Abstract:
A microfluidic device for performing a test on the reagent includes a fill port formed on the chip to inject the reagent into at least one of the channels, one or more heating portions for performing a test on the reagent injected into the channel, and a micropump. An inside of the micropump and a vicinity of the channel connecting to an inlet and an outlet of the micropump are filled with a drive solution that is driven by the micropump, a gas is sealed between the reagent and the drive solution in the channel to prevent the reagent from contacting the drive solution directly, and the micropump directly drives the drive solution in the forward and backward directions, so that the reagent is repeatedly moved to the test portions through the gas in an indirect manner or is repeatedly passed through the test portions through the gas.

Description:
[0001]     This application is based on Japanese Patent Application No. 2004-143108 filed on May 13, 2004, the contents of which are hereby incorporated by reference.  
       BACKGROUND OF THE INVENTION  
       [0002]     1. Field of the Invention  
         [0003]     The present invention relates to a microfluidic device for distributing a small amount of reagent in channels formed on chips to test the reagent. The present invention is used for, for example, gene amplification by a PCR method.  
         [0004]     2. Description of the Related Art  
         [0005]     Conventionally, Japanese Patent No. 3120466 proposes that a capillary is used as a channel for a reagent or a reaction solution for gene amplification by the PCR method.  
         [0006]     More specifically, three vessels containing three liquids whose temperatures differ from one another are prepared. The three liquids are adjusted so as to be a heat denaturation temperature (95° C., for example), an annealing temperature (55° C., for example) and a polymerization temperature (75° C., for example), respectively. One capillary, which is separately prepared, is placed in a manner to soak sequentially in each of the three liquids. A reagent is injected into the capillary and the injected reagent is transported in the capillary using a gas supplied from end portions of the capillary. A three-way valve is switched to control a supply of the gas so that the reagent is provided sequentially in a position of each of the three liquids for each predetermined time interval. The repetition of this operation gives the reagent a temperature cycle.  
         [0007]     In addition, another method is also proposed in which three large temperature portions having different temperatures are prepared, a meandering channel is formed to sequentially pass through the three temperature portions plural times and a reagent is transported unidirectionally within the channel.  
         [0008]     Meanwhile, in recent years, a μ-TAS (Micro Total Analysis System) has drawn attention that uses a micromachining technique to microfabricate equipment for a chemical analysis or a chemical synthesis and then to perform the chemical analysis or the chemical synthesis in a microscale method. Compared to the conventional systems, a miniaturized μ-TAS has advantages in that required sample volume is small, reaction time is short, the amount of waste is small and others. The use of the μ-TAS in the medical field lessens the burden of patients by reducing volume of specimen such as blood, and lowers the cost of examination by reducing reagent volume. Further, the reduction of the specimen and reagent volume causes reaction time to shorten substantially, ensuring that examination efficiency is enhanced. Moreover, since the μ-TAS is superior in portability, it is expected to apply to broad fields including the medical field and an environmental analysis.  
         [0009]     Japanese unexamined patent publication No. 2002-214241 discloses a technique in which such a μ-TAS is used to transport a reagent. According to the patent publication, two micropumps are used to transport two kinds of reagents which are subsequently joined together and the reagents after joining together are reciprocated within one channel after the confluence.  
         [0010]     According to an apparatus described in Japanese Patent No. 3120466 mentioned above, the three-way valve is switched to control a supply of the gas, so that a movement amount of the reagent, i.e., a position of the reagent is controlled. Accordingly, positioning of the reagent is far from easy and it is difficult that the reagent is brought to a standstill at a predetermined position correctly and a temperature process using a liquid is performed precisely. In addition, the use of the three vessels and the capillary imposes limitation on reduction in the size of the apparatus. In other words, downsizing and improvement in portability are difficult.  
         [0011]     Further, in the case where an apparatus has a meandering channel formed on a microchip and serves to transport a reagent unidirectionally, an amount of the reagent cannot be reduced and a pump is large. Accordingly, downsizing of the apparatus is far from easy.  
         [0012]     When a micropump is used to transport a reagent, it is necessary to fill an area extending from the micropump to a portion for a temperature process with the reagent. Accordingly, it is impossible to reduce an amount of the reagent.  
       SUMMARY OF THE INVENTION  
       [0013]     The present invention is directed to solve the problems pointed out above, and therefore, an object of the present invention is to provide a microfluidic device, a method for testing a reagent and a system for testing the same, all of which can perform a test using a small amount of reagent, can accurately control a movement amount of reagent and can perform a test precisely.  
         [0014]     According to one aspect of the present invention, a microfluidic device for distributing a reagent in a channel formed on a chip to perform a test on the reagent, the device includes a fill port formed on the chip to inject the reagent into at least one of the channels, one or more test portions for performing a test on the reagent injected into the channel, and a micropump capable of transporting a liquid in forward and backward directions in one end portion of the channel, wherein an inside of the micropump and a vicinity of the channel connecting to an inlet and an outlet of the micropump are filled with a drive solution that is driven by the micropump, a gas is sealed between the reagent and the drive solution in the channel to prevent the reagent from contacting the drive solution directly, and the micropump directly drives the drive solution in the forward and backward directions, so that the reagent is repeatedly moved to the test portions through the gas in an indirect manner or is repeatedly passed through the test portions through the gas in an indirect manner.  
         [0015]     Preferably, the chip includes a process chip in which a first channel for distributing the reagent is provided, and a drive chip in which a second channel for transporting the drive solution, the test portions and the micropump are provided, the process chip is removably attached to the drive chip, and the gas passes through a connection portion of the first channel and the second channel.  
         [0016]     Further, the test portions are three heating portions having different temperatures, and the reagent is repeatedly moved to the three heating portions in a sequential manner.  
         [0017]     The channel is provided with three reagent chambers corresponding to positions of the three heating portions, the reagent chambers being for containing the reagent, and the reagent is capable of being moved to the reagent chambers to be contained therein sequentially.  
         [0018]     Further, the reagent chambers are equal to one another in volume and the volume is set so as to be greater than a volume of the reagent that is injected at one time.  
         [0019]     A transport volume of the drive solution at one time by driving the micropump is set so as to be equal to a sum of the volumes of the reagent chambers and a volume of the channel connecting the two reagent chambers.  
