Abstract:
A reactor comprises a main body having a flow path substrate and a crystal substrate chemically bonded to the flow path substrate to form a flow path for running a sample to be measured and a reactor tank connected to the flow path. An adsorption film is disposed in the reactor tank for adsorbing a specific substance contained in the sample to be measured. A measuring device measures a physical quantity of the specific substance contained in the sample and adsorbed by the adsorption film.

Description:
The present invention relates to a reactor utilizing a crystal oscillator and a method for manufacturing the same. In particular, the present invention relates to a reactor, a micro-reactor chip, and a micro-reactor system for the measurement of a viscosity, density and the like of a sample and the detection of the mass of a specific substance contained in a sample, as well as methods for manufacturing the same. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a reactor utilizing a crystal oscillator and a method for manufacturing the same. In particular, the present invention relates to a reactor, a micro reactor chip, and a micro reactor system for the measurement of the viscosity, density and the like of a sample and the detection of the mass of a specific substance contained in a sample as well as their manufacturing method. 
     2. Description of the Related Art 
     Active developments of small analysis systems called lab-on-a-chip have been under way in recent years. Small analysis systems of this type integrate element structures such as flow paths, reactor tanks, valves, sensors and the like on a small substrate to analyze gases and liquids flowing through these element structures. Examples of systems of this type can include biochips, which perform clinical inspection on the blood flowing through a minute flow path provided in a resin chip (Refer to Non-patent Reference 1, for example). Using such a small analysis system allows the fast analysis of a small quantity of a sample and therefore the reduction of burden on the side that provides the sample. Therefore, particularly the application of such a system to a living body is attracting attention. 
     Sensors utilizing various principles have been proposed for the sensor section, which is one of the components of the small analysis system. Above all, the use of QCM (Quarts Crystal Microbalance) and SAW (Surface Acoustic Waves) sensors is anticipated because these sensors are small-sized and capable of platy configuration and assumed to be easy to mount in systems. 
     QCM and SAW sensors utilize the oscillation of piezoelectric oscillators (particular crystal oscillators) and employ technologies for measuring the viscosity of samples that are in contact with the surface of the piezoelectric oscillator and minute mass adherent to the oscillator. More specifically, the QCM sensor oscillates at a specific frequency determined by the material properties and shape of the piezoelectric oscillator when an AC voltage is applied to electrodes formed on opposite surfaces of the piezoelectric oscillator. When a substance adheres to any electrode of the piezoelectric oscillator, the resonant frequency of the entire oscillator changes in response to mass adherent thereto. Also, the SAW sensor generates an elastic wave of a specific frequency determined by the frequency of the AC voltage, the shape of the electrode, the material properties of the piezoelectric element and the like when an AC voltage is applied to one of two pairs of blind-line electrodes thereof. The elastic wave generated is detected as a cyclic current by means of the other electrode pair due to the piezoelectric effects of the element. When a substance is then adhered between the two pairs of electrodes, the speed of the elastic wave changes in response to adherent mass. The phase, the frequency and the input/output impedance ratio also change between the voltage applied and the cyclic current detected. The technologies employed measure the mass of the substance adhered to the electrode by detecting the above-mentioned changes. 
     However, the detection of a specific substance is not possible with such mass measuring means. Therefore, a configuration is used which detects only a specific substance with means fixed in position for adsorbing or capturing the specific substance. As an example of such a configuration, a technique is known for using antigen-antibody reaction for protein detection (Refer to Patent Reference 1, for example. The utilization of such a configuration in a QCM or SAW sensor makes it possible to measure the minute mass of a specific substance to be measured. The utilization of the QCM or SAW sensor in the sensor section of a small analysis system therefore allows the high-precision measurement of a desired substance and the realization of an analysis system of a small-sized configuration. 
     The mounting of a crystal oscillator in an analysis system therefore requires a crystal oscillator to be mounted without any sample leaks. There are few disclosures and almost no specific disclosures concerning a method for mounting a crystal oscillator in a small analysis system. Consequently, such a method is analogized with typical methods for mounting a crystal oscillator as for use as a reactor. Conventional crystal oscillator mounting methods employed include methods for providing an O-ring at the interface between an analysis system base material and a crystal oscillator and pressing and contacting the oscillator and the system base material (refer to patent Reference 2, for example) and methods for bonding the base material and the oscillator (refer to Patent Reference 3, for example), and methods for gluing the base material and the oscillator.
     [Patent Reference 1] JP-A-2000-338022   [Patent Reference 2] JP-A-11-14525   [Patent Reference 3] JP-T-2004-523150   [Non-Patent Reference 4] Proc. μTAS Symposium 2002, vol. 1, 187-189   

     However, methods that use an O-ring at the interface between the analysis system base material and the crystal oscillator require a mechanism for pressuring the crystal oscillator against the analysis system base material and have the problem that it is impossible to reduce the size of the analysis system itself. These methods also problematically require strict pressure adjustments because a small pressure on the crystal oscillator causes sample leaks between the system base material and the crystal oscillator while a large pressure on the crystal oscillator causes damage to the crystal oscillator. 
     Methods for gluing the analysis system base material and the crystal oscillator problematically require strict control of amounts of adhesive applied because the adhesive applied to the crystal oscillator or system base material reaches contaminates the inner walls of the flow path and reactor tank of the system and the sensing surfaces of sensors. 
     Methods for bonding the analysis system base material and the crystal oscillator require heat treatments at high temperatures and produce residual stresses in the crystal oscillator because of the different coefficients of thermal expansion of the crystal oscillator and the system base material after bonding. Consequently, these bonding methods problematically suffer from a drop in sensitivity of the crystal oscillator due to failure to operate at a desired frequency. 
