JP4410040B2 - Microfluidic control mechanism and microchip - Google Patents

Description

  The present invention relates to a microfluidic control mechanism and a microchip, and more particularly to a microfluidic control mechanism and a microchip that are suitable for use in a microchip used for an experimental operation of a chemical experiment or a biological experiment.

  In recent years, it has been proposed to perform chemical and biological experiment operations using a microchip, and it has become possible to actually perform experimental operations such as chemical analysis and cell measurement in a microchip.

  Here, the microchip means one formed by integrating microchannels (microchannels) on a flat substrate by fine processing.

  By using such a microchip, the sample can be saved, the speed of the experiment can be increased to shorten the time spent for the experiment, and the experiment operation can be automated. It was possible to achieve excellent effects such as

  By the way, in many cases, the samples and reagents used in the experiments are liquids. However, in the experiments using the above-described microchip, the liquids such as samples and reagents are contained in the microchannel formed on the microchip. Need to be introduced. As a method for introducing such a liquid into the microchannel, two types of methods (first method and second method) described below are generally used.

(1) First method: Method using a syringe pump This method is a method in which a tube is connected to a microchannel via a connector and liquid is pumped into the microchannel using a syringe pump. (Refer nonpatent literature 1).

(2) Second method: Method using electroosmosis phenomenon This method provides reservoirs at both ends of the microchannel, and when electrodes are inserted into the microchannel and a high voltage is applied, This is a technique in which a liquid flows into (see Non-Patent Document 2).

However, the first method and the second method described above have the following problems (Problem 1 to Problem 3).

(I) Problem 1
Since an external device such as a syringe pump or a high-voltage power supply is required, the configuration of the entire device including such an external device becomes complicated and expensive.

(II) Problem 2
Since it is necessary to connect a tube or insert an electrode into the microchannel, it takes time to prepare for the actual experiment, and the entire work time including the preparation takes a long time. It was supposed to be.

(III) Problem 3
In particular, with respect to the first method, most of the samples and reagents are used to transmit power, and the samples and reagents for that purpose are wasted, which is wasteful.

In order to solve these problems 1 to 3, almost no power from the outside of the microchip is required to introduce a liquid such as a sample or a reagent into the microchannel, or power from the outside. As methods that do not require any of the above, two methods (third method and fourth method) described below have been proposed.

(3) Third method: Method using compressed air In this method, a pressure chamber is provided through a fragile partition wall in the microchannel, and energy for sending liquid in the microchannel is used as compressed air in the pressure chamber. Save it. And when the partition of a pressure chamber is destroyed by an electric current, the air pressure of compressed air is released and the liquid flows into the micro flow path (see Non-Patent Document 3).

(4) Fourth method: Method using capillary action In this method, a predetermined molecule as a sample is immobilized in advance on the wall of the reaction chamber of the microchannel, and a plurality of solutions such as a chemical solution and a cleaning solution are used. In this reaction chamber, one by one is poured into the reaction chamber by a capillary phenomenon to cause a reaction (see Non-Patent Document 4).

However, the third method and the fourth method introduce new problems (problem 4 and problem 5) as described below.

(IV) Problem 4
In the third method, the liquid is introduced into the microchannel by compressed air stored in the pressure chamber of the microchip, that is, energy stored in the microchip in advance. In order to begin introducing liquid into the chamber, the current chamber must be destroyed by an electric current. That is, the third method requires power from the outside of the microchip as a trigger for starting to introduce liquid into the microchannel, and completely without power from the outside of the microchip. The liquid could not be introduced into it.

(V) Problem 5
In the fourth method, the liquid is introduced into the reaction chamber by capillary action, so it is possible to introduce the liquid into the microchannel without power from the outside of the microchip, but the liquid is introduced into the reaction chamber. Immediately after that, the liquid starts to react with the molecules fixed to the walls of the reaction chamber, so that a plurality of liquids could not be mixed in the microchannel.

M.M. Tokoshi et al. Analytical Chemistry 72 (2000) pp. 1711-1714 S. C. Jacobson et al. Analytical Chemistry 71 (1999) pp. 4455-4459 C. C. Hong, J. et al. W. Choi, and C.I. H. Ahn, “Disposable air-bursting detectors as an alternative on-chip power source,” Proceedings of the 15th IEEE International Conference on MicroElectric MicroElectricity. 240-243, Las Vegas, USA, January 20-24, 2002. D. Juncker, et al. “Autonomous microfluidic capillary system,” Analytical Chemistry 74, pp. 6139-6144, 2002.

  The present invention has been made in view of the above-described various problems of the prior art. The object of the present invention is to solve the problems 1 to 5 described above and to have a simple structure. An object of the present invention is to provide a microfluidic control mechanism and a microchip that do not require external power to introduce liquid into the microchannel.

  In order to achieve the above object, the present invention is formed by a polymer material, creates a decompressed space in the microchannel, and automatically introduces liquid into the microchannel. is there.

That is, the invention according to claim 1 of the present invention is formed in each of a microchannel formed in a predetermined shape and a plurality of end portions of the microchannel to introduce a liquid into the microchannel. A plurality of ports that are open to the outside, and the whole or part of the microchannel is formed of a polymer material, and the polymer material is deaerated in a low-pressure environment and then returned to the atmosphere. The air in the microchannel is dissolved in the polymer material, and a liquid is dropped into at least one of the plurality of ports and the other port is sealed to form a decompressed space in the microchannel. It occurs, and the liquid is sucked into the decompressed space .

According to a second aspect of the present invention, a microchannel formed in a predetermined shape is connected to a predetermined end side of a plurality of end portions of the microchannel, and the microchannel A decompression chamber having an internal space that communicates only with the inside and is shielded from the outside, and a remaining end portion excluding an end portion to which the decompression chamber is connected among a plurality of end portions of the microchannel. A plurality of ports opened to the outside for introducing liquid into the microchannel, and at least one of the microchannel or the decompression chamber is made of a polymer material. formed, dissolved by returning to the atmosphere, the air between the inner microchannel and the decompression chamber to the polymeric material after degassing the polymeric material in a low pressure environment And dropping the liquid into at least one of the plurality of ports and sealing the other port to create a decompressed space formed by the microchannel and the decompression chamber, and the decompression The liquid is sucked into the formed space .

  The invention according to claim 3 of the present invention is the invention according to any one of claims 1 or 2, wherein one or more narrow passages are formed in the microchannel. It is.

  The invention according to claim 4 of the present invention is the invention according to claim 3 of the present invention, wherein two of the narrow passages are formed.

Further, the invention according to claim 5 of the present invention includes a mixing channel in which a plurality of introduction channels are connected to one end and the other end is connected to a decompression chamber, and the plurality of channels A plurality of ports that are formed on an end portion side different from the end portion on the side communicating with the mixing flow channel of each of the introduction flow channels and open to the outside in order to introduce liquid into the introduction flow channel; Sealing means for communicating the interior space of the decompression chamber only with the inside of the mixing flow path and shielding the outside, and each of the sealing means, the decompression chamber, or the mixing flow path By forming at least one of the whole or a part of the polymer material and degassing the polymer material in a low-pressure environment and returning it to the atmosphere, the inside of the introduction channel and the mixing channel Air in the decompression chamber and the inside of the decompression chamber is dissolved in the polymer material, The liquid is dropped into at least one of the ports and the other port is sealed to create a decompressed space formed by the introduction channel, the mixing channel, and the decompression chamber. The liquid is sucked into the decompressed space .

  Further, the invention described in claim 6 among the present inventions is the invention described in claim 5 among the present inventions, wherein one or more narrow passages are formed in the mixing channel.