         [0020]     Further, each of the reagent chambers is provided with two electrodes for detecting whether or not the reagent is contained.  
         [0021]     Furthermore, an inner circumferential surface of each of the channels connecting the reagent chambers is treated with a water repellent or an oil repellent.  
         [0022]     According to another aspect of the present invention, a microfluidic includes a reagent chamber formed on the chip to contain the reagent, a plurality of process chambers divided within the reagent chamber, a plurality of test portions for performing a test on the reagent, the test portions corresponding to the process chambers, and a micropump capable of transporting a liquid in forward and backward directions in one end portion of the channel, wherein an inside of the micropump and a vicinity of the channel connecting to an inlet and an outlet of the micropump are filled with a drive solution that is driven by the micropump, a gas is sealed between the reagent and the drive solution in the channel to prevent the reagent from contacting the drive solution directly, and the micropump directly drives the drive solution in the forward and backward directions, so that the reagent is moved in the reagent chamber through the gas indirectly, causing the reagent to move to the plurality of process chambers sequentially.  
         [0023]     Preferably, the chip includes three heating portions so as to correspond to the reagent chamber, the reagent chamber is divided into three process chambers corresponding to the three heating portions, and the reagent is moved in the reagent chamber, so that the reagent moves to the three heating portions sequentially.  
         [0024]     In the present invention, a nitrogen gas, air or various other gases are used as a gas.  
         [0025]     The present invention enables a test using a small amount of reagent, accurate control of a movement amount of reagent and a test with a high degree of precision.  
         [0026]     These and other characteristics and objects of the present invention will become more apparent by the following descriptions of preferred embodiments with reference to drawings. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0027]      FIG. 1  is a front view of a microfluidic device according to a first embodiment of the present invention.  
         [0028]      FIG. 2  is an exploded perspective view of a structure of the microfluidic device.  
         [0029]      FIG. 3  is a plan view of a micropump shown in  FIG. 2 .  
         [0030]      FIG. 4  is a front sectional view of the micropump.  
         [0031]      FIGS. 5A-5H  show an example of a manufacturing process of the micropump.  
         [0032]      FIGS. 6A and 6B  show an example of waveforms of a drive voltage of a piezoelectric element.  
         [0033]      FIGS. 7A and 7B  show an example of waveforms of a drive voltage of a piezoelectric element.  
         [0034]      FIG. 8  is a plan view showing a structure of a microfluidic system according to the first embodiment.  
         [0035]      FIG. 9  is a plan view showing process chambers in a channel chip according to another example.  
         [0036]      FIG. 10  is a diagram showing a modification of a structure of gas chambers and liquid chambers.  
         [0037]      FIG. 11  is a diagram of a microfluidic device in which gas chambers according to another example are used.  
         [0038]      FIG. 12  is a diagram of a microfluidic device in which liquid chambers according to another example are used.  
         [0039]      FIG. 13  is a diagram showing a structure of a microfluidic device according to a second embodiment of the present invention.  
         [0040]      FIG. 14  is a diagram showing a structure of a microfluidic device according to a third embodiment of the present invention.  
         [0041]      FIG. 15  shows a modification of the microfluidic device according to the third embodiment.  
         [0042]      FIG. 16  is a diagram showing an example of a structure of a coaxial incident light optical device used for optical detection. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
     First Embodiment  
       [0043]      FIG. 1  is a front view of a microfluidic device  1  according to a first embodiment of the present invention,  FIG. 2  is an exploded perspective view of a structure of the microfluidic device  1 ,  FIG. 3  is a plan view of a micropump MP 1  shown in  FIG. 2 ,  FIG. 4  is a front sectional view of the micropump MP 1 ,  FIGS. 5A-5H  show an example of a manufacturing process of the micropump MP 1 ,  FIGS. 6A and 6B  as well as  FIGS. 7A and 7B  show examples of waveforms of a drive voltage of a piezoelectric element.  
         [0044]     Referring to  FIGS. 1 and 2 , the microfluidic device  1  includes two chips removably attached to each other. One of the two chips is a chip CS for liquid transport on which the micropump MP 1  is mounted, while the other is a chip CR for process into which a reagent (a specimen liquid) is injected for a PCR reaction.  
         [0045]     The liquid transport chip CS includes a pump chip  11  and a glass substrate  12 .  
         [0046]     The pump chip  11  has a structure in which the micropump MP 1 , liquid chambers RE 1 -RE 4 , gas chambers RK 2 -RK 3 , connection chambers RS 1 -RS 2  and channels RR 1 -RR 8  for connecting therebetween are formed on a surface of a silicon substrate  31 . The inner circumferential surface of each of the channels RR 1 -RR 8  is treated with an oil repellent.  
         [0047]     The liquid chambers RE 1 -RE 4  are equal to the gas chambers RK 2 -RK 3  in volume. Further, the liquid chambers RE 1 -RE 4  may be equal to the gas chambers RK 2 -RK 3  in diameter and depth. Each of the liquid chambers RE 1 -RE 4  and each of the gas chambers RK 2 -RK 3  have, for example, a diameter of 3.5 mm, a depth of 0.2 mm and a volume of approximately 2 μl. As long as the connection chambers RS 1 -RS 2  have dimensions needed to be in communication with connection holes AN 1 -AN 2 , which are described later, formed on the glass substrate  12 , the dimensions are sufficient. The channels RR 1 -RR 8  serve to distribute (run) a liquid or a gas in areas provided among the chambers. Each of the channels RR 1 -RR 8  has, for example, a width of 100 μm and a depth of 100 μm.  
         [0048]     Referring to  FIG. 3 , the micropump MP 1  includes a chamber  62  functioning as a pump chamber and openings  61  and  63  that are formed at an inlet and an outlet of the chamber  62  respectively. The openings  61  and  63  connect to the channels RR 5  and RR 4  respectively. The openings  61  and  63  have width dimensions or effective sectional areas smaller than that of the channel RR 5  or the channel RR 4 , and the openings  61  and  63  differ from each other in effective length. The differences in shape and dimensions allow the micropump MP 1  to operate as a micropump. The details are described later.  
         [0049]     With reference to  FIG. 4 , the micropump MP 1  is fabricated as follows. A photolithography process is used to form grooves or cavities on the silicon substrate  31 , the grooves or cavities eventually structuring the chamber  62 , the openings  61  and  63 , the channels RR 5  and RR 4  or others. Then, a glass substrate  32  as a bottom plate or a top plate is bonded to a lower surface or an upper surface of the silicon substrate  31 .  