     In any of the gluing and bonding methods, the fixed region of the crystal oscillator relative to the system base material is increased and the region fixed to the system base material acts as a fixed end. Oscillations reflected from the fixed region cause the crystal oscillator to oscillate in an unintended oscillation mode (spurious). Desired oscillations cannot be separated from spurious oscillations and there is also a problem of a drop in sensor sensitivity. When, on the other hand, the fixed region is reduced, a sufficient fixing strength cannot be obtained relative to the internal pressure generated from the supply of the reagent and there is also a problem of liquid leaks from the boundary between the system base material and the crystal oscillator. 
     SUMMARY OF THE INVENTION 
     To attain the above-mentioned problems, the present invention is characterized by a configuration described below. That is, in a reactor having a flow path for running a sample to be measured, a reactor tank connected to the flow path and having capture means for capturing a specific substance contained in the sample to be measured, and a liquid-phase sensor for measuring a physical quantity of the specific substance contained in the sample to be measured which has been caught by the capture means, the reactor tank includes a substrate formed with a concave portion and made of a material that chemically bonds to silicon contained and a crystal substrate disposed to cover the concave portion and bonding to the substrate through a chemical bond. The liquid-phase sensor includes the crystal substrate, a crystal oscillator disposed on a surface of said crystal substrate and having electrodes formed with said capture means and frequency measuring means connected to said electrodes for measuring a change in frequency of said crystal oscillator. 
     In addition, a micro reactor chip according to the invention comprises the reactor and includes a liquid introduction port formed on the substrate for introducing the sample to be measured to the reactor tank through the flow path and a liquid discharge port formed on the substrate for discharging the sample to be measured from the reactor tank through the flow path. 
     The micro reactor system according to the invention includes the micro reactor chip, pump means connected to the liquid introduction port or the liquid discharge port for feeding the sample to be measured, liquid feed control means for controlling the opening and closing of the valve mechanism and control means for controlling the pump means, the frequency measuring means, and the liquid feed control means. 
     In addition, a micro reactor chip manufacturing method according to the invention includes a first step of forming a concave portion in a substrate, a second step of forming an electrode on a crystal substrate, and a third step of causing the substrate and the crystal substrate to bond to each other through a chemical bond to form a reactor tank by laying the substrate and the crystal substrate over the concave portion. 
     According to a reactor, micro reactor chip, and micro reactor system and their manufacturing methods of the invention, the configurations and the manufacturing methods are simple but ensure that the sensor, chip base material, and crystal oscillator can be integratedly formed. the configurations and the manufacturing methods also realize high-sensitivity detection because of the absence of residual stresses on the crystal oscillator and unwanted oscillation modes. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       In the accompanying drawings: 
         FIG. 1A-1C  are an explanatory view of the configuration of a reactor according to one embodiment of the present invention; 
         FIG. 2A-2B  are a circuit diagram of a sensor section according to one embodiment of the present invention; 
         FIG. 3A-3G  are a diagram showing a process for manufacturing the reactor according to one embodiment of the present invention; 
         FIG. 4  is a perspective view of a micro reactor chip according to one embodiment of the present invention; 
         FIG. 5A-5B  are a plan view of the micro reactor chip according to one embodiment of the present invention; 
         FIG. 6  is a flow chart showing the detection operation of the micro reactor chip according to one embodiment of the present invention; 
         FIG. 7A-7D  are a cross sectional view showing the liquid feed state for the micro reactor chip according to one embodiment of the present invention; 
         FIG. 8  is a cross sectional view showing a micro reactor system according to one embodiment of the present invention; 
         FIG. 9A-9B  are a cross sectional view showing the operation of a valve of the micro reactor chip according to the invention; 
         FIG. 10A-10B  are a perspective view showing the configuration of a micro reactor chip according to another embodiment of the present invention; 
         FIG. 11A-11E  are a process diagram showing a configuration for holding a crystal substrate on a micro reactor chip according to another embodiment of the present invention; 
         FIG. 12  is a perspective view showing a micro reactor chip according to another embodiment of the present invention; 
         FIG. 13A-13B  are a plan view of a micro reactor chip according to another embodiment of the present invention; 
         FIG. 14  is a cross sectional view showing a micro reactor chip according to another embodiment of the present invention; and 
         FIG. 15A-15F  are a diagram showing a process for manufacturing a micro reactor chip according to another embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention will be described in detail with reference to the drawings. Note that the invention is not limited to the embodiments described below. 
       FIG. 1  is a diagram describing the configuration of a reactor  1000  according to the invention.  FIG. 1A  is an exploded view showing the partial configuration of the reaction  1000   FIG. 1B  is a cross sectional view of the reactor  1000  (the cross section of surface A in  FIG. 1A ).  FIG. 1C  is an explanatory view showing a configuration for feeding a reagent into the reactor  1000 . The reactor  1000  is designed to analyze the interaction of biomolecules such as protein, specifically, to bond analyte to a ligand and detect the state of the resulting bond reaction (such as bond strength, bond speed, and dissociation constant, for example). 
     First Embodiment 
     The reactor  1000  includes a main body having a crystal substrate  100  and a flow path substrate  200  bonded together. The crystal substrate  100  has a generally flat shape with opposed major surfaces and will be described first. A detection electrode  601  and an opposite electrode  602  are provided on both surfaces of the crystal substrate  100 . In addition, an adsorption film  60  for adsorbing only a specific substance is provided on a surface of the detection electrode  601 . The flow path substrate  200  is provided with a groove  201  as well as a liquid an introduction port  202  and a liquid discharge port  203 , which are through-holes provided in the groove  201 . A portion of the groove  201  is widened. 
     The crystal substrate  100  and the flow path substrate  200  are integrated to form the reactor  1000 . Specifically, one surface of the crystal substrate  100  provided with the detection electrode  601  is bonded to and integrated with one surface of the flow path substrate  200  provided with the groove, thereby forming a flow path  400  between the groove  201  and the surface of the crystal substrate  100 . In addition, the widened region of the groove  201  is then formed into a reactor tank  500  and the detection electrode  601  will be provided in the reactor tank  500 . 
