JP4159596B2 - Micro liquid dilution method - Google Patents

Description

  The present invention relates to a method for diluting a trace amount of liquid using a microfluidic device in which a microchannel structure for mixing microfluids is formed in a substrate. More specifically, the present invention relates to mixing samples, reagents, and the like for various analyses. The present invention relates to a trace liquid dilution method using a microfluidic device used for dilution.

  Conventionally, when analyzing a specimen or chemically reacting various substances, the specimen or reagent is often diluted. In particular, when a small amount of liquid is diluted, an operation method using a microplate and a dispensing pipette, or a method using an automatic liquid dispensing robot apparatus has been used. In the operation method using a microplate and a dispensing pipette, the operation is complicated and a skilled experimenter is required. In addition, it is difficult to easily mix specimens and reagents outside the laboratory or on the bedside during clinical examinations.

  On the other hand, in the dilution method using the automatic liquid dispensing robot apparatus, the apparatus has to be large, and it cannot be easily used outdoors or at the bedside.

  In recent years, microfluidic devices have attracted attention as analytical devices that handle a small amount of liquid. The microfluidic device has, for example, a substrate that is sized to be easily carried and handled by hand. A fine channel structure for conveying a specimen, a reagent, a diluent, and the like is formed in the substrate. The fine channel structure is appropriately provided with a reagent storage unit, a sample supply unit, a diluent storage unit, a reaction chamber, and / or a mixing unit.

The microfluidic device is usually formed using a substrate having a plane area of several hundred cm ² or less, and the thickness of the substrate is about 0.5 to 10 mm. In addition, the diameter of the flow path in the fine flow path structure is usually very thin, about 5 μm to 1 mm. Here, when the flow path is flat, the diameter of the fine flow path is defined by the narrower width of the cross section of the flat flow path. In addition, the microfluid to be transported is sent by air or the like and is often in the form of droplets.

  Therefore, when diluting a specimen or reagent in the microfluidic device, the microfluid is transported through a very narrow fine channel. The surface tension of the fluid and the wettability of the wall surface of the fine channel have a great influence. In addition, it is difficult to quantitatively measure such a small amount of microfluidic material, and there is a problem that a complicated circuit configuration is required.

  Patent Document 1 below discloses a method of generating protein crystals in a laminar flow using a microfluidic device. Non-Patent Document 1 described below discloses a method for strictly controlling temperature in a microfluidic device, thereby generating crystals from a small amount of liquid.

  However, in each method described in Patent Document 1 and Non-Patent Document 1, the reaction site is very small and the reaction can be controlled with high accuracy. Then, there was a problem that the dead volume could not be reduced.

The following Patent Document 2 discloses a micro liquid weighing structure that can solve the above-described problems and can weigh a small amount of liquid by a simple operation with a simple configuration. The trace liquid weighing structure described in Patent Document 2 is a trace fluid weighing structure using a passive valve. The microfluidic weigh structure includes a first microchannel and a second channel that extend in a predetermined direction, and a third channel that opens to the channel wall of the first microchannel. , Opening the flow path wall of the second flow path, connecting one end of the third flow path and the second flow path, and a fourth flow path that is narrower than the first to third flow paths. Have. The fourth flow path is less likely to get wet than the second flow path and the third flow path, or has a property that the capillary force is relatively difficult to work. Then, the liquid introduced into the first microchannel is drawn into the third channel through the opening of the third channel that opens to the channel wall of the first microchannel. After that, the liquid remaining in the first fine channel is removed, and the volume of the liquid corresponding to the volume of the third channel can be weighed.
US Pat. No. 6,409,832 JP 2004-163104 A “Analytical Chemistry” (2002), 74, p. 3505-3512

  However, in the microfluidic device having the trace liquid weighing structure described in Patent Document 2, there is a drawback that accurate and reproducible mixing cannot be performed when the mixing ratio is increased to 10 times or more. Furthermore, in analysis and reaction, it may be necessary to dilute the specimen or reagent progressively at a high magnification of 10 times, 100 times, or 1000 times.

  However, conventionally, this type of microfluidic device has not been able to function as a multi-stage fine channel structure in which a plurality of mixing units are connected. This is because in this type of microfluidic device, a very small amount of microfluid is transported in a very small flow path in the form of droplets, and the surface tension of the microfluid, the wettability of the flow path wall surface and the capillary In order to perform weighing and unification using the influence of the phenomenon, it was assumed that the timing at which the weighed multiple microfluids were pushed out from the weighing section to the merge section was the same. In connection, due to the restriction that the output of the first mixing unit is used by the second mixing unit, the timing at which the plurality of microfluids weighed in the second mixing unit are pushed out from the weighing unit to the merging unit This is because the second mixing unit does not function even if the first and second mixing units are simply connected. Therefore, it has been very difficult to simultaneously construct a dilution series including mixed solutions of various dilution ratios in the microfluidic device. Actually, a microfluidic device that satisfies such a demand has not been developed so far.

  In addition, as a conventional dilution method, there are a method of adding a large amount of buffer solution to the solution to be diluted at once and mixing the solution, a multistage dilution in which the solution is diluted in several steps, etc. . Among them, multi-stage dilution has been used to prepare a highly diluted solution having a uniform concentration. In the case of such a dilution operation, it is possible to collect and mix the solution quantitatively by an ordinary method. On the other hand, in order to prepare a uniform solution having a high dilution ratio in the microfluidic device, it was necessary to realize a multistage dilution method in the microfluidic device. To perform multi-stage dilution at such an accurate concentration and finally obtain a highly diluted solution, 1) Accurate weighing of the solution to be diluted and the buffer solution, 2) Uniform mixing of the solution to be diluted and the buffer solution Had to do. However, completing them in a microfluidic device has been a very difficult task.

  The object of the present invention is not only to be able to weigh a plurality of microfluids with high accuracy in view of the above-described current state of the prior art, but also to easily mix microfluids with various dilution ratios by mixing the plurality of microfluids. Another object of the present invention is to provide a method for diluting a trace amount of liquid using a microfluidic device provided with a fine channel structure that can be reliably provided.

A micro liquid dilution method using a microfluidic device according to the present invention includes a substrate and a fine channel structure that is provided in the substrate and conveys the microfluid, and the fine channel structure includes: One mixing unit and a second mixing unit connected downstream of the first mixing unit, each mixing unit including a first microfluid in order to weigh a certain amount of the first microfluid. In order to weigh a first amount of the second microfluid having a first weigher having a fine flow path having a volume equal to the volume of the predetermined amount of the first microfluid, the second amount of the second microfluid A second weighing unit comprising a fine channel having a volume equal to the volume of the microfluid, and a merging unit where the first and second microfluids weighed by the first and second weighing units are merged And the first and second microphones are connected downstream of the junction. A mixing section for mixing fluid, a discharge section for discharging the mixed microfluid obtained by mixing the first and second microfluids, and first to third inlet ports and first to third outlet ports A first fine flow path connecting the first inlet port and the first outlet port, a second inlet port and the second outlet port, and the merging portion, the mixing portion, and the discharge portion And a third fine channel connecting the third inlet port and the third outlet port, and one end of the first weighing portion has the first fine flow The other end is open to the joining portion provided in the second fine channel, and one end of the second weighing unit is connected to the third fine channel. And the other end is open to the merging portion provided in the second fine flow path, and the second outlet port is connected to the exhaust port. Parts are connected to the second outlet port of the first mixing unit, the second the first or the third mixing unit of which is connected to the inlet port, using a microfluidic device trace In the liquid dilution method, the first microfluid as a sample is weighed in the first or second weighing unit of the first mixing unit, and diluted in the second or first weighing unit. A step of weighing a second microfluid as a liquid, and in the first mixing unit, the first microfluid as the specimen and the second microfluid as a diluent are mixed, and mixed micro A step of discharging the first specimen diluent as a fluid; and at least one of the first mixed microfluids discharged from the first mixing unit in the first or second weighing unit of the second mixing unit. Weigh a part of the Weighing the diluent as the second or first microfluid in the second or first weighing unit of the second mixing unit; and, in the second mixing unit, the first specimen diluent , Mixing the dilution liquid to obtain a second specimen dilution liquid as a second mixed microfluid, and the second specimen dilution liquid as the second microfluid to the second mixing unit. A discharge section of each mixing unit, and further, n-2 (n is a natural number of 3 or more) mixing units are connected to the subsequent stage of the first and second mixing units. From the above, the mixed fluid as the specimen diluent is discharged, whereby n kinds of specimen diluents having different concentrations are obtained.

