 and the connector 550 for the component comparison biosensor chip 100. And, the terminal pin 350 of the biosensor module 500 is inserted and connected to electrically connect to the electronic circuit 660 through the wiring 400. In the component comparison biosensor 1000 of the present invention, a suction pump is used as the driving means 610 for the inflow and outflow of the liquid sample, and the flow of the liquid sample can be rapidly controlled by the solenoid valve as the valve 600 via the tank 620. I am trying to do it. These controls are controlled by a microcomputer of an electronic circuit 660 built in the component comparison biosensor 1000 so that timing and flow start and stop are performed according to predetermined rules. Further, from each output signal based on the thermal reaction between the enzyme 4 fixed in each flow path in the component comparison biosensor chip 100 and the substrate corresponding to them, the concentration of each substrate and the like are converted into calibration data prepared in advance. Based on this, the calculation is performed by the microcomputer, and the characters are displayed on the liquid crystal display as the display unit 670.

When the component comparison biosensor chip 100 used for the component comparison biosensor is manufactured using an SOI substrate which is a Si single crystal, the MEMS technique can be easily applied and is suitable. A power supply circuit, an amplifier circuit, an arithmetic circuit, and a control circuit can also be integrated on the sensor chip made of the SOI substrate by integrated circuit technology, or these can be integrated on another semiconductor substrate or the like to be modularized. By doing so, it is possible to provide an extremely compact component comparison biosensor 100. The power supply circuit is a circuit related to driving the driving means 610, the valve 600, etc. and supplying power to other circuits, and the amplifier circuit is a first temperature sensor 20A, a second temperature sensor 20B, and a reference temperature sensor. 20R, a circuit that amplifies the output of temperature sensors such as the absolute temperature sensor 23 and their differential signals. Of course, when the heater 25 is not operated, it is not necessary to drive or control the heater 25. The calculation circuit includes outputs from temperature sensors such as the first temperature sensor 20A, the second temperature sensor 20B, the reference temperature sensor 20R, and the absolute temperature sensor 23, subtraction and integration based on these, and output signals thereof. This is a circuit that performs arithmetic processing such as conversion to the amount of a specific substrate component in a liquid sample and calculation of a ratio to these under calibration data prepared in advance by further combining with a memory circuit. Further, the control circuit is a solenoid valve of the valve 600, which controls the inflow start and stop of the liquid sample and the cleaning liquid, controls the repetition and its cycle, and further controls the temperature such as setting the signal integration time. It is a circuit that also performs feedback control and the like as needed. FIG. 12 shows a block diagram of an embodiment of a schematic configuration diagram when the component comparison biosensor 1000 of the present invention is driven.

In the above description, urine is mainly used as the body fluid of the liquid sample, but similarly, using body fluids such as blood, sweat, saliva, and tears, the substrates of various specific substrate components and the enzyme of enzyme 4 are used. In addition, if necessary, by using a combination using a coenzyme or the like, it is possible to identify various specific substrate components and types of the enzyme 4, compare their amounts, compare these amounts, and calculate the ratio for a short time, for example, 10 seconds. It can be measured within a certain degree. Further, in the above description, human body fluid is used, but it is not necessarily limited to human and may be an animal. In addition to body fluid, the amount of a specific substrate in a solution such as vegetables, fruits and seaweed can be detected. This is made possible by measuring the temperature increase ΔT due to the thermal reaction generated by the combination of the substrate and the corresponding enzyme or coenzyme that specifically performs a selective catalytic reaction. Further, in the above-described embodiment, the case where several thin films 10, 10a, 10b, 10c and the like having a 12-shaped crosslinked structure are shown is shown, but it goes without saying that 10 or more thin films can be used if necessary. No. Further, in the above description, the case where the SOI substrate is used for the component comparison biosensor chip 100 is mainly shown, but an inexpensive material such as heat-resistant plastic is used as the substrate 1 and the thin film 10, and the first temperature sensor 20A is further used. As the second temperature sensor 20B and the reference temperature sensor 20R, a thermocouple or a thermopile may be formed by vapor deposition or sputtering of a thermoelectric material, or further by printing or the like.

