JP2019128261A - Disposable biosensor chip and biosensor fitted with the same - Google Patents

Abstract

To provide a disposable biosensor chip having a thermal sensor for measuring creatinine in urine and the like, and an ion sensor for measuring ions such as Na, and a biosensor for concentration analysis of various urine components by creatinine correction which can attach and detach this.SOLUTION: A biosensor includes a thermal sensor 2 and ion sensor 3, is equipped with a reaction unit 6A and B of the thermal sensor in a flow path through which the liquid sample passes, detection temperature sensors 20A, B are disposed in contact with or in the vicinity of the enzyme, at least either of the upper and lower surfaces of the flow path of a reaction unit is sandwiched by a material having a small thermal conductivity of less than a half of Si, a disposable chip-like disposable biosensor chip integrated bonding by overlapping a base substrate 1 with any one of a chip A having a thermal sensor, a chip B having both a thermal sensor and an ion sensor, a chip C having only an ion sensor, or a combination of these, and a biosensor chip attaching-and-detaching mechanism.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a disposable biosensor sensor chip for detecting a specific substrate component or ion component in a liquid sample such as urine, and a biosensor equipped with the biosensor sensor chip.

The inventors of the present invention have found that glucose, protein, and creatinine as substrates are enzymes having specific selectivities, for example, glucose oxidase, trypsin, and creadine, respectively, using a calorimetric (thermal detection method) biosensor using enzymes. By fixing the tininase to the reaction part, a catalytic catalytic thermal reaction (hereinafter referred to as a thermal reaction) between these minute substrates derived from living organisms in a liquid sample such as urine (detected) and the corresponding enzyme It has been demonstrated that it is possible to measure the temperature change due to) with high sensitivity and high accuracy. Compact, high-accuracy and inexpensive component for determining the concentration of a substrate in a liquid sample such as urine and the ratio of components of multiple substrates from simple measurement of reaction heat generated during the reaction between the substrate and enzyme It has been proposed to provide a comparative biosensor chip, a biosensor module on which this chip is mounted, and a component comparison biosensor using these chips (Patent Document 1). This thermal detection method has made it possible to correct creatinine such as the above-mentioned urinary protein and urinary sugar.

Detection of ions such as sodium (Na) and potassium (K) in liquid samples such as urine and blood has been required. Conventionally, an ion electrode method (ion selective electrode method) (Patent Document 2) or an ion 2. Description of the Related Art There has been an ion sensor using an ion sensitive layer containing an ionophore of sodium (Na) or potassium (K) using a sensitive FET (ISFET) (Patent Document 3) or the like. It is formed on the gate insulating film of ISFET, and the ion concentration in these liquid samples is measured from the current change between the source (S) and drain (D) of these ISFET samples by utilizing the extremely high ion selectivity of ionophore It has been reported. However, these ISFETs are not a thermal detection method, but a manufacturing process of an enzyme immobilization reaction part formed on a thin film thermally separated from a substrate using a thermal reaction using an enzyme which is the above-described protein of the thermal detection method. There is a problem in the integrity, and forming the sensing part of the thermal detection method on the same substrate has been a major issue.

When urine is used as a liquid sample to be detected, it is required to measure the concentration of each component such as urine sugar and urine protein as a substrate. For example, the concentration of protein (eg, albumin) and the concentration of creatinine There is a demand for these measurements from the information that the evaluation of the ratio is suitable for grasping the progress of diseases such as kidney disease. For example, the ratio (ratio) between the concentration of protein in urine and the concentration of creatinine There has been a demand for direct measurement. This is because there are some differences between men and women, but because "the daily adult creatinine excretion is approximately 1 g", based on creatinine in urine of any volume, proteins and glucose contained in it This is because it is possible to evaluate stable urinary proteins and the like that do not depend on the intake concentration of water, etc., by obtaining the ratio of the concentration of the above (correction of creatinine). In the measurement of urinary components by the conventional electrochemical method, hydrogen peroxide (H2O2) must be generated by an enzymatic reaction, and the concentration of urine sugar, etc., can be determined by measuring the redox current of the generated H2O2. Although it was possible, it was extremely difficult to detect proteins that are difficult to generate H2O2.

Recently, exercise to recommend salt reduction has become popular because of the relationship between salt intake and hypertension. Among them, by determining the ratio of sodium (Na) to creatinine in urine (creatinine correction of Na in urine), it is possible to evaluate the intake concentration of Na ion, that is, salt, using one drop of urine as urine at any time. It has become desirable to measure the urinary concentration of creatinine, which is a biological substance, and simultaneously measure the Na ion in the urine. Instead of measuring these creatinine and Na ion concentrations individually and independently, a thermal sensor for detecting creatinine as described above is used on the same substrate for simultaneous measurement using a compact sensor. It is more preferable to integrate an ion sensor for measuring Na and Na ions, for example, an ISFET. However, as described above, for the thermal detection method of creatinine using the enzyme creatininase, the thin film thermally separated from the substrate, the flow path, and the immobilization of the protein enzyme to the reaction part in the flow path There are significant hurdles in the fabrication process for ISFET and the fabrication process of an ISFET with an ion sensitive membrane layer that includes an ionophore for ion detection on the substrate. The inventor previously proposed an ion biosensor in which a thermal sensor and an ISFET are mounted on the same substrate (Patent Document 4).

In addition, when using urine as a liquid sample, there is a sense of resistance to the re-use of biosensors used by others for hygiene and sensitivity, problems of the degree of washing during the previous use, and further, enzymes and ionophores. There are issues such as the problem of deterioration of, and the demand for disposable biosensor chips and biosensors equipped with the same is increasing.

Japanese Patent Application No. 2016-255431 WO 2010/087383 Special Table 2009-532669 Japanese Patent Application No. 2017-160770

  The present invention is a thermal type based on a thermal detection method for detecting biologically derived substances such as creatinine in consideration of the tendency to seek a disposable biosensor chip, particularly in the case of handy biosensors, in analyzing conventional urine components. A disposable biosensor chip equipped with a sensor, an ISFET that measures the concentration of specific ions such as sodium (Na), and an ion sensor such as an ion electrode method, and a simple structure with a high-speed response that can be detachably mounted. An object of the present invention is to provide a biosensor that can perform concentration analysis of various urine components by creatinine correction using urine as needed.

  In order to achieve the above object, the disposable biosensor chip according to claim 1 of the present invention selectively responds to a specific substrate component and its concentration in the liquid sample to be detected to the substrate component. A chip A provided with a thermal sensor for detecting a temperature change due to heat of catalytic reaction with an enzyme by a temperature sensor for detection, an electrode for taking out an electric signal from at least the chip A, and a base substrate having electric wiring therefor The biosensor chip has a reaction part in a flow path through which the liquid sample passes, the enzyme is fixed to the reaction part, and the temperature sensor for detection is disposed in contact with or near the enzyme. In other words, at least the upper and lower surfaces of the flow path of the reaction section are sandwiched by a material (including gas) having a thermal conductivity smaller than half of the thermal conductivity of silicon (Si). In other words, the chip A and the base substrate are overlapped and joined together to form a single disposable chip.

In a biosensor for a thermal sensor, a reaction part having a temperature sensor for detection that detects a temperature change due to reaction heat of catalytic action between a substrate component and an enzyme that selectively responds to the substrate component is arranged in a flow path, It is better to form the temperature sensor for detection on a thin film having a bridge structure or a diaphragm structure in order to thermally separate it from the substrate of the chip A. This thin film flows to a liquid sample such as urine so that reaction heat is not easily released. It is good to make it a structure which does not contact from the outside other than a way. For this purpose, the chip A and the base substrate are integrated by being overlapped and joined so as to be watertight with at least a flow path through which the liquid sample passes. In the reaction part of the chip A, the liquid sample to be detected flowing in the flow channel is prevented from leaking outside, and the reaction heat is less likely to escape to the outside than Si, which is the main material of the chip A. Cover with a material that has as low thermal conductivity as possible, for example, a cavity, a foam or a plastic plate. In addition, when a disposable biosensor chip is mounted and analyzed for components in a liquid sample such as urine, it is formed at the end of the disposable biosensor chip, for example, a handheld biosensor body. To form a small thin chip with a size of, for example, about 1 cm square, about 1 mm, which can be detached from the biosensor main body so that an electrical signal can be sent through the electrode pad which is an external output terminal. Is good.

In addition, when silicon (Si) single crystal is used as the material of the chip A, the conventional MEMS technology can be easily used, and in particular, the SOI substrate can be used to fabricate the known technology using the SOI layer. It is better to configure the thermal sensor part to have a microscopic dimension, for example, a length of about 1 mm, a thickness of about 0.01 mm, a bridge structure with a width of about 0.2 mm, and a diaphragm structure with a diameter of about 1 mm. A highly sensitive semiconductor thermocouple as a temperature sensor for detection and a pn junction diode as an absolute temperature sensor can be formed, and it is also convenient for the formation of an enzyme immobilization reaction part and the formation of an ISFET. Integrated circuits to be the peripheral circuits can be formed on the same substrate.

As described above, “at least the upper and lower surfaces of the flow path of the reaction part are structured to be sandwiched between materials (including gas) having a thermal conductivity smaller than half of the thermal conductivity of silicon (Si)”. In the MEMS technology using silicon (Si), when a flow path through which a liquid sample passes is formed, the area near the enzyme generates heat due to the contact thermal reaction between the enzyme and the substrate, and the liquid sample in the flow path is Heating is performed from the vicinity of the enzyme, and heat propagates mainly to the liquid sample in the vicinity of the reaction section, resulting in a temperature increase in the reaction section region. This temperature increase is detected by the above-described temperature sensor for detection. In this case, it is preferable that the reaction area be surrounded by a heat insulating material, and the smaller the volume, the higher the temperature, the higher the sensitivity. However, since silicon (Si) has a thermal conductivity as high as that of metal, it is better that the flow path in the reaction region is relatively small in the region in contact with silicon (Si). For this reason, at least the upper and lower surfaces of the flow path of the reaction section are desired to be sandwiched by a material having a thermal conductivity as small as possible than that of silicon (Si). In particular, in the case where the flow path is provided in the chip A, a space (cavity) can be formed in the top and bottom in the structure, so that the heat insulation effect is enhanced by filling it with air (gas). be able to.