     When a sample liquid fed is to the liquid introduction port  202  by means of a pump  902  connected to the liquid introduction port  202 , the sample liquid can be made to flow through the flow path  400  and the reactor tank  500  and via the liquid discharge port  203  to a waste liquid tank  800 . The detection electrode  601  for measuring a specific substance contained in the sample liquid and the surface of the adsorption film  609  will therefore be soaked with the sample liquid. 
     A description will then be made of a technique for measuring the mass of a substance adhered to the detection electrode  601  through an electrical system connected to the detection electrode  601  and the opposite electrode  602  provided on the crystal substrate  100  and electric signals. the reactor  1000  described here uses a AT-cut crystal plate. The AT-cut crystal plate will generate thickness shear vibrations when a cyclic electric potential difference is provided across the thickness thereof. 
       FIG. 2A  shows an electrical configuration where a crystal substrate  100  is connected to a detection electrode  601  and an opposite electrode  602 . A variable-frequency AC power supply  701  and an ammeter  702  are connected in series to each other. One end is connected to the detection electrode  601  and the other end to the opposite electrode  602 . The application of an AC voltage to the detection electrode  601  and the opposite electrode  602  from the AC power supply  701  causes a current flowing through the ammeter  702  to change in response to the frequency of the voltage applied. The frequency of the voltage applied where the current reaches a maximum value is resonant frequency. When a substance to be measured adheres to the detection electrode  601 , the resonant frequency drops in response of the mass of the adherent substance. Therefore, the mass of the substance adherent to the detection electrode  601  can be measured by detecting a change in resonant frequency. As shown in  FIG. 2B , the mass of the substance adherent to the detection electrode  601  can also be similarly measured by the Kollwitz oscillation circuit to cause the crystal oscillator to oscillate and then measuring a change in resonant frequency using a frequency counter  703 . 
     A method for manufacturing a reactor  1000  according to the invention will then be described below. A thin film of chrome or titanium is first formed on an electrode-forming region on both surfaces of a AT-cut crystal plate  100  which is then deposited or sputtered with gold to prepare a detection electrode  601 , an opposite electrode  602  and wiring to both electrodes. As shown in  FIG. 3 , a resist  32  ( FIG. 3B ) is then formed on a cleaned silicon wafer  31  ( FIG. 3A ), which is then coated with Teflon  33  ( FIG. 3C ). Polydimethylsiloxane (PDMS)  34  is then poured onto the silicon wafer and allowed to cure ( FIG. 3D ). The PDMS  34  is then peeled from the silicon wafer  31  ( FIG. 3E ). This forms a groove in the PDMS  34 . The PDMS  34  is then laid on the crystal plate  100 . When the crystal substrate  100  side is then irradiated with ultraviolet light (light source: UV excimer lamp; wavelength: 172 nm), the silicon-carbon bond between the crystal and the PDMS is cut, thus causing the crystal and the PDMS to bond to each other by means of a siloxane bond (which bonds silicon and oxygen to each other) ( FIG. 3F ). The liquid introduction port and the liquid discharge port are then cut to form the reactor  1000  ( FIG. 3G ). 
     Because the crystal and the PDMS are bonded to each other by means of covalent bond, the crystal and the PDMS plate can be bonded to each other with a high strength. Because only ultraviolet light is used for irradiation in the covalent bond, none of these members are heated and no residual stresses are generated in the crystal after bonding. Because of its low rigidity, the PDMS plate  34  also never attenuates the oscillation of the crystal even when bonded to the crystal. In the configuration according to the invention, the flow path substrate is provided with an elongate bank structure and the upper of the bank structure is bonded to the crystal substrate  100  to hold a crystal oscillator. Because the crystal oscillator is held with a low-rigidity structure as described above, the low-rigidity structure never attenuates the oscillation of the crystal. The resonant frequency of the crystal oscillator can therefore be captured accurately, which allows the mass of a substance on the crystal oscillator to be measured with high sensitivity. 
     In the manufacturing method described above, ultraviolet light is used for irradiation when the crystal substrate  100  and the PDMS  34  are bonded to each other. If the bonding surfaces are clean enough, both of the members can be bonded to each other by simply pressing these members to bring one member into contact with the other. However, the two members, when positioned, can be bonded to each other in a position, not in a predetermined positional relationship. After ethanol is applied to one bonding surface, one member is therefore aligned with the other, when the crystal substrate  100  and the PDMS  34  are not bonded to each other because of the ethanol layer on the bonding surface. When the two members can be aligned with each other and are pressed to bring one member into contact with the other, the ethanol is carried away, thus allowing the planned bonding surfaces to be bonded to each other under pressure. 
     While the crystal substrate  100  and the PDMS plate  34  are bonded to each other by means of covalent bond, the electrode (gold) on the crystal substrate  100  cannot bond to and simply self-sticks to the PDMS plate  34 . Even if the electrode is not bonded to the crystal substrate  100 , however, there is no fear that the electrode section in the bonding region will have liquid leaks and the like because the electrode section has a width of a few hundred nm or so. 
     In addition, reactive ion etching (RIE) by oxygen is performed on the planned boding surfaces of the crystal substrate  100  and the PDMS plate  34  to activate the planned boding surfaces. After that, the crystal substrate and the PDMS plate can also be bonded to each other by laying the PDMS plate  34  on the crystal substrate  100  and using ultraviolet light for irradiation. The crystal substrate and the PDMS plate can also be bonded to each other by applying silicone oil to the crystal substrate  100 , laying the PDMS plate  34  on the crystal substrate  100 , and using ultraviolet light for irradiation. Using these methods allows a high bonding strength to be maintained even if there are contamination on and minute irregularities in the planning surfaces of the crystal substrate  100  and the PDMS plate  34 . 
     In addition, the formation of the flow path substrate uses a method for pouring liquid silicone resin onto the surface of a mold to transcribe mold irregularities, thus allowing the flow path substrate to be manufactured easily. Because the mold with a resist formed on a silicon wafer can be used many times, it is easy to mass produce the flow path substrate. 