         [0050]     For example, a silicon substrate  310  is prepared as shown in  FIG. 5A . A silicon wafer having a thickness of 200 μm, for example, is used as the silicon substrate  310 . Then, oxide films  311  and  312  are formed on the upper and lower surfaces of the silicon substrate  310  respectively, as shown in  FIG. 5B . Each of the oxide films  311  and  312  is coated by thermal oxidation so as to have a thickness of 1.7 μm. After that, the upper surface is coated with a resist, exposure and development of a predetermined mask pattern is performed, and the oxide film  311  is etched. Then, the resist on the upper surface is peeled off, and subsequently, coating of a resist, exposure, development and etching are performed again. In this way, portions  311   a  where the oxide film  311  is completely removed and portions  311   b  where the oxide film  311  is partly removed in the thickness direction are formed as shown in  FIG. 5C . In the resist coating process, for example, a resist such as OFPR800 is used to perform spin coating with a spin coater. The resist film has a thickness of, for example, 1 μm. An aligner is employed for exposure and a developer is used for development. For instance, RIE is used for etching of the oxide film. A stripper such as a mixture of sulfuric acid and hydrogen peroxide is used in order to separate the resist.  
         [0051]     Next, before completing silicon etching of the upper surface, the oxide film  311  is completely removed by the etching process. Then, silicon etching is performed again to form portions  311   c  where the silicon substrate  310  is etched by 170 μm in depth and portions  311   d  where the silicon substrate  310  is etched by 250 μm in depth, as shown in  FIGS. 5D and 5E . For the silicon etching, for example, Inductively Coupled Plasma (ICP) is used.  
         [0052]     As shown in  FIG. 5E , BHF is used, for example, to remove the oxide film  311  on the upper surface completely. Then, an electrode film  313  such as an ITO film is formed on the lower surface of the silicon substrate  310  as shown in  FIG. 5F . Subsequently, a glass plate  32  is attached to the upper surface of the silicon substrate  310  as shown in  FIG. 5G . For the attachment of the glass plate  32 , anodic bonding is performed under the condition of 1200 V and 400° C. Lastly, as shown in  FIG. 5H , a piezoelectric element  34  such as PZT (lead zirconate titanate) ceramics is adhered to a portion of a diaphragm of the chamber  17  for attachment.  
         [0053]     Note that, in  FIG. 5H , reference numerals in parentheses show portions corresponding to the portions denoted by the same reference numerals in  FIG. 4 . Referring to  FIG. 4 , the openings  61  and  63  are formed by reducing widths of grooves (the vertical direction with respect to the paper surface) compared to the channels RR 5  and RR 4  to serve as openings. Referring to  FIG. 5H , the openings  61  and  63  are formed by reducing depths of grooves (the vertical direction in a plan view) compared to the channels RR 5  and RR 4  to serve as openings. Further, note that the upper side and the lower side shown in  FIG. 4  are turned upside down in  FIG. 5H .  
         [0054]     The micropump MP 1  can be fabricated in the method described above. Instead, it is also possible to fabricate the micropump MP 1  by conventionally known methods or other methods, or by the use of other materials.  
         [0055]     The glass substrate  12  has a structure in which the connection holes AN 1 -AN 2  penetrating a glass plate  32  and heating portions KN 1 -KN 3  are formed on the glass plate  32 .  
         [0056]     The connection holes AN 1 -AN 2  are brought into communication with the connection chambers RS 1 -RS 2  respectively, when the pump chip  11  is bonded to the glass plate  32 . The heating portions KN 1 -KN 3  can be structures using various heating elements, such as heaters using nichrome wires or others, and structures in which resistance values are controlled using ITO films with different widths.  
         [0057]     The heating portions KN 1 -KN 3  are supplied with currents from a heating drive portion (not shown). The heating portions KN 1 -KN 3  are heated and controlled so as to be a temperature corresponding to denaturation of a PCR reaction, a temperature corresponding to extension thereof and a temperature corresponding to annealing thereof, respectively. For instance, the heating portion KN 1  has a temperature of 95° C., the heating portion KN 2  has a temperature of 75° C. and the heating portion KN 3  has a temperature of 55° C. However, since the temperatures are taken as one example, it is not necessarily that the heating portions KN 1 -KN 3  should have these temperatures, respectively. The arrangement order of the heating portions KN 1 -KN 3  can also be modified.  
         [0058]     To cite instances of dimensions, the pump chip  11  has outside dimensions of approximately 30 mm×30 mm×0.5 mm, the glass substrate  12  has outside dimensions of approximately 50 mm×30 mm×1 mm and the entire liquid transport chip CS has outside dimensions of about 50 mm×30 mm×1.5 mm. These dimensions and shapes are one example and other various dimensions and shapes can be adopted.  
         [0059]     Hereinafter, the operation of the micropump MP 1  is described.  
         [0060]     A drive circuit  36  shown in  FIG. 4  is used to apply a voltage having a waveform shown in  FIG. 6A  or  FIG. 7A  to the piezoelectric elements  34 , so that a diaphragm  31   f  that is a silicon thin film and the piezoelectric elements  34  perform flexion deformity in unimorph mode. The flexion deformity is used for increase or decrease of the volume of the chamber  62 .  
         [0061]     As discussed above, the openings  61  and  63  have effective sectional areas smaller than those of the channels RR 5  and RR 4 . The opening  63  is so set that the opening  63  has a lower rate of change in channel resistance when pressure inside the chamber  62  is raised or lowered, compared to the opening  61 .  
         [0062]     More specifically, the opening  61  has low channel resistance when the differential pressure between the both ends thereof is close to zero. As the differential pressure in the opening  61  increases, the channel resistance thereof increases. Stated differently, pressure dependence is large. Compared to the case of the opening  61 , the opening  63  has higher channel resistance when the differential pressure is close to zero. However, the opening  63  has little pressure dependence. Even if the differential pressure in the opening  63  increases, the channel resistance thereof does not change significantly. When the differential pressure is large, the opening  63  has channel resistance lower than the opening  61  has.  