  The invention according to claim 7 of the present invention is the microfluidic control mechanism according to claim 6 of the present invention in which two narrow passages are formed.

  The invention according to claim 8 of the present invention is any one of claims 1, 2, 3, 4, 5, 6, or 7. In the invention described in the item, the polymer material is rubber.

  In addition, the invention according to claim 9 of the present invention is the invention according to claim 1, claim 2, claim 3, claim 4, claim 5, claim 6, claim 7 or claim 8. A microchip having a microfluidic control mechanism according to any one of the inventions.

  The invention according to claim 10 of the present invention is the invention according to claim 9 of the present invention, wherein different types of liquids are introduced from the ports, and different types of liquids are formed in the narrow passages. An interface is formed.

  The invention according to claim 11 of the present invention is the solution according to claim 10 of the present invention, wherein at least one of the liquids is a solution containing a sample, and at least one of the other liquids interacts with the sample. It is made to be the solution containing the substance to be.

  The invention according to claim 12 of the present invention is the invention according to claim 11 of the present invention, wherein the sample is electrophoresed in the direction of the interacting substance to concentrate, separate, detect or detect the sample. A combination of these is performed.

  The invention according to claim 13 of the present invention is the invention according to any one of claims 11 or 12, wherein the sample is DNA and the interacting substance is This is a DNA-polymer complex.

  Since the present invention is configured as described above, it solves the problems 1 to 5 described above, has a simple structure, and uses external power to introduce liquid into the microchannel. There is an excellent effect that a microfluidic control mechanism and a microchip that are not required can be provided.

  Hereinafter, an example of an embodiment of a microfluidic control mechanism and a microchip according to the present invention will be described in detail with reference to the accompanying drawings.

  First, FIG. 1 shows a schematic top view of the structure of a microchip implementing a microfluidic control mechanism according to the present invention, and FIG. 2 (a) shows a cross section taken along line AA of the microchip shown in FIG. FIG. 2 is a diagram (schematic configuration longitudinal sectional view), and FIG. 2B is a sectional view (schematic configuration longitudinal sectional view) taken along line BB of the microchip shown in FIG.

  Here, the microchip 10 includes a first plate-like member 12, a second plate-like member 14, and a covering member 16 serving as a sealing unit disposed on the first plate-like member 12. It is configured.

  The first plate-like member 12 is formed with microchannels 20 that form a series of Y-shaped flow paths as viewed from above as microchannels.

  The microchannel 20 is a mixing channel 22 that is a microchannel that extends linearly when viewed from above, and a microchannel formed by bifurcating one end 22a of the mixing channel 22. The first introduction channel 24 and the second introduction channel 26 are provided.

  Here, the mixing flow path 22, the first introduction flow path 24, and the second introduction flow path 26 are formed in a groove shape in which the lower surface 12b side of the first plate-like member 12 is opened, and the lower surface 12b. The opening surface on the side is shielded by the second plate member 14.

  Further, one end 24 a of the first introduction flow path 24, that is, an end different from the end 22 a side of the mixing flow path 22 is formed on the upper surface 12 a side of the first plate-like member 12. In order to introduce a liquid such as a sample or reagent, the first sample port 30 communicates with the first sample port 30 that is open to the outside. On the other hand, one end portion 26 a of the second introduction flow channel 26, that is, the end portion on the side different from the end portion 22 a side of the mixing flow channel 22 is formed on the upper surface 12 a side of the first plate member 12. In order to introduce a liquid such as a sample or a reagent, the second sample port 32 communicates with the second sample port 32 that is open to the outside.

  Therefore, the end 24a of the first introduction flow path 24 and the end 26a of the second introduction flow path 26 are open to the atmosphere. On the other hand, the other end 24 b of the first introduction flow channel 24 and the end 26 b of the second introduction flow channel 26 communicate with one end 22 a of the mixing flow channel 22.

  The other end 22 b of the mixing channel 22, that is, the end on the side different from the side communicating with the first introduction channel 24 and the second introduction channel 26 is the side of the decompression chamber 40. It communicates with the opening 40a. That is, one end 22a of the mixing flow path 22 is connected to a plurality of introduction flow paths of the first introduction flow path 24 and the second introduction flow path 26, and the other end 22b is connected to the other end 22b. The decompression chamber 40 is connected.

  The decompression chamber 40 is formed in a cylindrical shape that opens at a substantially circular upper opening 40b on the upper surface 12a of the first plate-like member 12 and opens at a substantially circular opening on the lower surface 12b side. The opening on the lower surface 12 b side is shielded by the second plate member 14.

  On the other hand, the covering member 16 serving as a sealing means is disposed in the upper opening 40 b of the decompression chamber 40, and the upper opening 40 b is sealed by the covering member 16. Thereby, the internal space 40c of the decompression chamber 40 communicates only with the inside of the mixing channel 22 and is shielded from the outside. That is, the other end 22b of the mixing channel 22 is open to the internal space 40c of the decompression chamber 40, but is not open to the atmosphere.

  Here, as the material of the first plate member 12, the second plate member 14, and the covering member 16 constituting the microchip 10, a polymer material can be used.

  For example, rubber, which is one of the polymer materials, has a very small solid microstructure and a considerable degree of freedom of movement of solid molecules, so that gas can easily get into the solid, and so many It is a solid material in which gas dissolves. The first plate member 12, the second plate member 14, and the covering member 16 may be formed of a polymer material such as rubber, which is a solid material in which a large amount of gas can be dissolved.

  In this embodiment, the material of the first plate member 12, the second plate member 14 and the covering member 16 constituting the microchip 10 is rubber, more specifically, polydimethylsiloxane (polydimethylsiloxane). (Hereinafter referred to as “PDMS”).

here,
C: Gas concentration in solid (cm ³ (STP) / cm ³ )
P: Pressure of gas in contact with solid (atm)
S: Solubility coefficient (cm ³ (STP) / (cm ³ atm))
Henry's Law C = SP
Thus, the solubility coefficient of PDMS in air can be calculated. The gas concentration C is obtained by converting the amount of gas dissolved in a solid of 1 cm ³ into a volume in a standard state (STP, 0 ° C., 1 atm).

And when the temperature is 35 ° C., the air is 80% nitrogen, and the oxygen is 20%, the solubility coefficient of PDMS in air is
S = 0.11 cm ³ (STP) / (cm ³ atm)
It becomes. However, the solubility coefficient S of PDMS in the air depends on the degree of polymerization of PDMS.

The material of the first plate member 12, the second plate member 14, and the covering member 16 constituting the microchip 10 is preferably a solid material in which a large amount of gas can be dissolved. For example, the above-described PDMS As described above, a polymer material having a solubility coefficient S in air of S = 0.001 to 10 (cm ³ (STP) / (cm ³ atm)) can be used.

  Of course, the materials of the first plate-like member 12, the second plate-like member 14 and the covering member 16 constituting the microchip 10 are not limited to those described above. It can be appropriately selected according to the type of liquid, such as a sample or a reagent, when performing the experiment.

  The first plate-like member 12 of the microchip 10 has a rectangular parallelepiped shape having a length L1 of 30 mm, a length L2 of 30 mm, and a thickness L3 of 2 mm, for example. For example, the shape member 14 is set to a dimension that substantially coincides with the first plate member edge 12.

  The length L4 from the end 22a to the end 22b of the mixing channel 22 of the microchannel 20 is set to 14 mm, for example, and the width L5 of the mixing channel 22 is set to 100 μm, for example. The depth L6 of the mixing channel 22 is set to 25 μm, for example.

