JP4717738B2 - Microreactor and microreactor system - Google Patents

Description

  The present invention relates to a microreactor used for detecting a chemical substance or the like contained in a fluid flowing in a fine flow path, and a microreactor system using the microreactor.

  In recent years, development of small analysis systems called Lab-on-a-Chips, microreactor chips, and the like has been actively conducted. This is a configuration in which element structures such as flow paths, reaction vessels, valves, sensors, and the like are integrated on a small substrate (chip), and an analysis process is performed on a gas or liquid sample flowing in the inside. An example of such a small analysis system is a biochip that conducts a clinical test by flowing blood through a fine flow path provided in a resin chip (see, for example, Non-Patent Document 1). When such a small analysis system is used, the analysis can be performed at a high speed with a small amount of sample, so that the burden on the side providing the sample can be reduced. For this reason, application to a living body is attracting attention.

  Sensors using various principles have been proposed for the sensor unit which is one of the components of the small analysis system. In particular, the use of QCM (Quartz Crystal Microbalance), which is relatively easy to integrate with a substrate mounted on the system and enables a small system configuration, is expected.

  QCM is a technique for measuring a minute mass attached to a vibrator using a resonance phenomenon of a piezoelectric vibrator (particularly a quartz vibrator). More specifically, when an AC voltage is applied to the electrodes formed on both sides of the piezoelectric vibrator, resonance occurs at a specific frequency determined from the material characteristics and shape of the piezoelectric vibrator. Therefore, when a substance adheres to the electrode of the piezoelectric vibrator, the resonance frequency of the whole vibrator changes according to the attached mass. This is a technique for measuring the mass of a substance attached to an electrode by detecting a change in the resonance frequency.

  However, since such a mass measuring means cannot detect a specific substance, a configuration in which a means for adsorbing or capturing only a specific substance is fixed on an electrode and only the specific substance is detected is used. As an example, a technique using an antigen-antibody reaction for protein detection is known (see Patent Document 1). When the QCM having such a configuration is used, a minute mass of a specific measurement target substance can be measured. Therefore, if QCM is used for the sensor part of a small analysis system, the mass of the substance to be measured can be measured with high accuracy, and as described above, the structure integrated with the analysis system is possible, and the small size is small. The configuration can be maintained.

However, since the QCM uses the resonance phenomenon of the piezoelectric vibrator, it is necessary to consider that the vibration of the piezoelectric vibrator is not inhibited as much as possible when connecting the flow path for flowing the sample to the surface of the QCM. . Usually, the structure for fixing the piezoelectric vibrator is made flexible, and the fixing area with the piezoelectric vibrator is made small. For example, in general, only the outer periphery of the piezoelectric vibrator is bonded and fixed, or a thin O-ring is pressed into one wall surface of the flow path. However, since the region for fixing the piezoelectric vibrator is small, if the fixing is slightly insufficient, the sample in the channel leaks out of the channel. Therefore, in order to ensure the fixation, the fixing area is enlarged to allow some vibration attenuation and sensitivity reduction, or after the piezoelectric vibrator is fixed, the fluid is actually flowed into the flow path to check whether the fixing is complete. It was necessary to check for leaks.
JP 2000-338022 A J. et al. Aug et al. , Sensors and Actuators B, 26-27 (1995) 181-186.

  However, when the fixed region is increased, the vibration is surely attenuated and the sensitivity of the QCM is lowered. Also, when the fixed state of the piezoelectric vibrator is confirmed by flowing a fluid, the capturing means for the specific substance provided on the piezoelectric vibrator is contaminated by the fluid that has flowed into the flow path, and the detection operation performed after the fixing confirmation work is performed. There was a problem that sensitivity was lowered.

  Therefore, an object of the present invention is to provide a microreactor that does not require a fixing confirmation operation for flowing a fluid in a flow path and that can perform high-sensitivity detection without contamination of a specific substance capturing means.