         [0063]     The characteristics of channel resistance mentioned above can be obtained by any of the following: 1. Bringing a liquid flowing through a channel to be any one of laminar flow and turbulent flow depending on the magnitude of the differential pressure. 2. Bringing the liquid to be laminar flow constantly regardless of the differential pressure. More particularly, for example, the former can be realized by providing the opening  61  in the form of an orifice-like opening having a short channel length, while the latter can be realized by providing the opening  63  in the form of a nozzle-like opening having a long channel length. In this way, the characteristics of channel resistance discussed above can be realized.  
         [0064]     The channel resistance characteristics of the opening  61  and the opening  63  are used to produce pressure in the chamber  62  and a rate of change in pressure is controlled, so that a pumping action in a discharge process and a suction process respectively, such as discharging or sucking more fluids to/from either one of the openings  61  and  63  that has lower channel resistance can be realized.  
         [0065]     More specifically, the pressure in the chamber  62  is raised and the rate of change in pressure is made large, resulting in the high differential pressure. Accordingly, the channel resistance of the opening  61  is higher than that of the opening  63 , so that most fluids within the chamber  62  are discharged from the opening  63  (discharge process). The pressure in the chamber  62  is lowered and the rate of change in pressure is made small, which keeps the differential pressure low. Accordingly, the channel resistance of the opening  61  is lower than that of the opening  63 , so that more liquids flow from the opening  61  into the chamber  62  (suction process).  
         [0066]     To the contrary, the pressure in the chamber  62  is raised and the rate of change in pressure is made small, which keeps the differential pressure low. Accordingly, the channel resistance of the opening  61  is lower than that of the opening  63 , so that more fluids in the chamber  62  are discharged from the opening  61  (discharge process). The pressure in the chamber  62  is lowered and the rate of change in pressure is made large, resulting in the high differential pressure. Accordingly, the channel resistance of the opening  61  is higher than that of the opening  63 , so that more fluids flow from the opening  63  into the chamber  62  (suction process).  
         [0067]     The drive voltage supplied to the piezoelectric element  34  is controlled and the amount and timing of deformation of the diaphragm are controlled, which realizes pressure control of the chamber  62  mentioned above. For example, a drive voltage having a waveform shown in  FIG. 6A  is applied to the piezoelectric element  34 , leading to discharge to the channel RR 4  side. A drive voltage having a waveform shown in  FIG. 7A  is applied to the piezoelectric element  34 , leading to discharge to the channel RR 5  side.  
         [0068]     Referring to  FIGS. 6A and 6B  as well as  FIGS. 7A and 7B , a maximum voltage e 1  to be applied to the piezoelectric element  34  ranges approximately from several volts to several tens of volts and is about 100 volts at the maximum. Time T 1  and T 7  are on the order of 20 μs, time T 2  and T 6  are from approximately 0 to several microseconds and time T 3  and T 5  are about 60 μs. Time T 4  and T 8  may be zero. Frequency of the drive voltage is approximately 11 KHz. With drive voltages shown in  FIGS. 6A and 7A , the channel RR 4  provides flow rates, for example, illustrated in  FIGS. 6B and 7B . Flow rate curves in  FIGS. 6B and 7B  schematically show flow rates obtained by a pumping action. In practice, inertial oscillation of a fluid is added to the flow rate curves. Accordingly, curves in which oscillation components are added to the flow rate curves shown in  FIGS. 6B and 7B  show actual flow rates obtained by an actual pumping action.  
         [0069]     Each of the openings  61  and  63  in the present embodiment is structured by a single opening. Instead, a group of openings can be used in which plural openings are arranged in parallel. The use of the group enables pressure dependence to be further lowered. Accordingly, when the group of openings is substituted for the opening, especially for the opening  63 , the flow rate is increased and the flow rate efficiency is improved.  
         [0070]     Referring back to  FIGS. 1 and 2 , the process chip CR includes a channel chip  13  and a resin substrate  14 .  
         [0071]     The channel chip  13  has a structure in which process chambers RY 1 -RY 3 , a gas chamber RK 1 , gas chambers RK 4 -RK 6 , a connection chamber RS 3 , a connection hole AN 3  and channels RR 9 -RR 16  for connecting therebetween are formed on a surface of a resin plate  41  made of a synthetic resin. The inner circumferential surface of each of the channels RR 9 -RR 16  is treated with a water repellent.  
         [0072]     The process chambers RY 1 -RY 3  are equal to the gas chambers RK 1  and RK 4 -RK 6  in volume. Further, the process chambers RY 1 -RY 3  and the gas chambers RK 1  and RK 4 -RK 6  are respectively equal to the corresponding chambers formed on the pump chip  11  in volume. Accordingly, the three process chambers RY 1 -RY 3  have the same volume. In addition, each of the process chambers RY 1 -RY 3  is set so as to have a volume greater than a volume of a reagent that is injected at a time. The following mathematical expression shows the relationship among volumes Vy 1 -Vy 3  of the process chambers RY 1 -RY 3 . 
 
 Vy   1 = Vy   2 = Vy   3 = Vy&gt;Vk  
 
 where Vy 1 -Vy 3  denote volumes of the process chambers RY 1 -RY 3  respectively and Vk denotes a reagent amount used in one test. The establishment of the relationship prevents a reagent from extending over two of the process chambers RY, i.e., from extending over two temperature areas. Thus, it is possible to securely retain a reagent in one temperature area for an accurate test. 
 
         [0073]     The process chambers RY 1 -RY 3  are positioned so as to correspond to the positions of the heating portions KN 1 -KN 3  respectively when the process chip CR is attached to the liquid transport chip CS. More specifically, the heating portions KN 1 -KN 3  heat reagents filled in the process chambers RY 1 -RY 3  respectively.  
         [0074]     The whole or a part of the process chambers RY 1 -RY 3  and the vicinity thereof are transparent. Each of the process chambers RY 1 -RY 3  has a shape that enables a reagent filled in the process chamber RY 2  to be measured or observed optically, for example when the process chamber RY 2  is set to an extension temperature (75° C., for example).  
         [0075]     The connection hole AN 3  has the same size as the connection hole AN 2 . When the process chip CR is attached to the liquid transport chip CS, the position of the connection hole AN 3  matches the position of the connection hole AN 2 , so that the connection hole AN 3  and the connection hole AN 2  are in communication with each other.  
