JP2007218838A - Micro sample inlet method and microfluidic device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a micro sample inlet method for quantitatively introducing a trace amount of samples to channels in a microfluidic device, and to provide a microfluidic device provided with a micro chromatograph. <P>SOLUTION: The microfluidic device comprises a fist flow inlet, a first flow exit, a first channel connecting both, a first branch part for dividing the first channel into two, a second channel branching from the first branch part, and a second flow inlet provided for the other end of the second channel. In the micro sample inlet method, a first process and a second process are executed. In the first process, a second pump, connected to the first flow inlet for discharging a fluid for transfer, is brought into a halt or operating state, and a first pump, connected to the first flow outlet, is operated in a suction direction at a speed higher than the discharge speed of the second pump to transfer the fluid for transfer in the first channel, in the direction of the first pump, and simultaneously transfers a sample in the second channel to a downstream part of the first channel by negative pressure generated by the difference in speeds between both pumps. In the second process, the inflow of the sample is halted, by making the difference in the speeds of both pumps to be zero or make it reversed, when a prescribed amount of the sample has been introduced. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for quantitatively introducing a very small amount of sample into a flow path or mechanism in a microfluidic device, and a microfluidic device having a chromatograph.

  The microfluidic device is a device that performs reaction and analysis in a fine capillary channel provided inside. By using a microfluidic device, it is possible to speed up reactions and analysis, reduce the amount of analysis sample required, save resources and energy, and reduce waste. Using such a microfluidic device, for example, sample pretreatment units such as filtration, extraction, and reaction, separation units such as micro chromatography and micro electrophoresis, and detection units such as fluorescence and visible / ultraviolet absorption Attempts have been made to construct a micro total analysis system (μ-TAS) that analyzes a small amount of sample in the fields of medical diagnosis, biochemical analysis, and chemical analysis.

  However, the volume of the microfluidic device channel is much smaller than the volume of sample handling means and devices such as conventional pumps, switching valves, syringes, pipettes, etc. It was quite difficult to inject the sample quantitatively. For example, in micro-chromatography such as micro-liquid chromatography and micro-affinity chromatography, it is expected to perform high-resolution analysis quickly by shortening the development distance. It was necessary to inject into the separation column in short pulses. However, conventional sample injection means such as microsyringes and sampling valves tend to cause the volume of the injected sample to be excessive, which causes chromatogram peak broadening and tailing, and sufficient resolution can be obtained at short development distances. I couldn't.

  As a method for solving this, Patent Document 1 discloses the following method relating to electrophoretic analysis. That is, a sample is injected into one end of two intersecting channels, a voltage is applied to both ends of the channel, the sample is transferred by electrophoresis or electroosmotic flow, and the sample passes through the intersection. At this time, the application of the voltage is stopped, and then the voltage is applied to both ends of the other channel, whereby only the sample in the intersection is injected into the other channel.

  However, in this method, it is possible to inject a very small amount of sample corresponding to the volume of the branching portion into the other channel, but since the sample is transferred by electrophoresis, the sample is limited to a charged sample, Since the medium is also limited to the electrolyte medium, it could not be applied to many liquid chromatography and uses such as gas chromatography in which a sample is injected into the gas phase.

  Further, as a method based on the same idea, an apparatus including a device having two channels to be joined is disclosed (Patent Document 2) with respect to an analysis apparatus using a micro flow channel. In the device, a pressure gradient is applied to a channel in the device, the sample is caused to flow in the sample injection channel, and the sample is arranged at the junction (intersection) of the separation channel that is joined (intersect) with the sample injection channel. However, this method is insufficient as a sample injection method in the following points. (1) Since it is necessary to precisely control the pressure of the sample injection channel and the electrophoresis analysis channel and the timing of the control, the mechanism such as a pump and a valve tends to be complicated and large-scaled. ) Although there is no description of chromatographic sample injection in the known literature, according to the study by the present inventors, it is necessary to strictly control the pressure in the two flow paths, and the control device is large. (3) The injected sample tends to cause tailing, and more complicated mechanisms and pressure control operations are required to suppress it.

  On the other hand, in Patent Document 3 filed by the present inventors, a capillary-shaped upstream flow channel, a liquid injection portion located at the upstream end of the upstream flow channel, and one end of the upstream flow channel are formed in the member. A microfluidic device provided with a micro liquid chromatography mechanism (micro liquid chromatograph) having an adsorption column connected to the end of a side flow path and a downstream outlet for discharging the liquid that has passed through the adsorption column. The branch channel is provided on the upstream channel, and the branch channel is provided from the branch unit, and the branch channel passes through the gas-liquid separator that allows gas to permeate but does not allow liquid to permeate. A microfluidic device is disclosed that is in communication outside the device. However, the device has a low quantitative amount of sample introduction.

  Moreover, in patent document 4 which becomes an application of the present inventors, the first channel upstream portion, the first channel downstream portion branched at the branch portion in the middle thereof, and the first channel downstream portion near the branch portion. A valve provided on the flow path wall of the first flow path upstream portion in the vicinity of the branch portion, and a compression portion that reduces the cross-sectional area of the first flow path upstream portion by pressure from the outside of the first flow path upstream portion A microfluidic device is disclosed in which the valve is normally closed and is opened by pressing the compression portion, and the fluid in the upstream portion of the first flow channel flows into the downstream portion of the first flow channel. However, this also has a drawback in that it is necessary to form a valve, and the structure of the microfluidic device is complicated.

  Further, in Patent Document 5 filed by the present inventors, a sample adsorbing substance is immobilized on a channel sharing portion where at least two channels intersect or contact each other, and one of the channels is arranged. One is a flow path for transferring the sample and adsorbing the sample to the sample adsorbing substance, and the other flow path crossing or contacting the flow path is for transferring the sample desorbed from the sample adsorbing substance. There is disclosed a microfluidic device characterized in that However, this also has a low quantification of the sample introduction amount, and even in the chromatograph, it is necessary to provide a valve and a pump between the sample introduction port and the flow path sharing portion, and the structure becomes complicated. It was.

  In addition, in Patent Document 6 filed by the present inventors, a method for introducing a sample into a microfluidic device having a capillary channel provided with an introduction port and a discharge port opened to the outside is introduced into the channel. A sample introduction method is disclosed in which a sample is brought into contact with a liquid for introduction at the introduction port after filling the liquid for introduction, and the sample is introduced into the flow path by discharging the introduction liquid from the discharge port. However, this is a method that fills the sample with the entire flow path, and there is no description of a method for quantitatively introducing a very small amount of sample into a pal shape, and it cannot be used for a sample injection method of chromatography. It was.

  Further, Patent Document 7 filed by the present inventors discloses a chromatograph having an injection part for a microsyringe made of silicon rubber. However, as described above, there is a drawback of sample injection by a microsyringe, and even in the case of a chromatograph, the sample injection port is closed with silicon rubber and is not opened outside the microfluidic device.