  In order to achieve the above object, a microreactor according to the present invention has a measurement chamber provided with a specific substance capturing means for capturing a specific substance contained in a flowed sample, and is captured by the specific substance capturing means. A microreactor used for measuring a mass of a specific substance, wherein a detection substrate is formed on a module substrate having a plurality of first through holes through which the sample flows and a transparent piezoelectric substrate, and the detection The electrode is provided with the specific substance capturing means, and the detection electrode is disposed so as to face the module substrate, and is disposed between the module substrate and the piezoelectric resonator, A first surface to be bonded to the module substrate and a second surface opposite to the first surface, which is bonded to the piezoelectric vibrator and holds the piezoelectric vibrator A sensor holding portion having a second through hole penetrating from the first surface to the second surface, and the sensor holding portion is cylindrical and surrounds the second through hole. A cylindrical bank portion formed in a convex shape, and a plurality of sensor holding bases formed in a convex shape provided on the outer side of the cylindrical bank portion on the second surface; The portion is joined to the peripheral portion of the detection electrode to form the measurement chamber that holds the peripheral portion of the piezoelectric vibrator and communicates with the first through hole, and a plurality of the sensor holding bases Has a function of holding the piezoelectric vibrator so that the cylindrical bank portion is uniformly joined to the peripheral portion of the detection electrode, and a function of making it possible to visually determine the degree of joining with the piezoelectric vibrator. It is characterized by having.

  In addition, the microreactor according to the present invention has a flow path through which the sample flows in connection with the first through hole in the substrate, and is newly formed at a position facing the detection electrode of the module substrate. A flow path substrate that has a columnar convex portion that fits into the third through hole on the surface of the substrate, and is stacked by being fitted to the third through hole and the columnar convex portion. It is characterized by further having.

  The microreactor according to the present invention is a sheet that is disposed between the module substrate and the flow path substrate, and prevents liquid leakage of the sample from a fitting portion between the third through hole and the convex portion. It has a cylindrical packing.

  The microreactor according to the present invention has a silicon oxide film on a surface of the module substrate to be joined to the sensor holding portion, and the sensor holding portion is made of a silicone resin, while the piezoelectric substrate is made of a quartz substrate. The module substrate, the sensor holding portion, and the quartz substrate are bonded to each other by a siloxane bond.

  Further, the module substrate and the sensor holding portion are integrally molded products made of the same member, while the piezoelectric substrate is made of a quartz substrate, and the sensor holding portion and the quartz crystal substrate of the integrally molded product are each siloxane. It is characterized by being joined by bonding.

  Further, the microreactor system according to the present invention is connected to any one of the above microreactors and the detection electrode, and is captured by the specific substance capturing means by detecting a change in the resonance frequency of the piezoelectric vibrator. And a measuring means for measuring the mass of the specific substance.

  According to the microreactor and microreactor system of the present invention, it is not necessary to perform a confirmation operation for flowing a fluid into the flow path in order to confirm the fixed strength of the crystal resonator, and there is no contamination to the specific substance capturing means, so that the high sensitivity. Detection is possible. In addition, the crystal unit can be fixed securely, and the positioning accuracy when the crystal unit is mounted is improved.

  Hereinafter, the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited by the following embodiment.

  FIG. 1 shows the configuration of the microreactor 100 of the present invention, and FIG. 2 shows the structure of components of the microreactor 100. 1A is a plan view of the microreactor 100 of the present invention, and FIG. 1B is a cross-sectional view taken along line A-A ′. 2A is an exploded perspective view when the microreactor 100 of the present invention shown in FIG. 1 is disassembled, and FIG. 2B is a sectional view taken along the line BB ′ when disassembled. .

  The microreactor 100 of the present invention is composed of a flow path substrate 1 and a sensor module 2.

  First, each component will be described individually.