  In the microfluidic device used in the present invention, the second outlet port of the first mixing unit may be connected to the first or third inlet port of the second mixing unit. Thereby, the microfluid mixed in the first mixing unit is used as a certain amount of the first or second microfluid in the second mixing unit. In this case, since the first mixing unit and the second mixing unit are connected as described above, a dilution series with a higher magnification can be configured.

  In the microfluidic device used in the present invention, the first outlet port of the first mixing unit is connected to the first inlet port of the second mixing unit. The third outlet port of the mixing unit may be connected to the third inlet port of the second mixing unit. In this case, since the first and second mixing units are connected in parallel, the same dilution can be achieved by configuring the first and second weighing units of the first and second mixing units in the same manner. A plurality of microfluids with a magnification can be easily obtained. Further, if the dilution ratio is different between the first mixing unit and the second mixing unit, microfluids having different dilution ratios can be obtained.

  In the microfluidic device used in the present invention, at least one third mixing unit may be further connected to the downstream side of the second mixing unit, thereby increasing the number of dilution factors. This microfluid can be easily obtained.

  In the microfluidic device used in the present invention, in the first and / or second mixing unit, the outlet opening of the first weighing unit and the outlet opening of the second weighing unit include the merging unit. Are opposed to each other.

  However, in the first and / or second mixing unit, the outlet opening of the first weighing unit and the outlet opening of the second weighing unit are in the direction in which the microfluid flows in the junction. May be arranged at different positions.

  Preferably, the distance between the outlet opening of the first weighing unit and the outlet opening of the second weighing unit in the flow direction of the microfluid in the second microchannel is the first weighing unit. In order not to form bubbles between the first microfluid supplied from the first to the merging section and the second microfluid supplied from the second weighing section to the merging section, the first and second When the first and second microfluids are discharged from the weighing unit to the merging unit at different timings, the first and second microfluids are discharged from the outlet opening of the second weighing unit or the first weighing unit. Is selected so as not to contact the outlet opening of the part. In this case, even if the first and second microfluids are discharged from the first and second weighing units at different timings, bubbles are unlikely to be caught, and the first and second microfluids are prevented from being generated in the first unit. , The second microfluid is reliably united.

  Preferably, of the first and second weighing units, the microfluid discharged from the weighing unit on the side where the outlet opening is located downstream in the merging unit is The width of the second fine channel in the merging portion is larger than the dimension of the discharged microfluid along the width direction of the channel so that the size does not reach the wall on the opposite side of the outlet opening. It has been enlarged. Even in this case, it is difficult for air bubbles to bite between the first microfluid discharged from the first weigher and the second microfluid discharged from the second weigher to the junction. .

  In the microfluidic device used in the present invention, preferably, in the mixing section, the wall surfaces on both sides in the width direction of the flow path may be asymmetric and / or the wall surfaces on both sides in the substrate height direction of the flow path may be asymmetric. . In this case, in the mixing part, since the flow state of the microfluid is different on one side and the other side on at least one of both sides in the width direction of the flow path and both sides in the substrate height direction, vortices are generated in the microfluid. The first and second microfluids are mixed more uniformly.

  In still another specific aspect of the microfluidic device used in the present invention, the gas connected to the second inlet port and supplying the gas for transporting the first and second microfluids at the junction is supplied. In the first micropump device and the first and second weighers, a certain amount of microfluid is weighed, and the first and second weighers from the first and second weighers to the merging part. In order to discharge the microfluidic, a second and a third micropump device connected to the first and third microchannels may be further provided.

  By driving the first to third micropump devices, the first and second weighing units can weigh the first and second microfluids and discharge them to the merging unit. The discharged first and second microfluids can be transported to the mixing unit side.

  In the microfluidic device used in the present invention, in at least one microchannel, in order to realize a state where the microfluid can flow in each microchannel and a state where the transport of the microfluid is stopped, at least one microchannel is used. A flow path opening / closing device provided in the substrate in relation to one fine flow path, and when the fine flow path is opened by the flow path opening / closing apparatus, And the microfluidic movement is stopped when the microchannel is closed by the channel switching device. Therefore, by driving the flow path opening / closing device, the microfluid can be fed into the fine flow path, or the liquid feed can be stopped. Preferably, the flow path opening / closing device includes a stopper that can be moved between the open state and the closed state, and a stopper driving unit that moves the stopper between the open state and the closed state. It has been. In this case, by driving the stopper driving means provided in the substrate, the gas pressure in the flow path before the weighing unit is increased, and the microfluid is pushed out from the weighing unit to the mixing unit.

  In particular, if the second outlet port of the first mixing unit is connected to the first or third inlet port of the second mixing unit, thereby the micros mixed in the first mixing unit. Since the fluid is utilized as a certain amount of the first or second microfluid in the second mixing unit, a microfluid diluted at a higher magnification can be obtained in the second mixing unit.

  Conventionally, in this type of microfluidic device, a multi-stage fine channel structure in which a plurality of mixing units are connected has not been adopted. This is because in this type of microfluidic device, a very small amount of microfluid is transported in a very small flow path in the form of droplets, and the surface tension of the microfluid, the wettability of the flow path wall surface and the capillary In order to perform weighing and unification using the influence of the phenomenon, it was assumed that the timing at which the weighed multiple microfluids were pushed out from the weighing section to the merge section was the same. In connection, due to the restriction that the output of the first mixing unit is used by the second mixing unit, the timing at which the plurality of microfluids weighed in the second mixing unit are pushed out from the weighing unit to the merging unit This is because the second mixing unit does not function even if the first and second mixing units are simply connected.

  On the other hand, in the present invention, since the device is able to exert the function as a mixing unit even when the timings at which the plurality of microfluids are pushed out from the weighing unit to the merging unit are not the same, Even in the structure in which the second mixing unit is connected, the second mixing unit can exhibit its function. Therefore, various combinations of a plurality of mixing units are possible, and the microfluids mixed in each combination can be transported quickly and accurately.

  Therefore, the micro liquid dilution method of the present invention is suitable for use in various analyzes and chemical reactions that require multi-stage mixing / dilution with different mixing ratios. By quickly preparing the microfluidic fluid in the microfluidic device, it is possible to automate the operation required for analysis and reaction and shorten the operation time.

  Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention with reference to the drawings.

  FIG. 1 is a schematic plan view showing a microchannel structure of a microfluidic device according to a position embodiment of the present invention, and FIG. 2 is a front sectional view schematically showing a part of the microfluidic device of this embodiment. FIG.

  As shown in FIG. 2, the microfluidic device 1 has a substrate 2. The substrate 2 has a structure in which a transparent base plate 3, intermediate plates 4 to 6, and a top plate 7 are stacked. A gas generation chamber 8 is provided in the base plate 3. The gas generation chamber 8 is opened on the upper surface of the base plate 3, and a responsive gas generation member 9 that generates gas by light irradiation or heating is accommodated in the gas generation chamber 8. By accommodating the responsive gas generating member 9 in the gas generating chamber 8, a micro pump device as a driving source for driving the micro fluid is formed. As the responsive gas generating member, a photoresponsive gas generating member that generates gas by light irradiation is preferably used because of easy control of the amount of gas generated.

