JP3787578B2 - Liquid feeding method in micro channel of microchip - Google Patents

Description

  The present invention relates to a liquid feeding method for a liquid feeding device that allows a liquid to flow at a minute flow rate in a fine channel (channel) formed in a microchip or the like.

  Recently, as is known by the names such as Microscale Total Analysis Systems (μTAS) or Lab-on-Chip, a microchannel that forms a flow path of a predetermined shape in a substrate and Providing a fine structure such as a port and performing various operations such as chemical reaction, synthesis, purification, extraction, generation and / or analysis of substances within the fine structure has been proposed and partially put into practical use. A structure manufactured for such a purpose and having a fine structure such as a microchannel and a port in a substrate is generically called a “microchip”.

  Microchips can be used for a wide range of applications such as genetic analysis, clinical diagnosis, drug screening and environmental monitoring. Compared with the same type of equipment of the common size, the microchip is (1) significantly less sample and reagent usage, (2) shorter analysis time, (3) higher sensitivity, (4) carried on-site, It can be analyzed in the field and (5) can be disposable.

  The material, structure, and manufacturing method of the microchip are proposed in, for example, Patent Document 1, Patent Document 2, and Non-Patent Document 1. In the conventional microchip 100, for example, as shown in FIG. 6, at least one channel 104 is formed on a substrate 102 such as a synthetic resin (for example, polydimethylsiloxane or acrylic resin). A well 106 to be an input / output port is formed at least at one end, and a facing substrate 108 made of a transparent or opaque material (for example, glass or synthetic resin film) is bonded to the lower surface side of the substrate 102. The presence of the facing substrate 108 seals the bottom of the well 106 and the channel 104.

  The main use of the well 106 for the input / output port is injection of reagents and samples. As shown in FIG. 7, liquid is pumped into the channel 104 by connecting a tube or capillary 112 to the well 106. In addition to the case where the tube or capillary 112 is detachably attached by the dedicated fitting 114, the tube or capillary 112 may be fixed to the well 106 with an adhesive or the like. The other end of the tube or capillary 112 is connected to a liquid delivery device (not shown).

  There are not many uses for flowing a micro flow rate of the order of 0.1 μL / min through a channel (micro flow path) having a width of several tens μm to several hundreds μm, such as a microchip. For this reason, there are many cases in which existing simple syringe pumps or high-performance micropump systems developed for liquid chromatography are used as liquid delivery devices used there.

  In the case of using a syringe pump, a small flow rate is selected by selecting a small inner diameter of the syringe used there. However, the flow rate accuracy is low, and it does not have a function of controlling the pressure of the fluid with a single function that allows a constant flow rate to flow.

  On the other hand, a high performance micropump system for liquid chromatography is an expensive instrument developed to achieve high flow accuracy even in a micro flow region. However, the purpose of liquid chromatography is to keep flowing at a constant flow rate for a long time. For this reason, the high-performance micropump system does not take into consideration the instability of the flow rate at the start or stop of the liquid feeding.

  In addition, there are various problems such as dead volume and volume / volume change when liquid such as a reagent or sample is fed to a microchip channel by a liquid feeding device such as a micropump. For example, the volume of a pipe connecting a liquid delivery device such as a micropump to a channel such as a microchip is extremely large relative to the volume of a channel through which a liquid actually flows. First, in order to physically connect objects having different sizes such as a liquid delivery device and a microchip, the pipe length becomes long. For example, the channel cross section has a rectangular shape with a width of 100 μm and a depth of 30 μm. The length becomes mm. In addition, the cross-section in the pipe must be much larger than the channel. As a result, it takes a lot of time to fill the piping volume with a liquid (eg, reagent or sample). For example, assuming that the pipe length is 100 mm and the pipe cross section is 10 times the channel cross section, a flow rate of 0.1 μL / min requires about 30 minutes. In order to solve this problem, for example, a means for increasing the flow rate until the pipe volume is filled and switching to a desired flow rate when starting to flow into the channel can be considered. However, it is difficult to determine the timing for switching, and automation is also difficult. In addition, if a large flow rate is accidentally flowed through the channel, a large pressure increase occurs, and there is a risk of damaging the liquid delivery device or the channel itself.