         [0076]     The resin substrate  14  has a connection hole AN 4  and a fill port AT 1  formed on a resin plate  42  made of a synthetic resin. The position of the connection hole AN 4  matches the position of the connection chamber RS 3  when the resin substrate  14  is bonded to the channel chip  13 , so that the connection hole AN 4  and the connection chamber RS 3  are in communication with each other. The fill port AT 1  is used for injecting a reagent into the process chambers RY 1 -RY 3 . The fill port AT 1  has a diameter of, for example, 0.5-2 mm, preferably on the order of 1 mm. The position of the fill port AT 1  matches the position of the process chamber RY 1  and a reagent injected from the fill port AT 1  is supplied to the process chamber RY 1  directly.  
         [0077]     The resin substrate  14  and the channel chip  13  are aligned with each other and are joined to each other by, for example, laser fusion or other methods. The process chip CR clings to the liquid transport chip CS. Further, the process chip CR has a packing (not shown) and thereby channels are sealed.  
         [0078]     Next, a description is provided of operation of the microfluidic device  1  structured as discussed above.  
         [0079]      FIG. 8  shows a connection state of the chambers in the microfluidic device  1 .  
         [0080]     Referring to  FIG. 8 , in an initial state before starting a test, the inside of the micropump MP 1 , i.e., the inside of the pump chamber, the liquid chambers RE 1 -RE 2  and the channels RR therebetween are filled with a drive solution such as a mineral oil. The gas chamber RK 6  is filled with a sealing solution such as a mineral oil. The mineral oil prevents a reagent (a specimen liquid) from evaporating and also serves to prevent contamination.  
         [0081]     A reagent is injected from the fill port AT 1  to be supplied to the process chamber RY 1 . For example, approximately 2 μm of a specimen liquid for which gene amplification is intended is injected. Then, a plug FT 1  is put in the fill port AT 1  for closing the same. Note that, after completing a test, the plug FT 1  can be pulled out and the reagent can be removed from the fill port AT 1 .  
         [0082]     At the time point when the plug FT 1  is put in the fill port AT 1 , a gas with a pressure equivalent to an atmosphere pressure is present in each of the gas chambers RK 1 -RK 5 , the liquid chambers RE 3 -RE 4  and the process chambers RY 2 -RY 3 . As the gas, a nitrogen gas, air or various other gases are used. The gas present in each of the gas chambers RK 1 , RK 2 , RK 4  and RK 5  and the process chambers RY 2 -RY 3  is sealed by the sealing solution or the drive solution. In addition, no reagent in the process chamber RY 1  comes into contact with the sealing solution in the gas chamber RK 6  and the drive solution in the liquid chamber RE 1 . In other words, the gas is present in areas among the process chamber RY 1 , the gas chamber RK 6  and the liquid chamber RE 1 .  
         [0083]     The drive circuit  36  is used to drive the micropump MP 1  until, for example, the liquid chamber RE 3  is filled with the drive solution. This drive moves the drive solution contained in the liquid chamber RE 1  to the liquid chamber RE 2  and moves the drive solution contained in the liquid chamber RE 2  and the drive solution in the micropump MP 1  to the micropump MP 1  and the liquid chamber RE 3  respectively. Stated differently, the drive solution moves by one liquid chamber RE.  
         [0084]     Then, along with the movement of the drive solution, the reagent contained in the process chamber RY 1  moves through the gases contained in the gas chambers RK 1 -RK 2  and in the process chambers RY 2 -RY 3  and all the reagent contained in the process chamber RY 1  is supplied to the process chamber RY 2 . The sealing solution contained in the gas chamber RK 6  is supplied to the gas chamber RK 5 . In such a case, amount Vs of liquid transport using the micropump MP 1  is derived from the following equation. 
 
 Vs=Vy+Vr  
 
 where Vr represents a volume of one channel RR neighboring the process chamber RY. Accordingly, each of the channels RR 3 -RR 6 , RR 11 , RR 12 , RR 14  and RR 15  is preferably formed so as to have the same volume. Especially, it is necessary to equalize the volumes of the channels RR 11  and RR 12 , each of which is directly connected between the process chambers RY. 
 
         [0085]     Then, the micropump MP 1  is further driven, until, for example, the liquid chamber RE 4  is filled with the drive solution contained in the liquid chamber RE 3 . This drive moves the reagent contained in the process chamber RY 2  to the process chamber RY 3  through the gas, similar to the foregoing case.  
         [0086]     The control of the drive amount of the micropump MP 1  enables the reagent contained in the process chamber RY 1  to move to the process chamber RY 3  at one time.  
         [0087]     In the case where the liquid transport direction by the micropump MP 1  is reversed to move the drive solution to the direction opposite to the above-mentioned direction, the reagent contained in the process chamber RY 3  can be moved to the process chamber RY 2  or the process chamber RY 1 .  
         [0088]     More specifically, the control of the drive amount and of the drive direction of the micropump MP 1  permits the reagent to reciprocate between the process chambers RY 1 -RY 3 . The reagent is contained in a predetermined process chamber RY and the state is maintained for a predetermined period of time. This repetition enables the reagent to be subjected to a cycle of a temperature process necessary for the PCR method. Thereby, gene amplification is performed.  
         [0089]     In the meanwhile, no sealing solution and no drive solution leak out. No reagent comes into contact with the sealing solution and the drive solution directly. Accordingly, diffusion or mixing of a reagent or a liquid does not occur. Further, the provision of the gas chambers RK 1 -RK 3  prevents the drive solution from getting in another chip or from outflowing from a chip, even if the drive solution moves excessively. Accordingly, each of the chips or of the chambers is not contaminated by other liquids.  
         [0090]     The reagent is made to reciprocate between the process chambers RY 1 -RY 3 , for example, 20 through 30 times and, the reagent is made to remain in the process chamber RY 2  ultimately. The reagent retained in the process chamber RY 2  is optically measured or observed with an appropriate measurement device or sensor. In this way, for example, an amplification state of a gene under an extension temperature can be measured. This measurement can be made for one cycle or for every plural cycles. Accordingly, an amplification state of a gene can be easily measured in real time, i.e., a real-time PCR can be realized and the result thereof can be obtained without delay.  
         [0091]     Since it is sufficient that the reagent has an amount enough to fill one process chamber RY, a needed amount of the reagent can be substantially reduced compared to conventional cases.  
