JP3749991B2 - Micro liquid weighing structure and microchip having the structure - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a micro liquid weighing structure. More specifically, the present invention relates to a micro liquid weighing structure suitable for use in performing analysis or chemical reaction using various samples, and a microchip having the structure in a substrate.
[0002]
[Prior art]
Conventionally, various apparatuses for performing analysis by electrophoresis, chromatography, and the like are known, and in such an apparatus, an accurate analysis result can be obtained by quantitatively weighing a liquid such as a sample to be used. It is something that can be done.
[0003]
For this reason, various methods for quantitatively weighing a liquid such as a sample in various apparatuses used for such electrophoresis and chromatography have been proposed. For example, Patent Document 1 describes a microchip electrophoresis apparatus, and Patent Document 2 describes an apparatus and method for electrophoretic separation of a fluid substance mixture. However, in any of these conventional methods, there is a problem that a sample more than the amount necessary for the analysis is actually required, and the dead volume of the sample cannot be reduced.
[0004]
Furthermore, in a chemical reaction or analysis using a liquid such as a small amount of sample, a microchip composed of a microchip may be used. Even when such a microchip is used, an accurate result can be obtained by quantitatively weighing the liquid such as the sample to be used. However, in the analysis using the microchip, the liquid such as the sample to be handled is used. Since the volume of the liquid is extremely small, it is difficult to weigh the liquid quantitatively, and various complicated structures are required for that purpose, and there is a problem that the operation for handling the structure becomes complicated.
[0005]
On the other hand, batch methods, dialysis methods, vapor diffusion methods, gel methods, and the like have been known as protein crystallization methods. However, each of these methods has a problem that a large amount of protein is required, and particularly when searching for a crystallization condition of a protein whose crystallization condition is unknown, a problem that a huge amount of protein is required. Had.
[0006]
In addition, in order to reduce the amount of protein to be used, a protein solution is dispensed by inkjet (for example, see Non-Patent Document 1) or electrospray (for example, see Patent Document 3), and the protein crystallization conditions are searched. The attempt was known. However, this method has a problem that since the droplets are exposed to the air, the droplets evaporate and dry or concentrate.
[0007]
Furthermore, attempts to produce crystals in a laminar flow using a microchannel device (for example, see Patent Document 4) or attempts to obtain crystals by strictly controlling temperature (for example, see Non-Patent Document 2) are known. It was. However, this method has a small reaction field and excellent reaction control. However, the introduction method to the crystallization site of the protein solution is the same as the conventional method, and the ratio of dead volume is extremely large. Had.
[0008]
[Patent Document 1]
Japanese Patent Laid-Open No. 10-148628 (see FIGS. 1 and 2)
[Patent Document 2]
JP-A-7-198680 (see FIGS. 1 and 2)
[Patent Document 3]
International Publication No. WO01 / 92293 Pamphlet
[Patent Document 4]
US Pat. No. 6,409,832
[Non-Patent Document 1]
“Journal of Applied Crystallography” (2002), 35, p. 278-281
[Non-Patent Document 2]
“Analytical Chemistry” (2002), 74, p. 3505-3510
[0009]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a trace liquid weighing structure that can quantitatively weigh a trace amount of liquid by a simple operation with a simple configuration.
[0010]
Another object of the present invention is to reduce the dead volume of the sample in various apparatuses that require quantitative liquid handling, and to realize space saving and cost reduction of the entire apparatus. It is an object of the present invention to provide a micro liquid weighing structure that can be used.
[0011]
[Means for solving problems]
The above-described problem is solved by the micro liquid weighing structure of the present invention. The trace liquid weighing structure of the present invention is made by taking advantage of the capillary phenomenon (capillary repulsive force) generated by the liquid with respect to the flow path by paying attention to the surface tension of the liquid.
[0012]
According to the first embodiment of the trace liquid weighing structure according to the present invention, the first flow path and the second flow path that are each extended in a predetermined direction, and the flow path wall of the first flow path The third flow path that opens and the flow path wall of the second flow path opens to connect one end of the third flow path and the second flow pathIn addition, it has a property that the capillary attraction is less likely to work than the third flow path.The third flow path has a fourth flow path, and the liquid introduced into the first flow path opens through the opening portion of the third flow path that opens in the flow path wall of the first flow path. The liquid remaining in the first flow path after being drawn into the flow path is removed, and a volume of liquid corresponding to the volume of the third flow path is weighed.
[0013]
According to the second embodiment of the trace liquid weighing structure according to the present invention, the first flow path and the second flow path that are each extended in a predetermined direction, and the flow path wall of the first flow path The third flow path that opens and the flow path wall of the second flow path opens to connect one end of the third flow path and the second flow pathIn addition, it has a property that the capillary attraction is less likely to work than the third flow path.And a liquid introduced into the first flow path through the opening of the third flow path that opens in the flow path wall of the first flow path. And at least two systems for removing the liquid remaining in the first flow channel after being drawn into the third flow channel and weighing the liquid having a volume corresponding to the volume of the third flow channel. The first flow path or the second flow path is shared.
[0014]
According to the third embodiment of the trace liquid weighing structure according to the present invention, two or more of the fourth flow paths are connected to the third flow path, or the second flow path is 2 It is characterized by branching into two or more.
[0015]
According to a fourth embodiment of the trace liquid weighing structure of the present invention, a plurality of sets of the third and fourth flow paths connected to the third flow path are formed.
[0016]
According to a fifth embodiment of the trace liquid weighing structure according to the present invention, the flow path wall of the fourth flow path is opened,It has the property that capillary attraction is less likely to work than the fourth channel.It further has the 5th channel.
[0017]
According to a sixth embodiment of the trace liquid weighing structure according to the present invention, the second channel has a channel width narrower than a normal channel width of the channel in a part of the channel. Having a narrow channel portion, the narrow channel portion being more than a normal channel width portionRelatively hard to work with capillary attractionIt is characterized by that.
[0018]
According to the seventh embodiment of the trace liquid weighing structure according to the present invention, the liquid weighed in a volume corresponding to the volume of the third channel is transferred to the second channel through the fourth channel. It further has outflow means for flowing out into the flow path.
[0019]
According to an eighth embodiment of the trace liquid weighing structure according to the present invention, when the second flow path in the vicinity of the opening of the fourth flow path is filled with liquid, the third flow It further has an outflow means for causing the liquid weighed in a volume corresponding to the volume of the path to flow out to the second flow path through the fourth flow path.
[0020]
According to a ninth embodiment of the trace liquid weighing structure according to the present invention, the first channel, the second channel, the third channel, the fourth channel, and the fifth channel Each of the flow paths is a channel formed on the substrate.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of a trace liquid weighing structure according to the present invention will be described in detail with reference to the accompanying drawings.