  The length from the end 24a to the end 24b of the first introduction flow path 24 and the length L7 from the end 26a to the end 26b of the second introduction flow path 26 are, for example, 4 mm. Is set. Note that the widths of the first introduction flow path 24 and the second introduction flow path 26 are set, for example, to dimensions that substantially match the width L5 of the mixing flow path 22, and the first introduction flow path 24 and the second introduction flow path 24 The depth of the introduction channel 26 is set to a dimension that substantially matches the depth L6 of the mixing channel 22, for example.

  The diameter L8 of the first sample port 30 and the second sample port 32 is set to 2 mm, for example. The diameter L9 of the upper opening 40b of the decompression chamber 40 is set to 1 mm, for example.

  In addition, the dimension of each above-mentioned component part shows an example of the magnitude | size of each component part, Comprising: This invention is not limited to the magnitude | size of the said filled-in dimension. Accordingly, the dimensions of the above-described components may be appropriately changed according to the required reaction time, flow rate, etc. For example, when it is desired to ensure a relatively long reaction time, the length L4 of the mixing channel 22 is extended. What is necessary is just to make the flow velocity comparatively slow, and what is necessary is just to make small the cross-sectional area of the flow path 22 for mixing.

  In the above configuration, in order to degas the microchip 10, it is placed in a vacuum chamber having a pressure of 10 kPa for 0.5 to 3 hours (see FIG. 3A). In this way, by placing the entire microchip 10 in a low-pressure environment, the air dissolved in the entire microchip 10 can be removed (see broken line arrows in FIG. 3A). That is, the gas dissolved in the PDMS forming the microchip 10 is removed.

  Then, after a predetermined time has elapsed since the start of deaeration, the microchip 10 is taken out from the vacuum chamber into the atmosphere (100 kPa) (see FIG. 3B). Thus, after the deaeration in the vacuum chamber is finished and the microchip 10 is taken out to the atmosphere and returned to the atmospheric pressure, the re-dissolution of the air into the PDMS forming the microchip 10 is started (FIG. 3 ( (See broken line arrow in b)).

  Such re-dissolution of air into PDMS is a phenomenon based on Henry's law regarding gas solubility. That is, silicone rubber such as PDMS has an equilibrium solubility proportional to the partial pressure of the contact gas like a liquid. Thus, by returning from low pressure to atmospheric pressure, the equilibrium solubility rises again and the system begins to redissolve air into PDMS towards this new equilibrium.

As described above, PDMS having a solubility coefficient S = 0.11 (cm ³ (STP) / (cm ³ atm)) is a solid material in which a large amount of gas can be dissolved. When the external environment of the chip 10 is returned to the atmospheric pressure from the low pressure environment, a lot of air is newly dissolved in the entire microchip 10.

  After being taken out to the atmosphere in this way, the first liquid is dropped into the first sample port 30 of the microchip 10.

  Here, among the three flow paths constituting the microchannel 20, that is, the mixing flow path 22, the first introduction flow path 24, and the second introduction flow path 26, the end 22b side of the mixing flow path 22 is provided. Is not previously opened to the atmosphere by the covering member 16, and the end 24 a of the first introduction flow path 24 is sealed with the first liquid dropped on the first sample port 30. As a result, when the first liquid is introduced into the microchannel 20, only the end portion 26a of the second introduction flow path 26 is open to the atmosphere.

  Next, the second liquid to be mixed with the first liquid is dropped into the second sample port 32 of the microchip 10.

  As a result, the end portion 26a of the second introduction flow channel 26 is sealed by the second liquid dropped on the second sample port 32, so that each flow channel formed in the microchannel 20 is exposed to the outside. On the other hand, the operation of closing the first liquid and the second liquid into the mixing channel 22 is started.

  More specifically, in a state where the first liquid is dropped on the first sample port 30 and the second liquid is dropped on the second sample port 32, each flow path formed in the microchannel 20 is exposed to the outside. Since the air in the mixing channel 22 and the air in the internal space 40c of the decompression chamber 40 in which the end 22b of the mixing channel 22 is open are closed, the first sample port 30 and the second sample port 30 are closed. Loss of communication with the outside of the microchip 10 via the sample port 32 and is located in a space (hereinafter referred to as “closed space”) formed by the closed flow paths of the microchip 10 and the decompression chamber. Will come to do.

  Here, since the microchip 10 is taken out from the vacuum chamber into the atmosphere as described above, the re-dissolution of air into the PDMS forming the microchip 10 has started (see FIG. 3B). The air located in this closed space dissolves in PDMS from the inner wall 22c of the mixing flow path 22 and the inner wall 40d of the decompression chamber 40 (see the broken line arrow in FIG. 3D).

  As a result, the pressure of the closed space formed by the mixing channel 22 and the internal space 40c of the decompression chamber 40 decreases, and the first liquid in the first introduction channel 24 and the second liquid in the second introduction channel 26 are reduced. The second liquid flows into the mixing flow path 22 at the same time (see FIG. 3C). Thus, the two liquids of the first liquid and the second liquid are mixed in the mixing channel 22.

  As described above, in the microchip 10 in which the microfluidic control mechanism according to the present invention is implemented, the first plate member 12, the second plate member 14, and the covering member 16 are formed by PDMS and deaerated. When the microchannel 20 is closed to the outside by the two liquids to be mixed in the state where the re-dissolution of the air into the PDMS is started, a decompressed space is created in the microchannel 20 and the liquid is decompressed. The suctioned space is automatically introduced into the mixing channel 22.

  Thus, the present invention has a simple structure and does not require external power to introduce the liquid into the microchannel. For this reason, according to the present invention, Problem 1, Problem 2, and Problem 3 shown in the section “Prior Art” are solved, and the overall apparatus configuration can be simplified. It is possible to shorten the entire work time including the preparatory operation for the experiment, and it is possible to reduce the usage amount of the sample and the reagent.

  Furthermore, the present invention does not require power from the outside of the microchip as a trigger for starting to introduce liquid into the microchannel, so that the micro flow is completely without power from the outside of the microchip. A liquid can be introduced into the passage, and the problem 4 shown in the section “Prior Art” can be solved.

  Further, according to the present invention, since the two liquids of the first liquid and the second liquid can be mixed in the mixing flow path 22, the problem 5 shown in the section “Prior Art” is solved. can do.

  Here, in the conventional microchip, when the liquid flows in the microchannel, an air bag may adhere to the inner wall of the microchannel, thereby blocking the flow of the liquid.

  On the other hand, in the microchip 10 in which the microfluidic control mechanism according to the present invention is implemented, the first plate member 12 and the second plate member 14 are formed by PDMS. That is, all the inner walls of the mixing flow path 22, the first introduction flow path 24, and the second introduction flow path 26 are formed by PDMS. As a result, when the liquid flows through each of the mixing channel 22, the first introduction channel 24, and the second introduction channel 26, the mixing channel 22, the first introduction channel 24, and the first introduction channel 24. 2 Even if bubbles adhere to each inner wall of the introduction channel 26, the gas is immediately dissolved in PDMS, and the bubbles are sucked and disappear. Therefore, the liquid flow is not blocked in the microchannel 20, a stable liquid flow with good reproducibility can be obtained, a good operating state can be maintained, and a highly accurate experimental result can be obtained. it can.

Here, in order to visualize the flow, the results of experiments using an aqueous solution of fluorescent fine particles will be described. The average diameter of the fluorescent fine particles is 2 microns, and the concentration is 6.5 × 10 ⁷ particles / mL.

  FIG. 4 shows a photomicrograph showing the flow of the solution when the two liquids are mixed in the microchannel 20. The two liquids introduced by the photographed fluorescent fine particles reach from the first sample port 30 and the second sample port 32 to one end portion 22a of the mixing flow path 22 which is a merging point (FIG. 4). A left arrow and a right arrow in FIG. 4) and a flow proceeding toward the decompression chamber 40 (see a total arrow in FIG. 4) are observed.