In the disposable biosensor chip according to claim 2 of the present invention, the flow path is provided in the chip A.

When the chip A uses the SOI substrate as described above, the heat capacity and the heat conduction floating in the air consisting of the SOI layer in the region of the reaction portion where the detection temperature sensor for the thermal type sensor is formed A small cross-linked structure, a diaphragm structure, a temperature sensor for detection, and an absolute temperature sensor to be described later can also be advantageously formed by a highly sensitive semiconductor sensor. Furthermore, when a fine channel made of a photoresist such as SU-8 is formed on the thin film having such a crosslinked structure or diaphragm structure by the above-mentioned MEMS technology, the crosslinked structure, the diaphragm structure and the channel are integrated. In addition, it is possible to provide a gap (cavity) outside these, and this area can be filled with air so that heat generated by the exothermic reaction cannot be easily released from the flow path to the outside. Can provide a fast response thermal sensor.

In the disposable biosensor chip according to claim 3 of the present invention, the flow path is provided between the chip A and the base substrate.

The flow path is provided with a reaction unit as a thermal sensor, and in the reaction unit, it is necessary to fix an enzyme that generates heat in contact with the substrate. Immobilization of the enzyme in the reaction zone in the closed channel is limited to the immobilization method such as placement in the reaction zone with bead immobilized enzyme, enzyme immobilization by electroclick method, etc. If the path is still open, it is often convenient because the enzyme can also be immobilized directly from above the reaction zone. The released state refers to a state in which the reaction part of the chip A is exposed before the chip A is stacked and integrated on the base substrate.

A disposable biosensor chip according to claim 4 of the present invention comprises a reaction part on which the enzyme is immobilized and a reference part having the same shape as that of the reaction part but on which the enzyme is not immobilized, It is a case where the reference part is provided with the detection temperature sensor and the reference temperature sensor, respectively.

It is desirable that the reference part has a thermal characteristic other than the exothermic reaction by the enzyme having the same thermal characteristics as the reaction part with respect to the reaction part where the enzyme is immobilized. Therefore, it is desirable not to immobilize the enzyme, but to make the environment for the other shapes and materials and the liquid sample equal to the reaction part. The detection temperature sensor and the reference temperature sensor provided in the reaction unit and the reference unit need to have the same shape on the same measurement principle.

In the disposable biosensor chip according to claim 5 of the present invention, the temperature sensor for detection and the temperature sensor for reference are temperature difference sensors.

Regarding the temperature sensor of the disposable biosensor chip of the present invention, a temperature sensor such as a high-sensitivity and high-accuracy temperature sensor for detection and a temperature sensor for reference is created by a thermocouple which is a temperature difference sensor, and the SOI layer is also used for this thermocouple. When used as one of the thermal conductors, it is often convenient because it can be easily manufactured and a highly sensitive semiconductor thin film thermocouple can be constructed. Of course, a thermopile in which thermocouples are connected in series may be used. In addition, in the semiconductor thin film thermocouple, since the Seebeck coefficient is larger when the resistivity is larger, a large thermoelectromotive force is generated with respect to the same temperature difference, so that the resistivity is, for example, 0.5 Ωcm rather than 0.01 Ωcm. The degree is high sensitivity detection. However, if the resistivity is too large, it will be difficult to form an ohmic electrode, so an appropriate resistivity may be selected.

In the disposable biosensor chip according to claim 6 of the present invention, the reference part is formed in a branched flow path different from the reaction part.

The reference part can also be formed in a form connected in series with the reaction part in one flow path, but here, it is formed in a form connected in parallel to a different flow path branched from the flow path with the reaction part. This is the case. In the case where a plurality of reaction parts for different enzymes are formed in the branch flow path, it is preferable when one reference part is used as a common reference part.

A disposable biosensor chip according to a seventh aspect of the present invention includes a reaction section that fixes each enzyme corresponding to a plurality of the substrate components, and the flow path and the detection temperature sensor corresponding to each reaction section. Is the case.

In the reaction part, an enzyme having high selectivity, which specifically reacts with a specific substrate, is immobilized. For example, for creatinine as a substrate, creatininase, which is an enzyme that selectively and specifically reacts with this specific substrate, is preferably immobilized. Of course, not only creatinine but also proteins and sugars may be used as substrates. Furthermore, not only a single substrate but also a plurality of substrates can be measured simultaneously, and a plurality of reaction units are provided to correspond to each of them. Enzymes such as trypsin and glucose oxidase can also be immobilized. In such a case, the liquid sample may be diverted and distributed, for example, to the flow path having the respective reaction parts on a plurality of thin films in a plurality of cross-linked structures. In addition, one flow path through which the liquid sample passes is formed so as to pass through a plurality of thin films having a bridge structure, and a region of the first reaction part including the first temperature sensor for detection is further provided. The structure of passing through the area of the second reaction portion provided with the temperature sensor for detection, ie, from the front of the substrate to the opposite position across the cavity constituting the flow path on the cross-linked structure of the thin film It returns to the front through the channel formed in the adjacent crosslinked structure thin film, and further returns to the substrate at the opposite position through the channel formed in the adjacent crosslinked structure thin film. With a structure, a liquid sample to be detected such as urine containing a specific substrate component can pass through a single flow path formed on a substrate provided with a thin film having a plurality of cross-linked structures zigzag.

For example, when two specific substrate components are protein and creatinine which are substrates in urine as a liquid sample, it is preferable to use trypsin and creatininase which are enzymes respectively corresponding to them as enzymes. Then, protein and creatinine, which are substrates in the liquid sample introduced through the channel, trypsin (for example, immobilized on the first reaction part) and creatininase (for example, the second enzyme) corresponding to each of them. The first detection temperature sensor and the second detection temperature sensor formed in the reaction part (or in the vicinity) are increased in temperature by catalytic catalytic thermal reaction with the reaction part (fixed in the reaction part), Based on the reference temperature sensor provided in another channel, detection is performed based on the magnitude of the signal including the time course, and the concentration of each of the substrate protein and creatinine and their concentration ratio, eg, protein / The creatinine ratio can also be calculated by using those differential amplified signals.

The disposable biosensor chip according to claim 8 of the present invention further comprises a chip B further comprising an ion sensor provided with an ion sensitive layer selectively sensitive to a specific ion component in the liquid sample. This is the case of mounting in place of the chip A.

The chip A had a thermal sensor but no ion sensor. The chip B is a case where the chip A is also provided with an ion sensor, and is superposed on the base substrate, for example, a concentration based on the temperature rise of the reaction part due to an enzyme thermal reaction such as creatinine or protein by a thermal sensor. This is a case where the measurement and further the concentration measurement of ions such as sodium (Na) ions and potassium ions can be simultaneously performed by the ion sensor. It is preferable to attach the chip B to the base substrate because electrical wiring and electrical insulation thereof are easy, and such a compact disposable biosensor chip can be provided.

The disposable biosensor chip according to claim 9 of the present invention is an ion sensor according to ISFET or ion electrode method as the ion sensor.

An ion sensing ISFET in which an ion sensitive layer selectively sensitive to a specific ionic component in a liquid sample is formed on a gate insulating film is used, or similarly, an ion sensitive to selectively sensitive to a specific ionic component This is a case where a layer is formed on the electrode and an ion electrode method (ion selective electrode method) is used to measure a potential change with respect to the reference electrode with respect to the ion concentration of the specific ion.

The ISFET has a structure equivalent to that of the ion detection ISFET and has a reference ISFET that shares the reference gate electrode with the ion detection ISFET. An ion sensitive layer is formed on the gate insulating film of the reference ISFET. It is good to not have a structure. Only one ISFET may be used to detect Na ions or K ions, and this may be used as an ion detection ISFET having an ion sensitive layer containing an ionophore selective to Na ions or K ions. This correction is often difficult due to the potential or voltage drop of the liquid sample at the electrode. For this purpose, it is better to provide a reference ISFET not formed with an ion sensitive layer in addition to the ion detection ISFET and arrange at least one shared reference gate electrode. By the differential amplification operation of the outputs of the detection ISFET and the reference ISFET, the potential and voltage drop components at the above-mentioned reference gate electrode are erased. The reference gate electrode is a conductive material such as a stable metal that is resistant to a liquid sample at a position near the middle of the ion detection ISFET and the reference ISFET on the same substrate. Can be formed.

In an ion sensor based on the ion electrode method, as in the case of ISFET, an ion detection electrode whose detection electrode is covered with a detection ion-sensitive layer, and a reference electrode (reference electrode) whose detection electrode is covered with a reference film layer for reference And, as with the ion detection electrode and the reference electrode, may also be provided with a common electrode immersed in the same liquid sample. In this case, the potential difference between the ion detection electrode and the reference electrode with respect to the common electrode may be differentially amplified using three terminals with the common electrode as ground (common ground). Note that the reference electrode (reference electrode) may be replaced with a common electrode and the two terminals may be used to measure the potential difference of the ion detection electrode with respect to the reference electrode. In addition, the reference film layer is the same component as the ion sensitive layer coated on the ion detection electrode, but the thickness is different from that of the ion sensitive layer coated on the detection electrode, for example, a reference film The layer can also be used as thin as about one tenth of the film thickness on the detection electrode. Silver chloride films are recommended as the ion detection electrode, reference electrode, and common electrode, but stable metals such as gold (Au) and platinum (Pt) may be used, and one ion sensor is the same. The use of this metal is preferable because an unnecessary potential difference can be offset. In the above description, as commonly used as an ion sensor based on the ion electrode method, the potential difference between the ion detection electrode and the reference electrode is measured with respect to the common electrode. You may make it measure by the electric current difference which flows.

A disposable biosensor chip according to claim 10 of the present invention comprises a chip C different from the chip A and the ion sensor that selectively reacts with a specific ion component in the liquid sample. This is a case where the chip C is mounted on the same base substrate and hybridized.