         [0092]     All materials required for a test of a reagent are incorporated into the microfluidic device  1 , the entire structure thereof is simple and significant downsizing thereof can be attempted. Since channels where a reagent or the like moves are short and sectional areas thereof are small, there are no wasted volumes and responsiveness is good. Accordingly, positioning after movement of a reagent can be accurately performed with a high degree of precision. Since the microfluidic device  1  also has a good compliant property with reagent temperature, a reaction time can be shortened.  
         [0093]     The liquid transport chip CS is removably attached to the process chip CR. Accordingly, replacement of process chips allows for tests using different reagents or under different conditions many times using the same liquid transport chip CS. Since the process chip CR is inexpensive, the process chip CR is disposable. This eliminates the need for washing the process chip CR and the possibility of mix of other reagents accidentally. Further, the process chip CR is provided with the gas chamber RK 1  which serves as a buffer when unforeseen circumstances occur, preventing the reagent from getting in the liquid transport chip CS and the liquid transport chip CS from being contaminated.  
         [0094]     The micropump MP 1  has a property that liquid transport characteristics change depending on a viscosity of a liquid to be transported. However, only the drive solution is supplied inside the micropump MP 1  and only one kind of a liquid is transported by the micropump MP 1 . Accordingly, physical properties such as a viscosity do not change and liquid transport characteristics are always constant. This allows for stable liquid transport of any kind of reagents and an accurate test.  
         [0095]     Additionally, since the inner circumferential surface of each of the channels RR 1 -RR 8  and RR 9 -RR 16  is treated with an oil repellent or a water repellent, a liquid can be stopped securely for each chamber, leading to the more accurate liquid transport compared to conventional cases.  
         [0096]     In the present embodiment, each of the channels RR 1 -RR 8  is treated with an oil repellent because a mineral oil is used as the drive solution. If the drive solution is of a water type, each of the channels RR 1 -RR 8  may be treated with a water repellent.  
         [0097]     According to the microfluidic device  1  described above, stable liquid transport can be realized by the micropump MP 1 . Further accurate liquid transport with a high degree of precision can be realized by the following method.  
         [0098]      FIG. 9  is a plan view showing process chambers RY 1 B-RY 3 B in the channel chip  13  according to another example.  
         [0099]     As shown in  FIG. 9 , inside each of the process chambers RY 1 B-RY 3 B, two detection electrodes DK 1   a  and DK 1   b , DK 2   a  and DK 2   b , or DK 3   a  and DK 3   b  are provided in the vicinity of an inlet and an outlet of each of the process chambers RY 1 B-RY 3 B. The detection electrodes DK are formed by patterning platinum or titanium. The detection electrodes DK may be formed by print on the surface of the resin substrate  14 .  
         [0100]     When a voltage Ek is applied between the two respective detection electrodes and a reagent remains in each of the process chambers RY 1 B-RY 3 B so as to wet the two detection electrodes DK therein, a current Ik flows between the two respective detection electrodes DK, and then, the current Ik is detected. In other words, the current Ik flowing between the two detection electrodes DK or the magnitude of the current Ik is detected, and thereby, it is judged that the reagent is supplied to the process chamber RY. Detection signals from the detection electrodes DK are fed back to the drive circuit  36 . For example, the micropump MP 1  is stopped by the detection electrodes DK. Thus, liquid transport among the process chambers can be performed even more accurately.  
         [0101]     Note that the voltage Ek in  FIG. 9  is depicted as a principle and, in practice, an electronic component or an IC circuit is used to detect a microcurrent or others. Further, it is possible to judge whether the reagent is supplied to the process chamber RY by optical detection of the reagent in the process chamber RY, instead of by provision of the detection electrodes DK.  
         [0102]     A sealing solution moves among the gas chambers RK 4 -RK 6  to prevent atmospheric contamination. The sealing solution, however, is omitted because influences of the atmospheric contamination on the liquid transport chip are low due to low heating temperature. Nevertheless, when measures for the atmospheric contamination are needed, it is possible to provide a structure as same as the gas chambers RK 4 -RK 5 , the channel RR 15  and the gas chamber RK 6 , the structure being substitute for the gas chamber RK 1 , between the channels RR 9  and RR 10  and to supply the structure with the sealing solution.  
         [0103]      FIG. 10  is a diagram showing a modification of a structure of the gas chambers RK and the liquid chambers RE.  
         [0104]     As shown in  FIG. 10 , one large unseparated gas chamber RK  7  is provided instead of the gas chambers RK 4 -RK 6  shown in  FIG. 8 . Similarly, one large liquid chamber RE 6  is provided instead of the gas chambers RK 1 -RK 2  and the liquid chamber RE 2  and, one large liquid chamber RE 7  is provided instead of the liquid chambers RE 3 -RE 4  and the gas chamber RK 3 . Under such a structure, a sensor using the detection electrodes DK shown in  FIG. 9  or others may be used to control a liquid transport amount or timing.  
         [0105]     Next, a description is provided of a structure of the gas chambers RK and the liquid chambers RE according to another example.  
         [0106]      FIG. 11  is a diagram showing a connection state of chambers in the microfluidic device  1  in which a gas chamber RK 11  in another example is used and  FIG. 12  is a diagram showing a connection state of chambers in the microfluidic device  1  in which a liquid chamber RE 11  in another example is used.  
         [0107]     Referring to  FIG. 11 , the gas chamber RK 11  is structured by a bag  71  made of a soft film-like material such as a resin film. A plurality of corrugations is formed in the bag  71  that has little resistance to gas moving in and gas moving out. The volume of the bag  71  expands depending on an amount of a gas that has moved therein. The bag  71  contracts when a gas moves out thereof. The gas chamber RK 11 , however, is cut off from outside air. Stated differently, the bag  71  serves to trap a gas within the gas chamber RK 11  and to maintain a pressure in the gas chamber RK 11  equal to an atmosphere pressure.  
         [0108]     Accordingly, in the case where a reagent in the process chamber RY 1  moves to the process chamber RY 2 , a gas in the gas chamber RK 11  is supplied to the process chamber RY 1 . When the reagent further moves to the process chamber RY 3 , the gas is supplied to the process chambers RY 1  and RY 2 . When the reagent returns to the process chamber RY 1 , the gas returns to the gas chamber RK 11 .  