  Even in a state where the pipes and channels are all filled with liquid, the volume of the pipes and channels changes due to the pressure fluctuation of the liquid accompanying the liquid feeding. Furthermore, a slight volume change of the gas mixed in the inner wall of the pipe or in the liquid also occurs. These volume changes and volume changes are very large for a fine channel. Therefore, once the liquid delivery of the liquid delivery device is stopped and then restarted, the discharge flow rate or suction flow rate does not immediately match the flow rate in the channel, the fluid pressure is stabilized, and the flow rate in the channel is There is a long response delay before it matches the flow rate of the device. This tendency becomes more prominent as the channel becomes finer and the flow rate becomes smaller.

  The problem as described above also occurs when the liquid feeding is stopped. The flow in the channel does not stop immediately even when the liquid delivery device is stopped, and the flow rate gradually decreases until the liquid pressure in the liquid delivery device, the pipeline, and the channel is released. The flow stops with a certain delay. As a result, samples and reagents are gradually wasted.

JP 2001-157855 A US Pat. No. 5,965,237 David C. Duffy et al., Rapid Prototyping of Microfluidic Systems in Poly (dimethylsiloxane), Analytical Chemistry, Vol.70, No.23, December 1, 1988, pp.4974-4984

Accordingly, an object of the present invention is to provide a liquid feeding method that shortens the time until the liquid feeding in the channel reaches a desired flow rate at the start of the liquid feeding.
Another object of the present invention is to provide a liquid feeding method that shortens the time until the liquid feeding in the channel stops when the liquid feeding is stopped.

  As a means for solving the above-mentioned problems, the feature of the invention according to claim 1 is that at least one substrate having a predetermined fluid micro-channel disposed on one surface, and the fluid micro-fluid In a microchip consisting of a facing substrate that seals a path, a method of feeding a liquid to the fine channel, applying a predetermined pressure from a liquid feeding device at the start of feeding, and setting the liquid After reaching the pressure, a constant flow rate is sent at a predetermined speed.

  As a means for solving the above-mentioned problems, the feature of the invention according to claim 2 is that at least one substrate having a predetermined fluid micro-channel disposed on one surface, and the fluid micro-fluid In a microchip comprising a facing substrate that seals a path, a method for feeding a liquid into the fine channel, wherein the liquid is sent when a constant flow rate is being delivered at a predetermined speed at the end of the liquid feeding. The fluid pressure is reduced to zero or a value close to it.

As a means for solving the above-mentioned problems, a feature of the invention according to claim 3 is that at least one substrate having a predetermined fluid micro-channel disposed on one surface, and the fluid micro-fluid In a microchip composed of a facing substrate that seals a path, a method of feeding a liquid to the fine channel,
At the start of liquid delivery, a predetermined pressure is applied from the liquid delivery device to deliver the liquid, and after reaching the set pressure, a constant flow rate is delivered at a predetermined speed.
At the end of liquid feeding, when feeding a constant flow rate at a predetermined speed, the liquid feeding pressure is reduced to zero or a value near it.
That is.

  As a means for solving the above-mentioned problems, the invention according to claim 4 is characterized in that, in the inventions according to claims 1 to 3, the liquid feeding device is a micropump provided with a plunger, and a syringe pump provided with a syringe. Or it is a piston pump provided with a piston.

  In the present invention, even when a micropump having a conventional plunger is used, a predetermined pressure is applied at the start of liquid feeding, and when the set pressure is reached, the plunger is driven at a predetermined speed to start a fine flow at the start of liquid feeding. It is possible to reduce the time until the liquid flow in the channel (channel) reaches a desired flow rate, and when the liquid feed is stopped, the plunger is retracted to reduce the liquid feed pressure to zero. The time until the liquid feeding in the flow path (channel) stops can be shortened.

  Hereinafter, preferred embodiments of the liquid feeding method of the present invention will be specifically described with reference to the drawings.

  FIG. 1 is a partial schematic perspective view of an example of a liquid flow feeding device for minute flow rate used for carrying out the liquid feeding method of the present invention, and FIG. 2 is a partial schematic sectional view thereof. For example, a plunger-type micropump 1 is used as the microflow rate liquid-feeding device used to carry out the liquid-feeding method of the present invention. The plunger drive unit 3 moves the plunger 5 forward and backward by, for example, a stepping motor and a screw / nut mechanism (not shown). The liquid contact portion 7 has a sufficient volume necessary for accumulating fluid, and the tip of the plunger 5 sealed from the plunger drive portion 3 moves forward and backward. A first port 9 is provided in a part of the liquid contact portion 7, and a pipe 11 is connected to the tip of the first port 9. Liquid is fed through the pipe 11. In order to detect the pressure during liquid feeding, a pressure detector 13 can be connected to the liquid contact portion 7. In FIG. 2, a second port 15 is disposed in the liquid contact portion 7. This second port 15 is used not only for injecting fluid into the wetted part 7 or extracting unnecessary air, but also for plugging or actually detecting pressure when the liquid is actually sent. Used to connect the device 13.