  Since the base plate 3 has transparency, gas can be generated from the photoresponsive gas generating member 9 by irradiating light from the lower surface side of the substrate 2. This gas serves as a pressure source for driving the microfluidic in a microchannel described later.

  The photoresponsive gas generating member 9 is not particularly limited, and an appropriate photoresponsive composition that generates gas when irradiated with light can be used. As such a photoresponsive composition, for example, a composition containing a binder resin and a gas generating agent that decomposes by light irradiation to generate gas is suitably used. Examples of such a gas generating agent include an azide compound, an azo compound, a polyoxyalkylene resin, a mixture of a photoacid generator and sodium hydrogen carbonate, and the like.

  The intermediate plate 4 is formed with discharge holes 4a for discharging gas. The discharge hole 4 a penetrates from the lower surface to the upper surface of the intermediate plate 4, and its lower opening faces the gas generation chamber 8.

  The intermediate plate 5 is provided with an opening 5 a penetrating the intermediate plate 5. The opening 5a constitutes a part of a fine channel having a fine channel structure. Further, the intermediate plate 6 is formed with a through hole 6a opened in the opening 5a. The upper opening of the through hole 6 a is open to a fine channel 7 a formed on the lower surface of the top plate 7. The fine flow path 7a forms a fine flow path structure together with the opening 5a and the through hole 6a described above.

  The intermediate plates 4 to 6 and the top plate 7 are made of an appropriate synthetic resin sheet or synthetic resin.

  FIG. 2 schematically shows a part of the microfluidic device 1 in which a micropump device for generating a gas pressure for driving the microfluid is configured and a part of the fine channel structure. The fine flow path of the microfluidic device is disclosed in Patent Document 3 described above.

Generally, as described above, the microfluidic device 1 has a size that can be carried by hand, and is configured using a small substrate 2 having a plane area of several hundred cm ² or less, preferably 100 cm ² or less. The thickness of the substrate 2 is about 0.5 to 10 mm. In the substrate 2, not only the driving portion for transporting the microfluid but also various fine channels for transporting the microfluid as the specimen and the diluent are formed. Such a fine channel structure usually includes a supply section for supplying a specimen and a diluent, a mixing section for mixing them, a reaction section for reacting them, and the like. The supply unit, the mixing unit, the reaction unit, and the like are formed as a space having a certain volume in the substrate 2, and are connected to a thin fine channel, for example, a fine channel 7a.

  A feature of the microfluidic device 1 used in the present embodiment is that a fine channel structure 10 shown in a schematic plan view in FIG. The fine channel structure 10 includes a first mixing unit 11 and a second mixing unit 21 as essential components. A second mixing unit 21 is connected to the downstream side of the first mixing unit 11. In the present embodiment, one third mixing unit 31 is further connected downstream of the second mixing unit 21.

  The structure of the 1st mixing unit 11 is demonstrated in detail with reference to the typical enlarged plan view of FIG.

  The first mixing unit 11 has a first fine channel 11a. A gas supply hole 11b is connected to one end of the first fine channel 11a. The other end of the first microchannel 11a is connected to the first microfluidic supply hole 11c. The first microfluid supply hole 11 c is opened to the outside of the substrate 1 and is a portion that supplies the first microfluid to the fine channel structure of the substrate 1.

  The gas supply hole 11b is connected to a gas generation drive source such as the above-described micropump device, and is configured to be able to open and close as appropriate.

  A second microchannel 12 is arranged in parallel with the first microchannel 11a. In the second microchannel 12, the microfluid flows in the direction of the arrow. A merging portion 12a is provided on the upstream side, and a mixing portion 12b is provided on the downstream side. A discharge unit 12c is connected to the downstream side of the mixing unit 12b.

  A third microchannel 13a is provided on the opposite side of the second microchannel 12 from the first microchannel 11a. The third microchannel 13a has one end connected to the gas supply hole 13b and the other end connected to the liquid supply hole 13c. The gas supply hole 13b and the liquid supply hole 13c are configured similarly to the gas supply hole 11b and the gas supply hole 11c.

  Moreover, the 1st weighing | measuring part 11d which consists of a microchannel branched from the 1st microchannel 11a is provided. The volume of the first weighing unit 11d is equal to the volume of the microfluid to be weighed.

  Similarly, a minute channel having a certain volume is formed by branching from the third minute channel 13a, and the second weighing unit 13d is formed by the minute channel.

  The volume of the second weighing unit 13d is set equal to the volume of the microfluid to be weighed by the second weighing unit 13d.

  Accordingly, one end of each of the first and second weighing units 11d and 13d is connected to the first fine channel 11a and the third fine channel 13a, and the other end is connected to the second fine channel 11a. An opening is formed in the junction 12 provided in the fine channel 12.

  The volumes of the first and second weighing units 11d and 13d are not particularly limited, but are usually very small in the order of picoliters to microliters. That is, as described above, in the microfluidic device of the present invention, such a very small volume of microfluid is transported in the microchannel.

  A gas supply hole 12 d and a gas discharge port 12 e are arranged on the upstream side of the second fine channel 12. The gas supply hole 12d and the gas discharge port 12e are formed in the same manner as the gas supply hole 11b and the liquid supply hole 11c described above.

  A structure in which a certain amount of microfluid is weighed in the first weighing unit 11d will be described with reference to FIGS.

  Micro fluid is injected from the liquid supply hole 11c. In this case, the inside of the first fine channel 11a is opened to the atmosphere. That is, the gas supply hole 11b may be opened to the atmosphere. When injecting the microfluid from the liquid supply hole 11c, a microsyringe or the like may be used to press-fit from the liquid injection hole 11c. As a result, as shown in FIG. 5, the microfluid 14 is fed into the first microchannel 11a and fills the first weighing unit 11d composed of the branched microchannel.

  In the present embodiment, a connecting fine channel 11e having a diameter smaller than that of the fine channel constituting the first weighing unit 11 is provided on the distal end side of the first weighing unit 11d. The diameter of the connection microchannel 11e is very thin. Therefore, the microfluid 14 cannot flow through the connection microchannel 11e at the injection pressure level due to the surface tension, but at the inlet or outlet of the connection microchannel. Stop.

  Next, gas is supplied from the gas supply hole 11b to the first microchannel 11a. In this case, the liquid supply hole 11c is opened to the atmosphere. As a result, as shown in FIG. 6, the first microfluid 14a remains as a certain amount of microfluid in the first weighing unit 11d. Thus, in order to leave the microfluid 14a in the first weighing unit 11d, it is desirable that the second flow path 12 side be closed and not released to the atmosphere when supplying gas. However, when the connection microchannel 11e is sufficiently thin and a capillary reaction force acts in the connection microchannel 11e, the second microchannel 12 side may be sealed.

  Next, a part of the first fine channel 11a on the liquid supply hole 11c side is closed by a valve or the like of a later-described channel opening / closing device, and in this state, the gas is supplied from the gas supply hole 11b to the first micro channel 11a. Supply. As a result, the first microfluid 14a weighed by the first weigher 11d is discharged into the second microchannel 12 as shown in FIG.

  Since the volume of the first microfluid 14a weighed in the first weighing unit 11d is the same as the volume of the first weighing unit 11d, according to the present embodiment, a certain amount of the first microfluid 14a is measured. One microfluid 14 a can be reliably discharged into the second microchannel 12.

  The channel opening / closing device can be formed by an appropriate valve capable of switching between a state in which a part of the microchannel is opened and a state in which it is closed. As such a valve, it is possible to use a structure in which a driving element such as an electromagnetic valve or a piezoelectric element is connected with a stopper that can move between a state where the flow path is narrowed and a state where the flow path is opened.

  Further, as shown in FIG. 4A, a flow path opening / closing device in which a viscoelastic stopper 41 is combined with a stopper driving source 42 made of gas or liquid may be used. Here, the viscoelastic stopper 41 can be made of an appropriate material that has elasticity and can move due to an increase in pressure in the flow path, such as an elastomer or a gel. When the microfluid to be fed is an aqueous solution, a water-insoluble viscoelastic body is preferably used, and when it is an organic solvent, it is desirable to use a non-organic solvent-soluble viscoelastic body.