  The result of analyzing the fluorescent fine particle image shown in FIG. 4 and calculating the flow rate of the solution is shown in FIGS.

  FIG. 5 shows a graph showing the change over time in the total flow rate when the deaeration time is changed. The “deaeration time” is the time for placing the microchip 10 in a vacuum chamber with a pressure of 10 kPa, and was changed to 0.5 hours, 1 hour, 2 hours and 3 hours. The “total flow rate” is a flow rate corresponding to the total arrow in FIG. 4, that is, a flow rate of the solution flow in the mixing channel 22 of the microchip 10. The “volume flow rate (nL / s) of the total flow rate” is equal to the product of the “average flow velocity (mm / s) of the total flow rate” and the cross-sectional area of the mixing flow path 22, The flow rate corresponds to the Total arrow. The “interval before solution injection” means that the first liquid is dropped into the first sample port 30 of the microchip 10 taken out into the atmosphere after the deaeration in the vacuum chamber is finished, This is the time until the second liquid is dropped into the sample port 32, and is 5 minutes. The “time” is an elapsed time after the second liquid is dropped on the second sample port 32.

  From the result shown in FIG. 5, the total flow rate in the mixing channel 22 after the two introduced liquids merge at one end 22a of the mixing channel 22 which is the merging point decreases with time, and It can be seen that it hardly depends on the deaeration time. That is, according to the microchip 10 of the present invention, a very small flow rate, for example, a flow rate of 1 mm / s can be obtained with good reproducibility.

  In FIG. 5, the total flow rate in the mixing flow path 22 decreases with time because the intake capacity of the PDMS forming the microchip 10 decreases with the passage of time and the flow of liquid proceeds. This is considered to be due to the fact that the inside of the mixing channel 22 and the internal space 40c of the decompression chamber 40 are filled with the liquid, and the intake area of the PDMS is reduced.

  FIG. 6 shows a graph showing the change over time in the ratio of the flow rate of one of the tributaries to the total flow rate. Note that “deaeration time”, “interval before solution injection”, and “time” in FIG. 6 are the same as those described in FIG. The “total flow rate” is a flow rate corresponding to the total arrow in FIG. 4, that is, a flow rate of the solution flow in the mixing channel 22 of the microchip 10. The “left branch flow rate” is a flow rate corresponding to the Left arrow in FIG. 4, that is, a flow rate of the solution in the first introduction flow path 24 of the microchip 10 and a flow rate of one of the tributaries. is there.

  From the result shown in FIG. 6, the ratio of the left branch flow rate / total flow rate is close to 0.5. According to the microchip 10 of the present invention, the first liquid and the second liquid in the first introduction flow path 24 The second liquid in the introduction flow channel 26 is evenly mixed, and two liquids that are ideally balanced can be realized.

  In the case shown in FIG. 6, when the elapsed time after dropping the second liquid on the second sample port 32 is 6 minutes or more, the ratio of the left tributary flow rate / total flow rate is 0.5. There is a tendency to deviate. As the elapsed time becomes longer, the mixing of the two liquids progresses and the flow rate becomes smaller, so the deviation of the left branch flow rate / total flow rate ratio from 0.5 appears significantly.

  Here, in order to maintain the ratio of the left tributary flow rate / total flow rate close to 0.5 even after a predetermined time has elapsed, for example, the shape of the first sample port 30 and the second sample port 32 and the inner wall surface It is better to improve the machining accuracy. Further, in each of the first introduction flow path 24 and the second introduction flow path 26, the liquid surface shapes of the first liquid and the second liquid are maintained so that the pressures of the two liquids become equal. Then, the flow of each of the two liquids is symmetric, and the ratio of the left tributary flow rate / total flow rate can be maintained close to 0.5 even when the flow rate decreases after a predetermined time has elapsed.

  FIG. 7 shows a graph showing the change over time in the total flow rate when the interval before solution injection is changed. Note that “degassing time”, “volume flow rate of total flow rate (nL / s)”, “average flow rate of total flow rate (mm / s)”, “interval before solution injection”, and “time” in FIG. This is the same as that described in FIG. The “degassing time” was fixed at 1 hour, and the “interval before solution injection” was changed to 3, 5, 10 and 15 minutes.

  From the results shown in FIG. 7, it can be seen that the interval before the solution injection greatly affects the total flow rate. Thus, since the interval before the solution injection greatly affects the total flow rate, the second liquid is dripped into the second sample port 32 in a relatively short time, for example, 5 minutes to 10 minutes before the solution injection. Good.

  Next, the manufacturing method of the microchip 10 which implemented the microfluidic control mechanism by this invention is demonstrated.

  Here, the outline of the manufacturing process of the microchip in which the microfluidic control mechanism according to the present invention is implemented is shown in time series in the order of FIGS. 8 (a), (b), (c), and (d).

  First, the first plate-like member 12 of the microchip 10 is manufactured by the following molding technique. That is, in order to form a reversal pattern for forming a microchannel 20 having a predetermined height, a super-thick film photoresist (SU-8: manufactured by Microchem, USA) is spin-coated on a silicon substrate, and manufactured. Process according to the manufacturer's instructions. Thereafter, the reverse pattern is exposed and developed.

After development, in order to strengthen the adhesion, it is baked in an oven for 4 minutes at 150 ° C. and then slowly cooled to room temperature over 1-2 hours. In order to improve the mold release, the silicon substrate was subjected to a fluorocarbon layer polymerized by CHF ₃ plasma in a reactive ion etching (RIE) machine (RIE-10NR: manufactured by Samco International Laboratories, Japan). Film is formed for 2 minutes. The conditions at that time are a CHF ₃ gas flow rate of 50 sccm, a pressure of 20 Pa, and an electric power of 200 w.

  Further, an unpolymerized solution of PDMS (Sylgard 184: manufactured by Dow Corning, USA) is poured onto the silicon substrate using a mold that holds the solution (see FIG. 8A). On the other hand, a first cure at 65 ° C. for 1 hour and a second cure at 100 ° C. for 1 hour are performed. The cured PDMS chip is peeled from the silicon substrate.

  The PDMS chip manufactured in this way is punched using a metal pipe, so that round holes corresponding to the first sample port 30, the second sample port 32, and the decompression chamber 40 are formed (FIG. 8). (See (b)).

  The first plate-like member 12 manufactured as described above is reversibly bonded by simply contacting the surface of the second plate-like member 14 that is also made of a PDMS chip (see FIG. 8C). .

  Then, when a tape made of PDMS is applied to the upper surface 12a of the first plate member 12 so as to cover the opening of the decompression chamber 40 (see FIG. 8D), the microchip 10 is completed. In this case, a commercially available adhesive tape may be used in place of the tape made of PDMS.

  The embodiment described above can be modified as shown in the following (1) to (11).

  (1) In the above-described embodiment, the microchip 10 is manufactured by the manufacturing process described above with reference to FIGS. 8A, 8B, 8C, and 8D. Of course, the microchip may be manufactured by an appropriate manufacturing process according to the type of material forming the microchip.

  (2) In the above-described embodiment, the microchip 10 is configured by the first plate-like member 12, the second plate-like member 14, and the covering member 16, but is not limited thereto. Of course, the portions corresponding to the first plate-like member 12, the second plate-like member 14, and the covering member 16 are all integrally formed by using an appropriate manufacturing process. Alternatively, the first plate member 12 and the covering member 16 may be integrally formed (see the microchip 10 ′ shown in FIG. 9). As described above, when the covering member 16 is formed integrally with the first plate-like member 12, a region that shields the internal space 40c of the decompression chamber 40 from the outside is a sealing means.