When an ion sensor such as an ISFET for ion detection is formed on the chip A on the same substrate as the thermal sensor unit, only one chip is required, so that it is possible to introduce a liquid sample such as urine, to form a flow path, and to be detached. Therefore, there is an advantage that the problem of electrical insulation of the wiring to the electrode pad from the liquid sample can be solved and a compact disposable biosensor chip can be provided. However, the problem of the yield of the product based on the difference in the manufacturing process of ion sensors, such as ISFET, and a thermal-type sensor part remains. For this reason, if a disposable biosensor chip that is hybridized by providing an ion sensor such as an ISFET or an ion electrode method ion sensor on a semiconductor chip C different from the chip A, the chip A having a thermal sensor is provided. If the semiconductor chip C including an ion sensor such as ISFET is manufactured independently and hybridized, it leads to an improvement in the product yield, and thus the form increases, but an inexpensive disposable biosensor chip can be provided.

In the disposable biosensor chip according to the eleventh aspect of the present invention, the liquid sample introduced from at least one inflow port is divided into each branched flow path, and the reference part of the thermal sensor and each flow It is a case where it is made possible to flow out from at least one outlet via each reaction part in a channel.

The ion sensor does not necessarily have to install the ion sensitive part in the flow channel, and the ion sensitive part or the reference gate electrode may be in contact with the liquid sample such as urine, but the reaction part or reference part of the thermal sensor The heat response in the thermal reaction is less dependent on the flow rate of the liquid sample, and therefore it is necessary to form it in the controllable channel. . Therefore, the liquid sample is introduced from at least one inflow port, and is allowed to flow through at least one outlet so that it can be diverted to each flow path via an ion sensor if necessary.

The disposable biosensor chip according to claim 12 of the present invention is a case where the liquid sample introduced from the inflow port passes through each flow path by capillary action.

When the main body of the biosensor to which the disposable biosensor chip is attached is a handy type, there is a demand for miniaturization as much as possible. Pumping liquid samples, such as urine, not only increases the size, but also has problems with power consumption. Therefore, this is a case where the inner wall of the flow path through which the liquid sample passes is made hydrophilic so as to pass through each flow path by capillary action.

The disposable biosensor chip according to claim 13 of the present invention is the case where a suction area for the liquid sample is provided at the outlet.

As described above, when the liquid sample passes through the flow path due to capillary action, the flow of the liquid sample stops unless it flows out from the outlet. In the above-mentioned thermal type battle s name, a reaction product is generated by an enzyme reaction, and if this is not removed, the sensitivity is lowered. Even if a thermal reaction occurs in the reaction section, the flow is stopped until a predetermined measurement time elapses. It is desirable to avoid it. For this purpose, a suction region of the liquid sample to be detected is provided at the outlet, and the size and structure of the suction region are determined so that the flow of the liquid sample does not stop until the predetermined measurement time has elapsed. is there. For example, a hydrophilic porous film such as blotting paper may be formed in the blotting area, or it may simply have a shape such as a minute space chamber having a hydrophilic wall. However, for example, a vent may be formed in the chamber so as not to be sealed.

The disposable biosensor chip according to claim 14 of the present invention has a plurality of the ion sensors formed, and in the ion sensitive portion of each ion sensor, an ion sensitive layer that selectively senses different ion components is formed. It is the case where the ions in the liquid sample corresponding to each ion sensitive layer can be detected.

As ionophores that selectively adsorb sodium (Na) ions or potassium (K) ions, biscrown ethers are preferred because of their strong affinity for alkali ions due to the cavity size of the crown ring. Bis (12-crown-4) is generally used for ions, and 15-crown-5-ether and dibenzo-18-crown-6 (DB18C6) are generally used for K ions. Also, valinomycin, which is an ionophore for potassium, is known for its good selectivity for Na. In the case of urine as a liquid sample, the concentration of sodium (Na) is generally several times higher than that of potassium (K) ion, so detection of the concentration of sodium (Na) is easier to measure. As an ion sensitive layer, for example, a plasticized polyvinyl chloride (PVC) film or a plasticizer-dispersed photoresist film containing an ionophore selective for Na ions or K ions as described above is used. In ISFET, ion detection is an ion sensitive part. In the ion sensor, it is formed on the gate insulating film of the ISFET for application and on the detection electrode which is the ion sensitive part. In the ISFET, for example, the above-described ion sensitive membrane ion detection ISFET is used, the ion sensitive layer and the reference gate electrode are brought into contact with a liquid sample to be detected such as urine, and a predetermined reference gate electrode / source of the ion detection ISFET. The reference gate voltage Vrg between S and the source S / drain D under the voltage Sd between source S and drain D, for example, Na in the liquid sample to be detected for the conductivity (or channel resistance) of n channel The concentration of Na ions and K ions in the liquid sample to be detected is obtained from calibration data prepared in advance from the change in the drain current Id based on the ion and K ion concentration dependency. Similarly, in the ion sensor, as described above, the ion sensor is prepared in advance from the change in the drain current Id based on the Na ion or K ion concentration dependency in the liquid sample to be detected from the change in the potential of the detection electrode with respect to the reference electrode. The calibration data is used to determine the concentration of Na ions or K ions in the liquid sample to be detected.

In the ISFET, when the region of the gate insulating film of the ion detection ISFET and the reference ISFET is exposed to a liquid sample to be detected such as urine, the reference gate electrode shared as described above is used. It is preferable that the detection liquid sample having a constant concentration distribution of detection ions (for example, Na ions) be disposed close to the gate insulating films of the ion detection ISFET and the reference ISFET. Similarly, in the ion sensor, it is better to arrange the ion detection electrode, the reference electrode, and the common electrode in close proximity. For example, they may be overlapped via a minute gap, or may be disposed close to each other on a plane.

The disposable biosensor chip according to claim 15 of the present invention is a case where the enzyme for detecting creatinine is used as the enzyme.

In order to increase sensitivity, the thermal sensor generally fixes a predetermined enzyme in a reaction part in a flow path formed in a thin film thermally separated from a substrate, but as one type of enzyme, so that creatinine correction can be performed, Enzymes that are specifically selective for the substrate creatinine in liquid samples such as urine, such as creatininase, are fixed in the reaction part of the thermal sensor unit, and the temperature rise due to contact thermal reaction is referenced by the temperature sensor for detection. It is a case where it measures by differential with the temperature sensor output.

In the disposable biosensor chip according to claim 16 of the present invention, sodium (Na) ion is used as a detection ion of the ion sensor.

Since the ion sensor is also formed on chip B on the same substrate as the thermal sensor, or on chip C different from chip A, the concentration measurement of creatinine with the thermal sensor and Na ions that are specific ions are simultaneously performed. The present invention provides a disposable biosensor chip which can be measured and can also be used for creatinine correction of Na ion.

Simultaneously detecting the output from each of these thermal sensor units and the output corresponding to the concentration from the ion sensor of Na ions that are specific ions, the concentration of Na ions in the liquid sample to be detected such as urine, So-called creatinine correction of Na ions, which is a ratio to the concentration of creatinine, for example, a Na ion / creatinine (concentration) ratio, can also be obtained. Of course, as a biosensor output for a thermal sensor, in addition to creatinine, for example, protein in urine is simultaneously measured to obtain a protein / creatinine (concentration) ratio, so that the thermal sensor of a disposable biosensor chip Trypsin, which is a specific enzyme for protein detection, may be fixed to the reaction unit.

In order to form an ion sensor such as ISFET on the substrate body forming the thermal sensor part, the enzyme immobilized on the reaction part of the thermal sensor part is extremely weak against heat and chemicals, and the reaction part is Forming a mechanically weak thin film of a cross-linked structure or diaphragm structure floating in the air thermally separated from the substrate, and further, the ion sensitive portion of the ion sensor is weak to heat including an ionophore and has a thickness There is a need for a manufacturing process based on a manufacturing order or a device for problems such as formation of an ion sensitive layer requiring control. In addition, since the ion sensor is not a thermal sensor, it is not necessary to manufacture it on a bridge structure that is thermally separated from the substrate, and a dedicated ion sensor chip as an ion sensor unit is provided on the base substrate body that is mechanically durable. Preferably, they are formed as hybrids.

A disposable biosensor chip according to claim 17 of the present invention is a case where an absolute temperature sensor is mounted on at least one of the chip A, chip B and chip C.

A thermocouple or a thermopile, which is a temperature difference sensor, is suitable for the detection temperature sensor or the reference temperature sensor of the thermal type sensor described above, because only a temperature rise is detected. Since the temperature difference sensor measures only the temperature difference based on the absolute temperature of the reference point, it is necessary to know the absolute temperature. Also, the thermal reaction of the enzyme is temperature dependent, and furthermore, since the enzyme is a protein, it rapidly inactivates at temperatures above 50 ° C. Further, also in the ion sensor, in particular, in the ion electrode method, since it is a detection principle using the Nernst equation, it is necessary to have direct absolute temperature dependency and to perform temperature calibration. Thus, it is necessary to measure the absolute temperature, and this absolute temperature sensor is formed in any one of the chip A, chip B, and chip C.

A biosensor according to an eighteenth aspect of the present invention includes a biosensor chip detachment mechanism that allows the disposable biosensor chip according to any one of the first to seventh aspects to be detachable, and the specific substrate component and a predetermined Measuring the temperature change based on the heat of reaction caused by the contact with the enzyme with a differential output according to the concentration of the specific substrate component of the reference temperature sensor and the detection temperature sensor Using a certain calibration data, a differential output corresponding to the concentration of the specific substrate component in the liquid sample is used, and an output related to the concentration of the substrate component can be performed. It is.

In the present invention, when the chip A or the chip B having the thermal type sensor unit is used and an output related to the concentration of a specific substrate component in the liquid sample is made as a thermal type sensor, The present invention provides a biosensor having a biosensor chip detachment mechanism that allows detachment of a biosensor chip having the base substrate including the chip A and the chip B.

A biosensor according to a nineteenth aspect of the present invention includes a biosensor chip detachment mechanism that enables the disposable biosensor chip according to the eighth to seventeenth aspects to be detachable, and using the discarded biosensor chip, Measuring the output of the specific ion in the liquid sample according to the concentration of the ion sensor, and using the calibration data prepared in advance, according to the concentration of the specific substrate component in the liquid sample to be detected The differential output and the output corresponding to the specific ion concentration of the ion sensor are used to output the ratio thereof.