  As shown in FIG. 4A, a stopper channel portion 43 in which the viscoelastic stopper 41 can be accommodated is formed in a part of the liquid microchannel 11a that is opened and closed. In the present embodiment, the stopper flow path portion 43 has a shape arranged in a direction perpendicular to the direction in which the fine flow path 11a extends so that the three circles partially overlap each other. Among the circular portions 43a to 43c of heat, one circular portion 43a is provided in the middle of the first fine channel 11a, and the remaining circular portions 43b and 43c are on the side of the first fine channel 11a. It is arranged in the direction. In FIG. 4A, the viscoelastic stopper 41 is filled in the second and third circular portions 43b and 43c, and in this state, the fine channel 11a is in an open state.

  As shown in FIG. 4B, gas is generated from the driving source 42, and the viscoelastic stopper 41 is moved to the side by the pressure. As a result, the viscoelastic body stopper 41 moves toward the first and second circular portions 43a and 43b, and comes into close contact with the inner surface of the first circular portion 43a. Therefore, the first fine channel 11a is closed.

  In this way, the first fine channel 11a can be switched from the open state to the closed state. In order to return to the open state again, for example, a second drive source is connected to the first circular portion 43a, gas is supplied again from the second drive source side, and the viscoelastic stopper 41 is moved. What is necessary is just to move to the state of Fig.4 (a).

  Alternatively, a gas suction source may be connected instead of the gas drive source 42 from the state shown in FIG. 4B, and the viscoelastic stopper 41 may be returned to the open state shown in FIG. 4A by suction. Good.

  Returning to FIG. 3, by providing the flow path opening / closing device as described above in the middle of the first fine flow path 11a, the pressure of the gas supplied from the gas supply hole 11b is increased as described above. A fixed amount of the first microfluid 14a can be weighed in the weighing unit 11d and discharged to the second microchannel 12 as described above.

  Similarly, the second microfluidic fluid corresponding to the volume of the second weighing unit 13d is weighed by the second weighing unit 13d in the third fine channel 13a in the same manner as described above. It can discharge to the 2nd microchannel 12.

  The second weighing unit 13d is formed so as to have a volume corresponding to the volume of the second microfluid. In this embodiment, as shown in a schematic partial enlarged plan view in FIG. The second weighing unit 13d also has a connecting fine channel 13e that is narrower than the fine channel that forms the second weighing unit 13d at the tip.

  By providing the connection microchannels 11e and 13e, the microfluid leaks to the second microchannel 12 before discharging the first and second microfluids 12 to the second microchannel 12. It can be surely suppressed. When the microfluid is an aqueous solution and the wall surface of the connecting microchannel is hydrophilic, the microfluid stops at the outlet of the microchannel. Further, when the microfluid is an aqueous solution and the wall surface of the connecting microchannel is hydrophobic, the microfluid stops at the inlet of the microchannel. Further, when the microfluid is an oily solution and the wall surface of the connection microchannel is hydrophilic, the microfluid stops at the inlet of the microchannel, and the microfluid is an oily solution and the wall surface of the connection microchannel Is hydrophobic, the microfluid stops at the exit of the microchannel.

  However, in place of the connecting fine flow channels 11e and 13e, the wettability of the wall surfaces of the portions opened to the second flow channel 12 of the first and second weighing units 11d and 13d is lowered, thereby The first microfluid 14a and the second microfluid may be prevented from leaking from the portion opening in the second microchannel 12. As a measure for reducing the wettability, a known partial water-repellent processing means can be used.

  In the present embodiment, the opening 13f that opens to the second fine channel 12 of the second weighing unit 13d and the second fine channel 12 of the first weighing unit 11f open. The openings 11 e are arranged at different positions in the microfluid flow direction in the second microchannel 12. That is, the opening 13f and the opening 11f are separated by a distance R in FIG. The distance R is the distance between the centers of the opening 13f and the opening 11f.

  The opening 11f of the first weighing unit 11d and the opening 13f of the second weighing unit 13d may be arranged to face each other in the second microchannel 12. However, it is very difficult to dispose the two in opposition and to discharge the first and second microfluids from the first and second weighing units 11d and 13d to the second microchannel 12 at substantially the same timing. Have difficulty. Even when the flow path opening / closing device or the like is driven at the same timing, the timing at which the first and second microfluids are discharged may be slightly shifted in practice.

  If the timing at which the first and second microfluids are discharged is slightly shifted, one microfluidic material adheres to the other weigher opening, and the microfluidic material weighed in the other weigher leaks. Therefore, it becomes difficult to mix the first and second microfluids at an accurate volume ratio.

  In contrast, in the present embodiment, the opening 11e of the first weighing unit 11d and the opening 13f of the second weighing unit 13d are separated by the distance R. Therefore, when the discharge timing of the first microfluid 14a from the first weighing unit 11d is different from the discharge timing of the second microfluid from the second weighing unit 13d, for example, downstream When the first microfluid 14a is discharged first to the part, the first microfluid 14a is unlikely to adhere to the opening 13e of the second weighing part 13d. Therefore, the leakage of the microfluidic weighed by the second weighing unit 13d is unlikely to occur. In other words, the distance R is desirably a distance long enough to prevent the discharged microfluid from coming into contact with the opening of the other weighing unit.

  However, if the distance R is too large, as schematically shown in FIG. 8 (b), the first microfluidic fluid 14a discharged from the first weighing portion 11d and the second weighing The droplets of the second microfluid 13b discharged from the portion 13d are very separated from each other, and an air layer X is formed therebetween. Therefore, air bubbles may be caught between the microfluidic fluid 14a and the microfluidic fluid 14b, which may not be sufficiently mixed.

  Therefore, as shown in FIG. 8A, it is desirable that the distance R be close enough that no bubbles are involved between the first microfluid 14a and the second microfluid 14b.

  However, even if the distance R is large, air entrapment can be prevented by sufficiently increasing the width W of the joining portion 12a of the second microchannel 12. That is, as shown in FIG. 8C, when the discharge of the first microfluidic fluid 14a is completed, the first microfluidic fluid 14a has a second micro flow on the side opposite to the opening 14e. It is desirable that the width W of the second microchannel 12 in the junction 12a is larger than the dimension in the width W direction of the discharged first microfluid 14a so as not to contact the inner wall of the road. In that case, even if an air layer is formed between the first microfluidic fluid 14a and the second microfluidic fluid 14b, when the second microfluidic fluid 14b is fed, the air is Air passes between the microfluid 14a and the inner wall of the channel on the opposite side. Therefore, it is difficult for air to be caught.

  Returning to FIG. 3, by supplying gas from the gas supply hole 12d provided on the upstream side of the second microchannel 12, the first and second microfluids 14a and 14b are joined together in the downstream. Will be swept to the side. As shown in FIG. 9, in the mixing unit 12b, the planar shape of the fine channel is asymmetric on both sides in the width direction of the second fine channel 12b. Will be mixed. That is, a vortex is generated in the mixed microfluid 14c due to the above asymmetry, and the mixed microfluid 14c is stirred and reliably mixed. Here, the taper is given so that one inner wall 12e of the 2nd fine flow path 12 may approach as it goes downstream toward the inner wall 12f on the opposite side. Accordingly, a vortex is generated in the mixed microfluid 14, and the first and second microfluids are sufficiently mixed by the stirring action.

  Therefore, in this embodiment, it is not necessary to provide a large mixing chamber in the mixing unit 11 or on the downstream side of the mixing unit 11. The absence of a mixing chamber is advantageous in the integration of microfluidic devices, short processing times, and interconnection.