  Non-Patent Document 1 reports a slide-type microphone valve provided in a glass microfluidic device, which can be used for quantitative introduction of a sample. However, it is necessary to form a valve having a complicated structure in a microfluidic device, and an extremely high precision work is necessary to prevent leakage from a sliding portion of a small sliding valve, which is inevitably expensive. It became a device.

JP-A-11-271273 Japanese translation of PCT publication No. 2002-542489 JP 2004-144568 A JP 2006-10327 A JP 2005-274405 A JP 2004-191256 A Japanese Patent Laid-Open No. 2005-091135 Proceedings of the 11th Chemistry and Micro / Nano System Study Group, 2005, 88 pages

  A problem to be solved by the present invention is a sample introduction method for quantitatively introducing a very small amount of sample into a flow path in a microfluidic device, in which not only a charged sample but also a nonionic sample is introduced. In addition, the present invention is to provide a sample introduction method that can introduce a sample into a non-aqueous solvent, does not require a complicated and precise device, and can be injected without a tailing with a simple mechanism. It is to provide a method for injecting a sample into a minute chromatograph (that is, a mechanism for chromatography). Moreover, it is providing the microfluidic device provided with such a chromatograph.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention connected the two pumps connected to both ends of the branched flow path by controlling the extrusion amount and the suction amount. It has been found that the sample can be quantitatively introduced from the flow path and transferred, and the present invention has been completed.

That is, the present invention is a method for quantitatively introducing a small amount of sample into a fine channel of a microfluidic device,
The microfluidic device includes a first inlet, a first outlet, a first channel connecting the first inlet and the first outlet, and the first channel provided upstream of the first channel. A first branch portion that is divided into a first portion and a downstream portion of the first flow passage, a second flow passage that branches off from the first flow passage at the first branch portion, and a second provided at the other end of the second flow passage Using a microfluidic device having an inlet and
(1) The first flow path is filled with a transfer fluid, the second flow path is filled with a sample or a transfer fluid,
The transfer fluid is discharged into the first flow path from the second pump connected to the first inlet, and the first pump connected to the first outlet is larger than the discharge speed of the second pump. By driving in the suction direction at a speed or by stopping the first pump and further driving the second pump in the suction direction,
The transfer fluid filled in the first flow path is transferred toward the first pump, and at the same time, due to the negative pressure generated by the difference between the discharge speed of the first pump and the suction speed of the second pump, A first step of transferring the sample from the second inlet to the downstream portion of the first channel through the second channel;
(2) When a predetermined amount of the sample is introduced into the downstream portion of the first flow path, the first flow rate at which the value obtained by subtracting the discharge speed of the second pump from the suction speed of the first pump is zero or negative. By operating one pump and the second pump,
A second step of stopping the sample from flowing into the downstream portion of the first flow path from the second flow path through the first branch portion;
The present invention provides a method for introducing a trace amount sample characterized in that

Further, the present invention is a microfluidic device in which a separation column for chromatography is provided in a part of a fine flow path,
The microfluidic device is provided in the middle of the first flow path, the first flow path, the first flow path connecting the first flow path and the first flow path, and the first flow path on the first flow path. A first branch part that divides into a flow part and a downstream part of the first flow path, a second flow path that branches off from the first flow path at the first branch part, and that is not provided with a valve or pump, and the second flow path A second inlet provided at the other end of the channel, a liquid receiving tank connected to the second inlet and opened to the outside of the microfluidic device, and a chromatograph provided in the downstream of the first channel. A microfluidic device having a chromatographic separation column is provided.

  The method for introducing a micro sample according to the present invention is a sample introducing method capable of quantitatively and pulse-introducing a very small amount of sample into a flow path in a microfluidic device. Since non-tailored samples and non-aqueous solvent solution samples can be introduced without tailing, they can be applied to various microfluidic devices such as microchromatographs and various microreactors such as polymerase chain reaction (PCR). It is. Of course, a sample for electrophoresis analysis can also be introduced. Since the micro sample introduction method of the present invention can be implemented using a micro fluid device having a simple structure, the micro fluid device can be manufactured at low cost and can be made disposable. Moreover, the microfluidic device provided with such a chromatograph can be provided.

  Furthermore, in the method for introducing a small amount of sample according to the present invention, the sample is simply dropped into the sample introduction port, and after that, a predetermined amount is automatically sucked and introduced into the flow path and transferred to a predetermined mechanism in the microfluidic device. Therefore, a fully automatic analyzer for biological samples such as blood can be easily constructed. Alternatively, in a microfluidic device for a microreactor, it is possible to quantitatively extract a part of the reaction solution flowing in the flow path and introduce it into the next-stage reaction flow path or analysis mechanism. A research microreactor with an analysis part and an analysis part can be easily constructed. In addition, a microreactor having such a chromatograph can be provided.

Hereinafter, the present invention will be described in detail.
[Microfluidic device]
A microfluidic device is a microfluidic device, a microfabricated device, a microfluidic device, a microchannel, a lab-on-chip, etc. A device that carries out chemical and biochemical reactions, chemical engineering processes such as dispersion, crystallization, mixing, separation, and concentration, as well as detection and measurement within a channel and various mechanisms.

  1 and 2 are a plan view and a side view showing an example of a microfluidic device used in the present invention. The microfluidic device used in the present invention has a capillary channel having two inlets, one outlet, and one branch. That is, the first inlet 11, the first outlet 12, the first channel 10 connecting the first inlet 11 and the first outlet 12, and the first channel 10 are provided in the middle of the first channel 10. A first branch portion 15 that is divided into a flow portion 10a and a first flow passage downstream portion 10b, a second flow passage 20 that is branched from the first flow passage 10 in the first branch portion 15, and the second flow passage 20 The second inflow port 21 is provided at the other end.

  FIG. 1 is a plan view and a side view showing a first embodiment of a microfluidic device used in the present invention. In the first embodiment, the second inlet 21 is a sample inlet 21 opened outside the microfluidic device. That is, the microfluidic device of the first embodiment has a T-shaped channel structure, and introduces the sample arranged in the sample introduction port 21 into the first channel downstream portion 10b through the second channel 20. Used in the sample introduction method (first embodiment of the present invention). In the first embodiment, the microfluidic device (that is, the chromatograph) in which the separation column for chromatography is formed in the middle of the downstream portion of the first flow path is the first embodiment of the chromatograph of the present invention. It is said.

  FIG. 2 is a plan view and a side view showing a second embodiment of the microfluidic device used in the present invention. In the second form, the microfluidic device further includes a third inlet 31, a second outlet 32, a third flow path 30 connecting the third inlet 31 and the second outlet 32, and the third flow. A second branch part 16 provided in the middle of the passage 30 and dividing the third flow path 30 into a third flow path upstream part 30a and a third flow path downstream part 30b; Connected to the bifurcation 16. That is, the microfluidic device of the second embodiment has an H-shaped channel structure. In the microfluidic device according to the second embodiment, the sample flowing through the third flow path 30 from the third inflow port 31 to the second outflow port 32 is transferred from the second branch portion 16 through the second flow path 20 to the first flow. It is used in the sample introduction method (second embodiment of the present invention) for introduction into the downstream path portion 10b. In the second embodiment, a microfluidic device (chromatograph) in which a separation column for chromatography is formed in the middle of the downstream portion of the first channel is a second embodiment of the chromatograph of the present invention. The

  The microfluidic device used in the method for introducing a micro sample of the present invention will be described in more detail.