In the drawings of the above-described embodiment of the present invention, the parts having the same concept described in the claims are designated by the same reference numerals. Further, in the examples of the present invention, preferred embodiments of the present invention have been described with reference to the accompanying drawings, but the present invention is not limited to the present examples, and the gist, action and effect of the present invention are the same. However, of course, there can be various modifications. It is clear that a person skilled in the art can come up with various modifications or modifications within the scope of the technical ideas described in the claims, and of course, the technical scope of the present invention also includes them. It is understood that it belongs to.

In the component comparison biosensor chip 100 of the present invention, a first temperature sensor 20A, a second temperature sensor 20B, etc., which are connected to a thin film 10 thermally separated from the substrate 1 by a MEMS technique by a flow path 17 through which a liquid sample flows, are reaction portions. The reference temperature sensor 20R is formed in the reference portion 7, and further extends from the inflow port 16 of the substrate 1 via the reaction portion 6 and the reference portion 7 of the thin film 10. A flow path 17 extending to an outlet 18 formed in the same substrate 1 region straddling the cavity 40 and facing the cavity 40 is provided, and different specific enzymes corresponding to each formed in different reaction portions 6 in the flow path 17 are provided. 4 is fixed so that the temperature rise due to the enzymatic reaction can be measured from an external electrode pad provided around the substrate 1. Then, the temperature sensor of the reaction unit 6, for example, the first temperature sensor 20A and the reference temperature sensor 20R of the reference unit 7 are arranged to have the same shape and symmetry so as to be thermally balanced. Therefore, even if a liquid sample such as urine, which is significantly deviated from the ambient temperature, is poured into the flow path 17, high-precision temperature difference measurement can be realized by these differential detections. Further, since the inside of the flow path 17 can be cleaned with a cleaning liquid, a component comparison biosensor that can be used many times can be provided. The biosensor module 500 of the present invention modularizes the component comparison biosensor chip 100 so that it can be easily incorporated into and replaced with the component comparison biosensor 1000 with a single touch. In addition, the component comparison biosensor 1000 of the present invention contains substrates and enzymes that are substances to be detected contained in body fluids such as urine, sweat, saliva and blood, which are substances derived from living organisms, and the amount of temperature increase due to these thermal reactions. There is an advantage that many substrates and enzymes can be measured at the same time within a few seconds, and since the enzyme is not consumed, the catalytic activity is reduced, but it can be used many times after washing, so it is cheap daily health. It can be useful for diagnosis. Further, since the component comparison biosensor 1000 of the present invention can easily quantify the amount of a substrate or enzyme which is a specific substrate component and the ratio thereof, and can be made compact for portable use, it can be installed in a household toilet or used. It can be used in a wide range of fields, such as being able to be used in place of a urine test strip for examining color changes in mass examinations.

1 Substrate 4, 4A, 4B, 4C Enzyme 6 Reaction part 6A First reaction part 6B Second reaction part 6C Third reaction part 7,7A, 7B, 7C Reference part 10, 10a, 10b, 10c Thin film 11 SOI Layer 12 Bridge structure 15 Base substrate 16 Inlet 17 Flow path 18 Outlet 20A First temperature sensor 20B Second temperature sensor 20C Third temperature sensor 20R, 20RA, 20RB, 20RC, ... Reference temperature sensor 23 Absolute Temperature sensor 25 Heater 26 Measurement point (warm contact)
27 Reference point (cold contact)
40 Cavity 50 Insulation film 51 BOX layer 60 Ohm contact 70P, 70AP, 70BP, 70CP Temperature sensor electrode pad 70R, 70RAP Reference temperature sensor electrode pad 71P Heater electrode pad 72 Enzyme fixing electrode 72P Enzyme fixing electrode Pad 73 Counter electrode 73P Counter electrode pad 74 Reference electrode 74P Reference electrode pad 75P Common electrode pad for SOI layer
76 P Electrode pad for absolute temperature sensor
80 Unevenness 81 Pillar 90 Surface enzyme fixing beads 100 Component comparison Biosensor chip 105 Notch 110 Insulation separation groove for SOI layer 120a, 120b Thermoelectric conductor 150 Cover 160 Injection port 161 Inflow part 180 Discharge part
310 Bump electrode 350 Terminal pin 400 Wiring 500 Biosensor module 510 Module member 520 Sensor chip fixing plate 530 Packing 550 Connector 560 Adapter 570 Case 580 Connector fixing plate 600 Valve 610 Drive means 620 Tank 650 Battery 660 Electronic circuit 670 Display 1000 Component comparison Biosensor