  (3) The microchip 10 according to the above-described embodiment is used after being deaerated in the vacuum chamber and then taken out from the vacuum chamber to the atmosphere. However, the present invention is not limited to this. Of course. For example, if the microchip 10 that has been previously deaerated is stored in a sealed container (box or bag) that can be kept in a vacuum, it can be easily stored and transported, and can be mass-produced or marketed. In use, the user can immediately use the container by opening the container and taking out the microchip.

  (4) In the microchip 10 of the above-described embodiment, the first plate member 12, the second plate member 14, and the covering member 16 are all formed by PDMS (FIG. 10 ( Of course, the present invention is not limited to this, and the whole or part of the first plate-like member 12, the whole or part of the second plate-like member 14, the whole of the covering member 16 or Some of these may be formed by PDMS.

  For example, as shown in FIG. 10 (b), the entire first plate member 12 and the entire covering member 16 may be formed by PDMS, or as shown in FIG. 10 (c), Only the entire covering member 16 may be formed by PDMS, or only a part of the first plate-like member 12 may be formed by PDMS as shown in FIG. As shown in FIG. 10E, a part of the first plate member 12 and a part of the second plate member 14 may be formed by PDMS. In 10 (a), (b), (c), (d), and (e), the region indicated by the pear ground indicates the region formed by PDMS.

  That is, the region formed of the polymer material such as PDMS is not limited to the entire microchip 10, but the entire or a part of the covering member 16 and the entire or one part of the mixing channel 22 of the microchannel 20. Part, the whole or a part of the decompression chamber 40, or at least one of them.

  However, since the capacity of the entire region formed by the polymer material such as PDMS affects the gas concentration inside the polymer material, the microchannel 20 is within a range where a decompressed space can be generated in the microchannel 20. The region formed of the polymer material may be appropriately changed according to the overall dimensions or the overall dimensions of the decompression chamber 40.

  Furthermore, only a portion formed of the polymer material may be deaerated in advance, and then assembled with other portions not formed of the polymer material to form a microchip. For example, in the case shown in FIG. 10C, since the first plate member 12 and the second plate member 14 are not formed by PDMS, only the covering member 16 formed by PDMS is deaerated. What is necessary is just to assemble | attach to the upper surface 12a of the 1st plate-shaped member 12 after processing.

  (5) In the above-described embodiment, the first introduction channel 24 and the second introduction channel 26 are formed as the introduction microchannels. However, the present invention is not limited to this. Yes, the number of introduction flow paths connected to one end of the mixing flow path may be three or more.

  (6) In the microchip 10 of the above-described embodiment, the liquid is dropped into all the ports of the microchannel 20, that is, the first sample port 30 and the second sample port 32, and the microchannel 20 is closed to the outside so that the first liquid and the second liquid flow into the mixing flow path 22 at the same time. However, the liquid is limited to dropping a predetermined liquid in all of the plurality of ports. It is not a thing.

  For example, in the microchip 10 according to the above-described embodiment, a predetermined molecule serving as a sample is immobilized in the mixing flow path 22 in advance, and a predetermined liquid is dropped onto the first sample port 30, and then the second sample. When a tape is applied to the upper surface 12a of the first plate member 12 so as to cover the use port 32, the microchannel 20 is closed to the outside, and the solution dropped into the first sample port is used for mixing. The solid phase reaction can be performed by flowing into the flow path 22.

  In addition, when three or more introduction channels are formed, it is not necessary to use all the introduction channels to mix two or more liquids. If a predetermined liquid is dropped on any one of the three or more ports formed and the remaining ports are shielded from the outside, the microchannel is closed to the outside, Two or more solutions flow into the mixing channel 22 and are mixed.

  (7) In the above-described embodiment, a series of Y-shaped flow paths are formed as the microchannel 20 as viewed from above, but the present invention is not limited to this. As the shape, an appropriate shape can be adopted depending on the total number of the mixing channels, the size of the port, and the like.

  Further, the total number of microchannels formed on a single microchip is not limited to one as in the microchip 10 of the above-described embodiment, and a plurality of microchannels may be formed. .

  (8) Without being limited to the microchip 10 of the above-described embodiment, for example, the microfluidic control mechanism according to the present invention is provided in the microchip 100 (see FIGS. 11 and 12), and the cross is viewed from above. If a series of microchannels 120 are formed, the microchip 100 can be used as a microchip for electrophoresis.

  Specifically, the microchip 100 includes a first plate member 112 and a second plate member 114. The first plate member 112 is made of PDMS, and the second plate member 114 is made of glass.

  The cross-shaped microchannel 120 includes a first channel 122 having both ends 122a and 122b, and a second channel 124 having both ends 124a and 124b. It is what has. The first flow path 122 and the second flow path 124 are formed so as to be substantially orthogonal at the intersection 120 a, and the total length of the first flow path 122 is larger than the total length of the second flow path 124. Has been made short. 11 and 12 show an example of the size of each component, but it is of course not limited to the size of the entered dimension. .

  Compared with the microchip 10 (see FIGS. 1 and 2) of the above-described embodiment, the microchip 100 has a different total number of ports because the shape of the entire microchannel is different. The covering member 16 that shields the decompression chamber 40 and the internal space 40c of the decompression chamber 40 from the outside is not provided.

  When performing electrophoretic separation using the microchip 100 having the above-described configuration, first, in the same manner as the microchip 10 of the above-described embodiment, in order to degas the microchip 100, a vacuum chamber of 10 kPa pressure is set to 0. Put in 5 to 3 hours (for example, 1 hour). As a result, the gas dissolved in the PDMS forming the first plate-like member 112 of the microchip 100 is removed.

  Then, after a predetermined time has elapsed from the start of deaeration, the microchip 100 is taken out from the vacuum chamber into the atmosphere (100 kPa). As a result, re-dissolution of air into the PDMS that forms the first plate-like member 112 of the microchip 100 starts.

  A liquid as a separation material for electrophoretic separation is dropped onto all the ports of the microchip 100 thus taken out into the atmosphere. More specifically, a port 131 that communicates with the end 122a of the first flow path 122, a port 132 that communicates with the end 122b, a port 133 that communicates with the end 124a of the second flow path 124, and an end 124b. A polymer (polymer) solution such as an aqueous polyacrylamide solution that serves as a sieve for separating DNA and the like is dropped into each of the four ports of the communicating port 134.

  As a result, all of the plurality of ports formed at each of the plurality of ends of the microchannel 120 are sealed with the polymer solution dropped onto each of the ports, so that each flow path formed in the microchannel 120 Is closed to the outside. As a result, the air in the first flow path 122 and the air in the second flow path 124 are located in a space formed by each closed flow path of the microchip 100. The air located in this space is the inner wall formed by the first plate member 112 of the first flow path 122 or the inner wall formed by the first plate member 112 of the second flow path 124. To PDMS.

  As a result, the pressure in the space formed by each closed channel of the microchip 100 decreases, and the end 122a and end 122b of the first channel 122 and the end 124a and end of the second channel 124 are reduced. From each of the parts 124b toward the intersection 120a, the polymer solution flows into the first flow path 122 and the second flow path 124, and the microchannel 120 is filled with the polymer solution.

  After the polymer solution is filled in the microchannel 120 in this way, a sample is introduced from the port 131 through the platinum electrode attached to each of the ports 131, 132, 133, and 134, and the first flow path 122 is used for sample injection. Electrophoretic separation is performed using the second channel 124 as a separation channel.

  As described above, according to the microchip 100 provided with the microfluidic control mechanism according to the present invention, a part of the microchannel 120 is formed by PDMS and degassed, so that a decompressed space is formed in the microchannel. The microchannel 120 can be automatically filled with a polymer solution for electrophoretic separation.