(First inlet 11)
The first inflow port 11 is opened on the surface of the microfluidic device, and serves as a connecting portion of piping for connecting the pump. The first inflow port 11 is a connecting portion of piping for connecting a pump (referred to as a second pump). Of course, the first inlet 11 may be connected to the second pump via a filtration mechanism or the like. When a pump (referred to as an internal pump) incorporated in the microfluidic device is used as the second pump, the first inlet 11 is connected to the outlet of the internal pump.

(First flow path upstream portion 10a)
The first channel upstream portion 10a has a role as a channel for transferring the transfer fluid introduced from the first inlet 11 to the first channel downstream portion 10b. Although the pressure loss of the first flow path upstream portion 10a is arbitrary, it is preferable that the pressure loss is small because the pressure resistance of the pump may be low. The cross-sectional area of the first flow path upstream portion 10a is preferably 100 μm ² or more, more preferably 400 μm ² or more, most preferably 1000 μm ² or more, preferably 10 mm ² or less, more preferably 1 mm ² or less, and most preferably 0.00. 1 mm ² or less.

(First branch 15)
In the first branch portion 15, the second flow path 20 is connected to the first flow path 10.

(1st flow path downstream part 10b)
The first channel downstream portion 10b is provided with a target mechanism for introducing a trace amount sample, such as a chromatographic column or a reaction tank.
In the case where a chromatography column is provided, the length of the column is arbitrary, and can be a suitable length depending on the purpose of use. For example, 1 mm to 500 mm is preferable, and 5 mm to 200 mm is more preferable. By setting it to the above lower limit or higher, sufficient separation can be obtained, and by setting the upper limit or lower, the microfluidic device can be miniaturized.
The pressure loss of the first flow path downstream portion 10b, that is, the pressure loss between the first branch portion 15 and the first outlet 12 is less than 100 kPa, preferably 30 kPa or less in use, including the mechanism as described above. More preferably, it is 10 kPa or less. The lower limit of the pressure loss may be as close to zero as possible. The pressure loss can be calculated by a formula known in the field of chemical engineering, for example, a formula described in “New Theory and Calculation of Chemical Machinery”, edited by Saburo Kamei, Sangyo Tosho Co., Ltd. (1959). In order to make the pressure loss within the above range, the cross-sectional area of the first flow path downstream portion 10b is preferably 100 μm ² or more, more preferably 400 μm ² or more, and most preferably 1000 μm ² or more. The larger the upper limit of the cross-sectional area of the first flow path downstream portion 10b, the smaller the pressure loss. However, in order not to lose the merit of the microfluidic device being small, it is preferably 10 mm ² or less, more preferably 1 mm ² or less. Most preferably, it is 0.1 mm ² or less. Although the pressure loss depends on the flow rate that is the method of use, by setting the flow path cross-sectional area in the above range, the allowable range of the flow rate is widened, and operation restrictions are reduced.

(First outlet 12)
The 1st outflow port 12 is opened to the microfluidic device surface, and is made into the connection part of piping connected with a pump (it calls a 1st pump). Of course, the first outlet 12 may be connected to the first pump via a filtration mechanism or the like. Moreover, when using the internal pump integrated in this microfluidic device as this 1st pump, the 1st outflow port 12 is connected to the suction port of this internal pump.

(Second inlet 21)
The second inlet 21 is an inlet for a sample. In the first form of the microfluidic device used in the present invention, the second inlet 21 opens outside the microfluidic device, and has a shape in which a liquid receiving tank is provided in the opening. Has been. However, in general, any structure can be used as long as the sample is introduced from the pipe, and in addition to the above, for example, a pipe connection portion to which a pipe communicating with the outside of the microfluidic device is connected, other mechanisms in the microfluidic device, For example, it may be a flow path other than the first flow path or the second flow path, a storage tank, a connection portion with a filtration mechanism, or the like. Moreover, in the 2nd form of the microfluidic device used by this invention, it connects with the 2nd branch part 16 provided in the middle of the 3rd flow path 30 provided in the microfluidic device. In any case, the second inflow port 21 communicates with atmospheric pressure or has a sufficient volume of gas so that a liquid sample can be introduced by suction from the first flow path downstream portion 10b side. It is necessary to be sent out as much as it is in contact or sucked by a pump or the like.

(Second flow path 20)
The second channel 20 has a role as a channel for transferring the sample introduced from the second inlet 21 to the first channel downstream portion 10b. The pressure loss of the second channel 20 is the same as that of the first channel downstream portion 10b. The second flow path 20 may be a simple transfer flow path, and there is no need to provide an on-off valve, a check valve, or a pump on the way. Therefore, the structure of the microfluidic device can be greatly simplified. Although the length of the second flow path 20 is arbitrary, it is preferable that the length of the second flow path 20 is short because a small amount of sample is required.
In the case where the sample is repeatedly introduced, for example, in the second aspect of the method for introducing a trace amount of the present invention, when the sample in the third channel 30 is repeatedly introduced into the first channel downstream portion 10b, the previous sample introduced It is preferable to make the length of the second flow path 20 substantially zero, that is, to join the first branch portion 15 and the second branch portion 16.

(First pump)
The first pump is a pump connected to the first outlet 12 and is a suction type metering pump, that is, a pump capable of suctioning at a set flow rate. As the metering pump, one having a small dependency of the suction speed on the fluid pressure is preferable. Examples of such pumps include syringe pumps, plunger pumps, gear pumps, and ironing pumps. It is more preferable that the first pump can perform both suction and discharge because it is easy to fill the flow path of the microfluidic device with the transfer fluid.
The first pump may be an independent pump, or may be an internal pump incorporated in the microfluidic device. The internal pump can be incorporated at an arbitrary position in the middle of the first flow path downstream portion 10b.
The first pump sucks the first transfer fluid pre-filled in the flow path of the microfluidic device, but after sucking the first transfer fluid in the flow path, the first pump sucks the sample, and then A second transfer fluid is aspirated. In order to prevent contamination of the first pump with the sample and the second transfer fluid, it is preferable to insert a buffer tank between the first outlet 12 and the first pump. The pipe itself connecting the first outlet 12 and the first pump can also serve as the buffer tank.