  The flow path substrate 1 is provided with a protruding portion 16 formed in a columnar shape and two fine flow paths 13a and 13b formed inside the structure. More specifically, a flow path 13 a extending from the inflow port 11 to the sensor inflow port 12 and a flow path 13 b extending from the discharge port 14 to the sensor discharge port 15 are provided. Further, a convex portion 16 is provided between the sensor inflow port 12 and the sensor discharge port 15. The outer dimensions of the channel chip 1 are about 20 to 100 [mm] in length, about 10 to 20 [mm] in width, and about several [mm] in thickness.

  The sensor module 2 includes a module substrate 21, a crystal resonator 23, and a sensor holding unit 22 that is interposed between the module substrate 21 and the crystal resonator 23 and bonded to each of them. The module substrate 21 has a through hole (first through hole) 211a, a (third through hole) 211b, and a (first through hole) 211c, and a silicon oxide film on the surface to be joined to the sensor holding unit 22 have. The sensor holding part 22 is made of silicone resin (polydimethylsiloxane, hereinafter referred to as PDMS), and is formed by surrounding a through hole (second through hole) 221 and surrounding the through hole 221. A bank portion 222 and a sensor holding base 223 are provided. The crystal resonator 23 is provided with a detection electrode 231 and a counter electrode 232, and further, a specific substance capturing means 233 is provided on the detection electrode 231.

  When the convex portion 16 of the flow path substrate 1 and the hole (third through hole) 211b of the sensor module 2 are fitted and the flow path substrate 1 and the sensor module 2 are integrated, the microreactor 100 is obtained. Due to this integration, the microreactor 100 is formed from the inflow port 11 to the flow path 13 a, the sensor inflow port 12, the hole 211 a of the sensor module 2, the crystal unit 23, and the convex portion 16 of the flow path substrate 1. A single flow path 13 that passes through the measured measurement chamber 10 and reaches the hole 211c of the sensor module 2, the sensor discharge port 15, the flow path 13b, and the discharge port 14 is formed.

  Therefore, when the sample flows into the inflow port 11, the detection electrode 231 for measuring the specific substance contained in the sample and the specific substance capturing means 233 formed on the detection electrode are immersed in the sample. In addition, a power source is required to perform this liquid feeding, and a liquid is dropped into the inflow port 11 and is sucked and fed by a suction pump connected to the discharge port, or a pressure pump between the tank and the inflow port 11 Can be connected, and the method of sucking the liquid from the tank and feeding it to the inflow port can be selected. Further, in order to prevent liquid leakage between the sensor inflow port 12 and the through hole 211a, the sensor discharge port 15 and the through hole 211c, and the convex portion 16 and the through hole 211b, as shown in FIG. 3 is preferably installed between the module substrate 21 and the flow path substrate 1. Even if the packing is not in the form of a sheet, the same effect can be expected even if three O-rings are individually installed.

  As will be described in detail later, the size of the through hole 221 is such that when the sensor holding part 22 is joined to the module substrate 21, the first through holes 211a and 211c and the third through hole are formed as shown in FIG. When the hole 211b has a size that can be disposed in the second through hole 221, and the flow path substrate 1, the module substrate 21, the sensor holding unit 22, and the crystal resonator 23 are sequentially stacked, 23, the second through-hole 221, the third through-hole 211b, the cylindrical bank portion 222, and the convex portion 16 are formed, and the first through-hole through which the sample flows into the measurement chamber 10 is also formed. It is formed so as to communicate.

  The sensor holding pedestal 223 is provided at a plurality of locations outside the cylindrical bank portion 222, becomes a convex pedestal, and is formed at substantially the same height as the cylindrical bank portion 222. Has a function of holding the crystal resonator so that the convex end portion of the cylindrical bank portion 222 is uniformly joined to the peripheral portion of the detection electrode 231. In addition, since the crystal substrate constituting the crystal resonator 23 is a transparent substrate, when the crystal substrate and the sensor holding base 223 are bonded, the degree of bonding strength between the piezoelectric resonator and the piezoelectric resonator is determined by visual observation as described later. It also has functions that enable it.