     An example of the reactor manufacturing process has been described above. However, it is also possible to use, as a system base material, other types of silicon resin and silicon-free resin with the surface thereof coated or sputtered with silicon dioxide. 
     A method for forming an adsorption film to be provided on a detection electrode  601  will be described below. A method for preparing an adsorption film  609  with a self-assembled monolayer (hereinafter referred to as SAM) will be described below as an example of the method. Pure water is first poured into the flow path  400  for cleaning purposes. A SAM reagent (carboxyl-terminated disulfide type) is then poured on the detection electrode  601  to form the SAM thereon, which is then cleaned with phosphoric acid buffer. Hydroxysuccinic acid imide is then poured onto the SAM to activate the SAM, which is cleaned with phosphoric acid buffer again. An immobilization antibody is then mixed with the phosphoric acid buffer and the resultant mixture is poured onto the SAM to immobilize the antibody on the SAM. A method for forming the adsorption film  609  after the gluing and bonding of two substrates has been described above. However, it is also possible to form an adsorption film  609  during formation of the crystal wafer and then glue or bond the two substrates to each other. 
     A process for detecting a specific substance in a sample flowing through a reactor  1000  will be described below. The detection of a biopolymer, particularly a protein, will be described below as an example of a specific substance. 
     A sample liquid is poured into the liquid introduction port  202  of the reactor  1000 . The sample liquid flows through the flow path  400  and to the reactor tank  500 . The reactor tank  500  is filled with the sample liquid and an adsorption film  609  provided on the surface of a detection electrode  601  is soaked with the sample liquid, at which time an antibody in the adsorption film  609  captures and fixes a specific antigen contained in the sample liquid. An resultant increase in mass of the antigen on the detection electrode  601  causes a change in the resonant frequency on the detection electrode  601  side. An ammeter  702  can measure the change to measure the mass of the substance fixed to the adsorption film  609 . Before the change in the resonant frequency can be measured, the frequency of an applied signal from a power supply  701  is first changed gradually with no mass on the detection electrode  601 , under which condition, the resonant frequency is then measured. 
     The frequency of the applied signal is then limited to a frequency close to the resonant frequency to repeat a gradual change in the frequency of the applied signal. Each time the gradual change is repeated, the ammeter  702  is used to determine the frequency at which the current reaches a maximum value. With the applied signal from the power supply  701  as a white noise, it is also possible to break a current value measured on the ammeter  702  into frequency components using FFT and determine a change in the resonant frequency. With a clear relation between the frequency of the applied signal and the current value from the ammeter, it would also be possible to estimate the resonant frequency through a curve fit and the like after the measurement of the frequency of the applied voltage and the current value at several points. 
     The configurations and manufacturing method described above, though simple, makes it possible to maintain a high sensitivity without residual stresses on the crystal oscillator and unwanted oscillation modes and to ensure that the system base material and the crystal oscillator can be integrated with each other. 
     Second Embodiment 
       FIG. 4  is an exploded perspective view of a micro-reactor chip (hereinafter “micro reactor chip”)  2000  according to the invention.  FIG. 5A  and  FIG. 5B  show a schematic plan view of the micro reactor chip and a cross sectional view of the micro reactor chip (the cross section taken along the surface B in  FIG. 5A ), respectively. The same descriptions as in the first embodiment will not be made below. 
     The micro reactor chip  2000  includes a generally flat crystal substrate  100  having opposed major surfaces, flow path substrates  220   a  and  220   b , and a holding substrate  310 , all of which are laminated together. Each of the members will be described below. The crystal substrate  100  is configured in the same way as in the first embodiment. The flow path substrates  220   a  and  220   b  are formed with a minute concave portion and a through-hole while the holding substrate  310  and the substrate  300  are formed with a through-hole. These members are laminated together and integrated with one another, thereby providing a reactor tank  223 , where reaction occurs, a sample liquid supply path  225  for feeding a sample liquid for analysis, a buffer liquid supply path  226  for feeding a buffer liquid having a flow path cleaning function and a buffer function (dissociation function), and a waste liquid path  227  leading to a liquid discharge port  203   a . Specifically, the buffer liquid supply path  226  and the reactor tank section  223  are connected to each other, thereby forming a first flow path. The sample liquid supply path  225  and the waste liquid path  227  are connected to each other, thereby forming a second flow path. In addition, a branch flow path section  229  connected to the upstream side away from the reactor tank section  223  of the first flow path is provided starting on the second flow path. The crystal substrate  100  and the flow path substrates  220   b  to form the reactor tank section  223 , which is almost the same way as in the first embodiment described earlier. Moreover, the flow path substrates  220   b  is then integrated with the reactor tank section  223 , thereby forming a flow path. The flow path and the reactor tank section  223  will then be connected to each other. 
     A port  202   a  for sample liquid introduction, shaped like a cup to which drops of a sample liquid is fed, is provided at an end of the sample liquid supply path  225  (the end on the opposite side of the waste liquid path  227 ). Similarly, a port  202   b  for buffer liquid introduction, shaped like a cup to which drops of a buffer liquid is fed, is provided at an end of the buffer liquid supply path  226  (the end on the opposite side of the reactor tank section  223 ). While the waste liquid path  227  is connected to a liquid discharge port  203   a , the reactor tank section  223  is connected to a liquid discharge port  203   b.    
     The second flow path including the sample liquid supply path  225  and the waste liquid path  227  is provided with a valve  212   a  and a valve  212   c  on the upstream side of the branch flow path section  229  and on the downstream side of the branch flow path section  229 , respectively. The first flow path including the buffer liquid supply path  226  and the reactor tank section  223  is also provided with a valve  212   b  and a valve  212   d  on the upstream side of a portion connected to the branch flow path section  229  and on the downstream side of a portion connected to the branch flow path section  229 , respectively. 