(Second pump)
The second pump is a pump connected to the first inlet 11 and is a discharge-type metering pump, that is, can discharge at a set flow rate, and is substantially discharged even if the discharge port is sucked when the pump is stopped. The pump is not. The pressure at which the discharge port is sucked at this time is the pressure at which the sample is sucked from the second inlet 21 in the first step of the present invention, and when used under atmospheric pressure, the maximum is minus 100 kPa. (Gauge pressure). Moreover, as this metering pump, a pump whose discharge speed dependency on the back pressure is small is preferable. Examples of such pumps include syringe pumps, gear pumps, and ironing pumps.
The second pump may be an independent pump or may be an internal pump built into the microfluidic device. The internal pump can be incorporated at an arbitrary position in the middle of the first flow path upstream portion 10a. The second pump may discharge the second transfer fluid, or may discharge another transfer fluid that comes in contact with the second transfer fluid previously filled in the first flow path upstream portion 10a.

(Third inlet 31)
In FIG. 2, the third inflow port 31 included in the microfluidic device of the second form used in the present invention is open to the surface of the microfluidic device, and is used as a connection part of a pipe connecting a discharge type pump. Yes. However, generally, the third inlet 31 only needs to be connected to a fluid transfer mechanism for flowing a sample through the third flow path 30. An internal transfer mechanism incorporated in the microfluidic device is used as the fluid transfer mechanism. When used, the third inlet 3 is connected to the outlet of the internal transfer mechanism. The fluid transfer mechanism is arbitrary, and may be an arbitrary pump such as a diaphragm pump in addition to the same pump as the second pump, or a mechanism that transfers by pressure gas or gravity. Further, when the sample is transferred to the third flow path 30 by sucking the second outlet 32, it may be connected to a liquid storage tank.

(Third channel upstream portion 30a)
In the second embodiment of the microfluidic device used in the present invention and the third flow path 30 of the second form of the chromatograph of the present invention, the upstream side from the second branch section 16 is the third flow path upstream section. 30a. The third flow path upstream portion 30a may be any flow path, and may be, for example, a simple communication flow path for flowing a sample, a reaction tube of a microreactor, a separation mechanism, or the like. There is no restriction | limiting in particular in the pressure loss of the 3rd flow path upstream part 30a, You may be the same as that of the said 1st flow path upstream part 10a.

(Second branch 16)
In the second mode of the microfluidic device of the second form used in the present invention and the chromatograph of the present invention, the flow of the second flow path 20 to the third flow path 30 in the second branching section 16. An inlet 21 is connected.

(Third channel downstream portion 30b)
The third flow path downstream portion 30b, which is the downstream portion of the third flow path 30 from the second branch section 16, discharges excess sample that has not been introduced from the second branch section 16 into the second flow path 20. It can be any channel. There is no restriction | limiting in particular in the pressure loss of the 3rd flow path downstream part 30b, You may be the same as that of the said 1st flow path upstream part 10a.

(Second outlet 32)
In FIG. 2, the second outlet 32 provided in the microfluidic device of the second form used in the present invention is open to the surface of the microfluidic device, and the liquid discharged from the third flow path downstream portion 30b. The reservoir is open to atmospheric pressure so that it can be stored. However, in general, the form of the second outlet 32 is arbitrary. For example, the second outlet 32 may be connected to a reaction channel in the next stage in the microreactor, or may be a pipe connection. , May be connected to a suckable fluid transfer mechanism. The fluid transfer mechanism is arbitrary, and may be an arbitrary pump such as a diaphragm pump in addition to the same pump as the first pump, or a reduced pressure gas or a porous absorbent.

[Sample and fluid for transfer]
A sample targeted by the sample introduction method of the present invention and a transfer fluid to be used will be described.

(sample)
In the present invention, the sample introduced into the flow path of the microfluidic device is a fluid, and the fluid may be a gas, a liquid, or a supercritical fluid. Further, it may be a dispersion, steam, or mist. Among these, a liquid (including a dispersion, the same applies hereinafter) is preferable because the accuracy of the amount of introduced sample is improved.
When the sample is a solution or dispersion, the solvent or dispersion medium is arbitrary, and may be an aqueous liquid, non-aqueous liquid, electrolyte, or non-electrolyte. For chemical substances, organic substances, inorganic substances, volatile substances, non-volatile substances, nonionic substances, cationic substances, anionic substances, zwitterionic substances, hydrophilic substances, hydrophobic substances, both Shinma isomers, etc. possible.
The dispersion chamber when the sample is a dispersion is also arbitrary, and may be an organic compound, an inorganic compound, a biological material such as a cell or an intracellular tissue, a charged material, an uncharged material, or the like.

(Transfer fluid)
In the sample introduction method of the present invention, the sample is introduced and transferred via the transfer fluid sucked into the first pump and the transfer fluid discharged from the second pump. That is, the sample is introduced and transferred to the first flow path downstream portion 10b in a state where the front and the rear in the transfer direction are sandwiched in contact with the transfer fluid. These transfer fluids before and after the sample may be the same or different. The front transfer fluid in the moving direction of the sample is referred to as a first transfer fluid, and the rear one is referred to as a second transfer fluid.

(First transfer fluid)
The first transfer fluid is optional and may be a gas, liquid, or supercritical fluid, but it is preferred to use a liquid. By using a liquid as the first transfer fluid, the accuracy of the sample introduction amount is improved. The first transfer fluid may or may not be mixed with the sample or the second transfer fluid, and can be selected according to the purpose of use. When the second transfer fluid described later is a liquid, the same one as the second transfer fluid is preferably used as the first transfer fluid.

(Second transfer fluid)
The second transfer fluid is also arbitrary and may be a gas, a liquid, or a supercritical fluid, but is preferably a liquid because the effects of the present invention are more exhibited. When it is a liquid, it may be mixed with the sample or not, and can be selected according to the purpose of use. For example, when the sample introduction method of the present invention is sample injection into a microchromatograph, a chromatographic developing solution is used as the second transfer fluid.

[Microfluidic device of the present invention]
Among the microfluidic devices used in the sample introduction method of the present invention, the microfluidic device of the present invention has a chromatograph in the middle of the first channel downstream portion 10b, the third channel upstream portion 10a, or both. It is provided. What has a chromatograph in the microfluidic device of the above first form is referred to as the first form of the microfluidic device of the present invention, and what has the chromatograph in the microfluidic device of the above second form, This is referred to as a second embodiment of the microfluidic device of the present invention.

[Sample introduction method]
The sample introduction method of the present invention is based on the following procedure.
(1) First step The second transfer fluid is operated or stopped in the discharge direction from the second pump connected to the end of the first flow path upstream portion, and the end of the first flow path downstream portion The first pump connected to the first pump is operated in the suction direction at a speed greater than the discharge speed of the second pump, so that at least the first transfer fluid filled in the downstream portion of the first flow path is At the same time as transferring in the direction of one pump, the sample is transferred from the second inlet to the downstream of the first channel through the second channel and the first branch by the suction force of the transfer fluid. To do.
At this time, the second flow path may be pre-filled with the first transfer fluid or the sample, but it may be filled with the first transfer fluid that is a liquid, This is preferable because the quantitativeness of the sample introduction amount is increased.