In the present invention, when the chip B or the chip C having the ion sensor unit is used and an output related to the concentration of a specific ion component in the liquid sample can be performed as the ion sensor, the chip B And a chip C. The present invention provides a biosensor provided with a biosensor chip desorption mechanism capable of detaching the biosensor chip having the base substrate provided with the chip C.

The biosensor according to claim 20 of the present invention is a case where creatinine is used as a specific substrate component in the liquid sample and sodium is used as a specific ion component.

The daily excretion of creatinine (substrate) in adult urine is almost fixed at around 1 g, and based on this, one drop of urine (arbitrary urine) is used to examine the concentration of ions in other substrates and urine. Is so important that it can be known as creatinine correction. When the concentration of Na ions in urine is examined, the intake amount of salt (salt) which is the cause of high blood pressure can be determined, measurement of Na ion concentration by ion sensor such as ISFET or ion electrode method ion sensor, creatinine by thermal type sensor As a result of the concentration measurement, the Na ion creatinine correction, which is the ratio of “Na ion concentration / creatinine concentration”, can be corrected by one handy biosensor of the present invention.

A biosensor according to claim 21 of the present invention comprises at least a power supply circuit, an amplifier circuit, an arithmetic circuit, and a control circuit, and relates to the concentration of a specific substrate component and the concentration of a specific ion component in the liquid sample, and a ratio thereof. It is a case where information can be obtained.

When the chips A, B, and C of the disposable biosensor chip are manufactured using an SOI substrate that is a Si single crystal, the MEMS technology is easily applied and is preferable. The biosensor main body also integrates a power supply circuit, an amplifier circuit, an arithmetic circuit, and a control circuit, and an extremely compact, for example, handy type biosensor can be provided.

A Joule heater is installed in the reaction part of the thermal sensor of the disposable biosensor chip of the present invention so that the catalytic catalyst thermal reaction is performed in an optimum temperature environment or in a temperature environment where thermal reaction is easily observed. In addition, the temperature of the reaction part can be set to an optimum temperature for the enzyme, and a biosensor that can be used for calibration such as evaluation of the degree of inactivation of the reaction heat in the thermal reaction with the enzyme. It can also be provided. In particular, the activity of the enzyme often decreases depending on time and environment, and the heater formed in the reaction part is also used to check and correct the activity of the enzyme. As described above, for example, the temperature increase by adding 10 μW (microwatt) which is a predetermined power to the heater is measured in advance, and the reaction of a specific enzyme by the enzyme thermal reaction by flowing a standard liquid sample By measuring the temperature rise of the heat from time to time, it is possible to check the degree of enzyme activity according to the change over time, and based on this, it is also possible to correct or calibrate the concentration of specific substrate components in the liquid sample or creatinine become.

When the biosensor of the present invention is used for measurement of urinary protein or urine sugar and creatinine as specific substrate components with a thermal sensor, and further with an ion sensor for measurement of Na ion or the like as specific ions, these urinary protein or urine Creatine correction of sugar concentration and creatinine correction of Na ion concentration are possible, and information on the concentration of specific substrate components and ions from an individual's ion biosensor, and the ratio including these creatinine corrections, Not only that, it is important to know the daily changes and trends in everyday life. It is important to accumulate past data as well as numerical values at the time of measurement, and to graph or forecast daily changes, and it may be necessary to contact medical institutions as well. It is more convenient to be able to transmit information to an external computer wirelessly or by wire, so that various processes can be performed.

A biosensor according to claim 22 of the present invention is a case where the disposable biosensor chip according to claims 1 to 17 is inserted and fixed to the biosensor using a dedicated sensor chip stacker prepared separately. is there.

If a large amount of the disposable biosensor chip is stored in the biosensor body from the beginning, the shape of the biosensor chip becomes large. Therefore, it is unsuitable for the handy type, and the enzyme immobilized on the sensor chip also loses its time. It is expected to live. This is a case where the disposable biosensor chip is separated from the biosensor main body of the present invention and prepared separately, and is suitable for a handheld biosensor. Prepare a dedicated sensor chip stacker so that a plurality of sensor chips can be stored here, and the shape of the sensor chip take-out port of the dedicated sensor chip stacker is compatible with the sensor chip insertion port of the biosensor body. It is good to design like this. Then, it is desirable that the disposable biosensor chip can be attached to the biosensor body with one touch, and the concentration can be measured immediately.

The disposable biosensor chip of the present invention has a flow path through which the (sample to be detected) liquid sample passes, and a reaction section for immobilizing an enzyme is provided in the flow path. Since at least the upper and lower surfaces of each are made into one disposable chip shape sandwiched between materials (including gas) having a thermal conductivity smaller than half of that of silicon (Si). Since the heat of reaction due to the contact reaction between the enzyme and the corresponding substrate hardly escapes from the reaction part to the outside, it has an advantage of being a highly sensitive thermal type sensor.

In the disposable biosensor chip of the present invention, the heat of the contact exothermic reaction with the enzyme substrate in the thermal sensor escapes to the liquid sample to be detected if there is no channel, and the sensitivity is reduced. By providing, a structure hermetically sealed with a heat insulating material including a cavity, further preventing evaporation of the liquid sample and eliminating heat generation, there is an advantage that a minute temperature change can be stably measured.

In the disposable biosensor chip of the present invention, the chip A on which the thermal sensor is mounted and the base substrate are overlapped and joined to form a single disposable chip. It has the advantage of being a compact disposable biosensor chip.

In the disposable biosensor chip of the present invention, since the flow path of the thermal sensor can be provided between the chip A and the base substrate, there are less restrictions on the method of immobilizing the enzyme to the reaction part, There is an advantage that the amount and fixation of enzymes can be facilitated, such as being able to be fixed by dropping directly from the top.

In the disposable biosensor chip of the present invention, since the temperature sensor for detection and the temperature sensor for reference are temperature difference sensors, there is an advantage that only the temperature rise of the enzyme exothermic reaction in the reaction part of the thermal sensor can be measured.

The disposable biosensor chip of the present invention includes a thermal sensor that detects the heat of reaction between a specific substrate component in a liquid sample and a specific selectivity enzyme and a sensor that detects a specific ion component in the liquid sample. Two types of sensors based on different principles with certain ISFETs and ion sensors of the ion electrode method can be provided on the same substrate by devising the manufacturing process, and it is a specific substance in a liquid sample such as a drop of urine There is an advantage that a specific substrate and a specific ion concentration can be measured simultaneously.

In the disposable biosensor chip of the present invention, an enzyme that selectively reacts to each of the thermal sensors is fixed to each reaction part so that a plurality of substrates such as creatinine, protein, and sugar can be measured. Furthermore, as an ion sensor, an ion-sensitive layer containing each ionophore capable of detecting multiple ions such as Na ions and K ions is provided, so that the creatinine correction of each substrate concentration and ion concentration and the ratio between them are also obtained. It has the advantage of being able to

In the disposable biosensor chip of the present invention, the Na ion / creatinine concentration ratio (creatinine correction) can be easily obtained by using creatinine as a thermal sensor and Na ion measurement as an ion sensor such as ISFET. There is an advantage that simple measurement of salt intake is possible.

In the disposable biosensor chip of the present invention, a flow path through which a liquid sample to be detected such as urine passes is formed on the substrate. Therefore, even in an ion sensor, if the ion sensing portion is in the flow path, the concentration of ions is reduced. There is an advantage that variation can be reduced and highly accurate ion concentration measurement becomes possible.

In the disposable biosensor chip of the present invention, the liquid sample to be detected that has flowed in from one inflow port passes through one or a diverted flow path, passes through an ion sensor or a thermal type sensor, and has one outflow port. Because it flows out of the sensor, it requires only a small amount of liquid sample to be detected, and is suitable as a handy disposable biosensor chip.

In the disposable biosensor chip of the present invention, when each chip A, chip B, and chip C is formed on a semiconductor substrate such as Si crystal, conventional MEMS technology can be used, and power circuit, amplification circuit, arithmetic in integrated circuit technology The circuitry and control circuitry can also be integrated, thus providing a very compact, for example, handheld type of biosensor.

In the disposable biosensor chip of the present invention, each chip A, chip B, and chip C can be hybridized and mounted on the base substrate, so that there is an advantage that the yield in the manufacturing process can be increased.

The biosensor of the present invention includes a biosensor chip detachment mechanism that allows the disposable biosensor chip to be detached, so that there is an advantage that the disposable biosensor chip can be replaced with one touch.

In the biosensor of the present invention, the differential output according to the concentration of the specific substrate component in the liquid sample and the output according to the specific ion concentration of the ion sensor can be used to output the ratio thereof. Therefore, there is an advantage that the ratio of the concentration of a specific substrate component to the concentration of a specific ion can be easily displayed.

In the biosensor of the present invention, the specific substrate component is creatinine, and the disposable biosensor chip is a sodium (Na) ion as the specific ion in the liquid sample, for example, in urine as a liquid sample. There is an advantage that creatinine correction of sodium (Na) ions can be easily performed.

In the biosensor of the present invention, since the disposable biosensor chip is inserted and fixed using a dedicated sensor chip stacker prepared separately, the disposable biosensor chip can be stored in large quantities, for example, under refrigeration storage. In addition, there is an advantage that the biosensor body can be inserted and fixed or replaced with one touch.