     The configuration of a micro-reactor system (hereinafter “micro reactor system”)  2500  using a micro reactor chip  2000  will be then described below with reference to a configuration diagram in  FIG. 8 . The micro reaction chip  200  is first provided on a stage  2510 . A contact pin  2511  is fixed to the state  2510 . When the micro reactor chip  2000  is provided, the contact pin  2511  is brought into contact with wiring from a detection electrode  601  of a crystal substrate  100  and an opposite electrode  602  under a constant pressure, thereby providing electrical conduction. 
     A drop port  2520  for feeding drops of a sample liquid and a buffer liquid is also disposed directly above each of the port  202   a  for sample liquid introduction and the port  202   b  for buffer liquid introduction of the micro reactor chip  2000  provided on the stage  2510 . A linear actuator  2530  for valve opening and closing is disposed directly above the valve  212 . A suction port  2540  is connected to each of liquid discharge ports  203   a  and  203   b . The suction port  2540  on the liquid discharge port  203   a  side is connected to a waste liquid tank  800  while the suction port  2540  on the liquid discharge port  203   b  side is connected through a trace liquid feed pump  901  to the waste liquid tank  800 . In addition, a pump  902  is connected to the waste liquid tank  800 . The trace liquid feed pump  901 ′ and the pump  902  can suction a fluid from the waste liquid path  227  and the reactor tank section  223 . 
     Moreover, a control circuit  2550  is connected to each of the contact pin  2511 , drop port  2520 , linear actuator  2530 , trace liquid feed pump  901 , and pump  902 . 
     Liquid feeding for the micro reactor system  2500  will be described below. A drop port  2520  is disposed directly above a port  202   a  for sample liquid introduction and a port  202   b  for buffer liquid introduction. This causes a constant amount of a sample liquid or buffer liquid to drop and be fed to a chip without any contact. The liquid flowing through a valve  212  is fed under the control of the valve  212 . The valve is opened and closed by a linear actuator  2530  disposed directly above the valve. The valve  212  has an elastic deformation portion. When pressed by the linear actuator  2530 , the elastic deformation portion shuts off the flow path ( FIG. 9B ). The elastic deformation portion deforms elastically and ceases to deform if a pressure is relieved, thereby opening the flow pass (Refer to  FIG. 9A ). 
     Consequently, the valve can be open and closed by simply providing a force to the valve from outside the chip. A liquid is discharged from the chip through liquid discharge ports  203 . A suction port  2540  is connected to each of the liquid discharge ports  203 . Because an O-ring is fit in a stepped portion in an outside surface, the O-ring becomes deformed when the suction port  2540  is brought into contact with the liquid discharge port  203  under pressure. The trace liquid feed pump  901  and the pump  902  can therefore communicate with the chip without any negative-pressure loss, thus allowing an accurate liquid feed. The liquid from the chip flows through the suction port  2540 , being ultimately accumulated in a waste liquid tank  800 . A control circuit is responsible for all of the liquid feed described above and detection by sensor operation at a predetermined timing. 
     With the configuration as described above, the micro reactor system  2500  can be connected to the micro reactor chip  2000  through only contact under pressure without utilizing screws. This allows the system to be easily connected to and disconnect from the chip. The configuration described above is an example of the configuration of the system. The connection between the chip and the system is not limited to the above configuration. 
     A method for manufacturing a micro reactor chip  2000  will be then described below. As with the first embodiment, a crystal substrate  100  is formed by forming an electrode on a polished AT-cut crystal plate  100 . As with the first embodiment, flow path substrates  220   a  and  220   b  are formed with a minute concave portion by using polydimethylsiloxane (PDMS) and photolithography. After the application of a silica solution to the surface of an acrylic plate, a holding substrate  310  is heated, thereby forming thin glass the surface thereof. A substrate  300  is a glass plate. 
     In the bonding process, silicon oil is first to the planned bonding surfaces of a flow path substrates  220   b  and a crystal substrate  100 . One member is then brought into contact with the other. The crystal substrate  100  side is then irradiated with ultraviolet light to bond flow path substrates  220   b  and a crystal substrate  100  to each other. The flow path substrates  220   a  and  220   b  are then brought into contact with the crystal substrate  100  for bonding purposes. The planned bonding surfaces of the flow path substrates  220   b  and a substrate  300  are then irradiated with oxygen plasma and the flow path substrates  220   b  and a substrate  300  are brought into contact with each other for bonding purposes. Finally, the planned bonding surfaces of a holding substrate  310  and the flow path substrates  220   a  are irradiated with oxygen plasma and the holding substrate  310  and the flow path substrates  220   a  are brought into contact with each other for bonding purposes. 
     A port  202   b  for buffer liquid introduction and a port  202   b  for buffer liquid introduction, both fabricated of polycarbonate resin, are connected to a plate-like chip that includes these four members laminated together, thereby providing a complete micro reactor chip  2000 . Note that other resin materials or more desirably any material with good heat resistance can be substituted for acrylic and polycarbonate mentioned as member materials. 
     An analysis method according to the invention using the micro reactor system  2500  will be described specifically with reference to a flow chart in  FIG. 6 . 
     A reactor tank section  223  is first modified with a ligand (Step  21 ). Specifically, an adsorption film  609  is formed through a detection electrode  601  provided in a crystal substrate  100  in the reactor tank section  223 . Air relief is then done to discharge air from each flow path. A buffer liquid is then dropped a port  202   b  for buffer liquid introduction and a sample liquid is dropped into a port  202   a  for sample liquid introduction. Valves  212   a  and  212   d  are first closed and valves  212   b  and  212   c  are opened before a pump  902  is operated. This causes the buffer liquid dropped into the port  202   b  for buffer liquid introduction to be suctioned and fed through a buffer liquid supply path  226 , a branch flow path section  229 , and a waste liquid path  227  to a waste liquid tank  800 . Air is then relieved of the buffer liquid supply path  226 , the branch flow path section  229 , and the waste liquid path  227  (Step  22 ) to fill these components with the buffer liquid as shown in  FIG. 7A . The valve  212   c  is then closed and the valve  212   d  is opened to guide the buffer liquid in the buffer liquid supply path  226  to the reactor tank section  223  and to cause the buffer liquid to be suctioned and fed to the waste liquid tank  800 . 