  Here, in a microchip used for conventional electrophoresis analysis, a syringe is connected to the microchannel, and the polymer solution is pumped to fill the microchannel. For this reason, a syringe or the like is required as an external device, and the configuration of the entire device is complicated and expensive. Further, in the case of pressure feeding using a conventional syringe, it is difficult to fill the polymer solution so that bubbles do not occur in the microchannel, particularly when the surface of PDMS is hydrophobic.

  On the other hand, the microchip 100 having the microfluidic control mechanism according to the present invention does not require external power to introduce liquid into the microchannel. Compared with the microchip used for the above, the overall device configuration can be simplified. Furthermore, according to the present invention, even if the microchannel is formed by PDMS or has a complicated shape, there is no air bubble in the polymer solution automatically filled in the microchannel, which is good. Electrophoretic separation can be performed in the operating state.

  As described above, the microfluidic control mechanism according to the present invention generates a decompressed space in the microchannel as in the microchip 100 described above without the decompression chamber 40 and the covering member 16 of the microchip 10. This is useful for filling each flow path of the microchannel with a predetermined single type of liquid without bubbles.

  The entire microchannel 120 of the microchip 100 described above may be formed of a polymer material such as PDMS, or instead of the port 132 with which the end 122b of the first flow path 122 communicates, A decompression chamber may be connected to the end 122b.

  (9) In the embodiment described above, the microfluidic control mechanism according to the present invention is provided in the microchip. However, the present invention is not limited to this. For example, various systems for handling a small amount of liquid In addition, the microfluidic control mechanism according to the present invention may be employed.

  Further, when an enzyme solution is used as the first liquid to be mixed in the microchip 10 and a sample is used as the second liquid, the enzyme reaction can be observed, and the present invention is not limited to this. Various solutions can be used.

  (10) In the above-described embodiment, the microchannels 20 and 120 are formed in a straight line and the cross-sectional area thereof is substantially constant. However, the cross-sectional area of the microchannels 20 and 120 is not limited to this. You may make it form the narrow channel | path which is a location which becomes rapidly small. Thus, when a narrow passage is formed where the cross-sectional area of the microchannels 20 and 120 is abruptly reduced, the cross-sectional area is abruptly reduced, and the narrow passage acts as a resistance to the movement of the solution. It will be.

  For example, FIG. 13 shows an example in which the protruding portion 200 is formed so as to face the inner wall of the mixing flow path 22 in the microchannel 20 of the microchip 10 and the narrow passage 202 is formed. 14 shows an example in which the narrow passage 302 is formed by forming the protruding portion 300 so as to face the inner wall of the second flow path 124 in the microchannel 120 of the microchip 100.

  Here, FIG. 15 shows another shape of the microchannel of the microchip having such a narrow passage.

  That is, the microchip 400 shown in FIG. 15 differs from the above-described microchips 10 and 120 only in the shape of the microchannel 402, and the material and manufacturing method are the same as those of the above-described microchips 10 and 120. The detailed explanation is omitted.

  The microchannel 402 includes a first flow path 404 and a second flow path 406 formed in an inverted T shape. That is, one end 406 a of the second flow path 406 is connected so as to be orthogonal to the first flow path 404.

  One end 404a of the first flow path 404 communicates with the first port 408, which is an opening that opens to the outside, and the other end 404b of the first flow path 404 is connected to the outside. The other end 406b of the second flow path 406 communicates with the third port 412 that is an opening that is open to the outside. .

  Further, in this microchannel 402, two narrow passages, a first narrow passage 414 and a second narrow passage 416, are formed in the first flow path 404, and the first narrow passage 414 includes the first narrow passage 414 and the first narrow passage 414. Formed between the connection portion between the first flow path 404 and the second flow path 406 and the connection portion between the first flow path 404 and the second flow path 406. On the other hand, the second narrow passage 416 is located between the connection between the first flow path 404 and the second flow path 406 and the second port 410 and closer to the second port 410. Is formed.

  Here, the first narrow passage 414 is configured by arranging two protrusions 418 on the inner wall of the first flow path 404 so as to face each other with a predetermined pitch shift. Similarly, the second narrow passage 416 is configured by arranging two protrusions 420 on the inner wall of the second flow path 406 so as to face each other with a predetermined pitch shift. Therefore, the first narrow passage 414 and the second narrow passage 416 are crank-shaped flow paths.

Next, a technique for concentrating DNA and performing sequence-specific separation using affinity microchip electrophoresis using the microchip 400 shown in FIG. 15 will be described. When the microchip according to the present invention such as the microchip 400 is used, the sample DNA can be concentrated and its sequence-specific separation can be performed simply and quickly in one operation.

  Specifically, according to the experiment conducted by the present inventor described below, the sample DNA was successfully concentrated 110 times or more and sequence-specific separation was successful. These can be done in one operation, and the time is only within 15 seconds. In addition, this method enables simple device production and detection operation for detection of single-base mutations in a diluted sample DNA.

  The dimensions of each part of the microchip 400 are as described in FIG. 15, and the cross-sectional shapes of the first channel 404 and the second channel 406 of the microchannel 402 are a width of 100 μm and a depth of 22 μm. The cross-sectional shape of the portion where the cross-sectional area of the first narrow passage 414 and the second narrow passage 416 is sharply reduced is a rectangular shape having a width of 10 μm and a depth of 22 μm. Further, the PDMS portion of the microchip 400 was produced by a casting method and was reversibly bonded to a slide glass.

In the experiment, an equimolar mixture of two types of fluorescently labeled single-stranded DNAs (K-ras gene codons 10 to 13 and a single nucleotide variant thereof) was used as a specimen (normal body (N): FITC-5′- GGA GCT GGT GGC-3 ′, mutant (M): FITC-5′-GGA GCT A GT GGC-3 ′). A DNA-poly (N, N-dimethylacrylamide) (DNA-PDMA) complex for concentration and separation of sample DNA is a single-stranded ligand DNA (5'-CAC) having N, N-dimethylacrylamide and a methacryloyl group. It was obtained by radical polymerization of CAG C-3 ′ (complementary to the central part of a normal body).

  FIGS. 16 (a), (b), (c), (d), and (e) show schematic diagrams of experimental operations performed by the inventors of the present application. The experimental operations will be described with reference to these drawings.

  First, the microchip 400 is held in the vacuum vessel 500 set to 10 kPa for 5 minutes, and the air dissolved in PDMS is degassed (FIG. 16 (a): degassing).

  Then, after the microchip 400 is taken out into the atmosphere, 3 μl of the sample DNA and the DNA-PDMA complex are dropped into the first port 408, the second port 410, and the third port 412 respectively (FIG. 16B). : Sample dropping).

  The solution dropped into the first port 408, the second port 410, and the third port 412 spontaneously flows into the microchannel 402, that is, the first flow path 404 and the second flow by the operation of the present invention described above. A liquid-liquid interface is formed in the vicinity of the first narrow passage 414 and the second narrow passage 416 (FIG. 16C: spontaneous solution filling and interface formation).

  Here, the plug length of the sample is determined by the distance between the two narrow passages, that is, the first narrow passage 414 and the second narrow passage 416, and the length thereof is 11 mm. After the platinum electrode was attached to the first port 408, the second port 410, and the third port 412, electrophoresis was performed by applying a high voltage (250 V / cm).

FIG. 17 shows a concentration profile, and FIG. 18 shows a fluorescence micrograph thereof (applied voltage 250 V / cm, 25 mM Tris-borate buffer solution (pH 7.4), 10 mM MgCl ₂ , initial DNA concentration 0.1 μM each, plug length 11 mm). ). The concentrations in FIG. 17 were determined with known concentration samples.