The top view schematic diagram (A) of the transparent form seen from the upper direction of one Example of the disposable biosensor chip | tip 100 of this invention, and the cross-sectional schematic diagram (B) in the XX line are shown. Example 1 1 is a schematic diagram of an ion electrode method ion sensor 80 in one embodiment of the ion sensor (part) 3 of the disposable biosensor chip 100 of the present invention. FIG. Example 1 FIG. 3A shows another embodiment of the disposable biosensor chip 100 of the present invention. FIG. 3A is a schematic plan view of the base substrate 1, and FIG. 3B is a cross section taken along line XX. Schematic view, (C) is a schematic plan view of the chip A 101 to be bonded to the position where the thermal sensor (part) 2 is fixed, (D) is a schematic plan view of the chip C 103 provided with the ion sensor (part) 3 Indicates. (Example 2) A schematic diagram (FIG. 4A) of a chip C103 provided with an ion electrode method ion sensor 80 as the ion sensor (part) 3 of the disposable biosensor chip 100 of the present invention and the basics of the ion electrode method ion sensor 80. It is a measurement circuit diagram (FIG. 4 (B)). (Example 2) FIG. 5 (A) and FIG. 5 (B) show an embodiment of the ion electrode method ion sensor 80 shown in FIG. 4 as the ion sensor (part) 3 of the disposable biosensor chip 100 of the present invention. ). (Example 2) A basic measurement circuit diagram when an ISFET 90 is used as the ion sensor (part) 3 of the disposable biosensor chip 100 of the present invention is shown. (Example 2) An experiment of one embodiment in which Na ions in an aqueous solution in which sodium chloride (NaCl) as a liquid sample was dissolved was measured using the measurement circuit of one embodiment of the ion electrode method ion sensor 80 of the disposable biosensor chip 100 of the present invention. A graph of the data is shown. (Example 2) Another embodiment of the chip A101 provided with the thermal sensor (part) 2 in the disposable biosensor chip 100 of the present invention is shown. 8A is a schematic plan view of the chip A101, FIG. 8B is a schematic cross-sectional view taken along the line XX of FIG. 8A, and FIG. 8C is a longitudinal section taken along the YY line. FIG. (Example 3) An example of the schematic shape of the base substrate 1 on which the chip A101 of another example of the disposable biosensor chip 100 of the present invention is mounted is shown, FIG. 9A is a schematic plan view thereof, FIG. ) Is a schematic cross-sectional view taken along line XX in FIG. 9A, and FIG. 9C is a schematic cross-sectional view taken along line YY. (Example 3) FIG. 10 shows a cross-sectional schematic view of one embodiment of the disposable biosensor chip 100 of the present invention shown in FIG. (Example 3) The other example of chip | tip A101 provided with the thermal-type sensor (part) 2 of the disposable biosensor chip | tip 100 of this invention is shown, The same figure (A) is the plane schematic, The figure (B) The cross-sectional schematic along the XX line, and the same figure (C) is the longitudinal cross-section schematic along the YY line. (Example 4) FIG. 12A shows an example of a main body of a biosensor 1000 to which the disposable biosensor chip 100 of the present invention is attached. FIG. 12A is a schematic surface view seen from above, and FIG. FIG. (Example 5) 1 is a schematic cross-sectional view of an embodiment of a biosensor chip stacker 2000 in which the disposable biosensor chip 100 of the present invention is stored and supplied to the biosensor 1000. FIG. (Example 5)

  Chips A, B, and C of the disposable biosensor chip of the present invention can be formed of a silicon (Si) substrate using MEMS technology. Some examples in the case where these chips A, B, and C are manufactured using a silicon (Si) substrate, particularly an SOI substrate, will be mainly described, and these chips A, B, and C are further described. An embodiment of a disposable biosensor chip mounted on the base substrate 1 is shown. Further, embodiments of the biosensor of the present invention will be described with reference to the drawings. In the present specification and the drawings, components having substantially the same functional configuration will be assigned the same reference numerals and redundant description will be omitted.

1 (A) and 1 (B) are a schematic plan view (A) of a perspective view seen from above of an embodiment of the disposable biosensor chip 100 of the present invention and a schematic cross-sectional view taken along line XX (FIG. 1). B) shows the case where the thermal sensor (part) 2 and the ion sensor (part) 3 are provided on the base substrate 1 in the chip B 102 which is one sensor chip and integrated. The chip B 102 is manufactured using a silicon (Si) substrate having the SOI layer 11. Here, as the thermal sensor (part) 2, a hollow flow path 17 is provided on the thin film 10 mainly composed of the SOI layer 11 thermally separated from the substrate of the chip B102 by the cross-linking structure 12, for example, SU- A photoresist film such as 8 is formed, and a liquid sample for detection such as urine flows here. And in the flow path 17 located on the said bridge | crosslinking structure 12, it has the reaction part 6A and the reaction part 6B in two of the branched flow paths 17 of the three equivalent shapes, respectively, In this case, enzyme 4A and enzyme 4B, which are enzymes 4 that show catalytic catalytic reactions with different specific substrates in the liquid sample, are respectively immobilized. In addition, in FIG. 1 (A), the shape display of these enzyme 4A and enzyme 4B was abbreviate | omitted from the complexity of drawing. The state of immobilization of the enzyme 4A is shown in FIG. 1 (B). These enzymes 4A and 4B are substrate components in a liquid sample to be detected, such as urine, for example, Claire which is an enzyme 4 that selectively shows a thermal contact reaction with creatinine, protein, sugar, etc. The desired two kinds of tininase, trypsin, glucose oxidase and the like are selected and fixed, and the heat generated by the contact thermal reaction between these enzymes 4 and the corresponding substrate is converted into the reaction parts 6A and the reaction parts. The detection temperature sensor 20A and the detection temperature sensor 20B, which are respectively formed in 6B, are used to detect a temperature change. In addition, the thermal sensor (part) 2 is provided with a reference part 7 of a flow path 17 that has a shape equivalent to that of the reaction part 6A and the reaction part 6B and branches to become a reference part with respect to the temperature of the liquid sample. In this case, the enzyme 4 is not immobilized here, and instead of the enzyme 4, a reference membrane 5 is provided to adjust the flow of the liquid sample in the flow path to be equal to that of the reaction units 6A and 6B. doing.

In the disposable biosensor chip 100 of the present invention shown in FIGS. 1 (A) and 1 (B), the ion sensor (part) 3 is also mounted on the same chip B 102. Here, the ion sensor (part) 3 As a case, ISFET 90 is used. Here, the ISFET 90 includes an ion detecting ISFET 90A and a reference ISFET 90R, and an ion sensitive layer 95 is formed on the gate insulating film 93 of the MOS structure to be the ion sensitive portion of the ion detecting ISFET 90A. In this case, the ion insensitive layer 96 is formed on the gate insulating film 93 of the corresponding reference ISFET 90R. Further, as a reference electrode common to the ion detection ISFET 90A and the reference ISFET 90R, the reference gate electrode 97 is placed on the same chip B102 so that the reference gate electrode 97 is in contact with the liquid sample to be detected such as urine with the same electrode material. Forming. As a chip B102, a chip substrate having a p-type SOI layer 11 of Si semiconductor (for example, a thickness of about 10 μm) is used as a MOS gate serving as an ion sensitive portion, and the gate width is, for example, about 10 μm. However, in order to increase the drain current Id and reduce the peripheral effect, this is a case where the ISFET 90 having an extremely long ion detection ISFET 90A and a reference ISFET 90R having a gate length of about 1000 μm is formed. In the thermal sensor (part) 2, a liquid sample to be detected is caused to flow in the flow path 17, and heat is generated by contact with the specific substrate in each of the enzymes 4 A and 4 B in the reaction parts 6 A and 6 B. In order to minimize heat diffusion and to consider the contact amount and flow rate dependence of the liquid sample, reaction sections 6A and 6B to which the enzymes 4A and 4B are fixed are provided in the flow path 17. However, since the reaction principle of the ion sensor (unit) 3 does not measure the thermal reaction, it is not necessarily formed in the flow path 17. However, since there is a concentration distribution of detected ion components such as sodium (Na) and potassium (K) in the liquid sample, the same liquid sample as much as possible is the ion sensor (part) 3 and the thermal sensor (part) 2. It is more convenient for the evaluation of urine from time to time. Therefore, in the present invention, the ion sensor (part) 3 is formed in the vicinity of the inlet 161 which is the inlet of the flow path to the disposable biosensor chip 100 of the liquid sample.

In the disposable biosensor chip 100 of the present invention, in order to be able to provide a disposable and inexpensive sensor chip, the introduction of a liquid sample to the ion sensor (part) 3 and the thermal sensor (part) 2 is carried out It leads to a sensing part by capillary action via the path | route 17, and devises so that a flow may be produced. Therefore, first, the wall surface of the flow path 17 is made hydrophilic, and the enzyme can be fixed using a hydrophilic material. The flow channel 17 is also about 200 micrometers (μm) wide and about 100 μm high in internal cross section, so that it can be measured with a small amount of liquid sample and near the end of the flow channel 17 The case where the suction port 201 is provided in the outflow port 180 and the water sample is packed in the suction region 201 as needed is arranged so as to effectively pump the liquid sample. In the thermal sensor (part) 2, a reaction product different from the substrate is produced by the enzyme reaction. To maintain a stable enzyme reaction, this is removed immediately and brought into contact with a fresh substrate. It is important to ensure that a flow occurs in the liquid sample.

In the disposable biosensor chip 100 of the present invention, as described above, the base substrate 1 is provided with the thermal sensor (part) 2 and the ion sensor (part) 3 in the chip B 102 which is one sensor chip, In the case of being integrated, the thermal type formed in the chip B102 in order to send the electrical signals from the thermal type sensor (part) 2 and the ion sensor (part) 3 at that time to the biosensor 1000 body described later. Corresponding to each electrode pad such as the source electrode pad 191P and the temperature sensor electrode pad 70AP for detection and the base substrate 1 through the wiring 400 from the sensor (part) 2 and the sensing part of the ion sensor (part) 3 To make an electrical connection via the bump electrode 310 made of conductive paste or the like when overlapping with the electrode pads (identical symbols) Yes. Further, in the present embodiment shown in FIG. 1, the enzyme 4 is fixed to the reaction part 6 of the cross-linking structure 12, so that depending on the method of fixing the enzyme 4, the enzyme is fixed to the closed channel 17. For this purpose, an enzyme fixing window 99 is provided on the upper side of the flow path 17 at the position of the reaction unit 6, and for example, the enzyme 4 dissolved in hydrophilic polyvinyl alcohol is dropped to evaporate the solvent. It shows the case where it is allowed to solidify and fix the enzyme 4. And the case where it was made to block | close with the seal | sticker 210 which is a very thin sheet | seat so that the liquid sample which flows through the flow path 17 may not leak from this enzyme fixed window 99 is shown. Further, the bottom plate 220 is chipped for the purpose of preventing heat dissipation due to air turbulence in the cavity 40 forming the bridge structure 12 including the reaction part 6 and the reference part 7 and preventing the physical bridge structure 12 from being broken from the outside. It shows the case where it is attached to B102. Then, on the insertion end side of the base substrate 1 to the main body of the biosensor 1000, the external connection for positioning with each corresponding spring electrode 555 provided in the sensor chip insertion port 530 of the main body of the biosensor 1000 is performed. The electrode pads 150P are arranged.