  In the present embodiment, the sufficiently mixed microfluid is discharged from the discharge portion 12c provided on the downstream side of the mixing portion 12b. In the present embodiment, the mixed microfluid 14c discharged from the discharge unit 12c of the first mixing unit 11 is supplied to the first weighing unit of the second mixing unit 21 from a first inlet port described later. Will be. That is, the mixing result of the first mixing unit 11 is used in the second mixing unit 21. By connecting the second mixing unit 21 directly to the first mixing unit 11 that mixes the microfluidic structure to form a multistage configuration, it is possible to obtain a microfluid having a higher dilution ratio than the one-stage configuration.

  In the above-described embodiment, the opening of the second weighing unit 13d having a larger volume than the first weighing unit 11d is located on the upstream side in the merging unit 12a. On the other hand, you may arrange | position so that the 1st weighing | measuring part 11d may open to the confluence | merging part 12a upstream from the 2nd weighing part 13d like the modification shown in FIG. In this modified example, the volume of the upstream first weighing unit 11d may be larger than the volume of the downstream second weighing unit 12d.

  On the other hand, you may connect several 2nd weighing parts 13d and 13d to the confluence | merging part 12a like the other modification shown in FIG. In that case, the mixing ratio of the second microfluid can be increased.

  Further, as in the modification shown in FIG. 12, in the second fine channel 12, the wall surface on the opposite side of the opening portion 11f where the first weighing portion 11d is opened protrudes toward the opening portion 11f. As a result, the width of the second microchannel 12 at the portion where the first microfluid 14a is discharged may be narrowed. In this case, even if the volume of the first weighing unit is small, the first microfluid reaches the wall on the opposite side of the merging unit, so the volume ratio between the first weighing unit and the second weighing unit is Even if it is made small, stable operation can be achieved. For example, the first and second microfluids can be mixed at the junction 12a at a ratio of 1: 1.

  Furthermore, as in the modification shown in FIG. 12B, the second microchannel 12 has a narrow portion 12f that is relatively narrower than the remaining portion in the middle of the microfluid flow direction as a confluence 12a. It may be provided. Here, the merging portion 12a has a narrow width portion 12f at the center of the microfluid flow direction. And the opening part 11f of the 1st balance part 11d is opening in the upstream of the narrow part 12f, and the opening part 13f of the 2nd balance part 13d is opening in the downstream of the narrow part 12e. is doing.

  Also here, on the downstream side of the narrow width portion 12f, if the width of the second fine channel 12 is made larger than the diameter of the microfluidic droplet discharged from the second weighing portion 13d, The first and second microfluids can be combined at the merging portion 12a while preventing air from being caught. That is, when the first microfluidic fluid 14a discharged upstream is moved downward by the gas pressure from the gas supply hole 12d, the first microfluidic fluid 14a is positioned between the first microfluidic fluid 14b and the second microfluidic fluid 14b. Air is released to the downstream side. Therefore, the first and second microfluids 14a and 14b are united without interposing bubbles. However, after the first microfluid and the second microfluid are combined, it is necessary to form a droplet that fills the width of the second microchannel 12.

  One microfluidic device of the present invention has a structure in which at least a first and a second mixing unit are connected. In this case, the mixing unit has the first to third fine channels as described above, and the merging unit, the mixing unit, and the discharge unit are configured in the second fine channel from the upstream side. Such first and second mixing units can be connected in various forms.

  With reference to FIGS. 13-17, the modification of the connection form of a 2nd mixing unit is demonstrated to such a 1st mixing unit, respectively.

  FIG. 13 is a plan view schematically showing a structure in which the first and second mixing units are connected using the mixing result in the first mixing unit, similarly to the embodiment shown in FIG. It is. Here, the 2nd mixing unit 21 is connected to the back | latter stage of the 1st mixing unit 11 like the said embodiment. In the drawings of the fine channel structure below FIG. 13, the mixing unit, the channel switching device, and the like are appropriately shown as a block surrounded by a broken line.

  In the fine channel structure 61 shown in FIG. 13, the second mixing unit 21 is connected to the downstream side of the first mixing unit 11 as in the above embodiment. Here, the first mixing unit 11 can be represented as having first to third inlet ports A to C and first to third outlet ports D to F. That is, the first fine channel 11 a is connected between the first inlet port A and the first outlet port D. One end of the first weighing unit 11 d is connected to the first fine channel 11 a, and the other end of the first weighing unit 11 a is connected to the second fine channel 12. A second fine channel 12 is connected between the second inlet port B and the second outlet port E. Between the third inlet port C and the third outlet port F, the third fine channel 13 is connected. The 2nd exit port E is connected to the discharge part, and is equivalent to the portion which discharges the mixed micro fluid outside. Further, the third outlet port F is connected to the flow path opening / closing device 62.

  The first inlet port A of the second mixing unit 21 is connected to the first outlet port D of the first mixing unit 11, and the second inlet port B of the second mixing unit 21 is a gas Connected to the supply hole. The third inlet port C is connected to the second outlet port E of the first mixing unit 11. Therefore, the mixed microfluid mixed in the first mixing unit 11 is supplied from the third inlet port C of the second mixing unit 21, and the microfluid is supplied to the second balance of the second mixing unit 11. It is weighed by the collecting part 13d.

  Therefore, the second mixing unit 21 uses the result of mixing in the first mixing unit 11. Therefore, when it is set as the structure which supplies a dilution liquid from the 1st weighing part 11d, it can dilute to higher magnification by connecting the 1st, 2nd mixing units 11 and 21 as mentioned above. .

  A flow path opening / closing device 63 is also connected to the third outlet port of the second mixing unit 21. Similarly, the channel opening / closing device 64 is also connected to the first outlet port D of the second mixing unit 21, and the microfluid diluted at a high magnification is discharged from the second outlet port E, The solution is sent to a measurement unit and a reaction unit provided in the subsequent stage.

  FIG. 14 is a plan view schematically showing a structure in which a third mixing unit 31 is further connected to the downstream side of the first and second mixing units 11 and 21. As described above, one or more mixing units 31 may be further connected to the downstream side of the first and second mixing units 11 and 21. Further, in the structure shown in FIG. 14, the third outlet ports F of the first to third mixing units 11, 21, 31 are connected to the flow path opening / closing devices 62, 63, 65, respectively. A branch channel is formed between the outlet port F and the channel switch, and the branch channel is connected to the storage chamber 71. Instead of the storage chamber 71, a reaction cell may be configured.

  Therefore, in the storage chambers 71, 71, 71 connected to the third outlet ports F of the first to third mixing, microfluids having different dilution ratios are prepared. Further, the second opening port E connected to the discharge portion of the third mixing unit 31 is similarly connected to the flow path opening / closing device 66 and the storage chamber 71, and the storage chamber 71 is also connected to the second outlet port E. Microfluids with different dilution ratios are prepared.

  FIG. 15 is a schematic plan view showing a modification of the fine channel structure shown in FIG. In FIG. 14, the storage chamber 71 is connected between the third outlet port F of the first to third mixing units 11, 21, 31 and the flow path opening / closing device, but in FIG. 15, The storage chamber 81 is connected to the front stage of the third inlet port C of the first mixing unit, and downstream of the second outlet port E of the first to third mixing units 11, 21, 31, respectively. The storage chambers 82 to 84 are connected. In the storage chambers 82 to 84, the mixing results in the mixing units 11, 21, 31 at each stage are measured.

  By using the microfluidic device having the fine channel structure shown in FIG. 14 or FIG. 15, the trace liquid dilution method of the present invention can be carried out. For example, in the fine channel structure shown in FIG. 14, the diluent is weighed as the first microfluid in the first weighing unit 11 d of the first mixing unit 11. On the other hand, the sample to be diluted as the second microfluid is weighed in the second weighing unit 13d. The sample and the diluted solution are mixed by the first mixing unit 11 and discharged from the discharge portion of the second fine channel 12 of the first mixing unit 11 through the second outlet port E. In the second mixing unit 21, as in the case of the first mixing unit 11, the diluted liquid is weighed in the first weighing unit 11d as the first microfluid in the first weighing unit 11d. Is done.