  FIG. 3 is a schematic perspective view of an example of a microchip in which the liquid feeding method of the present invention is implemented. The flow path in which the liquid feeding method of the present invention is implemented is not particularly limited as long as it is fine. For example, a fused silica capillary used for liquid chromatography may be used. However, the liquid feeding method of the present invention is particularly effective at a minute flow rate on the order of 0.1 μL / min, and as its application, a fine flow path (channel) 22 in the microchip 20 as shown in FIG. 3 is particularly suitable. ing.

  In FIG. 3, the microchip 20 has a Y-shaped channel 22. The sample hole 24 and the reagent hole 26 are connected to separate micro pumps 1 (see FIG. 1 or FIG. 2) by the pipe 11, and the sample and the reagent are sent from the micro pump 1 into the channel 22 in the microchip 20. . The sent sample and reagent are mixed in the vicinity of the junction of the Y-shaped channel to cause a desired chemical reaction. This can be observed with a microscope (not shown). In the case of such a microchip, the flow rate of each sample must be precisely controlled in order to keep the mixing ratio of the sample and the reagent constant. Reference numeral 28 denotes a waste liquid hole.

  Specifically, when the cross section of the channel 22 is a rectangle with a length of 30 μm and a width of 100 μm, the pipe length is about several tens of mm, and the flow rates of the sample and the reagent are each 0.1 μL / min, the channel at this time The flow rate within 22 is about 560 μm / second for the sample and the reagent before mixing and about 1.1 μm / second for the mixing, respectively. The pressure required for this liquid transfer is 1 cp (centipoise) for the sample and the reagent, respectively. In this case, it is about several KPa. However, the pipe 11 connecting the micropump 1 and the microchip 20 has a sufficiently large pipe cross section compared to the channel 22 in the microchip 20, and the pipe resistance can be ignored.

  When the liquid is sent to the channel 22 of the microchip 20 shown in FIG. 3 using the micropump 1 shown in FIG. 1 and FIG. The flow rate liquid feed is a liquid feed at a target constant flow rate, and the pressure liquid feed is a liquid feed at a target constant pressure value.

  In the case of performing flow rate liquid feeding using the micropump 1 shown in FIGS. 1 and 2, the tip of the plunger 5 is moved to the end face (suction end) on the plunger driving unit 3 side in the liquid contact part 7 in advance. deep. The first port 9 and the target hole 24 or 26 of the microchip 20 are connected by the pipe 11. After injecting the liquid into the wetted part 7 through the second port 15, the second port 15 is plugged. In this state, the plunger 5 is moved at a constant speed in the direction of the tip (discharge end) of the liquid contact portion 7. Along with the movement of the plunger 5, the liquid pushed out from the liquid contact portion 7 is fed into the channel 22 from the target hole 24 or 26 through the pipe 11. At this time, for example, in order to achieve a flow rate of 0.1 μL / min, the plunger driving unit is controlled so that the plunger speed is set accordingly. If the outer diameter of the plunger 5 is 3 mm, the flow rate is 0.1 μL / min if the plunger 5 moves at a speed of about 15 μm / min.

  In pressure feeding, the pressure of the liquid is detected, and the position of the plunger is controlled so that the pressure becomes a target pressure. For this purpose, a pressure detector is required, which is connected to the second port 15 in the examples of FIGS. When pressure feeding is performed using the micropump 1 shown in FIGS. 1 and 2, it is necessary to perform feedback control based on a control block diagram as shown in FIG. A target pressure value is input to the control circuit 30 in advance. In response to the difference between the target value and the pressure detection value 32 of the pressure detector 13, the control circuit 30 transmits a control signal 34 that reduces the difference to the drive circuit 36. The drive circuit 36 drives the plunger 5 (see FIG. 2) of the micropump 1 based on the control signal 34. As a result of the plunger 5 of the micropump 1 being driven, a change in the pressure of the liquid occurs, and the control circuit 30 operates accordingly. This closed loop operation is repeatedly controlled at a time interval of, for example, 10 milliseconds (ms).