         [0109]     Such a bag  71  may be made of a soft rubber film or of an accordion-like material. Further, instead of the bag  71 , a constituent element in which a resin film or a rubber film flexibly covers an opening of a concave portion formed on a chip may be used.  
         [0110]     Referring to  FIG. 12 , the liquid chamber RE 11  is structured by a bag  72  made of a soft film-like material such as a resin film. A plurality of corrugations is formed in the bag  72  that has little resistance to liquid moving in and liquid moving out. The volume of the bag  72  expands depending on an amount of a liquid that has moved therein. The bag  72  contracts when a liquid moves out thereof. The liquid chamber RE 11 , however, is cut off from outside air. Stated differently, the bag  72  serves to trap a liquid within the liquid chamber RE 11  and to maintain a pressure in the liquid chamber RE 11  equal to an atmosphere pressure.  
         [0111]     Accordingly, a drive solution discharged from the micropump MP 1  is reserved in the liquid chamber RE 11 . In the case where the drive solution is discharged to the liquid chamber RE 2  side by the micropump MP 1 , the drive solution is supplied from the liquid chamber RE 11 . In short, the liquid chamber RE 11  functions as a tank of the drive solution.  
         [0112]     Similarly to the case of the bag  71  as mentioned above, such a bag  72  may be made of a soft rubber film. Further, instead of the bag  72 , a constituent element in which a resin film or a rubber film flexibly covers an opening of a concave portion formed on a chip may be used.  
         [0113]     Further, the bag  71  can be used as the gas chamber RK 11  and the bag  72  can be used as the liquid chamber RE 11 , i.e., the bag  71  and the bag  72  can be used in the same microfluidic device  1 .  
         [0114]     In the case where dirt or bubbles enter the chip for some reason, the drive solution is discharged from the connection holes AN 1 -AN 2 , so that the dirt or the bubbles can be discharged together with the drive solution, leading to the recovery to the normal state with ease.  
         [0115]     In the present embodiment, the description is provided of an example in which the microfluidic device  1  is structured as a device for conducting a test or an examination by the PCR method. In addition to the example, it is possible to use the present embodiment in order to move or transport various intended liquids through a gas by filling the micropump MP 1  with various drive solutions. The present embodiment can apply to, for example, a biochemical examination, an immunological examination, a genetic test, a chemical synthesis, drug development or an environmental measurement.  
       Second Embodiment  
       [0116]     In the foregoing first embodiment, the three process chambers RY 1 -RY 3  are individually provided corresponding to the three heating portions KN 1 -KN 3  that are separately provided. In a second embodiment, however, a structure is adopted in which a plurality of temperature areas is provided in one chamber having a constant sectional area.  
         [0117]      FIG. 13  is a diagram showing a structure of a microfluidic device  1 B according to the second embodiment of the present invention, mainly by a connection state of chambers therein.  
         [0118]     As shown in  FIG. 13 , one process chamber RY 11  is provided with extending over three heating portions KN 1 -KN 3 . Three chambers Y 1 -Y 3  are provided inside the process chamber RY 11 . The chambers Y 1 -Y 3  are provided at portions corresponding to the heating portions KN 1 -KN 3 , respectively. When being heated, the three chambers Y 1 -Y 3  function as temperature areas of the heating portions KN 1 -KN 3 , respectively. Each of the three chambers Y 1 -Y 3  has a volume greater than an amount of a reagent used for one test. The three chambers Y 1 -Y 3  are separated from one another by gap chambers SP 1 -SP 2 . Heat insulation in the heating portions KN 1 -KN 3 , e.g., slits between heater portions lead to a more preferable result.  
         [0119]     The amount of liquid transport using the micropump MP 1  at one time is so set that a reagent present in one chamber Y is entirely transported to the neighboring chamber Y. Sensors are provided for detecting the presence of a reagent in the chambers Y 1 -Y 3  or the gap chambers SP 1 -SP 2  and the drive circuit  36  is controlled based on detection signals from the sensors, ensuring that more accurate control can be realized.  
         [0120]     Referring to  FIG. 13 , the upper side of the chamber Y 1  included in the process chamber RY 11  is provided with a fill port AT 2  into which a reagent is injected. The reagent injected from the fill port AT 2  is supplied to the chamber Y 1  directly. After the injection of the reagent, the fill port AT 2  is plugged and sealed.  
         [0121]     Since the structures, operations and effects other than the process chamber RY 11  of the microfluidic device  1 B are similar to the case of the microfluidic device  1  in the first embodiment, descriptions thereof are omitted.  
       Third Embodiment  
       [0122]     In the foregoing first and second embodiments, an end portion of the channel RR 1  provided in the micropump MP 1  side, i.e., the connection chamber RS 1  is completely independent of an end portion of the channel RR 16  provided in the process chambers RY side, i.e., the connection chamber RS 3 . In short, the connection chamber RS 1  is not in communication with the connection chamber RS 3  in the first and second embodiments. Instead, in a third embodiment, a structure is adopted in which the both end portions are in communication with each other and all the channels RR form one closed loop.  
         [0123]      FIG. 14  is a diagram showing a structure of a microfluidic device  1 C according to the third embodiment of the present invention, mainly by a connection state of chambers therein.  
         [0124]     As shown in  FIG. 14 , the microfluidic device  1 C includes a liquid transport chip CSC and a process chip CRC.  
         [0125]     The liquid transport chip CSC includes two micropumps MP 1 -MP 2 , a liquid chamber RE 12 , a gas chamber RK 2 , liquid chambers RE 1 -RE 2 , a gas chamber RK 8 , liquid chambers RE 8 -RE 9  and connection chambers RS 21 -RS 22 . The liquid chamber RE 12 , channels RR 21 -RR 22  and the micropumps MP 1 -MP 2  are filled with a drive solution.  
         [0126]     The process chip CRC includes a process chamber RY 21 , gas chambers RK 21 -RK 22  and connection chambers RS 23 -S 24 . The process chamber RY 21  further includes three chambers Y 1 -Y 3  and gap chambers SP 1 -SP 2  for separating the three chambers Y 1 -Y 3 , similar to the case of the process chamber RY 11  described in the second embodiment. The chambers Y 1 -Y 3  are provided at portions corresponding to heating portions KN 1 -KN 3 , respectively. When being heated, the three chambers Y 1 -Y 3  function as temperature areas of the heating portions KN 1 -KN 3 , respectively.  