(2) Second step,
When a predetermined amount of the sample is introduced into the downstream portion of the first flow path, each pump is operated at a flow rate at which a value obtained by subtracting the discharge speed of the second pump from the suction speed of the first pump is zero or negative. As a result, the fluid flowing into the downstream portion of the first flow path is switched from the sample to the first transfer fluid.
Through the first step and the second step, a predetermined amount of sample is introduced into the first flow path downstream portion 10b in a pulsed manner sandwiched between the second transfer fluid and the first transfer fluid, To the position.

Hereinafter, the above procedure will be described in detail.
[First embodiment of the method for introducing a small amount of sample]
The first embodiment of the sample introduction method of the present invention uses the microfluidic device of the first form. If a gas that is a compressible fluid is present in the flow path, the quantitativeness of the sample introduction amount is lowered. Therefore, it is preferable to fill at least the first flow path downstream portion 10b with a liquid transfer fluid in advance. The second flow path 20 may be pre-filled with the first transfer fluid or may be pre-filled with the sample, but the liquid first transfer fluid is pre-filled. It is preferable because gas can be easily removed from the channel, and the quantitativeness of the introduced amount of a small amount of sample is increased. In this case, in order to improve the quantitativeness of the introduced amount of the sample, it should be ensured that no gas (bubbles) is left in the flow path, in each pipe connection part, and also in each pipe connecting the microfluidic device and the pump. Is preferred. When repeating this method of introducing a small amount of sample, if the flow rate difference between the first pump and the second pump in the second step described later is zero, the second flow path is used for the second and subsequent sample introductions. The first step is started in 20 with the sample filled.

  The method for filling the fluid for transfer into the flow path is arbitrary, and the fluid may be filled before the second pump or the first pump is connected, or transferred from the pump after both the pumps are connected. The working fluid may be discharged and filled. For example, when the transfer fluid is discharged from the second pump and the first pump, respectively, the transfer fluid discharged from the second pump is the first inlet 11, the first flow path upstream portion 10 a, the first branch portion 15, and the second The transfer fluid flowing through the flow path 20 and flowing out from the second inlet 21 and discharged from the first pump is the first outlet 12, the first flow path downstream portion 10b, the first branch portion 15, and the second flow path 20. It flows out from the 2nd inlet 21 via. Therefore, in this method, the first flow path downstream portion 10b is filled with the transfer fluid discharged from the first pump, and the first flow path upstream portion 10a is filled with the transfer fluid discharged from the second pump. A state is obtained. The transfer fluid that fills the second flow path 20 varies depending on the operation order of both pumps, and is filled with the transfer fluid that has flowed later. If flowing simultaneously, the mixed fluid is filled. The transfer fluid filled in the first flow path downstream portion 10b becomes the first transfer fluid, and the transfer fluid filled in the second flow path 20 becomes the second transfer fluid.

Instead of discharging the liquid for transfer from both the second pump and the first pump, both pumps were operated under the condition that the discharge speed of the second pump was higher than the suction speed of the first pump, and the air that was in the flow path It is good also by the method of driving | operating until it is suck | inhaled by the 1st pump and discharged | emitted from this pump. In this case, the whole of the first channel and the second channel are filled with the transfer fluid discharged from the second pump. When the second pump and the first pump are reversed, the entire flow path is filled with the transfer fluid discharged from the first pump.
The excess transfer fluid (liquid) accumulated in the liquid receiving tank provided at the second inlet 21 by the above-described filling operation may be removed by suction with a pipette, a syringe, or the like.

(1) First step The second pump is operated or stopped to discharge the second transfer fluid, and at the same time, the first pump is moved at a speed higher than the discharge speed of the second pump. By operating in the suction direction, the sample disposed in the liquid receiving tank provided in the second inlet 21 is sucked, and the sample passes from the second flow path 20 to the first branch portion 15 and passes through the first flow path. It introduces into the downstream part 10b. At this time, it is preferable to stop the second pump because the introduced sample is not diluted with the transfer fluid discharged from the second pump at the first branch portion 15. The above pump operation state may be carried out by operating only the first pump from the state where both of the two pumps are stopped, or the two pumps are operated at the same discharge speed and suction speed. You may implement by stopping only a 2nd pump from the state which is carrying out. Thus, when the discharge speed of the second pump is lower than the suction speed of the first pump, the sample is sucked into the second flow path 20 from the second inlet 21 at the difference flow rate, and passes through the first branch portion 15. It introduce | transduces into the 1st flow-path downstream part 10b. At this time, if the flow path cross-sectional area and the flow velocity are adjusted so that the pressure loss of each flow path falls within this range, the sample is introduced with high quantitativeness without any delay in suction due to flow path wall deflection.

(2) Second step When a predetermined amount of the sample is introduced into the first flow path downstream portion 10b, the value obtained by subtracting the discharge speed of the second pump from the suction speed of the first pump becomes zero or negative. Each pump is operated at a flow rate, that is, at a flow rate at which the discharge speed of the second pump is equal to or higher than the suction speed of the first pump. Then, instead of the sample flowing from the second flow path 20 to the first flow path downstream section 10b, the second transfer fluid is transferred from the first flow path upstream section 10a to the first flow path downstream section in the first branch section 15. 10b, the sample flow from the second flow path 20 stops. At this time, if the discharge speed of the second pump is made higher than the suction speed of the first pump, the second transfer liquid flowing from the first flow path upstream portion 10a to the first branch portion 15 enters the first branch portion 15. A part flows to the first channel downstream portion 10b, and the remainder flows to the second channel 20 to push the sample in the second channel 20 back to the second inlet 21. Therefore, tailing of the sample inflow to the first channel downstream portion 10b is prevented. It is also preferable to program-control the operating speed of the second pump so that it is greater than the flow rate of the first pump for a certain period of time after switching from the first step to the second step, and then equal.

  The introduction of a predetermined amount of the sample into the downstream portion of the first flow path can be performed by a method of detecting the amount of introduction of the sample by an arbitrary means in addition to the method of measuring the pump operation time. For example, a detection part (not shown) is provided in a part of the first flow path downstream part 10b that is separated from the first branch part 15 by a predetermined length, and it is detected that the sample has reached the detection part and switched to the second step. The method is preferred. The detection unit may be provided in a part of the branch part or the second flow path. When the detection unit is provided in a part of the branching unit or the second flow path 20, a method of switching to the second process after a predetermined delay time from the time when the sample reaches the part can be adopted. The sample detection means is arbitrary, and examples thereof include optical means such as ultraviolet-visible absorption, infrared absorption, and fluorescence intensity, and electrical methods such as electrical conductivity and electromotive force. It is also possible to introduce the sample a plurality of times by repeating the first step and the second step.