In this embodiment, in FIG. 1, as the thermal sensor (unit) 2, in order to thermally separate the single thin film 10 from the chip B102 of the Si substrate, for example, a cross-linked structure 12 having a length of about 1000 μm is used. In one embodiment, the detection temperature sensors 20A and 20B and the reference temperature sensor 20R provided in the reaction section 6 are formed of thermocouples. At that time, the SOI layer (p Type semiconductor layer) (the electrode pad is a common electrode pad 75P for the SOI layer), and the thermal conductor 120B is formed using a sputtering thin film of nichrome (NiCr) formed with the insulating film 50 interposed therebetween. These thermocouple measurement points (hot junctions) 26 are electrically connected by ohmic contacts 60 through the through-holes of the insulating film 50. For example, creatininase, which is an enzyme 4A that generates heat by selectively reacting with creatinine, which is a substrate in a liquid sample such as urine, is used. The contact with the enzyme that specifically reacts with the substrate causes an exothermic reaction to raise the temperature of the reaction part 6A on which the enzyme is immobilized, and the influence of the ambient temperature by the reference temperature sensor 20R with the detection temperature sensor 20A Are differentially amplified. Further, as an ISFET 90 for detecting specific ions, an ion detecting ISFET 90A, a reference ISFET 90R, and a reference gate electrode 97 are provided close to the main body portion of the Si substrate of the chip B102, and the gate insulating film 93 region and the reference gate electrode are provided. 97 is exposed in the inflow port 161 which is the entrance of the hollow channel 17 formed of a photoresist film such as SU-8, and touches the liquid sample flowing through the channel 17 (to be detected). Designed. In this example, the heater 25 was not drawn in the reaction part 6 of the thermal sensor (part) 2 due to the complexity of the drawing, but the heater 25 was mounted for calibration such as deactivation of the enzyme reaction, It is preferable to be able to calibrate the change with time by comparing the temperature rise when applying a predetermined power and the calorific value of the enzyme reaction with a predetermined substrate.

In this embodiment, FIG. 1 shows a case where an ISFET 90 is used as the ion sensor (part) 3 of the disposable biosensor chip 100. However, as the ion sensor (part) 3, for example, an ion electrode as shown in FIG. The ion electrode method ion sensor 80 which is an ion sensor using a method (ion selective electrode method) may be used. In the example shown in FIG. 2, an insulating film 50 such as a thermal oxide film is formed on the surface of the SOI substrate on the ion sensor (portion) 3 of the chip B102 made of an SOI substrate, and the same shape as the ion detection electrode 80A. The reference electrode (reference electrode) 80R and the common electrode 81 are formed close to each other, and the ion detection electrode 80A is coated with, for example, an ionophore solution having extremely high ion selectivity to Na ions as specific detection ions. The ion-sensitive layer 85 that has been dried is formed to have a thickness of, for example, 100 μm. A reference film layer 86 is formed on the reference electrode (reference electrode) 80R, and the area in contact with the liquid sample to be detected is, for example, , 3 mm square, and exposed from the insulating film 50. The common electrode 81 that contacts the liquid sample to be detected in common is formed of the same metal as the ion detection electrode 80A and the reference electrode (reference electrode) 80R, for example, gold (Au). The reference film layer 86 is, for example, 10 minutes from the ion sensitive layer 85 formed on the ion detection electrode 80A while having the same composition as the ion sensitive layer 85 formed on the ion detection electrode 80A. It can be used as a layer having a thickness as thin as one, so-called composition is the same, but different in thickness. As a method of forming the ion sensitive layer 85 and the reference membrane layer 86, the solution of the ion sensitive layer 85 is diluted 10 times, applied in the same amount, formed, and dried. A thin film-like reference film layer 86 can be formed.

The gate insulating film 93 region of the ion detection ISFET 90A is a biscrown ether that can selectively incorporate sodium (Na) ions, for example, bis (12-crown-4) for Na ions. It is preferable to form an ion sensitive layer incorporating the ionophore. The ion sensitive layer 95 of the ISFET 90A for ion detection and the ion insensitive layer 96 of the ISFET 90R for reference are formed in the flow channel 17 at a position located on the gate insulating film 93 region of the ISFET 90A for ion detection and ISFET 90R for reference. Through the gate layer forming window, PVC (polyvinyl chloride) containing a plasticizer can be used to form a pattern such as the ion sensitive layer 95 in which the film thickness is adjusted by the gate layer forming frame 195, The ion sensitive layer 95 of the ion detecting ISFET 90A and the ion insensitive layer 96 of the reference ISFET 90R may be alternately formed in a thin film shape. The application process of the ion sensitive layer 95 and the like is easier to cope with the thermal problem of the enzyme if it is a manufacturing process prior to the enzyme fixation to the reaction part 6 of the thermal sensor biosensor 30. A plasticizer-containing photoresist can be used instead of PVC (polyvinyl chloride). When this photoresist is used, it can be easily patterned into an arbitrary shape, so the gate layer forming frame 195 is not necessarily required. Further, the same ionophore used for the ion detection ISFET 90A is also applied to the ion detection electrode 80A, which is a sensitive part of the ion sensor 80 using the ion electrode method (ion selective electrode method). It is good to use it as.

FIG. 3 shows another embodiment of the disposable biosensor chip 100 of the present invention, and FIG. 3 (A) is a schematic plan view of the base substrate 1 which is a component of the disposable biosensor chip 100. 3B shows a schematic cross-sectional view along the line XX, and FIG. 3C shows a position where the thermal sensor (part) 2 of the base substrate 1 is fixed. A schematic plan view of the chip A101 to be joined is shown, and FIG. 3D is a schematic plan view of a chip C103 provided with an ion sensor (unit) 3 that is similarly fixed to the base substrate 1. The chip A101 having the thermal sensor (part) 2 and the chip C103 having the ion sensor (part) 3 are turned upside down, and the respective electrode pads are formed on the base substrate 1 having the same reference numeral. When the electrode pads are integrated through the bump electrode 310 made of a conductive paste or the like as described above, the electrode pads are electrically connected to each other and connected to the external connection electrode pad 150P at the end of the base substrate 1. It is connected by the wiring 400. The chip A101 provided with the thermal sensor (part) 2 of this embodiment is the same as the thermal sensor (part) 2 in the chip B102 in the above-mentioned first embodiment, and the thin film 10 of the crosslinked structure 12 is hollow. The reaction unit 6 is fixed with, for example, creatininase, which is an enzyme 4 for detecting creatinine in a liquid sample introduced from the inlet 161. In the present embodiment, the ISFET 90 shown in FIG. 3D is used for the ion sensor (unit) 3 as an ion sensor, and these chips C103 are sensor chips provided separately from the chip A101. And the chip A101 are bonded to a predetermined region of the base substrate 1 so that the electrode pads such as the source electrode pad 191P and the detection temperature sensor electrode pad 70AP are aligned and overlapped to be integrated. This is a case where the disposable biosensor chip 100 is provided.

FIG. 3 of the present embodiment shows a case where the ion sensor (unit) 3 is provided with an ion sensor of ISFET 90 in the chip C103, but the ion electrode method (ion selective electrode method) as shown in FIG. ) And the chip C103 provided with the ion sensor 80. Here, the ion electrode method ion sensor 80 is obtained by forming the ion electrode method ion sensor 80 shown in FIG. 2 of the first embodiment into one chip. However, the ion electrode method ion sensor 80 may be formed on the electrical insulating film, and does not need to be formed on the Si substrate as shown in FIG. 2 of the first embodiment. For example, ions are formed on a glass substrate or a plastic substrate The detection electrode 80A or the reference electrode (reference electrode) 80R may be formed to be the ion electrode method ion sensor 80. FIG. 4B shows a basic measurement circuit diagram by the ion electrode method ion sensor 80. This measurement circuit is actually incorporated into a biosensor main body, which will be described later, that is mounted and measured with the disposable biosensor chip 100. The ion sensitive layer 85, the common electrode 81 and the reference membrane layer 86 of the ion electrode method ion sensor 80 are dipped in a liquid sample such as urine and measured. However, the liquid sample such as urine has high impedance when the ion concentration is small. By forming a high input impedance differential amplifier in which the potentiostat circuit is modified, the reference electrode (reference electrode) 80R and the common electrode 81 are rounded using a high input impedance circuit using an operational amplifier, and ionized. The differential output Vo between the detection electrode 80A and the reference electrode (reference electrode) 80R was measured. As described above, the potential can be detected without flowing a current to at least the ion detection electrode 80A and the reference electrode (reference electrode) 80R. In this way, the differential output Vo is measured, and the specific ion concentration such as Na ion in the liquid sample is calculated based on the calibration data acquired in advance. In this embodiment, as shown in FIG. 4, the ion electrode method ion sensor 80 is a case of measurement by a three-terminal method using a common electrode 81, but without forming the common electrode 81, for example, an ion detection electrode It is also possible to measure these potential differences directly by a two-terminal method in which only the 80A and the reference electrode (reference electrode) 80R are formed and the reference electrode (reference electrode) 80R is at least equivalent to the ground of the measurement circuit. However, according to experiments, more stable measurement could be realized in the case of the measurement by the three-terminal method using the common electrode 81 described above.

FIG. 5 shows a schematic longitudinal sectional view of the ion electrode method ion sensor 80 shown in FIG. 4 as shown in Example 1 using an ionophore of the same component as the ion sensitive layer 85 and the reference membrane layer 86. Further, the thickness is changed by about one digit or more, the ion sensitive layer 85 is thick, and the reference film layer 86 is thin. In FIG. 5A, an ion detection electrode 80A, a reference electrode (reference electrode) 80R, and a common electrode 81 are formed on the surface of an insulating substrate 89 of a chip C103 such as a glass substrate on a flat surface. When the membrane layer 86 is formed, the height of the ionophore fixing frame 88 is changed to set the thicknesses of the ion sensitive layer 85 and the reference membrane layer 86. Further, in FIG. 5B, as in the chip B102 of the first embodiment, by using the SOI substrate, the thickness of the SOI layer 11 and the ionophore fixing frame 88 can be used to increase the thickness of the ion sensitive layer 85. Shows one embodiment in the case of.