[0022]
1A, 1B, and 1C are conceptual explanatory diagrams for explaining the principle of the present invention. Four channels A (first channel), channel B (second channel), channel C (third channel), and channel D (fourth channel) are: Between A and B, the flow path C from the flow path A side toward the flow path B has a structure in which the flow path D bridges in series. A liquid is introduced into the channel C via the channel A.Since the channel D having the channel wall which is difficult to wet is thinner than the channel C, the capillary attraction is relatively difficult to work than the channel C.Since a larger force is required for the introduction, the liquid can be stopped at the boundary surface c2 between the flow path C and the flow path D by applying an appropriate pressure to the liquid (FIG. 1A and FIG. 1 (b)).
  In this specification, “The property that the capillary attraction is relatively difficult to work"For example,When the liquid is drawn into the channel C, it means that the channel cannot pass through the channel D with the same pressure, and the property of the channel where the liquid stops.That is, this is because the channel D having a channel wall that is difficult to wet is thinner than the channel C, or the channel D is not thinner than the channel C, but the channel wall of the channel D is not the channel C. This is achieved by being modified so as to be less wettable than the flow path wall.
[0023]
More specifically, when the liquid 100 (see the shaded area in FIG. 1) is introduced into the flow path A, the liquid 100 passes through the opening c1 of the thin flow path C that opens at the flow path wall aa of the flow path A. Can be introduced into the channel C.When the channel A and the channel C have a channel wall that easily wets, capillary attraction tends to work,If the flow path C is narrower than the flow path A, the liquid 100 spontaneously enters the flow path C via the opening c1 of the flow path C that opens to the flow path wall aa of the flow path A by stronger capillary attraction. Be drawn.On the other hand, when the channel A and the channel C have a channel wall that is difficult to wet, capillary attraction is difficult to work.The liquid 100 can be introduced into the channel C by applying an appropriate pressure to the liquid 100 from the end face of the channel A (see FIG. 1B).
[0024]
At this time, the liquid 100 reaching the end face c2 on the flow path C side of the flow path D (the flow path section between c2 and d1) is blocked by the capillary repulsion of the flow path D having a flow path wall that is difficult to wet. There is no entry into Road D. Even when the channel C is hard to get wet, the capillary repulsion of the channel D is stronger than the capillary repulsion of the channel C, so that the liquid 100 does not enter the channel D (FIG. 1B). )reference).
[0025]
Subsequently, the liquid 100 remaining in the flow path A is moved to a lower pressure side by causing an appropriate pressure difference at both ends of the flow path A, for example. It moves to the position which does not contact the part c1, or removes from the inside of the flow path A (refer FIG.1 (c)). At this time, the liquid 100 in the channel C does not normally enter the channel A back (see FIG. 1C). As a result, the end face 100a and the end face 100b, which are both end faces of the liquid 100 in the flow path C, are positioned on the opening c1 of the flow path C and the end face c2 on the side connected to the flow path A. It is possible to weigh a volume of liquid according to the volume of the interval between c1 and c2 (see FIG. 1C). However, the end face of the liquid is formed to the extent that it substantially matches the end face of the opening c1 or c2 due to the influence of surface tension, contact angle, etc., and may not exactly match the end face of the opening c1 or c2. Therefore, the volume of the liquid weighed in accordance with the volume of the sections c1 and c2 of the flow path C may not exactly match the volume of the sections c1 and c2 of the flow path C.
[0026]
Further, the liquid weighed in the flow path C flows by causing an appropriate pressure difference between the flow paths so that the pressure in the flow path A is slightly larger than the pressure in the flow path B. It is easy to introduce into the flow path B through the path D and its opening d1. As a result, this liquid can be used for the purpose of reaction or analysis, for example, by transferring it by air pressure.
[0027]
The invention according to claim 1 of the present invention includes a first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction, and the first flow path. A third channel (channel C) that opens to the channel wall of (channel A) and a third channel (channel C) that opens to the channel wall of the second channel (channel B) ( Connect one end of channel C) to the second channel (channel B)In addition, it has the property that the capillary attraction is less likely to work than the third channel (channel C).An opening portion of the third flow path having a fourth flow path (flow path D), in which the liquid introduced into the first flow path opens in the flow path wall of the first flow path. So that the liquid remaining in the first flow path is removed and the liquid having a volume corresponding to the volume of the third flow path is weighed. It is a thing.
[0028]
Therefore, according to the first aspect of the present invention, by introducing the liquid from the first flow path into the third flow path, the volume of the liquid according to the volume of the third flow path. With a simple configuration, the liquid can be weighed quantitatively with a simple operation, while reducing the dead volume of the sample, saving the entire space and reducing the cost. Can be realized.
[0029]
The invention according to claim 2 of the present invention includes a first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction, and the first flow path. A third flow path (flow path C) that opens to the flow path wall of the flow path (flow path A) and a third flow path that opens to the flow path wall of the second flow path (flow path B) Connecting one end of the channel (channel C) and the second channel (channel B)In addition, it has the property that the capillary attraction is less likely to work than the third channel (channel C).An opening portion of the third flow path that has a fourth flow path (flow path D) and in which the liquid introduced into the first flow path opens in the flow path wall of the first flow path. A system that removes the liquid remaining in the first flow path after being drawn into the third flow path through the first flow path and weighs a volume of liquid corresponding to the volume of the third flow path. There are at least two, and the at least two systems share the first flow path or the second flow path with each other (see FIGS. 2A and 2B). ).
[0030]
Therefore, according to the invention described in claim 2 of the present invention, since the two systems share the first flow path or the second flow path with each other, for example, two systems sharing the first flow path When the same type of liquid is quantitatively weighed in each of the systems, the different types of liquid weighed in the two systems and the second flow path of each system are combined, diluted by combining, Analysis by reaction, reaction after unification, etc. can be performed (see FIG. 2A). On the other hand, for example, when different types of liquids are quantitatively weighed in each of the two systems sharing the second flow path, a plurality of the created plurality of liquids are created in the second flow path shared by the two systems. It is possible to perform the analysis by the coalescence, the dilution by coalescence, the reaction by coalescence, the analysis by the reaction after coalescence, etc. (see FIG. 2B).
[0031]
Further, as in the invention described in claim 3 of the present invention, in the trace liquid weighing structure according to any one of claim 1 or 2, the fourth channel (channel D). May be divided into two or more flow paths, or may be branched into two or more (see FIG. 3A). This makes it possible to adjust the flow velocity of the fluid in the flow path D independently of the capillary repulsion.
[0032]
According to a fourth aspect of the present invention, there is provided the microfluidic weighing structure according to any one of the first, second, or third aspect, wherein the third flow path (flow path) C) and two or more fourth channels (channel D) are formed (see FIG. 3B). According to the fourth aspect of the present invention, a plurality of sets of the third flow path and the fourth flow path (for example, the flow path C and the flow path D in FIG. A plurality of different volumes of liquid can be weighed quantitatively and in parallel by the channels C ′ and D ′ and the channels C ″ and D ″).