According to the micro sample injection method of the present invention, by designing the flow path diameter in the microfluidic device to be used and the position of the detection portion as described above, a very small amount, particularly 0.1 pl (provided that pl = nm). ³ ) to 300 nl (provided that nl = pm ³ ), more preferably 1 pl to ³⁰ nl, and most preferably about 10 pl to 10 nl. Further, in the second step, the small amount of the sample is tailed by operating each pump at a flow rate at which a value obtained by subtracting the discharge rate of the second pump from the suction rate of the first pump is negative. It is possible to inject without pulse.

[Second embodiment of the method for introducing a small amount of sample]
The second aspect of the method for introducing a trace sample of the present invention uses the microfluidic device of the second form. The second aspect is the same as the first aspect except that the sample in the third flow path 30 is introduced from the second inlet 21 connected to the second branch portion 16.
The method for transferring the sample in the third flow path is arbitrary, and for example, a pump connected to the third inlet 31 or a pump connected to the second outlet 32 can be used. Of course, you may use the internal pump provided in the 3rd flow path upstream part 30a and the 3rd flow path downstream part 30b.

  In the second aspect of the method for introducing a small amount of sample of the present invention, a sample spot separated by chromatography, electrophoresis or the like can be selectively introduced into the first channel downstream portion 10b. For example, by providing a chromatography column in the third channel upstream portion 30a, the compound of the separated sample spot can be used for the reaction in the first channel downstream portion 10b. In addition, as described above, a chromatography column is also provided in the first channel downstream portion 10b, and the third channel chromatography and the first channel chromatography are performed under different conditions (for example, pH and temperature). It is possible to easily separate substances that are difficult to separate. At this time, a mechanism for detecting the sample in the third flow path 30 in the vicinity of the second branch portion 16 of the third flow path 30, for example, a microfluidic device provided with an optical measurement window is used. It is also preferable to detect the spot and introduce the sample spot into the first flow path downstream portion 10b.

  In the second aspect of the method for introducing a small amount of sample described above, a sample spot separated by chromatography or electrophoresis is selectively removed from the third flow path 30 and the remainder is transferred to the third flow path downstream portion 30b. It can also be used as a method. By this method, for example, a sample from which a specific sample spot has been removed can be collected from the second outlet 32, or the sample from which the sample spot has been removed can be subjected to a reaction in the third channel downstream portion 30b.

  In the second aspect of the method for introducing a trace sample, the sample in the third channel 30 can be repeatedly introduced into the first channel downstream portion 10b. In this method, the third channel 30 is a microreactor reaction channel, and the reaction solution in the reaction channel is introduced into the first channel downstream portion 10b as a sample at regular intervals for analysis. Useful for. In this case, for example, the following method (first method) can be employed in order to prevent the introduction sample at this time from being mixed with the remainder of the previous introduction sample remaining in the second flow path 20. That is, in the second step of the previous sample introduction, each pump is operated at a flow rate at which a value obtained by subtracting the discharge rate of the second pump from the suction rate of the first pump is negative. Then, a part of the transfer fluid discharged from the first pump flows to the second flow path 20, and the sample in the second flow path 20 is pushed back from the second branch portion 16 to the third flow path, so that the second flow The passage 20 is replaced with the transfer fluid. For this reason, the previous sample is not mixed in the sample introduction of this time. The sample pushed back to the third flow path 30 can be transferred to the third flow path downstream portion 30b so as not to be mixed at the time of the sample introduction. When a liquid is used as the transfer fluid, the second channel is filled with the liquid. Therefore, when the sample is introduced next time, the operation of refilling the second channel 20 with the liquid is unnecessary. In addition, since the first flow path downstream portion 10b is filled with the transfer fluid by the previous sample introduction operation, it is not necessary to refill the first flow path downstream portion 10b with liquid at the time of the sample introduction. is there.

  The second method of preventing the introduction sample remaining in the second flow path 20 from being mixed into the introduction sample of the second time is the microfluidic device used in the present invention (and the chromatograph of the present invention). In the second form of the microfluidic device having the above, the length of the second flow path 20 is made substantially zero. In other words, the first branch portion 15 and the second branch portion 16 are in contact with each other, and the first flow path 10 and the third flow path 30 are joined at the branch portion. As a result, the volume of the second flow path becomes zero. Therefore, in the second step, even if the suction speed of the first pump and the discharge speed of the second pump are the same, the previous introduced sample does not remain in the second flow path 20. Further, with respect to the first flow path downstream portion 10b, for the same reason as the first method, an operation for refilling the liquid is unnecessary.

EXAMPLES Hereinafter, although this invention is demonstrated further more concretely using an Example, this invention is not limited to the range of a following example.
The ultraviolet irradiation and fluorescence characteristic measurement methods in this example are shown below.

(Irradiation with ultraviolet lamp 1)
Using a UE031-353CHC type UV irradiation device manufactured by Eye Graphics Co., Ltd. using a 3000 W metal halide lamp as a light source, ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² were irradiated in a nitrogen atmosphere at room temperature unless otherwise specified.

(Irradiation with ultraviolet lamp 2)
Using a light source unit for multi-light 250W series exposure apparatus manufactured by USHIO INC. Using a 250W high-pressure mercury lamp as a light source, ultraviolet light with an ultraviolet intensity at 365 nm of 50 mW / cm ² is used at room temperature in an air atmosphere unless otherwise specified. Irradiated.

Example 1
In this example, a manufacturing method of a first mode of a microfluidic device having a chromatograph of the present invention and a first embodiment of a method of introducing a micro sample of the present invention using the same are shown. FIG. 1 shows a schematic plan view and a schematic side view of a microfluidic device manufactured in this example.

[Production of microfluidic devices]
[Preparation of composition (X1)]
70 parts of trifunctional urethane acrylate oligomer “Unidic V-4263” (manufactured by Dainippon Ink & Chemicals, Inc.) having an average molecular weight of 2000 as energy beam polymerizable compound (a), hexanediol diacrylate “New Frontier HDDA” (first 30 parts by Kogyo Seiyaku Co., Ltd., 3 parts by weight of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (Ciba Geigy) as a photopolymerization initiator, and 2,4-diphenyl-4-methyl-1- A composition (X1) was prepared by mixing 0.5 parts of pentene (manufactured by Kanto Chemical Co., Inc.).

[Preparation of composition (X2)]
Mix 80 parts of “Unidic V-4263” as an energy ray polymerizable compound, 20 parts of “New Frontier HDDA” and 2 parts of “Irgacure 184” as a photopolymerization initiator to prepare a composition for a lid. did.

[Preparation of film-forming solution (Y1)]
As the energy ray polymerizable compound (b), 72 parts by mass of the above “Unidic V-4263”, 18 parts by mass of dicyclopentanyl diacrylate “R-684” (manufactured by Nippon Kayaku Co., Ltd.), glycidyl methacrylate (sum) 10 parts by mass of Kojun Pharmaceutical Co., Ltd., 180 parts by mass of methyl decanoate (manufactured by Wako Pure Chemical Industries, Ltd.) as poor solvent (R), 10 parts by mass of acetone as volatile good solvent, UV polymerization initiator As described above, 3 parts by mass of “Irgacure 184” was uniformly mixed to prepare a film-forming solution (Y1).