  Further, when the crystal resonator 23 is placed on and joined to the cylindrical bank portion 222, the sensor holding pedestal 223 has crystal vibration at an unintended location due to the characteristics of the material of the crystal resonator 23 and the sensor holding portion 22. When the child 23 and the sensor holding part 22 are adhered to each other, the convex end part of the cylindrical bank part 222 is not uniformly joined to the peripheral part of the detection electrode 231 and liquid leakage occurs from the measurement chamber 10. In order to prevent this, it is formed in a convex shape. More specifically, it is formed in a thin column shape, has a small bonding area with the crystal unit 23, and does not inhibit the vibration of the crystal unit 23, so that more accurate measurement is possible.

  Further, since the cylindrical bank portion 222 can be firmly bonded with a small bonding area by siloxane bonding so as to surround the detection electrode 231, more accurate measurement can be performed without inhibiting the vibration of the crystal resonator 23. Is possible.

  In addition, when the sensor holding part 22 uses the material of the material which has elasticity and extensibility like PDMS, the height of the sensor holding base 223 and the height of the cylindrical bank part 222 are considered in consideration of the elasticity and extensibility. You just have to decide.

  The module substrate 21 has a role of forming a part of the measurement chamber 10 and a role of forming an inlet / outlet for allowing the sample to flow into and out of the measurement chamber 10. If it is formed, only the sensor module 2 can be removed from the flow path substrate 1 and transported. For example, cleaning in the measurement chamber 10 and formation of the specific substance capturing means 233 on the detection electrode 231 flow. It can be easily implemented in a state where it is detached from the road substrate 1.

  Further, in the case of this example, the flow path substrate 1 is incorporated in order to increase the flow path length, and does not form the third through hole 211b, but simply the module substrate 21, the sensor holding portion 22, the crystal vibration. A microreactor can be configured with only a sensor module including the child 23. Further, for example, if the module substrate 21 and the sensor holding part 22 are integrally formed of the same member, a microreactor 100 ′ composed only of a sensor module as shown in FIG. 4 can be formed.

  Note that the present invention is not limited to the quartz substrate and the quartz vibrator, and can be configured even by a piezoelectric vibrator made of a transparent piezoelectric substrate.

  Further, as shown in FIG. 5, the specific substance capturing means 233 captures the detection electrode 231 of the microreactor 100, 100 ′ configured as described above by detecting a change in the resonance frequency of the crystal resonator 23. The microreactor system 1000 of the present invention can be configured by connecting a measuring unit 300 that measures the mass of a specific substance.

  Next, a method for manufacturing the sensor module 2 will be described. The module substrate 21 is formed by injecting resin to form a plate having holes 211a, 211b, and 211c. Thereafter, silicon oxide is sputtered onto the molded plate to form a silicon oxide film on the plate surface. The quartz crystal resonator 23 is produced by vapor-depositing or sputtering both surfaces of an AT-cut quartz plate with gold to create a detection electrode 231, a counter electrode 232, and wiring to each electrode.

  Next, a method for manufacturing the sensor holding unit 22 will be described. When a resist is applied onto a silicon wafer and patterned using a photolithography technique, a fine structure is formed on the silicon wafer with the resist. On this, after forming a fluororesin film | membrane, PDMS is poured and PDMS is heat-hardened. Thereafter, the PDMS is irradiated with ultraviolet rays to activate the PDMS surface, and the PDMS surface is directly bonded to the silicon oxide film of the module substrate 21 by a siloxane bond. The PDMS and the silicon wafer are peeled off, and the quartz vibrator 23 is positioned on the peeled surface of the PDMS so that the crystal resonator 23 is positioned at a predetermined position. Then, ultraviolet rays are irradiated from above, and the siloxane bond between the PDMS and the quartz substrate is utilized. The sensor module 2 is manufactured by bonding.