[0033]
In addition, the invention according to claim 5 of the present invention is the microfluidic weighing structure according to any one of claims 1, 2, 3, or 4. Open to the channel wall of the channel (channel D), Has a property that capillary attraction is less likely to work than the fourth flow pathA fifth channel (channel E) is further provided (see FIG. 4).
[0034]
FIG. 4 is a conceptual explanatory diagram for explaining the principle of the trace liquid weighing structure according to the fifth aspect of the present invention. As shown in FIG. 4A, the five channels A, B, C, D, and E are connected between the channels A and B. The road D is connected in series and bridges. When liquid is introduced from channel A to channel CThe channel D has a property that the capillary attraction is less likely to work than the channel C.Therefore, the liquid stops at the interface c2 between the flow path C and the flow path D, and a larger force is required to introduce the liquid into the flow path D.
[0035]
When the liquid 100 (see the shaded area in FIG. 4) is introduced into the thick channel A, the liquid 100 flows through the opening c1 of the narrow channel C that opens at the channel wall aa of the channel A. (See FIG. 4B). This phenomenon may occur in any one of claims 1, 2, 3, and 4 when the liquid is not filled in the vicinity of the opening d1 of the channel D in the channel B. Although it is the same structure as the trace liquid weighing structure described, when the liquid 200 (gray area in FIG. 4) is introduced into the flow path B in advance, the first, second, and second aspects are described. In the trace liquid weighing structure according to any one of claims 3 and 4, the introduction of the liquid 100 into the flow path C occurs in an incomplete form and is not completed. This is because there is no escape route for the gas remaining in the channel C.
[0036]
According to the invention described in claim 5, when the liquid 200 (gray region in FIG. 4) has been introduced into the flow path B, the flow of the flow up to that time with the introduction of the liquid 100 into the flow path C. The gas in the interior is purged to the flow path E having the opening e1 on the flow path wall of the flow path D (the end surface of the flow path E opposite to e1 is open to the atmosphere). (See FIG. 4 (b)). Also in this case, the liquid 100 that has reached the end surface c2 between the channel C and the channel D isIt has the property that capillary attraction is less likely to work than channel CIt is blocked by the capillary repulsion of the flow path D (flow path section between c2 and d1), and does not enter the flow path D (see FIG. 4B).
[0037]
Subsequently, the liquid 100 remaining in the flow path A is removed from the flow path A, for example, by causing an appropriate pressure difference at both ends of the flow path A to move to a lower pressure side. As a result, it is possible to weigh a volume of liquid corresponding to the volume of the flow path C (section between c1 and c2) (see FIG. 4C).
[0038]
Further, the liquid formed in the channel C causes an appropriate pressure difference between the two channels so that the pressure in the channel A is slightly larger than the pressure in the channel B, and so on. It becomes possible to introduce into the liquid 200 in the flow path B via the. At this time, the end surface of the flow path E opposite to the end surface e1 needs to be closed (see FIG. 4D).
[0039]
Therefore, if the invention according to claim 5 of the present invention is used, a small amount of sample or reaction reagent can be introduced quantitatively in various analyzers or reactors used for electrophoresis or chromatography. A practical approach can be provided.
[0040]
5 (a) and 5 (b) are plan views showing the trace liquid weighing structure of the present invention according to claim 6, and FIG. 5 (c) is a sectional view taken along the line cc in FIG. 5 (b). is there. As shown in FIG. 5A, the second flow path (flow path B) is a narrow flow having a flow width narrower than the normal flow width of the flow path in a part of the flow path. It has a path portion (flow path b1 and flow path b2). The narrow channel part is a normal channel width part.It has the property that capillary attraction is relatively difficult to work than. One end of the second flow path (flow path B) itself and the narrow flow path portion (flow path b) is open to the atmosphere or adjusted to an appropriate pressure. Therefore, a volume-limited section can be formed in a part of the second channel by sandwiching a part of the second channel between the two narrow channel parts. The third channel (channel C) can be communicated with the volume-limited section of the second channel via the fourth channel (channel D). For example, when the first liquid weighed in one third flow path (flow path C) is pushed into the volume-limited section of the second flow path via the fourth flow path (flow path D). The air in the limited volume section is released into the atmosphere through the narrow channel portion, and the extruded first liquid remains in the limited volume section. Further, the second liquid weighed in the other third flow path (flow path C ′) is passed through the other fourth flow path (flow path D ′) to limit the volume of the second flow path. When pushed in, the remaining air in the volume-limited section is also released into the atmosphere through the narrow channel portion, and the first liquid and the second liquid can be filled in the volume-limited section. Therefore, a volume-limited section sandwiched between two narrow channel portions can be used as a reaction chamber, and the amount of reaction solution to be used can be minimized. For this reason, the volume of the volume limited section of the second flow path can be determined in consideration of the total amount of liquid supplied in the limited section. For example, it is preferable to set a value that is substantially the same as or slightly larger than the sum of the volumes of one or more third flow paths connected in the limited section.
  FIG. 5B shows an embodiment in which one third flow path is connected to the volume-limited section of the second flow path, and the second flow path includes two narrow channels. It has a flow path part (flow path b1 and flow path b2), and the other part has a normal flow path width. For example, the first liquid is supplied from the second flow path having the normal flow path width to the volume-limited section through the first narrow flow path portion (flow path b1), and then the third flow path (flow The second liquid weighed in the path C) is supplied into the volume limited compartment through the fourth flow path (flow path D).
  Therefore, the second flow path does not necessarily mean only a flow path in which the liquid moves (flows), but is also used as a flow path for moving air. In particular, it is only necessary to form a volume-limited section sandwiched between two narrow channel portions that can allow the liquid weighed in the third channel to flow out. The planar shape of the limited section is not limited to the illustrated rectangular shape, and may be various shapes such as a circle, an ellipse, and a triangle.
  As shown in FIG. 5 (c1), the narrow channel portion (channel b1 and channel b2) of the second channel is formed on the lower substrate 1, and the normal width portion of the second channel is formed. It can be formed on the upper substrate 3 or, as shown in FIG. 5 (c2), the narrow channel portions (channel b1 and channel b2) of the second channel and the second channel. All of the normal width portions can be formed on the upper substrate 3.
[0041]
The invention described in claim 7 among the present inventions is the micro liquid weighing method according to any one of claims 1, 2, 3, 4, 5, or 6. In the structure, the liquid weighed quantitatively in the third channel (channel C) is further passed through the second channel (flow channel D) via the fourth channel (channel D). An outflow means for flowing out from the opening of the fourth channel (channel D) that opens in the channel wall of the channel B) to the second channel (channel B) is provided.