[Formation of porous layer 3]
The composition X2 is applied to the acrylic resin substrate 1 having a thickness of 1 mm by using a spin coater, and the coating film is semi-cured by irradiating the ultraviolet ray with the ultraviolet lamp 1 for 1 second to form the adhesive layer 2. The film-forming solution (Y1) was applied to the film by using a spin coater, and the ultraviolet lamp 2 was applied to the area where the porous layer 3 was to be formed through a photomask for 40 seconds to cure the coating film. The porous layer 3 was formed by washing and removing the poor solvent.

[Formation of grooves to be flow paths]
On the substrate 1 on which the porous layer 3 is formed, the composition (X1) is applied using a bar coater, and the first channel upstream portion 10a, the first branch portion 15, the first channel downstream portion 10b, Further, a portion other than the portion to be formed with the second flow path 20 is irradiated with ultraviolet rays by the ultraviolet lamp 2 through a photomask for 120 seconds to form the composition (X1) as a semi-cured coating film that becomes the intermediate layer 4, and the non-irradiated portions The uncured composition (X1) was removed with ethanol to form grooves to be the first channel upstream portion 10a, the first branch portion 15, the first channel downstream portion 10b, and the second channel 20. At this time, the porous layer 3 is exposed on the bottom surface of the flow channel where the first flow channel downstream portion 10b and the porous layer 3 overlap, and the porous layer 3 in the other portion is composed of the composition (X1). Filled and cured. This member was a substrate side member.

[Immobilization of probe DNA]
(Amino group injection)
A 5% by mass polyallylamine (molecular weight: 15000, manufactured by Nittobo Co., Ltd.) aqueous solution is brought into contact with the porous layer 3 exposed on the bottom surface of the first channel downstream portion 10b of the substrate side member, and left at 60 ° C. for 1 hour. Then, a part of the amino group of polyallylamine was reacted with the epoxy group of the porous layer 3 and then washed with running water for 15 minutes to introduce the amino group into the porous layer.

(Introduction of aldehyde group)
After introducing the amino group, the substrate side member is immersed in a 5% by mass aqueous solution of glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) and kept at 50 ° C. for 2 hours, whereby almost all amino acids of polyallylamine are obtained. The group was reacted with one aldehyde group of glutaraldehyde and washed with running water for 10 minutes to introduce the aldehyde group into the porous layer.

(Immobilization of DNA)
2 μl of a 50 μM aqueous solution of 20-base-length DNA (named N0, manufactured by Espec Oligo Service Co., Ltd.) amino-modified at the 5 ′ end was dropped into the porous layer 3 introduced with the aldehyde group, and the humidity was 100%, 50 By maintaining at 15 ° C. for 15 hours, the terminal amino group of DNA (N0) was reacted with the aldehyde group of the porous layer, and then placed in a 0.2 mass% sodium tetrahydroborate aqueous solution, and subjected to a reduction reaction for 5 minutes. Next, rinsing with a 0.2 × SSC / 0.1% SDS solution, followed by rinsing with 0.2 × SSC and distilled water, followed by air drying, DNA (N0) as probe DNA in the porous layer 3 Fixed. (Here, 0.2 × SSC is a 0.03 M NaCl, 3 mM sodium citrate aqueous solution, and 0.1% SDS is a 0.1 mass% sodium dodecyl sulfate aqueous solution.)

[Fixing the cover layer 5]
A biaxially stretched polypropylene sheet (manufactured by Futamura Chemical Co., Ltd.) having a thickness of 30 μm, one side of which has been subjected to corona discharge treatment, is used as a temporary support (not shown), and the composition (X2) is coated thereon using a bar coater. Then, the ultraviolet lamp 1 was irradiated with ultraviolet rays for 2 seconds to form a semi-cured coating film of the composition to obtain a cover layer side member.
The cover layer side member was laminated and adhered to the intermediate layer 4 of the substrate side member, and the cover layer 5 was fixed by irradiating the ultraviolet ray with the ultraviolet lamp 1 for 40 seconds to be completely cured. Next, the temporary support is peeled and removed to provide a microfluidic device precursor having a capillary first channel upstream portion 10a, a first branching portion 15, a first channel downstream portion 10b, and a second channel 20. Formed.

[Formation of other structures]
Thereafter, a diameter of 0.5 mm is used by drilling at the end of the first channel upstream portion 10a, the end of the first channel downstream portion 10b, and the end of the second channel 20 through the cover layer 5. Holes 41, 42, 43 are formed to form the first inlet 11, the first outlet 12, and the second inlet 21, respectively, and luer fittings 51, 52, 53 are bonded to the respective mouths, A microfluidic device having was obtained.
The dimensions of each part of the microfluidic device are 75 mm × 25 mm × 1.145 mm, and the thickness of each layer is 1.0 mm for the substrate 1, 15 μm for the adhesive layer 2, 3 μm for the porous layer 3, and porous The thickness of the intermediate layer 3 excluding the material layer 3 is 30 μm, the cover layer is 100 μm, and the length of each flow path is 15 mm for the first flow path upstream part 10a, about 130 mm for the second flow path downstream part 10b, The length of the porous layer 3 exposed at a part of the bottom surface of the downstream path portion 10b is 100 mm, the second channel 20 is about 25 mm, and the widths of the first channel 10 and the second channel are both 100 μm. is there. Further, the range of the distance from the first branch portion of the first flow path downstream portion 10b is about 0 to 1 mm is set as the detection section 33 that is a window through which the first flow path 10 can be observed from the upper surface of the microfluidic device. The portion of 10b between the porous layer 3 forming portion and the first outlet 12 without the porous layer 3 was used as a chromatographic detection unit 34 that is a window through which the first flow path 10 can be observed from the upper surface of the microfluidic device.

[Sample introduction test]
(Preparation of materials)
(DNA sample complementary to probe DNA)
The 5 ′ end was modified with a fluorescent dye FITC, and a 20-base long DNA (referred to as F0) having a base sequence complementary to the probe DNA (N0) (manufactured by Espec Oligo Service Co., Ltd., concentration 500 μM) The sample was diluted with 2 × SSC solution to obtain a chromatographic measurement sample F0 having a concentration of 0.3 μM.

(DNA sample having single-base mismatch with probe DNA)
A sample F1 was prepared in the same manner as the sample F0 except that the DNA was a DNA having a base sequence that resulted in a one-base mismatch with the probe DNA (N0) (referred to as F1).