  On the other hand, in the second weighing unit 13d, the sample diluent, which is the mixing result of the first mixing unit 11, that is, the sample diluent as the first mixed microfluid discharged from the outlet port E is weighed. Is done. In FIG. 14, a storage chamber 71 is disposed at the third outlet port F of the second mixing unit 21, and the first mixed microfluid, that is, the first specimen diluent is stored in the storage chamber 71. The In addition, the first sample diluent and the diluted solution are mixed in the second mixing unit 21, and the obtained sample diluted solution as the second mixed microfluid is the outlet port E of the second mixing unit 21. And is supplied to the chamber 71 connected via the outlet port F of the third mixing unit 31. Accordingly, the first and second sample dilution liquids having different concentrations are supplied to the chambers 71 and 71 connected to the sides of the second mixing unit 21 and the third mixing unit 31, respectively.

  In the fine channel structure shown in FIG. 15, the first sample diluent is supplied to the second mixing unit 21 in the storage chamber 82 connected between the first mixing unit 11 and the second mixing unit 21. And the second sample dilution liquid is provided to the storage chamber 83 connected between the third mixing unit 31 and the third mixing unit 31.

  As described above, by using the fine channel structure shown in FIGS. 14 and 15, the trace liquid dilution method of the present invention can be carried out to prepare a plurality of specimen dilution liquids having different concentrations. 14 and 15, as described above, the third mixing unit 31 is further connected, so that three types of specimen dilution liquids having different concentrations can be provided.

  Further, as in the embodiments described later, more mixing units may be connected to the next stage of the first and second mixing units, in which case, downstream of the first and second mixing units, When n-2 (n is a natural number) mixing units are connected, n types of specimen dilution liquids having different concentrations can be prepared.

  Contrary to the case of FIG. 14 and FIG. 15, the sample may be weighed in the first weigher and the diluent may be weighed in the second weigher.

  FIG. 16 is a schematic plan view showing a fine channel structure in which more mixing units are connected in a matrix. Here, the first to third mixing units 11, 21, 31 are configured in the same manner as the fine channel structure shown in FIG. Of course, each third outlet port F of each mixing unit 11, 21, 31 is not only connected to the flow path opening / closing device and the storage chamber, but also to the third outlet port, A fine channel structure in which the fifth mixing units are connected in series is connected. That is, when the first to third mixing units 11, 12, 13 are set in both directions, the fourth and fifth mixing units 91, 92 are arranged in the column direction of the matrix composed of the rows and columns. 21 and 31 are connected. The connection structure of the fourth and fifth mixing units is the same as that of the first and second mixing units.

  That is, the fourth and fifth mixing units 91 and 92 are arranged such that the fifth mixing unit 92 uses the mixing result in the fourth mixing unit 91 in the fifth mixing unit 92. It is connected in the same way as the connection relationship. The flow path opening / closing device 93 and the storage chamber are connected to the second outlet port E of the fifth mixing unit. Similarly, the third opening ports F and F of the fourth and fifth mixing units 91 and 92 are connected to the flow path opening / closing device 93 and the storage chamber.

Therefore, in the fine channel structure shown in FIG. 16, if the same amount of microfluid is discharged from the first weigher 11d and the second weigher 13d in each mixing unit, in a matrix form The dilution ratios in the many storage chambers arranged are as follows. Note that the dilution ratio is, for example, when the stock solution is weighed in the second weighing unit 13d of the first mixing unit 11 and diluted in each of the following mixing units. The proportion of the stock solution shall be said. For example, since the original stock solution is prepared in the storage chamber 101a, the dilution rate is 1/1, and in the storage chamber 101b, the dilution rate of the stock solution is 1/3. That is, in the storage chambers 101a to 101c located on the sides of the fourth and fifth mixing units 91 and 92 connected to the first mixing unit 11, the dilution ratios are 1/1, 1/3 and 1 / 3 ² become. The dilution ratios in the storage chambers 102a to 102c arranged on the sides of the fourth and fifth mixing units connected to the second mixing unit 21 are 1/3 ³ , 1/3 ⁴ and 1 respectively. / ³ is ^5. Similarly, in the storage chambers 103a to 103c arranged on the sides of the fourth and fifth mixing units connected below the third mixing unit 31, the dilution ratios are 1/3 ⁶ and 1/3 ^7. And 1/3 ⁸ .

Further, in the storage chambers 104a to 104c arranged on the sides of the fourth and fifth mixing units connected to the second outlet port of the third mixing unit 31, the dilution rate is 1/3 ⁹ , 1 / 3 ¹⁰ and 1/3 ¹¹ . Therefore, by arranging the storage chambers 101a to 104c in a matrix as described above, a microfluidic device that automatically produces a series of dilution series in a short time can be produced. It is apparent that a different dilution series can be created depending on the selection of the mixing ratio of each mixing unit. For example, a 1/2 ⁿ dilution series can be obtained by replacing with a mixing unit capable of one-to-one mixing as shown in FIG. As described above, it is possible to easily provide various types of microfluids having a dilution ratio.

  FIG. 17 is a schematic plan view showing still another modification of the fine channel structure in the microfluidic device of the present invention. In the embodiment and the modification described above, the first and second mixing units are connected so that the mixing result in the first mixing unit is used. However, as shown in FIG. 111 and the second mixing unit 121 may be connected in parallel. Here, the first weighing unit 111d of the first mixing unit 111 and the first weighing unit 121d of the second mixing unit 121 are commonly connected by the first fine channel 111a.

  Similarly, the second weighing unit 113d of the first mixing unit 121 and the second weighing unit 123d of the second mixing unit 121 are also commonly connected by the third fine channel 113a. The storage chambers 131 and 132 are connected to the discharge portions provided on the downstream side of the second fine channels 112 and 122 of the first and second mixing units 111 and 121, respectively. Accordingly, microfluids having the same dilution ratio can be obtained from the storage chambers 131 and 132 connected to the first and second mixing units 111 and 121.

  In other words, the first and third outlet ports D and F of the first mixing unit are connected to the first and third inlets A and C of the second mixing unit 121, respectively. The microfluids having the same dilution ratio are discharged from the second outlet ports E and E of 111 and 121, respectively.

  Further, in the present invention, both the parallel connection for obtaining the microfluid of the same dilution rate shown in FIG. 17 and the connection form as shown in the above-described embodiment and modification may be used in combination.

Further, in the above-described embodiment, the mixing unit has an asymmetric wall surface located on both sides in the bundle direction in the second fine channel, and one wall surface approaches the other wall surface toward the downstream side. Tapered like this. The shape of the mixing portion is not limited to such a shape. For example, as shown in FIG. 18 (a), in the mixing section 12b, one wall surface 12b ₁ is spread one responsible as toward the downstream side, and then linearly to approach the wall 12b ₂ on the opposite side The other wall surface 12b ₂ may also be formed so as to bulge outward at a portion different from the wall surface 12b ₁ and then approach the opposite wall surface 12b ₁ .

Further, as shown in FIG. 18B, the planar shape of both wall surfaces 12b ₁ and 12b ₂ may be a shape like a sine curve having a different phase. The wall surfaces on both sides are arranged asymmetrically.

  Furthermore, instead of the structure in which the wall surfaces on both sides in the width direction are arranged asymmetrically, the wall surfaces located on the upper surface and the lower surface of the fine channel may be arranged asymmetrically, and the wall surfaces on both sides in the width direction are arranged asymmetrically. And a structure in which the wall surfaces located on both sides and the lower surface, that is, both sides in the thickness direction of the substrate are arranged asymmetrically may be used in combination.

  In any case, since the fine flow path is asymmetric on both sides in the width direction and on both sides in the thickness direction of the substrate, vortices are generated when the microfluid flows and the microfluid is sufficiently mixed. Therefore, it is not necessary to form a separate large mixing chamber or coil-shaped mixing portion, and the microfluidic device can be downsized.