[0042]
Specifically, in order to cause the liquid in the third flow path formed quantitatively to flow out to the second flow path, the pressure in the first flow path is slightly lower than the pressure in the second flow path. An appropriate pressure difference may be generated between the two flow paths or a centrifugal force may be applied in the outflow direction so as to increase. As a means for causing the pressure difference, for example, a known and commonly used pressure mechanism or pressure reduction mechanism is preferably used.
[0043]
Therefore, according to the invention described in claim 7 of the present invention, the liquid in the third flow path quantitatively weighed can be discharged to the second flow path. In various analytical devices or reaction devices used for chromatography or the like, it is possible to quantitatively introduce a small amount of sample or reaction reagent.
[0044]
Further, the invention according to claim 8 of the present invention is the trace liquid weighing structure according to claim 7, wherein the second flow path (flow path B) is further filled with liquid. The liquid weighed quantitatively in the third channel (channel C) is opened in the channel wall of the second channel through the fourth channel (channel D). An outflow means for flowing out from the opening of the third flow path to the second flow path is provided.
[0045]
Specifically, according to the invention described in claim 8 of the present invention, the liquid in the third channel quantitatively formed using the micro liquid weighing structure according to claim 5 An appropriate pressure difference is generated between the two flow paths so that the pressure of the first flow path is slightly higher than the pressure of the second flow path with the end face of E opposite to e1 closed. Or it can be made to flow out to the 2nd channel by performing operations, such as giving centrifugal force in the outflow direction.
[0046]
Therefore, according to the invention described in claim 5 of the present invention, the liquid in the third flow path quantitatively weighed can be discharged to the second flow path. In various analytical devices or reaction devices used for chromatography or the like, it is possible to quantitatively introduce a small amount of sample or reaction reagent. In addition, by applying the micro liquid weighing structure of the present invention, sample introduction (injection) in electrophoresis of nucleic acids and proteins, making the surface in the channel and necessary places easy to wet or difficult to wet, Alternatively, it is possible to adapt to protein synthesis and separation, or chemical synthesis and screening, etc. by performing each required treatment corresponding to the surface properties of protein, DNA, and the like.
[0047]
The invention described in claim 9 among the present inventions is any one of claim 1, claim 2, claim 3, claim 4, claim 5, claim 7, claim 7 or claim 8. In the invention described in the item, the first flow path (flow path A), the second flow path (flow path B), the third flow path (flow path C), the fourth flow path ( Each of the flow path D) and the fifth flow path (flow path E) is a channel formed in the substrate.
[0048]
Therefore, according to the invention described in claim 9 of the present invention, it is possible to quantitatively weigh a small volume of liquid with a simple configuration and only by a simple operation, and further dead samples can be obtained. It is possible to realize volume reduction, space saving and cost reduction of the entire apparatus.
[0049]
Further, the invention according to claim 10 of the present invention is claimed in claim 1, claim 2, claim 3, claim 4, claim 5, claim 7, claim 8, claim 9 or claim 9. In the invention described in any one of the above, the volume of the third flow path is formed in a size of picoliter to microliter order.
[0050]
Therefore, according to the micro liquid weighing structure of the present invention, it is possible to quantitatively weigh micro volumes of liquids of various sizes ranging from picoliters to microliters with a simple configuration and simple operation. it can.
[0051]
The trace liquid weighing structure of the present invention can be formed in a chip. A plurality of trace liquid weighing structures of the present invention may be formed on the same chip with the same structure or a combination of different structures. In the chip of the present invention, each formed flow path is preferably a microchannel. Here, the microchannel means a channel having a cross-sectional shape in which a micro effect appears when a liquid is introduced into the channel (flow path), that is, some behavior change appears in the liquid. The manifestation of the micro effect varies depending on the physical properties of the liquid, but the cross-sectional shape, that is, the length of the shortest interval (corresponding to the short side for a rectangle and the short axis for an ellipse) of the shape perpendicular to the main flow direction of the channel Is usually 5 mm or less, preferably 500 μm or less, more preferably 200 μm or less. The lower limit of this length is not particularly limited as long as it has a function as a microchannel.
In the present invention, the chip means a part of a member including a trace liquid weighing structure. The microchip means a chip having a trace liquid weighing structure composed of the microchannel. The size and shape of the chip are not particularly limited, and can be arbitrarily set according to the application.
The chip of the present invention is used for, for example, separation / analysis of substances, biochemistry or chemical reaction, or protein crystallization. The chip in the present invention is desirably replaced after use for a single use or a limited number of times, but may be used permanently. In this case, it may be integrated with a device such as a dispenser or a measuring device, but in this case, the part of the member including the flow path structure is called a chip.
The material of the chip in the present invention is not limited as long as the above channel structure can be realized. Examples of the material include polydimethylsiloxane (PDMS), glass, silicon, photoreactive resin and other resins, metals, ceramics, and combinations thereof.
The method for producing the trace liquid weighing structure in the present invention may be any method as long as the trace liquid weighing structure can be realized. For example, transfer technology represented by machining, injection molding and compression molding, dry etching (RIE, IE, IBE, plasma etching, laser etching, laser ablation, blasting, electrical discharge machining, LIGA, electron beam etching, FAB), wet Surface micro-machining, which forms fine structures by etching (chemical erosion), monolithic molding such as stereolithography and ceramics laying, and coating, vapor deposition, sputtering, deposition, and partial removal of various substances. It includes a method of forming a groove by forming an opening portion by using at least one sheet-like substance (film, tape, etc.), and a method of forming a channel by dropping and injecting a flow path constituent material by an inkjet or a dispenser.
In order to make the chip of the present invention, a mask may be used in the above method. The mask may have any design as long as the chip of the present invention can be finally produced, and a plurality of masks may be used. The mask is usually designed with a shape in which the flow path is projected onto a flat surface, but when processing on both sides of the flow path component to be bonded or when forming a flow path using multiple members, multiple masks are used. Therefore, the mask is not necessarily a projection of the final shape of the flow path because a part of the mask can be directly processed without using the mask. Examples of the electromagnetic wave shielding mask used for the photo-curing resin include those obtained by coating quartz or glass with chromium, or those obtained by baking a resin film with a laser.
The mask can also be created by drawing at least a part of the channel structure and printing it on a transparent resin film using a suitable software, for example. The present invention also includes a computer-readable recording medium used for creating the mask or master chip drawn by the software and storing the electronic information of at least a part of the flow channel structure. Examples of suitable recording media include magnetic media such as flexible disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, CD-RW, and DVD, and semiconductor memories. .