  As a result, it is uncertain at what flow rate during pressure feeding. There may be a case of moving to the discharge side or a case of moving to the suction side. The flow rate limit value may be set in consideration of pipe breakage. This pressure delivery is less common than flow delivery. Speaking of liquid feeding simply indicates flow rate liquid feeding. Pressure liquid feeding is a part of a highly functional micro pump system, and is only provided as an auxiliary function under the name of “constant pressure mode” in addition to flow rate liquid feeding.

  According to the liquid feeding method of the present invention, pressure liquid feeding is first performed at the start of liquid feeding. In this case, the target pressure is set to a pressure when the desired flow rate is constantly flowing or a value in the vicinity thereof. Specifically, it is preferable to measure the pressure during liquid feeding in advance or use an empirical value.

  During pressure liquid supply control, a large flow rate flows to reach the target pressure (if there is a flow rate limit value, the flow rate is set to be sufficiently larger than the desired flow rate), the pipe 11 between the micropump 1 and the channel 22 is filled, and the pipe line We will compensate for the increase in volume and reduction of mixed gas volume in a short time. As the liquid begins to flow into the intended channel 22, the pressure of the liquid increases significantly. Then, after reaching the target pressure, the flow rate is switched to a desired flow rate.

  The liquid feeding method of the present invention can make the flow rate in the channel a desired flow rate in a much shorter time than the conventional method in which liquid feeding is started at a constant flow rate from the start of liquid feeding. In addition, by appropriately performing pressure control (feedback control) during pressure feeding, pressure overshoot (instantaneously exceeding the target pressure) is prevented, and channel damage due to excessive flow rate is also prevented. Become.

  According to the liquid feeding method of the present invention, when the liquid feeding is stopped from the state where the liquid is fed into the channel at a certain flow rate, first, the pressure liquid feeding is performed with the target pressure set to zero or a value in the vicinity thereof. . The micropump 1 performs an operation of reducing the pressure generated at the time of liquid feeding to zero. For example, since the pressure is positive when the liquid is discharged, the micropump 1 attempts to reduce the pressure by performing a suction operation. Then, when the pressure reaches zero or a value close to the target pressure, the liquid feeding of the micro pump is stopped. Specifically, the pressure in the pipe 11 and the channel 22 can be reduced to zero in a very short time by reversing the micropump 1 to retract the plunger 5. This immediately stops the flow of liquid in the channel 22.

  The liquid feeding method of the present invention completely completes the liquid feeding in the channel in a much shorter time than the conventional method in which the liquid feeding cannot be completely stopped even if the liquid feeding is stopped when the liquid feeding is stopped. Can be stopped. As a result, not only samples and reagents are not wasted, but also the accuracy of analysis is improved.

(1) Verification experiment of time delay at the start of liquid feeding An experiment was conducted using a microchip having a Y-shaped channel as shown in FIG. Each of the Y-shaped channels 22-1 and 22-2 has a width of 100 μm and a depth of 25 μm, and the merge channel 22-3 has a width of 200 μm and a depth of 20 μm. Two different liquids are flowed through the Y-shaped channels 22-1 and 22-2 to be combined. The two liquids are selected so that the flow characteristics exhibited in the channel are equal and the interface between the two liquids can be observed. Specifically, red ink and blue ink having a viscosity of 1 cp (centipoise) are used, and the interface between them is determined by color.
FIG. 5A is a diagram illustrating a state in which both the red ink and the blue ink are stably flowing at 0.1 μL / min. The interface between the two liquids is clearly formed at the center of the merge channel 22-3 after the merge. This is because the liquid flow in the confluence channel 22-3 maintains a laminar flow and does not easily become a turbulent flow. As a result, the two liquids are not easily mixed.
If there is a difference between the flow rates (or flow velocities) of the two liquids, for example, as shown in FIGS. 5 (B) and 5 (C), the interface between the two liquids is one in the merge channel 22-3 after the merge. Will be biased. In addition, if the flow rates of the two liquids are unstable, it is observed that the interface also moves with it. By measuring the time from the start of feeding of the two liquids until the interface between the two liquids after merging is stabilized, the time delay until the desired flow rate is reached at the start of the liquid feeding can be measured.
The comparative experiment was performed as follows.
Connect the microchip and the micropump with a transparent Teflon (registered trademark) tube, and check the inside of the Teflon tube until just before the microchip port with a large flow rate (10 μL / min) while visually confirming in advance. Fill with liquid (red ink, blue ink). Then, liquid feeding of the micropumps is simultaneously started at a flow rate setting of 0.1 μL / min for each liquid, and an image of the merged portion of the Y-shaped channel is recorded on a video. After a sufficient time has elapsed, the recorded video image is replayed and examined to find a time when the interface between the two liquids in the merging portion is constantly stabilized, and the time from the start of liquid feeding to the time is measured.
Steam experiments were conducted using a conventional liquid feeding method (a method of feeding a liquid at a constant flow rate of 0.1 μL / min from the beginning) and a liquid feeding method according to the present invention (after first performing a pressure liquid feeding at a pressure setting of 5 KPa, The method of switching to a flow rate of 1 μL / min) was performed, and each time delay was measured. As a result, in the conventional liquid feeding method, there was a time delay exceeding 1 hour, whereas in the liquid feeding method according to the present invention, stable liquid feeding was obtained in about 10 minutes.
(2) Verification experiment of time delay at the time of liquid supply stop From the state where a stable flow is generated at a constant flow rate of 0.1 μL / min in the above-mentioned two liquid merge, the liquid feed of the micro pump is stopped simultaneously. By observing the video image of the merging portion, the time point when the flow stopped was searched, and the time delay when the liquid feeding was stopped was measured.
A comparison between a conventional method (a method for simply stopping the micropump immediately) and a method according to the present invention (a method for stopping the micropump after first performing pressure feeding at a set pressure of 0 KPa) Whereas there was a time delay exceeding 10 minutes, in the method according to the present invention, the fluid flow stopped in about 2 minutes.