  The microfluidic device can be used for, for example, separation / analysis of substances, biochemistry or chemical reaction, protein crystallization, etc., and is preferably disposable or exchanged for a limited number of times. You may use it permanently. In this case, it is possible to use it integrated with a device such as a dispenser or a measuring device.

  The materials that can be used in the present invention will be described below.

  As the substrate material of the microfluidic device, an inorganic material or an organic material can be used regardless of the type as long as the flow path structure can be realized. Examples of the material include polydimethylsiloxane (PDMS), glass, silicon, quartz, thermoplastic resin, curable resin and other resins by light and heat, metal, ceramic, and combinations thereof.

  The photoresponsive gas generating resin composition constituting the photoresponsive gas generating material used in the present invention is a resin composition mainly composed of a binder resin such as a thermoplastic resin and generating a gas upon irradiation with light. Although not limited, the resin composition which generate | occur | produces gas by the light irradiation of 330 nm to 410 nm is preferable.

  The resin composition may be a resin composition composed of a binder resin and a gas generating agent that generates gas when irradiated with light.

  Binder resin includes polyester, poly (meth) acrylate, polyethylene, polypropylene, polystyrene, polyether, polyurethane, polycarbonate, polyamide, acetal resin such as polyimide, acetal resin such as poval, butyral, etc. A polyoxyalkylene resin or the like can be selected.

  The gas generating agent that generates gas when irradiated with light is not particularly limited as the gas generating agent. For example, an azo compound, an azide compound, a polyoxyalkylene resin, a combination of a photo acid generator and sodium hydrogen carbonate. The azo compound and the azide compound are preferably used because of high gas generation efficiency.

  Examples of the azo compound include 2,2′-azobis- (N-butyl-2-methylpropionamide), 2,2′-azobis {2-methyl-N- [1,1-bis (hydroxymethyl)]. -2-hydroxyethyl] propionamide}, 2,2′-azobis {2-methyl-N- [2- (1-hydroxybutyl)] propionamide}, 2,2′-azobis [2-methyl-N— (2-hydroxyethyl) propionamide], 2,2′-azobis [N- (2-propenyl) -2-methylpropionamide], 2,2′-azobis (N-butyl-2-methylpropionamide), 2,2′-azobis (N-cyclohexyl-2-methylpropionamide), 2,2′-azobis [2- (5-methyl-2-imidazolin-2-yl) propane] diha Drochloride, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] disulfate Dihydrolate, 2,2′-azobis [2- (3,4,5,6-tetrahydropyrimidin-2-yl) propane] dihydrochloride, 2,2′-azobis {2- [1- (2 -Hydroxyethyl) -2-imidazolin-2-yl] propane} dihydrochloride, 2,2′-azobis [2- (2-imidazolin-2-yl) propane], 2,2′-azobis (2-methyl) Propionamidine) hydrochloride, 2,2′-azobis (2-aminopropane) dihydrochloride, 2,2′-azobis [N- (2-carb Boxyacyl) -2-methyl-propionamidine], 2,2′-azobis {2- [N- (2-carboxyethyl) amidyne] propane}, 2,2′-azobis (2-methylpropionamidoxime), Dimethyl 2,2′-azobis (2-methylpropionate), dimethyl 2,2′-azobisisobutyrate, 4,4′-azobis (4-cyancarbonic acid), 4,4′-azobis (4 -Cyanopentanoic acid), 2,2'-azobis (2,4,4-trimethylpentane) and the like. Among them, 2,2′-azobis- (N-butyl-2-methylpropionamide), 2,2′-azobis (N-butyl-2-methylpropionamide), 2,2′-azobis (N-cyclohexyl-) 2-methylpropionamide) is preferred. These azo compounds generate nitrogen gas when stimulated by light, heat, or the like.

  Examples of the azide compound include 3-azidomethyl-3-methyloxetane, terephthalazide, p-tert-butylbenzazide; and glycidyl azide polymer obtained by ring-opening polymerization of 3-azidomethyl-3-methyloxetane. Examples thereof include a polymer having an azide group.

  Photoacid generators include bis (cyclohexylsulfonyl) diazomethane, bis (t-butylsulfonyl) diazomethane, bis (p-toluenesulfonyl) diazomethane, triphenylsulfonium trifluoromethanesulfonate, dimethyl -4-methylphenylsulfonium trifluoromethanesulfonate, diphenyl-2,4,6-trimethylphenylsulfonium diazodisulfone such as p-toluenesulfonate, diazodisulfone such as triphenylsulfonium, triphenylsulfonium Series photoacid generators can be used.

  Further, a known sensitizer may be contained in the photoresponsive gas generating resin composition for the purpose of increasing the responsiveness.

  Examples of sensitizers include acetophenones, benzophenone, Michler's ketone, benzyl, benzoin, benzoin ether, benzyldimethyl ketal, benzoyl benzoate, α-acyloxime ester, tetramethylthiuram monosulfide, thioxanthone, aliphatic amine, aromatic group. Including amines, those in which nitrogen is part of the ring system, such as piperidine, allylthiourea, O-tolylthiourea, sodium diethyldithiophosphate, soluble salts of aromatic sulfinic acids, N, N-disubstituted-p- Aminobenzonitrile compounds, tri-n-butylphosphine, N-nitrosohydroxylamine derivatives, oxazolidine compounds, tetrahydro-1,3-oxazine compounds, condensates of formaldehyde or acetaldehyde and diamines Anthracene (or derivatives thereof), xanthine, N- phenylglycine, phthalocyanine, naphthocyanine, cyanine dyes porphyrins (or derivatives thereof) such as thiocyanine like. These sensitizers may be used independently and 2 or more types may be used together.

  When the optical window is irradiated with light, the photoresponsive gas generating resin composition in the gas generating chamber generates gas, but the surface of the photoresponsive gas generating resin composition irradiated with light is most active. It is. Therefore, an air layer is formed between the photoresponsive gas generating resin composition and the optical window in the gas generation chamber so that the gas is easily generated and the generated gas is easily discharged from the fine flow path. Is preferred.

  In addition, it is preferable that the surface of the photoresponsive gas generating resin composition has irregularities, since the surface area becomes large and the gas can be easily released, and the photoresponsive gas generating resin composition is further preferable in the gas generating chamber. It is preferable that the optical window is in partial contact with a plurality of points, and a large number of contact portions and air layers are formed.

  Since various samples, diluents, eluents, etc. are used in microfluidic devices, a large number of micropumps are required for one microfluidic device, so that multiple gas generation chambers are formed on the substrate. Is preferred. Further, since it is necessary to irradiate the gas generation chamber with light, the gas generation chamber is preferably formed on one surface of the substrate.

  The method for producing the trace liquid weighing structure in the present invention may be any method as long as the trace liquid weighing structure can be realized. For example, a transfer technique represented by machining, injection molding or compression molding, nanoimprint lithography, etc. , Cast molding, electroforming, dry etching (RIE, IE, IBE, plasma etching, laser etching, laser ablation, blasting, electrical discharge machining, LIGA, electron beam etching, FAB), wet etching (chemical erosion), stereolithography, Surface micro-machining that forms a fine structure by coating, evaporating, sputtering, depositing, and partially removing various substances in layers, such as ceramic molding, and one or more sheet-like substances (film, A method of forming a groove by forming an opening with a tape, etc., ink Dropping a flow duct forming material by jet or a dispenser, injecting method for forming, and the like.

  A mask may be utilized in the above method to create a microfluidic device. The mask may have any design as long as the microfluidic device can be finally produced, and a plurality of masks may be used. The mask is usually designed with a shape in which the flow path is projected onto a flat surface, but when processing on both sides of the flow path component to be bonded or when forming a flow path using multiple members, multiple masks are used. Therefore, the mask is not necessarily a projection of the final shape of the flow path because a part of the mask can be directly processed without using the mask. Examples of the electromagnetic wave shielding mask used for the photo-curing resin include those obtained by coating quartz or glass with chromium, or those obtained by baking a resin film with a laser.