In producing the chip of the present invention, the chip may be directly manufactured by the above method, or the chip may be shaped using this as a mold. Of course, it is also possible to shape the chip using this as a mold. In the present invention, a mold at each stage from a mold that is initially created to a chip that is actually used for operation may be referred to as a “master chip”. The master chip usually has a concave / convex pattern identical to that of an actual chip (positive) and is transferred twice, or has a concave / convex pattern opposite to that of an actual chip (negative). The master chip in the present invention is not necessarily limited to this, although it is used after being transferred twice.
These masks or master chips are also included in the present invention.
The chip in the present invention can be processed by bonding two or more materials. As bonding methods, adhesive bonding, resin bonding with primer, diffusion bonding, anodic bonding, eutectic bonding, thermal fusion, ultrasonic bonding, laser melting, bonding with solvent / dissolving solvent, adhesive tape, adhesive tape , Pressure bonding, bonding with a self-adsorbing agent, physical retention, and combination with unevenness.
In addition, a method of integrally forming the fluid branch portion and the independent flow path without requiring bonding is also possible. Specifically, it is possible to form a structure including a closed space by integral molding such as stereolithography.
The length, shape, and thickness of one side of the chip thus produced are not limited, and can be set to any value between 5 mm and 100 mm, for example.
[0052]
Note that when a microchip is manufactured by bonding a substrate using a mask and a master chip, the depth of the microchannel in the chip is, for example, that when the substrate is formed of two substrates, the channel is the upper substrate. Depending on whether it exists on the lower surface or the upper surface of the lower substrate, it can be partially set to one of two arbitrary values. The depth is not particularly limited, but preferably 1 to 200 μm. It can be set to any arbitrary value. Here, “the depth of the channel” corresponds to the height of a convex portion of a chip having a convex flow path serving as a channel mold, that is, a master chip. Further, the number of substrates is not limited to two, and a chip can be produced by laminating a plurality of arbitrary substrates as necessary.
[0053]
Various protein crystallization methods can be carried out using the trace liquid weighing structure of the present invention. For example, the first protein crystallization method is:
  (A) A protein solution or a precipitant solution is introduced into the first flow path of the flow path structure, and the third flow is made through an opening of a third flow path that opens to the first flow path. After the protein solution or precipitant solution is drawn into the path, the protein solution or precipitant solution remaining in the first flow path is moved to a position where it does not contact the opening of the third flow path, A step of causing a protein solution or a precipitant solution prepared in a volume corresponding to the volume of the third channel to flow out to the second channel through a fourth channel;
  (B) A precipitant solution or a protein solution is introduced into the first channel, and the precipitant solution is introduced into the third channel through an opening of the third channel that opens into the first channel. Alternatively, after the protein solution is drawn, the precipitant solution or protein solution remaining in the first flow path is moved to a position where it does not contact the opening of the third flow path, and the third flow path The precipitant solution or the protein solution prepared in a volume corresponding to the volume of the liquid is allowed to flow out to the second flow path through the fourth flow path, and the protein solution and the precipitant solution are allowed to flow in the second flow path. A process of contacting and uniting;
  (C) Precipitating protein crystals from the protein and precipitant solution combined in the second flow pathProcess.
  Also,As an alternative to the first method,Precipitation of protein crystals from the combined protein and precipitant solutionTheIn a state where the precipitant solution is disposed in the channel structure separated from the solution.It can also be made. In this second method,For example, the precipitant solution may be arranged in advance by being introduced into an appropriate flow path, for example, the second flow path, or after bringing the protein solution and the precipitant solution into contact with each other, The precipitant solution may be introduced into the first channel and placed in a state of being drawn into the third channel.
  In these first or second methods,Either the protein solution or the precipitant solution may be introduced into the second channel first, or the protein solution and the precipitant solution may be introduced into the second channel at the same time using two separate channel structures formed separately. Also good.
  Also, for example, another third method for crystallizing a protein is:
  (A) A protein solution is introduced into the first flow path of the trace liquid weighing structure, and the third flow path is opened through the opening of the third flow path that opens to the first flow path. After the protein solution is drawn, the protein solution remaining in the first flow path is moved to a position where it does not come into contact with the opening of the third flow path, and according to the volume of the third flow path. Creating a protein solution in a volume of
  (B) introducing a precipitant solution into the first channel, bringing the protein solution and the precipitant solution into contact at the opening of the third channel, and bringing the protein solution and the precipitant solution together;
  (C) Precipitating protein crystals from the combined protein and precipitant solutionProcess.
  In this method, since protein crystals are precipitated in the first and / or third flow paths, the combined solutions are allowed to stand for a suitable time as necessary, and then the presence or absence of the crystal precipitation is confirmed.
  The confirmation means of crystal precipitation may be performed by an appropriate detection means known per se, such as visual observation or microscopic observation. The precipitated protein crystals can be collected by an appropriate method known per se, for example, by introducing an appropriate liquid and allowing it to flow out.
  here,aboveThe physical property of the protein solution used for the protein crystallization method is not particularly limited as long as it can be used in the micro liquid weighing structure of the present invention. Specifically, examples of the solvent include water, salt solutions, buffer solutions, alcohol or glycerol and solutions thereof, synthetic or natural polymer solutions, and the like.
  The precipitant solution used means a solution that promotes protein crystal formation, and the physical properties of this solution are not particularly limited as long as they can be used in the trace liquid weighing structure of the present invention as described above. Specific examples include water, salt solutions, buffer solutions, alcohol or glycerol and solutions thereof, synthetic or natural polymer solutions, and the like. In addition, proteins and polymer gels, porous silicon (see Chayen et al., Journal of Molecular Biology, (2001) 312, 591-595) are known as substances that promote crystal formation. Good.
  Also,In the above method,When sealing or adjusting the vapor pressure is required, the volume of the gas phase in contact with the solution is preferably small. In this case, for example, it is preferable to use a microchip having a trace liquid weighing structure (see FIG. 5) having a volume-limited section formed by sandwiching the second channel between two narrow channel portions.
  Known methods for protein crystallization include, for example, a batch method, a vapor diffusion method, an interface contact method, and a gel method. Here, in order to perform crystallization by the batch method in the microchip having the flow channel structure of the present invention, for example,As one of the above methodsThe protein solution and the precipitant solution may be brought into contact with each other, and the flow path may be sealed by the above method as necessary.
  In order to perform the vapor diffusion method, for example,As described in the first method or the second method,After contacting and combining the protein solution and the precipitant solution in the second flow path, as described above, a liquid for adjusting the vapor pressure is introduced into the first flow path as necessary, and the contact and combining are performed. Vapor diffusion may be adjusted by separating the unified liquid and the third and / or fourth flow path. However, in the vapor diffusion method, the liquid that has been contacted and united and the liquid that adjusts the vapor pressure (precipitating agent) need only be arranged across the gas phase, and the arrangement of the liquid is not necessarily the above-described form. There is no need. A liquid for adjusting the vapor pressure may be introduced halfway through the first flow path or at the end thereof, and the vapor pressure may be adjusted across the first, third, and fourth flow paths. A liquid for adjusting the vapor pressure may be introduced to the middle of the flow path or at the end portion, and the vapor pressure may be adjusted across the second flow path.