     Air is then relieved of the reactor tank section  223  and the flow paths connected to the reactor tank section  223  as described above (Step  23 ) to fill the reactor tank section  223  with the buffer liquid as well, as shown in  FIG. 7B . The valves  212   b  and  212   d  are closed and the valves  212   a  and  212   c  are opened. This causes the sample liquid dropped into the port  202   a  for sample liquid introduction to suctioned and fed through a sample liquid supply path  225  and the waste liquid path  227  to the waste liquid tank  800 . Air is then relieved of the sample liquid supply path  225  and the waste liquid path  227  (Step  24 ) to fill these components with the sample liquid, as shown in  FIG. 7C . In this way, the air relief of each flow path is completed. At the time, the sample liquid supply path  225  and the waste liquid path  227  are, broadly speaking, filled with the sample liquid while the buffer liquid supply path  226 , the branch flow path section  229 , and the reactor tank section  223  are filled with the buffer liquid. 
     The valves  212   a  and  212   c  are then closed again and the valves  212   b  and  212   d  are opened to cause the buffer liquid in the buffer liquid supply path  226  to be suctioned and fed through reactor tank section  223  to the waste liquid tank  800 , when the suction force of a trace liquid feed pump  901  is also adjusted to cause the buffer liquid to flow at a predetermined flow rate (Step  25 ). Upon completion of the adjustment of the trace liquid feed pump  901 , the valve  212   b  is closed to stop feeding the buffer liquid. The valve  212   a  is then opened to feed the sample liquid through the sample liquid supply path  225  and the branch flow path section  229  to the reactor tank section  223 , as shown in  FIG. 7D . 
     Specifically, the sample liquid fed from the sample liquid supply path  225  carries away the buffer liquid that fills the branch flow path section  229  and the reactor tank section  223  and flows into the reactor tank section  223 . At the time, the ligand with which the reactor tank section  223  is modified reacts with an analyte in the sample liquidm, thus causing bond reaction. The prevailing change in the resonant frequency of a crystal oscillator is then measured and the state of reaction is detected (Step  26 ). Because, at Step  25 , the trace liquid feed pump  901  is under proper adjustment, the sample liquid is fed into the reactor tank section  223  at a predetermined flow rate and bond reaction is in progress on a predetermined condition. 
     Considering this flow rate, the valve  212   a  is closed at an appropriate timing and the suction operation of the trace liquid feed pump  901  is stopped and a predetermined amount of sample liquid is fed accurately to the reactor tank section  223 . This results in the completion of the bond reaction. As an example of analysis requirements, the amount of the sample liquid used in the reaction is 50 μl and the reaction time is, for example, 5 to 50 minutes. The flow rate of the sample liquid by the operation of the trace liquid feed pump  901  is also 0.1 to 10 μl/min. 
     Concentration equilibrium is then reached and there is not much bond between the analyte and the ligand, thus stopping a change in the resonant frequency. The supply of a predetermine of the sample liquid is then completed and the buffer liquid is fed into the reactor tank section  223 . The analyte and the ligand once bonded then undergoes partial dissociation, thus resulting in a small change in the resonant frequency. The detection of the state of the dissociation is effective for knowing the strength of the bond between the analyte and the ligand, for example. 
     As described above, using the micro reactor chip  2000  according to the second embodiment allows the detection of a substance contained in the sample liquid. However, the micro reactor chip, particularly the crystal substrate  100  and the reactor tank section  223  are not limited to the configurations described above. An example of a configuration for holding the crystal substrate  100  is, shown in  FIG. 11 . 
       FIG. 11A  shows a configuration in which a flow path substrates  220   a ′ is formed with a groove that will act as a reactor tank section  223 , a flow path substrate  220   b ′ is formed with a through hole, and the crystal substrate  100  and the flow path substrates  220   a ′ and  220   b ′ are integratedly formed with the crystal substrate  100  between the flow path substrates. The cross section of the configuration is shown in  FIG. 11B . The cross-direction (Y direction) end surface and the flow path substrates  220   a ′ are bonded to the upper surface of the crystal substrate  100  while the longitudinal (X direction) side surface of the crystal substrate  100  is bonded to the flow path substrates  220   b ′, thus holding the crystal substrate  100 . Any clearance between bonding surfaces would cause sample liquid leaks or air bubbles in the sample liquid, which demands high-accuracy x-directional dimensions of the through hole in the flow path substrates  220   b ′. If a determined dimensional accuracy cannot be obtained, it is also possible to apply a mold agent to the interface (D) between the flow path substrates  220   b  and the crystal substrate  100  for clearance sealing. Alternatively, a stepped through hole is formed in the flow path substrates  220   b ″ and the crystal substrate  100  is fit into the stepped portion. This allows the area where the flow path substrates  220   b ″ are bonded to the crystal substrate  100  to be increased to improve air-tightness between the crystal substrate  100  and the external world. See  FIG. 11C . 
     To increase detection sensitivity, it is also essential to increase the resonant frequency of the crystal oscillator. Increasing the resonant frequency requires a thinner crystal oscillator. However, a thinner crystal oscillator has a lower strength, thus raising a problem that the thinner crystal oscillator is difficult to mount. As a general method for solving the problem, a tray-like structure (mesa structure) having a thinner region including the electrode of the crystal oscillator is known. A crystal oscillator having such a mesa structure can realize a high resonant frequency while maintaining a high strength. An example of a configuration where a crystal substrate  110  having a mesa structure is shown in  FIGS. 11D and 11E . The introduction of a crystal substrate  110  having such a mesa structure allows a higher detection sensitivity. 
     In the examples of the configuration shown in  FIG. 11 , a sample liquid is fed parallel to the surface of the crystal substrate  100 , thereby producing a smooth flow the sample liquid. The sample liquid can therefore be fed to a adsorption film  609  at a stable flow rate and with a stable concentration, which allows high-accuracy detection. 