FIG. 6 shows a basic measurement circuit diagram when an ISFET 90 is used as the ion sensor (part) 3 of the disposable biosensor chip 100 of the present invention. This measurement circuit is actually incorporated into a biosensor main body, which will be described later, in which a disposable biosensor chip 100 is inserted and mounted in the same manner as the measurement circuit for the ion electrode method ion sensor 80. The source S91 of the ion detecting ISFET 90A and the reference ISFET 90R are shared by the ground of the measurement circuit, and the predetermined same DC voltage VD and the predetermined gate of the reference gate electrode 97 are provided to the drain 92 of the ion detecting ISFET 90A and the reference ISFET 90R. With voltage VG applied, drain current Ids1 and drain current flowing in ISFET 90A for ion detection and ISFET 90R for reference in such a manner that reference gate electrode 97, ion sensitive layer 95 and ion insensitive layer 96 are immersed in the liquid sample The difference from Ids2 is the change in voltage drop at the load resistors RD1 and RD2 connected to each, and the concentration of Na ions or the like in the liquid sample is measured.

In FIG. 7, Na ion of an aqueous solution in which sodium chloride (NaCl) as a liquid sample is dissolved is measured using the measurement circuit of one embodiment of the ion electrode method ion sensor 80 shown in FIG. 4 (B). The graph of the experimental data of an Example is shown. The data in FIG. 7 shows the differential output Vo between the ion detection electrode 80A and the reference electrode (reference electrode) 80R with respect to the common electrode 81, based on the value in fresh water in which salt (NaCl) is not dissolved. It is the data of the value at the time of subtraction, and shows the value of 100 times the actual potential difference (the magnification of the amplifier). Ion Electrode Method Since the measurement principle of ions such as Na ions in the ion sensor 80 uses a known Nernst equation, the concentration-dependent output is proportional to the logarithm of the ion concentration. As shown in the graph of FIG. 7, it can be seen that there is a linear relationship with the logarithm of the NaCl concentration, and that the Nernst equation is followed. Of course, the Na ion concentration is proportional to the NaCl concentration. As can be seen from the Nernst equation, the output of the ion electrode method ion sensor 80 is proportional to the absolute temperature of the solution, so temperature measurement is also important. As shown in FIG. 3 of the above-described embodiment, the absolute temperature sensor 23 is mounted on the chip A101 on which the thermal sensor (unit) 2 is mounted. Can measure the absolute temperature of

FIG. 8 shows another embodiment of the chip A 101 having the thermal sensor (part) 2 of the disposable biosensor chip 100. In the first embodiment, the thin film thermally separated from the Si substrate is shown. 10 is the cross-linked structure 12, and there are the flow path 17 and the reaction part 6 through which the liquid sample flows. In this embodiment, the diaphragm 4 is used as the thin film 10, and the enzyme 4 is fixed. This is a case where the reaction part 6 is also formed in this diaphragm structure 13. In the diaphragm structure 13, the heat generated by the enzyme 4 that generates a contact thermal reaction with the substrate is less susceptible to heat escape to the substrate such as Si than the crosslinked structure 12, but the sensitivity is reduced. There is an advantage that heat can be made difficult to escape and that the flow path 17 can be easily formed in the upper part of the diaphragm structure 13. In the present embodiment, when the space between the chip A 101 formed of a semiconductor substrate of Si or the like and the base substrate 1 is used as the flow path 17, at least in the region of the reaction portion 6 of the base substrate 1 In the present embodiment, the heat insulating material 230 having a thermal conductivity smaller than half that of Si of the semiconductor substrate is used and the heat generated by the enzyme 4 is difficult to escape to the outside. It is a case where it has the diaphragm structure 13, makes it one reaction part 6A, and makes the other the reference part 7. FIG. 8A is a schematic plan view of the chip A101, FIG. 8B is a schematic cross-sectional view taken along line XX of FIG. 8A, and FIG. And FIG.

FIG. 9 shows a schematic shape of one embodiment of the base substrate 1 on which the chip A101 having the thermal sensor (part) 2 of FIG. 8 is mounted, and FIG. 9A shows a schematic plan view thereof. FIG. 9B shows a schematic cross-sectional view along the line XX in FIG. 9A, and FIG. 9C shows a schematic cross-sectional view along the YY line in the same manner. When the base substrate 1 and the chip A101 are joined, the flow path 17 through which the liquid sample passes is mainly formed by the recess 41 formed in the base substrate 1 and is formed in a gap with the chip A101. A schematic diagram of a cross-sectional view of the disposable biosensor chip 100 thus formed is shown in FIG. As described above, the liquid sample to be detected introduced from the inlet 161 is branched by the flow path separation unit 235 formed in the base substrate 1, one passing through the reaction unit 6 </ b> A and the other passing through the reference unit 7. It passes through the flow path 17 and further merges into the suction area 201. Since the external connection electrode pad 150P and the operation of the disposable biosensor chip are the same as those in FIGS. 1 and 2 of the first embodiment, detailed description thereof is omitted here.

FIG. 11 shows another embodiment of a chip A101 provided with a thermal sensor (part) 2 in the disposable biosensor chip 100. FIG. 11 (A) is a schematic plan view thereof. FIG. (B) is a schematic cross-sectional view along the XX line, and FIG. (C) is a schematic vertical cross-sectional view along the YY line. In Example 3 described above, the thin film 10 thermally separated from the Si substrate is the diaphragm structure 13. However, in the present example, the thin film 10 is the crosslinked structure 12 and the flow path 17 is the example described above. 3, when the base substrate 1 and the chip A101 are joined, the concave portion 41 formed in the base substrate 1 shown in FIG. 9 of the third embodiment is mainly used, and the gap is formed between the chip A101 and the chip A101. In the case of The liquid sample to be detected flowing in the flow path 17 also enters the cavity 40 below the bridge structure 12 through the gap between the bridge structures 12, so that a heat insulating material 230 is put into the cavity 40 via the bottom plate 220, and further the reaction section The enzyme 4 (4A) is formed on the heat insulating material 230 of 6 (6A) so as to surround the temperature sensor 20 (20A) for detection formed in the reaction section 6 (6A). This is the case where the heat of reaction in (2) warms the direct detection temperature sensor 20 (20A) on the bridge structure 12. The reference unit 7 is also required to have the same thermal action and effect as the reaction unit 6 (6A) except for the heat generation of the enzyme 4, so that the enzyme fixing material not containing the enzyme 4 is the reaction unit. It is desirable to form in the same manner as 6 (6A). The other points are the same as in the case of the third embodiment, so the detailed description will be omitted.

FIG. 12 shows an embodiment of a main body of a biosensor 1000 on which the disposable biosensor chip 100 is mounted. FIG. 12 (A) is a schematic view of the surface seen from above, and FIG. Shows a schematic cross-sectional view thereof. The electronic circuit 660 of the biosensor 1000 includes at least a power supply circuit, an amplifier circuit, an arithmetic circuit, and a control circuit, and further includes a display unit 670, and the concentration and specific ions of a specific substrate component in the liquid sample. Information about the concentration of the components and their ratio can be displayed. In addition, data communication to the outside can be performed wirelessly. The button switch 520 is used to turn on / off the power and use other buttons to display and communicate various desired data. The disposable biosensor chip 100 is inserted and fixed in the chip insertion port 530, and is formed in each external connection electrode pad 150P of the disposable biosensor chip 100 and the chip insertion port 530 of the main body of the biosensor 1000. The disposable biosensor chip 100 and the main body of the biosensor 1000 are electrically connected with each other in contact with the respective spring electrodes 555. The disposable biosensor chip 100 is removed from the main body of the biosensor 1000 when the sensor chip attaching / detaching mechanism 500 is used. Although details are omitted, the disposable biosensor chip 100 can be detached from the main body of the biosensor 1000 by pressing the sensor chip attaching / detaching button 510.

FIG. 13 shows an example of a biosensor chip stacker 2000 in which the disposable biosensor chip 100 can be easily attached to the main body of the biosensor 1000 and a plurality of disposable biosensor chips 100 are mounted and stored. The cross-sectional schematic of an Example is shown. When the plurality of disposable biosensor chips 100 housed in the case 710 are pushed by the spring 750 and fixed one by one by the chip push rod 701 to the main body of the biosensor 1000 through the chip push hole 702, The disposable biosensor chip 100 is sequentially pushed out and can be used until the stored disposable biosensor chip 100 disappears, and the disposable biosensor chip 100 is replenished as necessary. It can be filled.

In the biosensor 1000, an enzyme specifically selective for creatinine, such as creatininase, is used so that creatinine can be corrected as one kind of enzyme in the thermal sensor (part) 2 of the disposable biosensor chip 100. The creatinine in the liquid sample to be detected such as urine is measured by the difference between the output of the detection temperature sensor 20A and the reference temperature sensor 20R by the temperature rise due to the contact thermal reaction, fixed to the reaction unit 6, and further ion An ion sensitive layer 85 of Na ions is formed by the sensor (unit) 3 so that Na ions can be measured, and an output corresponding to the concentration of Na ions is simultaneously detected to detect Na in a liquid sample to be detected such as urine. The ratio between the concentration of ions and the concentration of creatinine, for example, the so-called Na ion / creatinine (concentration) ratio, so-called Na ion creatinine correction, It is also possible to be able to indicate. Of course, in addition to creatinine, for example, protein in urine is simultaneously measured to obtain a protein / creatinine (concentration) ratio, and the thermal sensor unit 2 of the disposable biosensor chip 100 is used for protein detection. Trypsin, which is the specific enzyme 4B, may be fixed to the reaction part 6B.

As mentioned above, the concentration of sodium (Na) ion and potassium (K) ion can be measured simultaneously, so creatinine correction of sodium (Na) ion and potassium (K) ion can be performed, and sodium (Na) ion concentration / potassium (K) The concentration ratio of ion concentration can also be measured. It has been found that measurement of the concentration ratio of sodium (Na) ion concentration / potassium (K) ion concentration is important for antihypertensive measures, and a simple and handy biosensor can be provided.