         [0127]     The liquid transport chip CSC and the process chip CRC are formed on different substrates. When the liquid transport chip CSC and the process chip CRC are overlapped with each other to be integral with each other, the connection chambers RS 21  and RS 22  are connected to the connection chambers RS 23  and RS 24 , respectively, causing the channels RR to be closed for providing a closed loop. Thereby, a drive solution, a reagent and a gas within the microfluidic device  1 C are shut from outside air.  
         [0128]     The micropump MP 1  cooperates with the micropump MP 2  and thereby a reagent present in any of the chambers Y 1 -Y 3  within the process chamber RY 21  moves to the other chambers Y 1 -Y 3 . When the micropumps MP 1  and MP 2  are driven, pressures of gases present in front and in rear of the reagent can be separately adjusted, ensuring that movement or transport of the reagent can be smoothly performed in a precise manner.  
         [0129]     The liquid chamber RE 12  functions as a tank for reserving a drive solution. A part of the wall surface of the liquid chamber RE 12  is preferably structured by a soft material easily transforming, e.g., a resin film as mentioned above in order to prevent the interior of the liquid chamber RE 12  from providing a negative pressure when a drive solution in the liquid chamber RE 12  is reduced by driving the micropump(s) MP.  
         [0130]     Further, the liquid chamber RE 12  retains a drive solution having an amount that is sufficiently greater than a movement amount of the drive solution when the micropump(s) MP is driven. Then, a small amount of the drive solution is discharged from respective outlets of the connection chambers RS 21  and RS 22  at fixed intervals or every time when a test or an examination is carried out, leading to the improved maintenance.  
         [0131]     One liquid chamber RE 12  is shared by the two micropumps MP 1  and MP 2 . Instead, a structure is possible in which each of the micropumps MP 1  and MP 2  has a liquid chamber RE or a tank individually and the liquid chambers RE or the tanks are not in communication with each other.  
         [0132]     Since the two micropumps MP 1  and MP 2  are used, each of the micropumps MP 1  and MP 2  may transport a liquid unidirectionally. Alternatively, any one of the micropumps MP 1  and MP 2  may be omitted so that only one micropump MP, which is drivable bidirectionally, is used for drive.  
         [0133]     The microfluidic device  1 C according to the third embodiment shown in  FIG. 14  corresponds to the microfluidic device  1 B according to the second embodiment shown in  FIG. 13 . The microfluidic device  1 C according to the third embodiment shown in  FIG. 14  can be in the form corresponding to the microfluidic device  1  according to the first embodiment shown in  FIGS. 8 and 11 . Such an example is illustrated in  FIG. 15 .  
         [0134]      FIG. 15  shows a modification of the microfluidic device  1 C according to the third embodiment.  
         [0135]     As shown in  FIG. 15 , a liquid transport chip (a drive chip) CSC 2  and a process chip CRC 2  are formed on different substrates. The liquid transport chip CSC 2  and the process chip CRC 2  are overlapped with each other and integral with each other so as to be in communication with each other by connection holes AN 3  and AN 5 . The structure of the liquid transport chip CSC 2  is almost similar to that of the liquid transport chip CSC shown in  FIG. 14 . The structure of the process chip CRC 2  is similar to the structure extending from the gas chamber RK 1  to the gas chamber RK 4  including the process chambers RY 1 -RY 3  shown in  FIG. 8 . The process chip CRC 2  is provided with a heating portion if necessary.  
         [0136]     Various methods can be adopted for observation of a result after performing a test on a reagent or of a state during performing a test on a reagent. In the case where a part of the structure of the process chamber RY 2  is made transparent, a reagent is optically detected in the part. Fluorescence detection is generally used for the detection.  
         [0137]      FIG. 16  is a diagram showing an example of a structure of a known coaxial incident light optical device  3  used for optical detection of a reagent in the process chamber RY 2 .  
         [0138]     Referring to  FIG. 16 , the coaxial incident light optical device  3  includes a light source  101 , lenses  102 - 104 , a detector  105 , bandpass filters  106 - 107  and a dichroic mirror  108 .  
         [0139]     The light source  101  projects excitation light which is irradiated to a reagent in the process chamber RY 2  through the lens  102 , the bandpass filter  106 , the dichroic mirror  108  and the lens  103 . In response to the irradiated light, a fluorescent material included in the reagent produces fluorescence. The fluorescence is detected by the detector  105  through the lens  103 , the dichroic mirror  108 , the bandpass filter  107  and the lens  104 . The projected excitation light illuminates the interior of the process chamber RY 2 . A field stop (not shown) positioned right in front of the detector  105  sets a measurement field of a detection optical system so as to receive fluorescence from within an irradiation range of the projected excitation light.  
         [0140]     As discussed above, according to the microfluidic device  1 ,  1 B or  1 C in the first, the second or the third embodiment, it is possible to measure or observe a state or the course during performing a test on a reagent in addition to a test result of a reagent.  
         [0141]     According to each of the embodiments, the microfluidic devices  1 ,  1 B and  1 C for testing a reagent can be downsized. Since volumes of channels where a reagent or others moves can be reduced, a test is possible using a small amount of reagent and responsiveness to movement and to a temperature process is good. Positioning after movement of a reagent can be accurately performed with precision, which enables a test with precision.  
         [0142]     Additionally, the expensive liquid transport chip CS can be used permanently, while the inexpensive process chip CR is disposable. A trouble for washing the process chip CR can be saved, resulting in the reduced running cost.  
         [0143]     In the respective embodiments described above, constitutions, structures, shapes, dimensions, numbers and materials of each part or whole part of the microfluidic devices  1 ,  1 B and  1 C can be varied within the scope of the present invention.  
         [0144]     Structures, shapes, dimensions, numbers and materials of each part or whole part of the microfluidic system can be varied within the scope of the present invention.  
         [0145]     The microfluidic system discussed above can apply to test of reagents or processes thereof in various fields including environment, food product, biochemistry, immunology, hematology, a genetic analysis, a synthesis and drug development.  
         [0146]     While the presently preferred embodiments of the present invention have been shown and described, it will be understood that the present invention is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the scope of the invention as set forth in the appended claims.