     Using the micro reactor chip  2000  and micro reactor system  2500  according to the second embodiment described above allows a flow path configuration to be very simple and compact and the chip and system to be manufactured in a simply manner. The absence of a mixture of the sample liquid the buffer liquid makes it possible to realize detection with high accuracy. 
     Third Embodiment 
       FIG. 12 ,  FIG. 13A  and  FIG. 13B  show an exploded perspective view of a micro reactor chip  4000  according to the invention, a schematic plan view of the micro reactor chip  4000  and a cross sectional view of the micro reactor chip  4000  taken along the surface B-B, respectively. The same descriptions as with the first and second embodiments described earlier will be not be made below. 
     The micro reactor chip  4000  includes a crystal substrate  100 , a crystal holding substrate  101 , and flow path substrates  220   a  and  220   b , and a holding substrate  310 , all of which are laminated together. Each of the members will be described below. The crystal substrate  100  is configured in the same way as in the first embodiment. The crystal substrate  100  is bonded through a ring film  102  to the crystal holding substrate  101  having a through hole. The flow path substrate  220   a  is provided with minute irregularities, a through hole and through holed threaded while the flow path substrate  220   b  is a flat plate. 
     The flow path substrate  220   a  and the flow path substrate  220   b  are laminated together. The crystal holding substrate  101  is also integrated with the flow path substrate  220   a  by fitting a convex portion in the flow path substrate  220   a  into the through hole in the crystal holding substrate  101 , thereby providing a reactor tank section  223 , where reaction occurs, a sample liquid supply path  225  for feeding a sample liquid for analysis, a buffer liquid supply path  226  for feeding a buffer liquid having a flow path cleaning function and a buffer function (dissociation function), and a waste liquid path  227  leading to a liquid discharge port  203 . 
     A liquid introduction port  202   a , which is the end of the sample liquid supply path (the end opposite to the waste liquid path), a liquid introduction port  202   b , which is the end of the buffer liquid supply path (the end opposite to the reactor tank section  223 ), and a liquid discharge port  203  are provided with a connector  3520 , which can be fixed using a screw. 
     The configuration of a micro reactor system  4500  using the micro reactor chip  4000  will be then below with reference to a configuration view in  FIG. 14 . As with the second embodiment, the micro reactor chip  4000  is first provided on a stage  2510 . A lead wire from each of a detection electrode  601  and an opposite electrode  602  on the crystal substrate  100  is used to obtain electrical conduction with the system. 
     A tube is connected to each of three connectors  3520  of the micro reactor chip  4000 . Each connector can be easily connected to and disconnected from the tube because the elastic deformation of the tube itself is utilized for the connection between the connector  3520  and the tube. 
     Each of two tubes connected to the liquid introduction port  202   a  and  202   b  is connected to a sample liquid tank  3810  and a buffer liquid tank  3820  through a valve  212  that squeezes the tube to open and closes. A waste liquid tank  800  is also connected to the tube  3520  connected to the liquid discharge port  203 . A pump  902  is also connected to each of the sample liquid tank  3810  and the buffer liquid tank  3820 . When the pump  902  operates, a liquid stored in the sample liquid tank  3810  or the buffer liquid tank  3820  can be fed to the micro reactor chip. 
     In addition, a control circuit  2550  is connected to each of the pump  902 , valve  212 , and crystal substrate  100 . 
     Liquid feeding for the micro reactor system  4500  will be described below. The pump  902  is first operated to apply a pressure to the inside of the sample liquid tank  3810  and the buffer liquid tank  3820 . A sample liquid and a buffer liquid stored in the tanks try to flow through the tube and to a flow path in a micro reactor chip. However, a flow is produced in the micro reactor chip only with the valve  212  open. When the valve  212  provided on the buffer liquid supply path  226  is then opened, the buffer liquid flows from the buffer liquid tank  3820  through the tube, connector  3520 , and buffer liquid supply path  226  into the reactor tank section  223 . When the reactor tank section  223  is filled with the buffer liquid, the valve  212  connected to the buffer liquid supply path  226  is closed and the valve  212  on the sample liquid supply path  225  is opened. The sample liquid then flows from the sample liquid tank  3810  through the sample liquid supply path  225  into the reactor tank section  223 . Liquids fed to these reactor tank sections  223  flow through the waste liquid path  227  and are accumulated in the waste liquid tank  800 . The control circuit is responsible for all of the liquid feed described above and detection by sensor operation at a predetermined timing. The operation of the sensor for detection is performed as described for the second embodiment. 
     The configuration as described above allows the micro reactor system  4500  to be easily connected to and disconnect from the micro reactor chip  4000 . The configuration described above is an example of the configuration of the system. The connection between the chip and the system is not limited to the above configuration. 
     A method for manufacturing a micro reactor chip  4000  will be then described below. As with the first and second embodiments, a crystal substrate  100  has an electrode formed on a polished AT-cut crystal plate  100 . The crystal holding substrate  101  uses a glass plate and has a through hole, which has been provided by means of cutting. The flow path substrate uses a polycarbonate resin plate and has minute irregularities, a through hole, and a threaded hole, which have been formed by means of injection molding. 
     A process for bonding a crystal substrate  100  and a crystal holding substrate  101  to each other is shown in  FIG. 15 . A resist  32  is formed on a silicon wafer  31  ( FIG. 15A ) in a predetermined shape ( FIG. 15B ). A liquid PDMS  34  is then poured onto the wafer, which is then irradiated with ultraviolet light and the liquid PDMS is allowed to temporarily cure ( FIG. 15C ). The crystal holding substrate  101  is then placed on the temporarily cured PDMS  34  and the crystal holding substrate  101  side is irradiated with ultraviolet light, thereby causing the crystal holding substrate  101  and the PDMS plate  34  to bond to each other ( FIG. 15D ). The PDMS  34  is then removed from the through hole in the crystal holding substrate  101  ( FIG. 15E ). The PDMS  34  is then removed from the silicon wafer  31  and the crystal substrate  100  and the PDMS plate  34  are irradiated with ultraviolet light, thereby causing the crystal holding substrate  101  to be integrated with the crystal substrate  100  ( FIG. 15F ). A convex portion in the flow path substrate  220   a  is then fit into the through hole in the crystal holding substrate  101 , thus allowing the micro reactor chip  4000  to be fabricated. 