In the drawings of the above-described embodiments of the present invention, parts having the same concept described in the claims are denoted by the same reference numerals. In the embodiments of the present invention, preferred embodiments of the present invention have been described with reference to the accompanying drawings. However, the present invention is not limited to the embodiments, and the gist, operation, and effects of the present invention are the same. However, naturally, there can be various modifications. It is obvious for those skilled in the art that various changes or modifications can be conceived within the scope of the technical idea described in the claims, and of course, the technical scope of the present invention is also possible. It is understood to belong to

  The disposable biosensor chip 100 of the present invention includes a flow path 17 for supplying a liquid sample to the thin film 10 thermally separated from a semiconductor substrate of a chip A101 such as Si by the MEMS technology as the thermal sensor (part) 2. The first detection temperature sensor 20A, the second detection temperature sensor 20B, and the like connected to each other are formed in the reaction unit 6, the reference temperature sensor 20R is formed in the reference unit 7, and further from the inflow port 161. A flow path 17 extending to a suction area 201 formed in the same substrate area across the cavity 40 via the reaction part 6 of the thin film 10 and the reference part 7 is provided, and different reaction parts in the flow path 17 6, different specific enzymes 4 selectively corresponding to the respective ones are fixed, and the temperature rise due to the enzyme reaction can be measured as an electric signal from an electrode pad provided around the substrate. Unishi to have. Then, for example, the first temperature sensor 20A and the reference temperature sensor 20R of the reference unit 7 that are temperature sensors of the reaction unit 6 have an arrangement that has an equivalent shape and symmetry so as to be thermally balanced. Therefore, even if a liquid sample to be detected, which is greatly deviated from the ambient temperature, such as urine, is flowed into the flow path 17, it is possible to realize highly accurate temperature difference measurement by these differential detections. Further, an ion sensor (unit) 3 is mounted for detecting sodium (Na) ions or potassium (K) ions in a liquid sample such as urine. For example, an ISFET 90 or an ion electrode method ion sensor 80 is commonly used. Since it is arranged in the flow path 17 through which the liquid sample to be detected flows and the inlet 161 which is the inlet thereof, the concentration of the substrate in the thermal sensor (part) 2 and the ion concentration in the ion sensor (part) 3 are simultaneously measured. It can be measured. If the creatinine concentration is measured by the biosensor 1000, creatinine correction of various substrates and ions becomes possible, and the disposable biosensor chip 100 of the present invention and a handy biosensor 1000 device equipped with the same are provided. Thus, it is possible to contribute to the diagnosis of diabetes, a symptom of kidney disease, the prevention of high blood pressure, and the like with a short measurement time of several tens of seconds and, optionally, a drop of urine as a drop of urine.

1 Base substrate 2 Thermal sensor (part)
3 Ion sensor (part)
4, 4A, 4B Enzyme 5 Reference membrane 6, 6A, 6B Reaction part 7, 7A, 7B Reference part 10, 10A, 10B, 10R Thin film 11 SOI layer 12 Crosslinked structure 13 Diaphragm structure 15 Base substrate 17 Flow path 20, 20A, 20B Detection temperature sensor
20R Reference temperature sensor 23 Absolute temperature sensor 23P Absolute temperature sensor electrode pad 25 Heater 26 Measurement point (warm contact)
27 Reference point (cold junction)
40 Cavity 41 Recess 50 Insulating film 51 BOX layer 60 Ohmic contact 61 High concentration diffusion layer 70P, 70AP, 70BP Temperature sensor electrode pad 70RP for detection, 70RA P Temperature sensor electrode pad 71P for reference Electrode pad 75P for heater common to SOI layer Electrode pad 80 Ion electrode method ion sensor 80A, 80B Ion detection electrode 80R Reference electrode (reference electrode)
81 Common electrode 85 Ion sensitive layer 86 Reference membrane layer 87 Extraction electrode 88 Ionophore fixing frame 89 Insulating substrate 90 ISFET
90A, 90B ion detection ISFET
90R ISFET for reference
91 Source S
92 Drain D
93 Gate insulating film 94 Channel 95 Ion sensitive layer 96 Ion insensitive layer 97 Reference gate electrode 97P Reference gate electrode pad 98 Guard ring 99 Enzyme fixing window 100 Disposable biosensor chip 101 Chip A
102 Chip B
103 Chip C
110 SOI layer insulating separation groove 117 Channel member 120A, 120B Thermoelectric conductor 150P External connection electrode pad 161 Inlet port 180 Outlet port
191 Source electrode 191P Source electrode pad 192 Drain electrode 192P Drain electrode pad 194P Common substrate electrode pad 195 Gate layer forming frame 196 Electrode bump 201 Absorbing region 203 Water absorbing material 204 Vent hole 205 Enzyme fixing frame 210 Seal 220 Bottom plate 230 Heat insulating material 235 Flow Road separation part 310 Bump electrode 400 Wiring 500 Sensor chip attaching / detaching mechanism 510 Sensor chip attaching / detaching button 520 Button switch 530 Sensor chip insertion port 550, 551, 750 Spring 555 Spring electrode 560 Guide plate 570 Transmission rod 650 Battery 660 Electronic circuit 670 Display part 701 Chip Extrusion Bar 702 Chip Extrusion Port 710 Case 715 Case Cover 1000 Biosensor 2000 Biosensor Chip Stacker

Claims (22)

A chip equipped with a thermal sensor that detects a specific substrate component in a liquid sample to be detected and its concentration by a temperature sensor for detection of temperature change due to heat of catalytic reaction with an enzyme that selectively responds to the substrate component A biosensor chip comprising A, an electrode that extracts at least an electric signal from the chip A, and a base substrate having an electric wiring thereof, and having a reaction part in a flow path through which the liquid sample passes, the reaction part Is that the enzyme is immobilized, the temperature sensor for detection is in contact with or close to the enzyme, and at least the upper and lower surfaces of the flow path of the reaction section are thermal conductivity of silicon (Si) The chip A and the base substrate are superposed and bonded and integrated with each other, with a structure sandwiched between materials (including gas) having a thermal conductivity smaller than one half of that of the chip. A disposable biosensor chip characterized in that it is in the form of an individual disposable chip. The disposable biosensor chip according to claim 1, wherein the flow path is provided in the chip A. The disposable biosensor chip according to claim 1, wherein the flow path is provided between the chip A and the base substrate. A reaction part that fixes the enzyme and a reference part that has the same shape as the reaction part but does not fix the enzyme, and each of the reaction part and the reference part includes a detection temperature sensor and a reference temperature sensor, respectively. The disposable biosensor chip according to any one of claims 1 to 3, comprising: The disposable biosensor chip according to any one of claims 1 to 4, wherein the detection temperature sensor and the reference temperature sensor are temperature difference sensors. The disposable biosensor chip according to any one of claims 1 to 5, wherein the reference part is formed in a branched flow path different from the reaction part. The disposable biosensor according to any one of claims 1 to 6, further comprising: each reaction unit that fixes each enzyme corresponding to a plurality of the substrate components; and the corresponding flow path and the detection temperature sensor. Chip. The chip A further comprising an ion sensor provided with an ion sensitive layer that is selectively sensitive to a specific ion component in the liquid sample is mounted on the chip A instead of the chip A. A disposable biosensor chip according to any one of the preceding claims. The disposable biosensor chip according to claim 8, wherein the ion sensor is an ion sensor according to ISFET or ion electrode method. A chip C different from the chip A is provided with the ion sensor that selectively responds to a specific ion component in the liquid sample, and the chip A and the chip C are mounted on the same base substrate to provide a hybrid. The disposable biosensor chip according to any one of claims 1 to 9. The liquid sample introduced from the at least one inlet is diverted to each branched channel, and passes through the reference part of the thermal type sensor and the respective reaction parts in each channel to at least one. The disposable biosensor chip according to any one of claims 1 to 10, which can be discharged from the outlet. The disposable biosensor chip according to claim 11, wherein the liquid sample introduced from the inflow port passes through each flow path by capillary action. The disposable biosensor chip according to claim 12, wherein a suction area for the liquid sample is provided at the outlet. A plurality of the ion sensors are formed, and in the ion sensitive portion of each ion sensor, an ion sensitive layer selectively sensitive to different ion components is formed, and the liquid sample corresponding to each ion sensitive layer The disposable biosensor chip according to any one of claims 8 to 13, which can detect ions therein. The disposable biosensor chip according to any one of claims 1 to 14, wherein the enzyme is an enzyme for detecting creatinine. The disposable biosensor chip according to any one of claims 8 to 15, wherein sodium (Na) ion is used as a detection ion of the ion sensor. The disposable biosensor chip according to any one of claims 1 to 16, wherein an absolute temperature sensor is mounted on at least one of the chip A, the chip B, and the chip C. The disposable biosensor chip according to any one of claims 1 to 7, further comprising a biosensor chip desorption mechanism capable of desorbing, the respective temperatures based on the heat of reaction caused by the contact with the specific substrate component and a predetermined enzyme. The change is measured by the differential output according to the concentration of the specific substrate component of the reference temperature sensor and the detection temperature sensor, and the calibration data prepared in advance is used to measure the change in the liquid sample. The biosensor is characterized in that a differential output corresponding to the concentration of the specific substrate component is utilized to enable an output related to the concentration of the substrate component. The disposable biosensor chip according to any one of claims 8 to 17, further comprising a biosensor chip desorption mechanism capable of desorbing, using the disposable biosensor chip, the ion sensor for specific ions in a liquid sample to be detected. Measuring the output according to the concentration of the gas, and further using differential data prepared according to the concentration of the specific substrate component in the liquid sample using the calibration data prepared in advance, and the specific ion concentration of the ion sensor A biosensor characterized in that it is possible to output their ratio using an output according to the above. 20. The biosensor according to claim 19, wherein creatinine is a specific substrate component in the liquid sample, and sodium is a specific ion component. 18. At least a power supply circuit, an amplification circuit, an arithmetic circuit, and a control circuit are provided to obtain information on the concentration of a specific substrate component and the concentration of a specific ion component in the liquid sample and their ratio. 21. The biosensor according to any one of 1 to 20. The biosensor according to any one of claims 18 to 210, wherein the disposable biosensor chip according to claims 1 to 17 is inserted into and fixed to the biosensor using a separately prepared dedicated sensor chip stacker.

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