  The liquid feeding method of the present invention is applicable not only to the above-mentioned microchip but also to all uses as long as the liquid must be fed into a fine channel (channel). For example, it can also be used for capillary liquid chromatograph, capillary electrophoresis, liquid feeding with medical / diagnostic reagent measuring equipment, and liquid feeding / dispensing of reagents / serum etc. with analytical equipment. Moreover, as a liquid feeding apparatus, not only the said plunger type micro pump but a syringe type syringe pump or a piston type piston pump can also be used.

It is a partial outline perspective view of an example of the liquid feeding device for minute flow used for implementing the liquid feeding method of the present invention. FIG. 2 is a partial schematic cross-sectional view of the liquid delivery device shown in FIG. 1. It is a general | schematic perspective view of an example of the microchip by which the liquid feeding method of this invention is implemented. It is a control block diagram in the case of performing pressure liquid feeding using the micropump shown in FIG. In the microchip which has a Y-shaped channel, it is a schematic diagram which shows the state when two different liquids are made to flow with the same flow rate, and when it is made to flow with a different flow rate. It is a schematic sectional drawing of an example of the conventional microchip. FIG. 7 is a partial schematic cross-sectional view of an example in which liquid is dispensed into the open well of the microchip in FIG. 6.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Micropump 3 Plunger drive part 5 Plunger 7 Liquid contact part 9 1st port 11 Piping 13 Pressure detector 15 2nd port 20 Microchip 22 Fine flow path (channel)
24 Sample hole 26 Reagent hole 30 Control circuit 32 Pressure detection signal 34 Control signal 36 Drive circuit

Claims (4)

In a microchip comprising at least one substrate having a predetermined fluid micro-channel on one surface and a facing substrate that seals the fluid micro-channel, a liquid is provided in the micro-channel. The liquid is supplied by applying a predetermined pressure from a liquid supply device at the start of liquid supply, and after reaching the set pressure, a constant flow rate is supplied at a predetermined speed. A liquid feeding method in a fine flow path of a microchip. In a microchip comprising at least one substrate having a predetermined fluid micro-channel on one surface and a facing substrate that seals the fluid micro-channel, a liquid is provided in the micro-channel. Of the microchip characterized in that, at the end of the liquid feeding, the liquid feeding pressure is reduced to zero or a value close thereto when a constant flow rate is being delivered at a predetermined speed. Liquid feeding method in a fine channel. In a microchip comprising at least one substrate having a predetermined fluid micro-channel on one surface and a facing substrate that seals the fluid micro-channel, a liquid is provided in the micro-channel. A method of delivering a liquid,
At the start of liquid delivery, a predetermined pressure is applied from the liquid delivery device to deliver the liquid, and after reaching the set pressure, a constant flow rate is delivered at a predetermined speed.
At the end of liquid feeding, when feeding a constant flow rate at a predetermined speed, the liquid feeding pressure is reduced to zero or a value near it.
A liquid feeding method in a fine flow path of a microchip. 4. The liquid feeding method according to claim 1, wherein the liquid feeding device is a micro pump having a plunger, a syringe pump having a syringe, or a piston pump having a piston.

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