Claims (8)

A plurality of biosensor chips that detect a specific substrate component in a liquid sample flowing in from at least one inflow port by the heat of reaction of catalytic action with an enzyme corresponding to the substrate component, which is thermally separated from the substrate. At least one flow path through which the liquid sample passes is formed in the thin film, a reference portion is formed in at least one of the thin films in the flow path, and the remaining thin film. Each of the above has a reaction part that is substantially equivalent to the reference part and is formed in the same flow path, and is connected in series through the flow path. Different predetermined enzymes corresponding to the specific substrate components are fixed to the first reaction part, the second reaction part, and the like, but the enzyme is not fixed to the reference part, and further. The first reaction unit, the second reaction unit, and the reference unit are provided with a first temperature sensor for detecting the heat of reaction, a second temperature sensor, and a reference temperature sensor, respectively. The temperature change based on the heat of reaction at each of the reaction sections of the first temperature sensor, the second temperature sensor, etc. is measured with reference to the reference temperature sensor, and information on the amount of each of the substrate components, Alternatively, a component comparison biosensor chip characterized in that it is configured so that comparable information on those amounts can be obtained. The component comparison biosensor chip according to claim 1, wherein the reference unit is provided at a position substantially intermediate with the reaction unit such as the first reaction unit and the second reaction unit. The component comparison biosensor chip according to any one of claims 1 or 2, wherein the first temperature sensor, the second temperature sensor, and the reference temperature sensor are all temperature difference sensors. Pillars for placing the beads for fixing the surface enzyme are provided in the first reaction part, the second reaction part, etc. in the flow path formed on the same thin film, and the injection of the beads for fixing the surface enzyme is provided. The component comparison biosensor chip according to any one of claims 1 to 3 , wherein the inlet is formed at a position different from the inlet of the liquid sample on a substrate of the same flow path through which the liquid sample flows. Pillars having different intervals for placing beads for fixing surface enzymes are provided in the first reaction part, the second reaction part, and the like in the flow path formed on the same thin film, and the surface enzyme is fixed. Using the bead dimensions of different diameters of the beads, pillars are arranged and formed so that the surface enzyme-fixing beads of the predetermined different enzymes can be placed in the first reaction section, the second reaction section, and the like, respectively. The component comparison biosensor chip according to claim 1 or 2. The component comparison biosensor chip according to any one of claims 1 to 5 , wherein the shape of the flow path in each reaction section and the reference section is streamlined at least in the width direction. Of the component comparison biosensor chips according to any one of claims 1 to 6, the first reaction part and the second reaction part, which are at least two reaction parts, are specified to obtain the ratio in the liquid sample. A component comparison biosensor chip in which a predetermined enzyme corresponding to the substrate component of the above is fixed is mounted, and each temperature change based on the reaction heat is based on the reference temperature sensor formed on the component comparison biosensor chip. Then, the first temperature sensor and the second temperature sensor are measured by differential detection, and at least the amounts of the two substrate components or their ratios can be obtained based on predetermined calibration data. A component comparison biosensor characterized by the fact that it was made. At least the power supply circuit, amplifier circuit, the arithmetic circuit and the control comprises a circuit, the claims 7 to be able to obtain the amount or information regarding the amount of the ratio of at least the two matrix components specific matrix components in a liquid sample The described component comparison biosensor.

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