  Although the sample DNA is electrophoresed by applying a high voltage, the sample DNA is concentrated near the liquid-liquid interface due to a change in the solution composition near the liquid-liquid interface (FIG. 16 (d): concentration, FIGS. 17 and 18). 5s).

  Moreover, the normal body and the mutant were separated and detected only by continuing the electrophoresis without switching the voltage or the like (FIG. 16 (e): separation, 15s in FIGS. 17 and 18).

  A normal form having a large affinity with the ligand DNA was remarkably captured near the liquid-liquid interface by double helix formation. At this time, the peak concentration of the normal body was estimated to be 11 μM, which indicates that it was concentrated 110 times or more. On the other hand, the mutant had a small affinity with the ligand DNA, and thus migrated as it was without being captured by the ligand DNA, and was detected as a peak separated from the normal body.

  As described above, the above-described method using the microchip according to the present invention is easy to produce a device and is extremely useful as a rapid method for detecting a point mutation of a gene or a single nucleotide polymorphism.

Next, experimental results of DNA single-base mutation detection by affinity electrophoresis using a microchip having a form different from that of the microchip 400 will be described.

First, an experimental sample will be described with reference to FIG. 19. An equal amount mixture of two types of fluorescently labeled single-stranded DNAs (K-ras gene codons 10 to 13 and a single nucleotide variant thereof) was used as a specimen ( Normal body (N): FITC-5′-GGA GCT GGT GGC-3 ′, mutant (M): FITC-5′-GGA GCT A GT GGC-3 ′). A DNA-poly (N, N-dimethylacrylamide) (DNA-PDMA) complex for separation of sample DNA is a single-stranded ligand DNA (5′-CAC CAG C) having N, N-dimethylacrylamide and a methacryloyl group. -3 ', complementary to the central part of a normal body).

  Next, production of a device constituted by bonding a microchip according to the present invention and a glass substrate on which an electrode is formed will be described with reference to FIG.

  That is, the device is composed of an electrode portion patterned on a glass substrate and a PDMS chip (microchip according to the present invention) having a plurality of linear microchannels.

  For the electrode part, Cr / Au (10/100 nm) was vacuum-deposited on a glass substrate, and patterning was performed using a photolithography technique. The left and right electrode widths are 400 μm, and the center electrode width is 100 μm. FIG. 21 shows the shape of the electrode and the dimensions of each part.

  FIG. 22 is a schematic top view showing the arrangement relationship of microchannels, ports, and narrow passages in the PDMS chip. The PDMS chip is manufactured by a mold method, and six microchannels are formed. . Both ends of each of these six microchannels communicate with a port serving as an opening that opens to the outside.

  The cross-sectional shape of each microchannel is a rectangular shape having a width of 100 μm and a depth of 25 μm, and a narrow passage is formed near the center. The position of the narrow passage was set to a position of 500 μm from the center electrode when pasted to the glass substrate.

  Next, the experimental operation will be described with reference to FIG. 23. First, the device is held in a vacuum vessel (10 kPa) for 15 minutes, and air dissolved in PDMS is deaerated.

  Next, after being put in the atmosphere, 3 μl of the sample DNA is dispensed into each port on the left side of FIG. 22 and the DNA-PDMA complex is dispensed in 3 μl to each port on the right side of FIG.

  The solution is spontaneously filled into the microchannel by the action of the microchip of the present invention, and forms a liquid-liquid interface near the narrow passage. The plug length of the sample is determined by the distance between the central electrode and the narrow passage, and the length is 500 μm. After connecting the power source, electrophoresis was performed by applying a voltage (left: 10 V, center: 0 V, right: 10 V) to each electrode.

  FIG. 24 shows a case where the same specimen is simultaneously detected using six microchannels. In all the microchannels, normal and mutant were detected as two distinct bands 30 seconds after the start of electrophoresis.

In addition, FIG. Normal + mutant b. Mutants only c. Normal body only d. Experiments were performed using normal + mutants, a to c as DNA-PDMA complexes, and d as PDMA containing no ligand DNA.

  The “normal + variant” microchannel a was detected as two distinct bands after 60 seconds. Moreover, the expected result was obtained also about the microchannel of bc performed as control experiment.

In addition, a well-known document `` S. Kaneda and T. Fujii, Combining droplet-based liquid handling and on-chip capillary electrophoresis with a new sampling injection method (MA Northrup, KF Jansen, and DJ Harrison, Micro Total Analysis Systems 2003 , Vol. 2, Transducers Research Foundation, San Diego, 2003, pp. 1279-1282) ”, the interface between the DNA sample solution and the polymer solution is formed in the microchannel, followed by electrophoresis. A technique for separating a DNA sample is disclosed. The interface is formed by the technique of Japanese Patent Laid-Open No. 2000-27813 “Microfluidic control mechanism”, and it is not necessary to switch the voltage unlike the conventional microchip electrophoresis.

  However, in order to form the interface at a desired position by the method disclosed in the above document, the solution must be controlled using air pressure, and the structure and operation are complicated. Therefore, it is not easy to arrange a large number of channels on one chip and control them simultaneously, or to create two or more interfaces in one channel.

  Further, since the polymer solution does not contain immobilized DNA, it was impossible to concentrate 100 times or more and to perform sequence-specific separation as in the above-described method using the microchip according to the present invention.

  However, according to the above-described method using the microchip according to the present invention, it is easy to form a liquid-liquid interface at a desired position in the microchannel. There is no difficulty in creating two interfaces in one flow path.

  Here, by providing two interfaces, an electrophoresis port can be provided separately from the sample injection port, and a high voltage can be applied thereto. As a result, the analysis is speeded up.

  On the other hand, when there is only one interface, if it is assumed that the voltage is not switched, an electrode for electrophoresis must be provided in the flow path, and gas is generated by electrolysis when a high voltage is applied thereto. Therefore, there is a problem that the flow path is blocked and analysis cannot be performed.

  Further, the above-described method using the microchip according to the present invention can be combined with a known DNA-polymer complex technology. In this case, after forming an interface between the sample DNA solution and the DNA-polymer complex solution, a voltage is applied to cause the sample DNA to be electrophoresed in the direction of the DNA-polymer complex. The sample DNA is trapped by the DNA immobilized on the polymer and remarkably decelerated. In general, in the flow system, the concentration is inversely proportional to the mobility, so that the sample DNA is concentrated, for example, 100 times or more at the interface formed by the sample solution and the DNA-polymer complex solution.

  Furthermore, as another effect, known sequence-specific separation can be performed simultaneously with the concentration.

  That is, if the sample is a mixture of several DNAs and their sequences are slightly different, the mobility can be different due to the slightly different affinity for the immobilized DNA, so that they can be separated. It will be.

  In the present specification, the “interface” means a virtual plane in which the substance concentration changes abruptly at the boundary. Although the surface is considered to actually have a thickness of several μm to 100 μm by diffusion, it does not necessarily mean that it is not mixed at all.

  (11) The above-described embodiment and the modifications shown in (1) to (10) may be combined as appropriate.

  The present invention can be used when performing an experimental operation of a chemical experiment or a biological experiment, for example, an affinity capillary electrophoresis (ACE) method.