     In this process, a glass plate has been used for the crystal holding substrate  101 . It is also possible to prepare the micro reactor chip  4000  even by using a resin plate with glass coated with the surface thereof. The crystal holding substrate  101  and the flow path substrate  220   a  can be fixed to each other not only by means of fitting but also by means of bluing with a sealing tape and pressure welding using an O-ring. It is also possible to coat the surface of the flow path substrate  220   a  with glass, form a ring-like PDMS and bond the PDMS to the crystal holding substrate  101 , for example. 
     To prevent air bubbles from entering a flow path, which cause noise in detection, it is also possible to make the wall of the flow path hydrophilic. As an example of a method for doing this, the concave portion and through hole in the flow path substrate  220   a  and the bonding surface of the flow path substrate  220   b  are sputtered with a hydrophilic film such as parylene before the crystal holding substrate is integrated with the flow path substrates  220   a  and  220   b . Alternatively, After the crystal holding substrate is integrated with the flow path substrates  220   a  and  220   b , it will be possible to form a hydrophilic film on the wall of the flow path in the process of pouring a liquid glass coating agent into the flow pass and allow the coating agent to cure, for example. 
     Because the PDMS is gas-permeable, feeding a reagent at a negative pressure causes ambient air to enter the flow pass through the PDMS from outside the micro reactor chip, thus generating air bubbles in the flow path. Consequently, it is possible to prevent air bubble from entering the flow path by adding a process of coating the chip formed with a coating agent that has a high gas barrier property from the outside of the PDMS portion to form a gas barrier film. 
     The above-mentioned configuration of the micro reactor chip  4000  allows a micro reactor chip to be easily manufactured even on a thin crystal substrate having high-frequency properties. It is because there is no external force from bonding between the crystal substrate and the flow path substrate or no damage to the crystal substrate held in manufacturing the chip. The above-mentioned configuration also allows the crystal substrate  100  integrated with the crystal holding substrate  101  to be easily bonded to the flow path substrate after ligand modification. When the above configuration is used, a ligand with the amount predetermined can be fixed on the crystal substrate  100  and without unwanted ligand on the wall of the flow path, thus allowing high-accuracy interaction analysis. 
     Fourth Embodiment 
       FIG. 10  shows a reactor  3000  according to the invention. Specifically,  FIG. 10A  shows an exploded perspective view of the reactor  3000  and  FIG. 10B  shown a cross sectional view of the reactor  3000  (the cross section taken along the surface C in  FIG. 10A ). The same descriptions as in the first embodiment described earlier will be not be made below. 
     The reactor  3000  includes a crystal substrate  103 , a cell substrate  323 , and a holding substrate  313 , all of which are bonded to one another. The crystal substrate  1031  will first be described below. There are nine concave portions, each called a mesa structure, formed on one surface of the crystal substrate  103 . A detection electrode  601  is provided inside each mesa structure. An opposite electrode  602  is provided on the opposite surface of each detection electrode  601 . In addition, an adsorption film  609  for adsorbing only a specific substance is provided on the surface of the detection electrode  601 . The cell substrate  323  is provided with nine through holes  324 . Similarly, the holding substrate  313  is also provided with nine through holes  314 . 
     These crystal substrate  103 , cell substrate  323 , and holding substrate  313  are integrated with one another, thus forming a reactor  3000 . Specifically, the mesa structures of the crystal substrate  103  provided with the detection electrode  601  serve as a bottom and through holes  314  serve as openings and nine cells  3001 , substantially circular concave portions, are formed. 
     When a sample liquid to be analyzed is dropped onto one of these cells  3001 , the sample liquid is stored inside the cell  3001  and the adsorption film  609  provided above the detection electrode  601  becomes soaked with the sample liquid. At the time, an AC voltage is applied between the detection electrode  601  and the opposite electrode  602 , both of which are opposite to each other on the crystal substrate. The change in the resonant frequency is then measured. This allows the measurement of the mass of a substance fixed by the adsorption film  609 . 
     One cell has been described above. However, it is also possible to drop a sample of a different type onto each of a plurality of cells  301  provided on the reactor  300  and perform detection at the same time. It is also possible to provide each cell  3001  with each of adsorption films  609  of different types and drop one type of sample onto a plurality of cells  301  at one time for simultaneous detection. Note that the through hole  314  is smaller than the through  324  and the cell  3001  has a concave portion with a narrow opening. This is done to prevent a change in the concentration of the sample liquid due to evaporation during measurements. Any change in the concentration may hinder accurate detection. 
     The chemical reaction of a specific substance occurs only on the surface of the adsorption film  609 . It is therefore desirable that a mechanism for agitating the sample liquid in the cell  3001  (the agitating operation of the entire sensor due to vertical or horizontal oscillations, for example) should be available. 
     A method for manufacturing the reactor  3000  will be then described below. A mesa structure is first formed on one surface of an AT-cut crystal wafer  103  utilizing photolithography. Both surfaces of the mesa structure are deposited or sputtered with gold to prepare wiring to the detection electrode  601 , opposite electrode  602 , and each electrode. A through hole  324  is then formed in a polydimethylsiloxane (PDMS) plate to fabricate a cell substrate  323 . A through hole  314  is then formed on a silicon wafer to fabricate a holding substrate  313 . When these substrates are laminated together and pressed, a siloxane bond occurs at the interface among the substrates, thus allowing the three substrates to be bonded to one another. 
     As described above, providing a plurality of cells on a sensor allows the simultaneous detection of a plurality types of substances. The configurations and manufacturing method described above are simple and cause no residual stresses on the crystal oscillator or unwanted oscillation modes, thus allowing high detection sensitivity to be maintained.