  The mask can also be created by drawing at least a part of the channel structure and printing it on a transparent resin film using a suitable software, for example. A computer-readable recording medium used for creating the mask or master chip drawn with the software and storing at least part of the electronic information of the flow channel structure or a program for generating the flow channel structure pattern The code and its storage medium are also included in the present invention. Examples of suitable recording media include magnetic media such as flexible disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, CD-RW, and DVD, and semiconductor memories. .

  Moreover, when producing a microfluidic device, a chip may be directly manufactured by the above method, or a microfluidic device may be shaped using this as a mold. Of course, it is also possible to mold a microfluidic device using this as a mold.

  The microfluidic device in the present invention may have a two-layer structure in which an upper substrate and a lower substrate are bonded together. As bonding methods, adhesive bonding, resin bonding with primer, diffusion bonding, anodic bonding, eutectic bonding, thermal fusion, ultrasonic bonding, laser melting, bonding with solvent / dissolving solvent, adhesive tape, adhesive tape , Pressure bonding, bonding with a self-adsorbing agent, physical retention, and combination with unevenness. Moreover, it can also be realized by stacking multilayer substrates while maintaining the connection configuration.

  In addition, a method of integrally forming the fluid branch portion and the independent flow path without requiring bonding is also possible. Specifically, it is possible to form a structure including a closed space by integral molding such as stereolithography.

  The length, shape, and thickness of one side of the chip thus produced are not limited, and can be set to any value between 5 mm and 100 mm, for example.

FIG. 1 is a plan view schematically showing a fine channel structure of a microfluidic device used in one embodiment of the present invention. FIG. 2 is a front sectional view schematically showing a part of a microfluidic device used in one embodiment of the present invention. FIG. 3 is a schematic plan view for explaining a first mixing unit in the fine channel structure of the embodiment shown in FIG. 1. 4A and 4B are schematic plan views for explaining an example of the flow path switching device. FIG. 5 is a partially cutaway enlarged plan view for explaining a step of weighing a predetermined amount of the first microfluid in the first weighing unit in the embodiment shown in FIG. FIG. 6 is a partially cutaway enlarged plan view for explaining a step of weighing a predetermined amount of the first microfluid in the first weighing unit in the embodiment shown in FIG. FIG. 7 is a partially cutaway enlarged plan view for explaining a step of weighing a predetermined amount of the first microfluid in the first weighing unit in the embodiment shown in FIG. FIG. 8 is a schematic partial cutaway plan view showing a state in which the microfluid is discharged from the first and second weighing units to the second fine channel in the first embodiment. FIG. 9 is a partially cutaway plan view showing a state in which the microfluid passes through the mixing unit in the first embodiment. FIG. 10 is a schematic plan view for explaining the positional relationship between the first and second weighing units in the modified example of the first embodiment. FIG. 11 is a schematic plan view showing a modified example in which a plurality of second weighing units are provided in order to vary the mixing ratio in the merging unit. FIG. 12A is a schematic plan view showing another modified example in which the shape of the second fine channel is deformed, and thereby the mixing ratio in the joining portion is changed, and FIG. It is a typical partial notch top view for demonstrating the modification of the shape of a part. FIG. 13 is a plan view schematically showing another modification of the fine channel structure to which the first and second mixing units are connected. FIG. 14 is a schematic plan view showing still another modification of the fine channel structure of the microfluidic device used in the present invention. FIG. 15 is a schematic plan view showing a modification of the fine channel structure shown in FIG. FIG. 16 is a schematic plan view showing still another modified example of the microchannel structure of the microfluidic device used in the present invention, in which a large number of mixing units are arranged in a matrix. FIG. 17 is a schematic plan view showing still another modification of the fine channel structure of the microfluidic device used in the present invention. 18A and 18B are plan views showing modifications of the shape of the mixing unit.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Microfluidic device 2 ... Board | substrate 3 ... Base plate 4-6 ... Intermediate | middle plate 4a ... Discharge hole 5a ... Opening part 6a ... Through-hole 7 ... Top plate 8 ... Gas generating chamber 9 ... Photoresponsive gas generating member 10 ... Fine flow Path structure 11 ... first mixing unit 11a ... first fine flow path 11b ... gas supply hole 11c ... microfluid supply hole 11d ... first weighing section 11e ... connection fine flow path 11f ... opening 12 ... second micro-channel 12a ... merging portion 12b ... mixing section 12b _1, 12b 2 _... wall 12c ... discharge unit 12d ... gas supply holes 12e ... gas discharge port 13a ... third micro-channel 13b ... gas supply holes 13c ... liquid supply Hole 13d ... second weighing unit 13e ... connecting fine channel 13f ... opening 14a ... first microfluidic 14b ... second microfluidic 21 ... second mixing unit 31 ... 3. Mixing unit 41 ... Viscoelastic stopper 42 ... Stopper driving device 40 ... Fine channel opening / closing device 43a-43c ... Circular portion 62-66 ... Channel opening / closing device 71 ... Storage chamber 91, 92 ... Mixing unit 93 ... Channel opening / closing Apparatus 104a-104c ... Storage chamber 111, 121 ... Mixing unit 111a ... Fine flow path 111d, 121d ... Weighing part 113a ... Fine flow path 113d, 123d ... Weighing part 112, 122 ... Fine flow path 131, 132 ... Storage chamber

Claims (1)

A substrate,
A fine channel structure provided in the substrate and carrying a microfluid,
The fine channel structure has a first mixing unit and a second mixing unit connected to the downstream side of the first mixing unit;
Each mixing unit
In order to weigh a certain amount of the first microfluid, a first weigher comprising a fine channel having a volume equal to the volume of the constant amount of the first microfluid;
A second weigher comprising a microchannel having a volume equal to the volume of the fixed amount of the second microfluid in order to weigh a certain amount of the second microfluid;
The first and second microfluids are connected to a merging portion where the first and second microfluids weighed by the first and second weighing portions are merged, and downstream of the merging portion. A mixing unit for mixing,
A discharge portion for discharging the mixed microfluidic fluid obtained by mixing the first and second microfluidics;
A first fine flow path connecting the first inlet port and the first outlet port; a second inlet port; a second outlet port; A second fine flow path connecting the outlet port, having the merging section, the mixing section, and the discharge section; and a third fine flow path connecting the third inlet port and the third outlet port. ,
One end of the first weighing unit is connected to the first microchannel, and the other end is open to the junction unit provided in the second microchannel, and the second balance One end of the collecting portion is connected to the third fine channel, the other end is open to the joining portion provided in the second fine channel, and the second outlet port is the Connected to the discharge section,
A microfluidic dilution method using a microfluidic device, wherein a second outlet port of the first mixing unit is connected to the first or third inlet port of the second mixing unit,
A first microfluid as a specimen is weighed in the first or second weighing unit of the first mixing unit, and a second microfluid as a diluent in the second or first weighing unit. A step of weighing the fluid;
Mixing the first microfluid as the specimen and the second microfluid as the diluent in the first mixing unit, and discharging the first specimen diluent as the mixed microfluid;
At least a part of the first mixed microfluid discharged from the first mixing unit is weighed in the first or second weighing unit of the second mixing unit, and the second mixing unit Weighing the diluent as the second or first microfluid in the second or first weighing unit;
In the second mixing unit, the first specimen diluent and the diluent are mixed to obtain a second specimen diluent as a second mixed microfluid, and the second microfluid is used as the second microfluid. Discharging the second sample diluent from the discharge unit of the second mixing unit,
Further, n-2 (n is a natural number of 3 or more) mixing units are connected to the subsequent stage of the first and second mixing units, and the mixed fluid as the specimen diluent is respectively supplied from the discharge unit of each mixing unit. A method for diluting a small amount of liquid, wherein n kinds of specimen dilution liquids having different concentrations are obtained by discharging.

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