FIG. 1 is a top view of a schematic configuration of a microchip in which a microfluidic control mechanism according to the present invention is implemented. 2A is a cross-sectional view (schematic configuration longitudinal cross-sectional view) taken along line AA of the microchip shown in FIG. 1, and FIG. 2B is a cross-sectional view of the microchip shown in FIG. It is sectional drawing by a line (schematic structure longitudinal cross-sectional view). 3 is an explanatory view showing the operation of the microchip shown in FIG. 1, FIG. 3 (a) is an explanatory view showing a deaeration process, and FIG. 3 (b) is an explanatory view showing dropping of a liquid. FIG. 3 (c) is an explanatory view showing mixing of two liquids, and FIG. 3 (d) is a partially enlarged explanatory view of FIG. 3 (c). FIG. 4 is a photomicrograph showing the experimental results of the present inventors. FIG. 5 is a graph showing the time change of the total flow rate when the deaeration time is changed. FIG. 6 is a graph showing the change over time in the ratio of the flow rate of one of the tributaries to the total flow rate. FIG. 7 is a graph showing the time change of the total flow rate when the interval before solution injection is changed. FIGS. 8A, 8B, 8C, and 8D are schematic configuration explanatory views showing a manufacturing process of a microchip in which the microfluidic control mechanism according to the present invention is implemented. FIG. 9 is a schematic longitudinal sectional view showing another example of a microchip in which the microfluidic control mechanism according to the present invention is implemented. FIGS. 10 (a), (b), (c), (d), and (e) are schematic configuration explanatory views showing other examples of microchips that implement the microfluidic control mechanism according to the present invention. FIG. 11 is a top view of a schematic configuration of another example of a microchip in which the microfluidic control mechanism according to the present invention is implemented. 12 is a cross-sectional view (schematic configuration longitudinal cross-sectional view) taken along line CC of the microchip shown in FIG. FIG. 13 is a schematic top view illustrating an example of a microchip in which a narrow passage is formed in a microchannel. FIG. 14 is a schematic top view for explaining an example of a microchip in which a narrow passage is formed in a microchannel. FIG. 14 is a schematic top view for explaining an example of a microchip in which a narrow passage is formed in a microchannel. FIGS. 16 (a), (b), (c), (d), and (e) are schematic views showing an experimental operation performed by the present inventor. FIG. 17 is a graph showing a concentration profile of an experimental result conducted by the inventor of the present application. FIG. 18 shows fluorescence micrographs of the results of experiments conducted by the inventors of the present application (applied voltage 250 V / cm, 25 mM Tris-borate buffer solution (pH 7.4), 10 mM MgCl ₂ , initial DNA concentration 0.1 μM each, plug length) 11 mm). FIG. 19 is an explanatory diagram of an experimental sample in an experiment conducted by the inventor of the present application. FIG. 20 is an explanatory view showing a method of manufacturing a device constituted by bonding a microchip according to the present invention and a glass substrate on which an electrode is formed. FIG. 21 is an explanatory view showing the shape of the electrode and the dimensions of each part. FIG. 22 is a schematic top view showing the arrangement relationship of microchannels, ports and narrow passages in the PDMS chip. FIG. 23 is a schematic diagram showing an experimental operation performed by the inventor of the present application. FIG. 24 is a fluorescence micrograph of the experimental results of simultaneous measurement of a plurality of samples. FIG. 25 shows that “normal body + mutant”, “mutant only”, “normal body only”, “normal body + mutant” are used as specimens, and “normal body + mutant”, “mutant” are used as separation media. “Only” and “normal body only” are fluorescence micrographs of the results of an experiment using a DNA-PDMA complex, and “normal body + mutant” is a PDMA containing no ligand DNA.

Explanation of symbols

10, 10 ', 100 Microchip 12, 112 First plate member 12a Upper surface 12b Lower surface 14, 114 Second plate member 16 Cover member 20, 120 Microchannel 22 Mixing flow channel 22a, 22b End portion 22c Inner wall 24 first introduction flow path 24a, 24b end 26 second introduction flow path 26a, 26b end 30 first sample port 32 second sample port 40 decompression chamber 40a side opening 40b upper opening 40c inside Space 40d Inner wall 120a Intersection location 122 First flow path 122a, 122b End 124 Second flow path 124a, 124b End 131, 132, 133, 134 Port 200, 300 Protruding portion 202, 302 Narrow path 400 Microchip 402 Microchannel 404 First flow path 406 Second flow path 408 1 port 410 second port 412 third port 414 first narrow passage 416 second narrow passage 418, 420 protrusions 500 vacuum container

Claims (13)

Microchannels formed in a predetermined shape;
A plurality of ports formed at each of a plurality of ends of the microchannel and open to the outside for introducing a liquid into the microchannel;
All or part of the microchannel is formed of a polymer material;
By degassing the polymer material in a low pressure environment and returning it to the atmosphere, the air in the microchannel is dissolved in the polymer material,
A liquid is dropped into at least one of the plurality of ports and the other port is sealed to create a decompressed space in the microchannel, and the liquid is sucked into the decompressed space. microfluidic control mechanism, characterized in that that. Microchannels formed in a predetermined shape;
A decompression chamber having an internal space that is connected to a predetermined end side of the plurality of end portions of the microchannel and that is connected only to the inside of the microchannel and shielded from the outside;
A plurality of ports formed at each of the remaining end portions excluding the end portion to which the decompression chamber is connected, among the plurality of end portions of the microchannel, and open to the outside to introduce liquid into the microchannel And
Forming at least one of the whole or a part of each of the microchannel or the decompression chamber with a polymer material;
By degassing the polymer material in a low pressure environment and returning it to the atmosphere, the air in the microchannel and the decompression chamber is dissolved in the polymer material,
By dropping liquid into at least one of the plurality of ports and sealing the other port, a decompressed space formed by the microchannel and the decompression chamber is created, and the decompressed A microfluidic control mechanism , wherein the liquid is sucked into a space . The microfluidic control mechanism according to any one of claims 1 and 2,
A microfluidic control mechanism, wherein one or more narrow passages are formed in the microchannel. The microfluidic control mechanism according to claim 3,
A microfluidic control mechanism characterized in that two narrow passages are formed. A plurality of introduction channels connected to one end, and a mixing channel connected to the decompression chamber on the other end;
Each of the plurality of introduction flow paths is formed on an end portion side different from the end portion on the side communicating with the mixing flow path, and a plurality of openings are opened to introduce liquid into the introduction flow path. Port,
Sealing means for communicating the internal space of the decompression chamber only with the inside of the mixing flow path and shielding it from the outside;
Forming at least any one of the whole or a part of each of the sealing means, the decompression chamber or the mixing flow path with a polymer material;
By degassing the polymer material in a low-pressure environment and returning it to the atmosphere, the air in the introduction channel, the mixing channel, and the decompression chamber is dissolved in the polymer material,
A pressure-reduced space formed by the introduction flow path, the mixing flow path, and the decompression chamber by dropping liquid into at least one of the plurality of ports and sealing the other ports. And the liquid is sucked into the decompressed space . The microfluidic control mechanism according to claim 5,
A microfluidic control mechanism, wherein one or more narrow passages are formed in the mixing channel. The microfluidic control mechanism according to claim 6,
A microfluidic control mechanism characterized in that two narrow passages are formed. In the microfluidic control mechanism according to any one of claims 1, 2, 3, 4, 5, 6, or 7,
The polymer material is rubber. A microfluidic control mechanism. The microfluidic control mechanism according to any one of claims 1, 2, 3, 4, 5, 6, 7, or 8 is provided. Microchip. The microchip according to claim 9, wherein
A microchip, wherein different types of liquids are introduced from the ports to form an interface composed of different types of liquids in the narrow passage. The microchip according to claim 10, wherein
At least one of the liquids is a solution containing a sample, and at least one of the other liquids is a solution containing a substance that interacts with the sample. The microchip according to claim 11, wherein
A microchip, wherein the sample is electrophoresed in the direction of the interacting substance, and the sample is concentrated, separated, detected, or a combination thereof. The microchip according to any one of claims 11 and 12,
The microchip, wherein the sample is DNA, and the interacting substance is a DNA-polymer complex.