[Chromatography of sample F0]
A microfluidic device is fixed on a heat block whose temperature is adjusted to 40 ° C., a microsyringe pump (not shown) as a first pump at the first outlet 12, and a microsyringe pump as a second pump at the first inlet 11 (Not shown) was connected. As the piping, a tube made of polytetrafluoroethylene (PTFE) having an inner diameter of 500 μm was used. A pipe was not connected to the second inlet, and the luer fitting 53 was used as a sample receiving tank.
First, the buffer solution (0.2 × SSC) was discharged at 2 μl / min from both the first pump and the second pump, and the buffer solution was filled in all the channels. The buffer solution accumulated in the luer fitting 53 at the second inlet was removed by suction with a pipette.

(First step)
Next, when 100 μl of the sample was dropped with a pipette inside the luer fitting 53 and the first pump was operated in the suction direction at 0.2 μl / min, the sample was sucked from the second inlet 21 and passed through the second flow path 20. Then, it was transferred from the first branch portion 15 to the first flow path downstream portion 10b.

(Second step)
When the fluorescence of the sample in the detection unit 33 is observed using a Nikon fluorescent stereomicroscope (not shown), the sample passes through the first branching unit 15 and enters the first flow path downstream unit 10b by about 0.5 mm. When the second pump was operated in the discharge direction at 1.1 μl / min, the transfer fluid flowed from the first channel upstream portion 10a to the first channel downstream portion 10b instead of the sample. Then, the sample having a length of about 0.5 mm that had already been introduced into the first channel upstream portion 10a was continuously transferred in the downstream direction in the first channel downstream portion 10b. On the other hand, the sample that was in the second flow path 20 was slowly pushed out through the second flow path 20 toward the second inlet 21. In this way, a very small amount of sample was injected into the first channel downstream portion 10b in a pulsed manner.
Thereafter, both pumps were operated at 1 μl / min, and a change in fluorescence intensity was measured using a Nikon fluorescent stereomicroscope in the chromatograph detection unit 34 of the first flow path downstream portion 10b. As a result, a chromatogram having a peak at about 105 seconds for the DNA (F0) sample and a chromatogram without tailing having a peak at 88 seconds for the DNA (F1) sample were obtained. The sample injection amount was calculated to be about 1.5 nl from the peak area of the chromatogram and the calibration curve.

(Example 2)
In this example, a manufacturing method of the second form of the microfluidic device of the present invention and a second embodiment of the method of introducing a micro sample of the present invention using the same will be described.

[Production of microfluidic devices]
In the present embodiment, as shown in FIG. 2, the microfluidic device includes a third inlet 31, a third channel upstream 10a, a second branch 16, a third channel downstream 30b, and a second outlet. 32 except that the luer fittings 54 and 55 are bonded to the third inlet 31 and the second outlet 32, respectively, and that the second inlet 21 is connected to the second branch portion 16. Prepared in the same manner as in Example 1. The width of the third channel was 200 μm.

[Sample introduction method]
Except that a syringe pump (not shown) was connected to the luer fitting 54 of the third inlet 31 and that the sample was flowed from the syringe pump to the third flow path 30 at a constant flow rate of 1 mm ³ / min. The same sample introduction test as in Example 1 was performed, and the same result as in Example 1 was obtained.

It is (a) top view schematic diagram and (b) side view schematic diagram of the microfluidic device of this invention produced in Example 1. FIG. It is the (a) top view schematic diagram and (b) side view schematic diagram of the microfluidic device of this invention produced in Example 2. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 2 Adhesive layer 3 Porous layer 4 Intermediate layer 5 Cover layer 10 First flow path
10a First channel upstream portion 10b First channel downstream portion 11 First inlet 12 First inlet 15 First branch 16 Second branch 20 Second channel 21 Second inlet 30 Third channel 30a On the third channel Stream portion 30b Third flow path downstream portion 31 Third inlet port 32 Second outlet port 33 Detector 34 Chromatograph detector 41, 42, 43, 44, 45 Hole 51, 52, 53, 54, 55 Luer fitting

Claims (6)

A method for quantitatively introducing a small amount of sample into a fine channel of a microfluidic device,
The microfluidic device includes a first inlet, a first outlet, a first channel connecting the first inlet and the first outlet, and the first channel provided upstream of the first channel. A first branch portion that is divided into a first portion and a downstream portion of the first flow passage, a second flow passage that branches off from the first flow passage at the first branch portion, and a second provided at the other end of the second flow passage Using a microfluidic device having an inlet and
(1) The first flow path is filled with a transfer fluid, the second flow path is filled with a sample or a transfer fluid,
The transfer fluid is discharged into the first flow path from the second pump connected to the first inlet, and the first pump connected to the first outlet is larger than the discharge speed of the second pump. By driving in the suction direction at a speed or by stopping the first pump and further driving the second pump in the suction direction,
The transfer fluid filled in the first flow path is transferred toward the first pump, and at the same time, due to the negative pressure generated by the difference between the discharge speed of the first pump and the suction speed of the second pump, A first step of transferring the sample from the second inlet to the downstream portion of the first channel through the second channel;
(2) When a predetermined amount of the sample is introduced into the downstream portion of the first flow path, the first flow rate at which the value obtained by subtracting the discharge speed of the second pump from the suction speed of the first pump is zero or negative. By operating one pump and the second pump,
A second step of stopping the sample from flowing into the downstream portion of the first flow path from the second flow path through the first branch portion;
A method for introducing a minute sample, characterized in that The trace sample according to claim 1, wherein the passage of the sample is detected in a part of the second channel or the downstream portion of the first channel, and the first step is switched to the second step based on the information. Introduction method. The microfluidic device is a chromatographic microfluidic device in which a chromatography separation column is provided in the middle of the downstream portion of the first flow path, and the transfer fluid is a developing solution for chromatography. 2. A method for introducing a trace amount sample according to 2. The microfluidic device is further provided in the middle of the third flow path, the second flow outlet, the third flow path connecting the third flow inlet and the second flow outlet, and the third flow path; A second branch that divides the third channel into a third channel upstream portion and a third channel downstream portion;
The second inlet is connected to the second branch,
A part of the sample flowing from the third inlet to the second outlet in the third channel is connected to the second branch portion from the second inlet via the second channel. The method for introducing a trace amount sample according to claim 1, 2 or 3, which is introduced into the downstream portion. A microfluidic device in which a separation column for chromatography is provided in a part of a fine flow path,
The microfluidic device is provided in the middle of the first flow path, the first flow path, the first flow path connecting the first flow path and the first flow path, and the first flow path on the first flow path. A first branch part that divides into a flow part and a downstream part of the first flow path, a second flow path that branches off from the first flow path at the first branch part, and that is not provided with a valve or pump, and the second flow path A second inlet provided at the other end of the channel, a liquid receiving tank connected to the second inlet and opened to the outside of the microfluidic device, and a chromatograph provided in the downstream of the first channel. A microfluidic device having a chromatographic separation column. And a third inlet, a second outlet, a third channel connecting the third inlet and the second outlet, and a third channel provided in the middle of the third channel. 6. The microfluidic device according to claim 5, further comprising a second branch portion that is divided into a third channel upstream portion and a third channel downstream portion, wherein the second inlet is connected to the second branch portion.

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