[0054]
【Example】
Hereinafter, the micro liquid weighing structure of the present invention, the microchip having the structure, the qualitative / quantitative reaction using the micro chip, and the like will be specifically illustrated by examples.
Example 1 Separation of proteins by ion exchange chromatography
FIG. 6 (a) shows a microchip 10 having a trace liquid weighing structure according to the present invention. A microchip 10 shown in FIG. 6 (a) is a microchip for capillary ion exchange chromatography used for protein analysis, and a micro liquid weighing structure according to the present invention is provided in an injection portion of a sample.
[0055]
In FIG. 6A, port 11 and port 12 are ports for introducing a protein elution buffer. A buffer having a different ionic strength is introduced, a flow rate is changed, and a buffer having a different ionic strength is provided in the mixer 13. By mixing, a gradient of ionic strength can be formed. Therefore, the upper surfaces of the port 11 and the port 12 are open to the atmosphere, and the lower portion communicates with the channel.
[0056]
The microchannel 14 is a column for chromatography. From the damming unit 15 of ion exchange beads (as the ion exchange beads, for example, anion exchange beads manufactured by Pharmacia: particle size of 30 μm can be used). The ion exchange beads are filled in a section of 3 mm in the direction of the trace liquid weighing structure (b). A port 18 exists in the direction opposite to the damming portion 15.
[0057]
FIG. 6B is an enlarged view of the trace liquid weighing structure (b). The microchip 10 shown here is formed by stacking two plate-like substrates (upper: substrate 16, lower: substrate 17) formed of a polymer (polymer) material such as PDMS (polydimethylsiloxane) and bonding them. However, the length, shape, and thickness of one side of the microchip are not limited, and are set to any value between 5 mm and 100 mm, for example. be able to.
[0058]
Microchannels having different depths are formed on the lower surface of the upper substrate 16 and the upper surface of the lower substrate 17. The microchannel existing on the lower surface of the substrate 16 has a depth of 100 μm, and the microchannel existing on the upper surface of the substrate 17 has a depth of 10 μm. Furthermore, as a feature of PDMS, the hardened surface is hydrophobic. In addition, this surface is O₂By performing irradiation treatment with plasma, excimer UV laser, or the like, it is possible to easily bond PDMS and PDMS, PDMS and glass, or the like. From the above two points, PDMS is suitable for the production of this micro liquid weighing structure.
[0059]
Note that the depth of the microchannel in the microchip 10 is partially set to one of two arbitrary values depending on whether it exists on the lower surface of the upper substrate 16 or the upper surface of the lower substrate 17. The depth can be set to an arbitrary value of 1 to 200 μm.
[0060]
As shown in FIG. 6B and FIG. 6C1, the microchannel structure in the trace liquid weighing structure (b) is extended in the left-right direction in parallel with each other on the lower surface of the upper substrate 16. The first flow path 19 and the second flow path 20, the third flow path 21 extending perpendicularly from the first flow path 19 to the second flow path direction, and the upper surface of the lower substrate 17. A fourth flow path 22 that connects the second flow path 20 and the third flow path 21, and a fifth flow path 23 that is derived from the middle of the fourth flow path 22 and goes straight to the fourth flow path 22. Is formed. When a shallow and narrow channel is disposed on the upper surface of the lower substrate 17, each flow is partially overlapped with a deep and wide channel disposed on the lower surface of the upper substrate 16. Roads can communicate with each other. Alternatively, as shown in FIG. 6 (c2), when all the channels are arranged on the upper substrate 16 (or the lower substrate 17), it is not necessary to provide an overlapping portion in each channel. By appropriately selecting a synthetic resin material to be used as a substrate and / or making the surface of a specific flow path disposed in the substrate and a part or all of a necessary portion easy to wet or difficult to wet Accordingly, as shown in FIG. 6C2, all the flow paths can be provided only on one substrate.
[0061]
The width of the first flow path 19 and the second flow path 20 is 200 μm, the width of the third flow path 21 is 100 μm, the width of the fourth flow path 22, and the fourth flow in the fifth flow path 23. The width of the portion in contact with the path 22 is 20 μm. Note that the fourth flow path 22 needs to be thinner than the first flow path 19, the second flow path 20, and the third flow path 21 in order to make it difficult for liquid to enter the fourth flow path 22. In addition, the width of the fifth channel 23 needs to be equal to or less than the width of the fourth channel 22 in order to make it difficult for the liquid to enter the fifth channel 23.
[0062]
Next, the above-described microchip 10 is manufactured by the manufacturing process described below. Prior to the manufacturing process, first, the microchannels of the upper substrate 16 and the lower substrate 17 of the microchip 10 are arranged. Each layout pattern is printed on a transparent film at a high resolution (for example, 4064 dpi) in order to be used separately as a mask for photolithography.
[0063]
First, a silicon (Si) wafer is cut with a diamond cutter in accordance with the size of the microchip to be manufactured, ultrasonically cleaned, dried, and then removed using a plasma reactor.₂Plasma treatment is performed at 200 W for 30 seconds.
[0064]
Next, a negative photoresist SU-8 is spin-coated (when a 100 μm channel is prepared, SU-850 is used for 10 seconds at 2000 rpm, and when a 10 μm channel is prepared, SU-8 is used at 2000 rpm. 10 seconds) and then hold in an oven at 90 ° C. for 30 minutes.
[0065]
Next, using a mask aligner, the microchannel layout pattern on the upper substrate 16 and the lower substrate 17 in the microchip 10 printed on the film is photo-printed on separate silicon wafers coated with SU-8. After transferring using a lithography technique and holding in an oven at 90 ° C. for 30 minutes, the film is immersed in Developer (for example, 1-methoxy-2-propyl acetate can be used as Developer). Develop, wash with isopropyl alcohol, followed by distilled water and dry.
[0066]
The master thus fabricated has a convex structure that serves as a microchannel template for the upper substrate 16 and the lower substrate 17.
[0067]
Next, in order to facilitate the removal of the PDMS replica after molding, the surface treatment is performed by holding in a 3% dimethyloctadecylchlorosilane / toluene solution for 2 hours before pouring the PDMS prepolymer.
[0068]
Next, the PDMS prepolymer and a curing reagent (for example, “Sylgard 184: Dow Corning Co., MI” can be used as a curing reagent) are mixed at a ratio of 10: 1 and sufficiently stirred. Then, in order to install the master, it is poured into a frame made of acrylic and kept at 90 ° C. for 30 minutes for curing.