  At the time of joining the crystal resonator 23 and the sensor holding part 22 at this time, the mutual positioning and joining strength, that is, poor joining, etc. can be detected by visual observation of the sensor holding base 223. More specifically, first, water or ethanol is dropped onto the sensor holding unit 22, and then the crystal unit 23 is placed on the cylindrical bank portion 222 and the sensor holding base 223 of the sensor holding unit 22. At this time, since water or ethanol exists between the cylindrical bank portion 222, the sensor holding base 223, and the crystal unit 23, the crystal unit 23 can be moved in the plane.

  The image from the upper surface is observed, the crystal resonator 23 is positioned so that the relative position between the end of the crystal resonator 23 and the sensor holding base 223 is a predetermined position, and the crystal resonator 23 is pressurized. When the pressure is applied, water or ethanol is pushed to the surroundings, and the crystal resonator 23, the cylindrical bank portion 222, and the sensor holding base 223 are in direct contact with each other. If the ultraviolet ray is irradiated as it is, the crystal unit 23, the cylindrical bank portion 222, and the sensor holding base 223 are joined.

  After bonding, the bonding surface with the sensor holding base 223 is observed from the upper surface of the crystal unit 23. When joined as designed, the end face of the sensor holding base 223 can be clearly observed, but if the joining is insufficient, the end face is blurred. If all of the plurality of sensor holding bases 223 arranged on the outer periphery of the cylindrical bank portion 222 are joined, the cylindrical bank portion 222 and the crystal unit 23 having substantially the same height as the sensor holding base 223 are parallel to each other. From this, it can be confirmed that the cylindrical bank portion 222 and the crystal resonator 23 are joined. For this reason, it is possible to confirm the joining state without confirming the entire outer periphery of the cylindrical bank portion 222.

  Although an example of the manufacturing process is given here, the resin forming the module substrate can be acrylic, polycarbonate, cyclic olefin copolymer, etc. If possible, the peel strength from the silicon oxide film can be increased, and heat resistance・ It is desirable to have high chemical resistance.

  Next, a method for forming the specific substance capturing means 233 installed on the detection electrode 231 will be described. Here, as an example, a preparation method for immobilizing an antibody on a self-assembled membrane (hereinafter referred to as SAM) is shown. First, pure water is dropped and washed on the detection electrode 231, and then a SAM reagent (carboxyl terminal disulfide type) is dropped to form SAM on the detection electrode 231 and washed with a phosphate buffer.

  Next, hydroxysuccinimide is added dropwise to activate the SAM and again wash with phosphate buffer. Thereafter, the antibody to be immobilized on the phosphate buffer is mixed and added dropwise to immobilize the antibody on the SAM. Here, the specific substance capturing means 233 is formed directly on the crystal unit 23. However, even in the configuration of the sensor module 2, since there is a gap on the detection electrode 231, it can be formed by the same means. After the formation, the sensor module 2 and the flow path substrate 1 are fitted and integrated, and a detection operation can be performed. Therefore, the specific substance capturing means 233 is not contaminated without contaminating the flow path 13 used for detection. Can be formed.

  A process for detecting only a specific substance from a sample flowing in the microreactor 100 will be described. Here, detection of biopolymers, particularly proteins, will be described as an example.

  A sample flows into the inflow port 11 of the microreactor 100. The sample is immersed in the specific substance capturing means 233 on the crystal resonator 23 provided in the measurement chamber 10 through the flow path 13 from the inflow port 11. At this time, the antibody of the specific substance capturing means 233 captures and fixes the specific antigen contained in the sample, and the mass adhering to the detection electrode 231 increases, so that the resonance frequency of the crystal unit 23 changes. This change is measured by the frequency counter 5 connected to the crystal unit 23, and the mass of the captured substance can be measured. Since the resonance frequency of the crystal unit 23 changes depending on the ambient temperature, it is desirable to install the microreactor 100 in a heat retaining mechanism that keeps the temperature uniform.

  As described above, high-sensitivity detection can be performed without confirming the flow of the fluid into the flow path in order to confirm the fixed strength of the crystal unit and because there is no contamination of the specific substance capturing means. In addition, since the crystal unit can be securely fixed and the positioning accuracy when the crystal unit is mounted is improved, high detection sensitivity can be maintained.