[0069]
After the curing, the PDMS replica is peeled off from the master to obtain the upper substrate 16 and the lower substrate 17 of the microchip 10, respectively. Further, the lower surface of the upper substrate 16 and the upper surface of the lower substrate 17 are O₂The microchip 10 is obtained by treatment with plasma and bonding.
[0070]
A micro liquid weighing operation for sample injection actually performed using the microchip 10 obtained by the above method will be described below.
[0071]
First, the sample is filled into the first channel 19 and the third channel 21 by flowing the sample from one side of the first channel 19 by a pressure flow. At this time, the fourth flow path 22 isThan the third channel 21Since the capillary attractive force is relatively difficult to work and is thinner than the first flow path 19, the second flow path 20, and the third flow path 21, it is difficult to get wet with a hydrophilic liquid. 4 does not enter the channel 22.
[0072]
In a state where the second channel 20 is filled with liquid, if the fifth channel 23 does not exist, the sample in the third channel 21 is introduced when the sample is introduced into the third channel 21. Air leaks into the second flow path 20.
[0073]
However, the presence of the fifth flow path 23 causes air in the third flow path 21 to escape to the fifth flow path 23 even when the second flow path 20 is filled with liquid. Therefore, the sample can be introduced into the third channel 21 without air leaking into the second channel 20.
[0074]
Next, by introducing air into the first channel 19 by pressure flow, the sample in the first channel is pushed out, the sample remains only in the third channel 21, and the third channel The sample remaining in the flow path 21 does not flow back into the first flow path 19 again. In the example of the chip shown here, the amount of sample remaining in the third flow path 21 is designed to be 5 nl.
[0075]
Subsequently, by increasing the pressure in the first flow path 19, the sample in the third flow path 21 leaks into the fourth flow path 22, and the sample reaches the second flow path 20. A sample is introduced into the second flow path 20. At this time, since the outlet side of the fifth flow path 23 is closed, the sample does not leak into the fifth flow path 23.
[0076]
In order to reduce the possibility that the sample leaks into the fifth channel 23, the channel width of the fifth channel 23 is desirably equal to or less than the channel width of the fourth channel 22, Both are set to 20 μm.
[0077]
In order to prevent adsorption of a sample (protein or the like) for separation by chromatography on PDMS, only the first channel 19, the second channel 20, and the third channel 21 are used using HCl. Treatment to make it hydrophilic. However, the hydrophilization treatment is not limited to HCl, but O₂A method using plasma or a method using albumin is also possible. PDMS itself is a hydrophobic substance.
[0078]
Actually, in the chromatography using sample injection by the above-mentioned micro liquid control operation, a mixed solution of albumin and IgG labeled with FITC is introduced, and Tris / HCl Buffer (pH 8.0) is flowed at a flow rate of 1.0 μl / min. Then, using anion exchange beads (Pharmacia: particle size: 30 μm), after adsorption, they were successfully separated within 1 minute using a 0-1 M gradient buffer with sodium chloride.
[0079]
In the above embodiment, this trace liquid weighing structure is made of PDMS of silicon polymer. However, by making part or all of the inner surface and / or the end face of the channel easier to wet or less wet, If the internal wettability can be adjusted and the structure can be manufactured, the structure can be applied to any material (for example, silicon, polymer, glass, ceramics, metal, etc.). In addition, this micro liquid weighing structure can be used by a composite use of these different materials, or a material mixed with temperature, pH responsive substance PIPAAm (N-isopropylacrylamide) and the like.
[0080]
Example 2 Determination of glucose by enzymatic reaction
FIG. 7A is a plan view of a microchip 24 according to another embodiment of the trace liquid weighing structure according to the present invention. The microchip 24 shown in FIG. 7A is a microchip capable of individually performing up to 50 kinds of reactions or analyzes by mixing two liquids quantitatively weighed. Adopts a micro liquid weighing structure according to the present invention. The microchip 24 shown in FIG. 7A processes up to 50 types of reactions or analyses, but microchips that process a larger number of reactions or analyzes can naturally be produced.
[0081]
By using the microchip 24 as shown in FIG. 7 (a), for example, reaction or analysis by mixing one kind of reagent individually with respect to 50 kinds of samples, and very simple operation, and Can be done at the same time. Alternatively, for example, reaction or analysis by mixing 50 kinds of reagents can be performed individually and simultaneously with respect to one kind of sample.
[0082]
The microchip 24 as shown in FIG. 7A is similar to the microchip 10 shown in FIG. 5 in that two flat plates formed of a polymer material, for example, PDMS (polydimethylsiloxane). The substrate is formed by overlapping and bonding the substrate, and has a disk shape with a diameter of 90 mm and a thickness of 3 mm. Of course, other diameters and thicknesses can be used. The shape of the microchip 24 is not limited to the illustrated disk shape. It can also be formed using a rectangular or polygonal substrate.
[0083]
In the microchip 24 shown in FIG. 7A, microchannels having different depths are formed on the lower surface of the upper substrate 25 and the upper surface of the lower substrate 26, and exist on the lower surface of the substrate 25. For example, the depth of the microchannel is 100 μm, and the depth of the microchannel existing on the upper surface of the substrate 26 is 10 μm, for example. Depths other than these can of course be used.
[0084]
FIG. 7B is an enlarged view of the portion (b) of the microchip 24 in FIG. 7A, and FIG. 7C is an enlarged view of the portion (c) of FIG. 7B.
[0085]
In FIG. 7B, the first liquid supply channel 33 having a substantially annular shape in the center is cut at one place in the middle thereof, and one cut end is ported through a channel 27 extending radially outward. 28 is connected to and communicated with the port 30 via a channel 29 extending radially outwardly. Port 28 is the inlet for the first liquid to be mixed and port 30 is the outlet for the liquid. The liquid introduced from the port 28 enters the first liquid supply channel 33 through the channel 27, flows through the first liquid supply channel 33, and flows out from the port 30 through the channel 29. Alternatively, port 28 can be the outlet and port 30 can be the inlet.
[0086]
7B is an inlet for the second liquid to be mixed, and communicates with the outlet port 31b through the second liquid supply channel 35. Since the inlet port 31a and the outlet port 31b communicate with each other via the second liquid supply channel 35, the liquid injected from the inlet port 31a can be easily introduced into the second liquid supply channel 35 by an appropriate means such as air pressure. Can be satisfied. Adjacent to the port 31a and the port 31b, a port 32a and a port 32b are disposed through the mixing channel 34, respectively. The mixing channel 34 also serves as a reaction chamber for both mixed liquids. Since the port 32a and the port 32b communicate with each other through the mixing channel 34, air pressure is applied to the mixing channel 34 through the port 32a and / or the port 32b to promote mixing of the liquid in the channel. Alternatively, the reaction product in the mixing channel 34 can be used to take out.