It is a model diagram which shows the structure of the microreactor concerning this invention. It is an exploded view for demonstrating the structure of the microreactor concerning this invention. It is the schematic for demonstrating one structural example of the microreactor concerning this invention. It is the schematic for demonstrating the other structural example of the microreactor concerning this invention. It is a block diagram which shows the structure of the micro reactor system concerning this invention.

Explanation of symbols

100, 100 ′ Microreactor 1 Flow path substrate 2 Sensor module 3 Sheet packing 10 Measurement chamber 11 Inflow port 12 Sensor inflow port 13 Flow path 14 Discharge port 15 Sensor discharge port 16 Convex portion 21 Module substrates 211a and 211c Through holes (first Through-hole)
211b Through hole (third through hole)
22 Sensor holding part 221 Through hole (second through hole)
222 Cylindrical bank 223 Sensor holding base 23 Crystal oscillator 231 Detection electrode 232 Counter electrode 233 Specific substance capturing means 300 Measuring means 1000 Microreactor system

Claims (6)

A microreactor having a measurement chamber provided with a specific substance capturing means for capturing a specific substance contained in a flowed sample and used for measuring the mass of the specific substance captured by the specific substance capturing means. ,
A module substrate having a plurality of first through holes for flowing the sample;
A piezoelectric vibrator in which a detection electrode is formed on a transparent piezoelectric substrate, the specific electrode capturing means is provided on the detection electrode, and the detection electrode is disposed so as to face the module substrate; ,
A first surface that is disposed between the module substrate and the piezoelectric vibrator and is bonded to the module board; and a surface opposite to the first surface, the first surface being bonded to the piezoelectric vibrator. And a sensor holding part having a second surface for holding the piezoelectric vibrator, and a second through hole penetrating from the first surface to the second surface,
The sensor holding portion is provided in a cylindrical shape that is formed in a cylindrical shape and a convex shape surrounding the second through hole, and on the outer side of the cylindrical bank portion, and is formed in a convex shape. A plurality of sensor holding bases on the second surface;
The cylindrical bank portion is joined to the peripheral portion of the detection electrode to form the measurement chamber that holds the peripheral portion of the piezoelectric vibrator and communicates with the first through hole,
The plurality of sensor holding pedestals can visually recognize the function of holding the piezoelectric vibrator so that the cylindrical bank portion is uniformly joined to the peripheral portion of the detection electrode and the degree of joining with the piezoelectric vibrator. A microreactor having a function of enabling determination.   The substrate has a flow path through which the sample flows in connection with the first through hole, and is fitted with a third through hole newly formed at a position facing the detection electrode of the module substrate. 2. A flow path substrate having a columnar convex portion on a surface of the substrate and being stacked by being fitted to the third through hole and the columnar convex portion. 2. The microreactor according to 1.   A sheet-like packing which is disposed between the module substrate and the flow path substrate and prevents liquid leakage of the sample from a fitting portion where the third through hole and the convex portion are fitted. The microreactor according to claim 2, wherein   The module substrate has a silicon oxide film on a surface to be joined to the sensor holding portion, and the sensor holding portion is made of silicone resin, while the piezoelectric substrate is made of a quartz substrate, and the module substrate and the sensor holding portion The microreactor according to claim 1, wherein the quartz substrate and the quartz substrate are bonded to each other by a siloxane bond.   The module substrate and the sensor holding portion are integrally molded products made of the same member, while the piezoelectric substrate is made of a quartz substrate, and the sensor holding portion and the quartz crystal substrate of the integrally molded material are each bonded by a siloxane bond. The microreactor according to claim 1, wherein the microreactor is bonded. A microreactor according to any one of claims 1 to 5;
A microreactor connected to the detection electrode, and measuring means for measuring a mass of the specific substance captured by the specific substance capture means by detecting a change in a resonance frequency of the piezoelectric vibrator. system.

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