[0087]
Thirty structures for measuring and mixing a trace amount of liquid comprising ports 31a and 31b, a second liquid supply channel 35, ports 32a and 32b, and a mixing channel 34 are disposed radially outside the first liquid supply channel 33. Has been placed. On the other hand, 20 similar structures for measuring and mixing trace amounts of liquid are arranged on the inner side in the radial direction. The number of arrangement is not limited to the illustrated embodiment.
[0088]
The structure shown in FIG. 7 (c) is formed for the purpose similar to the structure shown in FIG. 2 (b), and includes a microchannel structure for weighing a small amount of liquid and two types. It consists of channels for mixing liquids. A first liquid supply channel 33 (flow path A in FIG. 2B) that supplies one liquid with a mixing channel 34 (corresponding to the flow path B in FIG. 2B) functioning as a reaction chamber interposed therebetween. And a second liquid supply channel 35 for supplying the other liquid (corresponding to the flow path A ′ in FIG. 2B). One liquid supplied from the first liquid supply channel 33 is weighed by a certain amount by the first weigh channel 36 (corresponding to the flow path C in FIG. 2B). Similarly, the other liquid supplied from the second liquid supply channel 35 is weighed by a certain amount by the second weighing channel 37 (corresponding to the flow path C ′ in FIG. 2B). The liquid in the first weigh channel 36 and the second weigh channel 37 passes through the first narrow channel 38 and the second narrow channel 39 which are narrower than these channels, respectively, using conventional means such as a pressure difference. It can be taken into the mixing channel 34 and mixed in the channel.
[0089]
In FIG. 7, the channel width of the first liquid supply channel 33 for supplying one liquid, the second liquid supply channel 35 for supplying the other liquid, and the mixing channel 34 is, for example, 200 μm, and the first weighing channel 36 and The channel width of the second weighing channel 37 is, for example, 100 μm, the length is, for example, 600 μm, and the channel width of the first narrow channel 38 and the second narrow channel 39 is, for example, 20 μm. Other channel widths and channel lengths may be used as a matter of course. For example, in FIG. 7C, the volume of the two liquids to be mixed is 6 nl, but this volume is equal to the volume of the first weighing channel 36 and the second weighing channel 37. Therefore, it can be arbitrarily adjusted within the range of 1 pl to 1 μl by changing the volume of each weighing channel.
[0090]
The microchip 24 of FIG. 7 can be manufactured by the same manufacturing process as the microchip 10 shown in FIG.
[0091]
The microchip 24 shown in FIG. 7 can be used, for example, for micro liquid weighing and mixing operations for quantitative analysis of glucose by the glucose oxidase / peroxidase method.
[0092]
The micro liquid weighing and mixing operation for quantitative analysis of glucose by the glucose oxidase / peroxidase method will be described below. In the glucose oxidase / peroxidase method, a phosphate buffer mixed with appropriate amounts of glucose oxidase, peroxidase, mutarotase, 4-aminoantipyrine and phenol is used as a reagent, and an aqueous glucose solution is used as a sample.
[0093]
First, about 1 μl of the reagent is introduced from the port 28 to the port 30 in FIG. 7A by pressure flow, and then air is introduced in order to allow excess reagent to flow. The introduced reagent and air flow in the first liquid supply channel 33 in the clockwise direction. In FIG. 7C, when the reagent is introduced into the first weighing channel 36, the first narrow channel 38 is hydrophobic and difficult to wet with the liquid, and the first liquid supply channel 33 and Since the channel width is narrower than the first weighing channel 36, the reagent stays in the first weighing channel 36 and does not enter the first narrow channel 38.
[0094]
By this operation, a reagent liquid having a volume equal to the volume of the first weighing channel 36 can be weighed in the first weighing channel 36. Also, a predetermined amount of reagent liquid is weighed at once in the 50 first weighing channels 36 by performing only one operation of introducing the reagent from the port 28 to the port 30 and subsequent air introduction. It is possible. By such an extremely simple operation, it is possible to further finely distribute a minute amount of liquid and simultaneously form a large number of extremely small amounts of liquid. Moreover, in such an operation, it is expected that the amount of residual liquid that is not distributed after the operation will be extremely small.
[0095]
Subsequently, similarly to the introduction of the reagent, the sample is introduced from the port 31a toward the port 31b, and a predetermined amount of the sample liquid is weighed into the second weighing channel 37 through the second liquid supply channel 35. Can do.
[0096]
Subsequently, the reagent in the first weighing channel 36 and the sample in the second weighing channel 37 are respectively increased by increasing the pressure of air in the first liquid supply channel 33 and the second liquid supply channel 35. It is pushed into the mixing channel 34 and mixed in the channel. Since the two mixed solutions exhibit a red color by a chemical reaction, quantitative analysis of glucose can be performed by evaluating the red value. Needless to say, the microchip 24 shown in FIG. 7 can also be used for other qualitative and / or quantitative analysis.
[0097]
Example 3 Crystallization of glucose isomerase (unification of liquids)
Protein (glucose isomerase) was crystallized using the microchip 41 shown in FIG. FIG. 8B is an enlarged view of the micro liquid weighing structure structure part (b) of the microchip 41 used. The microchip 41 is basically substantially the same as the microchip 24 shown in FIG. 7A except for the shape and the number of arranged micro liquid weighing structures.
A microchip 41 shown in FIG. 8 (a) is a microchip for protein crystallization, and a micro liquid weighing structure according to the present invention is provided in an injection portion of a solution (protein solution and precipitant solution). Yes. Using PDMS as a material, it was prepared by the method according to Example 1. The size of the chip is 24 × 46 mm and the thickness is 60 μm. Each of the first liquid supply channel 33 and the second liquid supply channel 35 has a width of 200 μm, and the mixing channel 34 has a width of 300 μm. The first weigh channel 36 and the second weigh channel 37 each have a width of 100 μm and a length of 1000 μm. The first narrow channel 38 and the second narrow channel 39 each have a width of 20 μm and a length of 400 μm.
[0098]
In FIG. 8A, ports 28 and 30 are ports for introducing a protein solution, and ports 31a and 31b are ports for introducing a precipitant solution.
In the micro liquid weighing structure flow path structure formed on the microchip, the first liquid supply channel (first flow path) 33 has a first balance for weighing the first liquid (protein solution). Seven take channels (third flow paths) 36 are connected, and a first narrow channel (fourth flow path) 38 is connected to each of the seven channels, and the first narrow channels 38 are mixed channels (second channels). The flow path 34) is open. The mixing channel (second flow path) 34 formed at each of the seven locations has a second liquid supply channel 35 and a second weighing channel 37 for weighing the second liquid (precipitant solution). And the flow path structure which consists of the 2nd narrow channel 39 is connected.
First, in order to weigh the first liquid (protein solution), glucose isomerase (Hampton) adjusted to a concentration of 20 mg / mL in water using positive pressure from the port 28 to the first liquid supply channel 33. Research HR7-102) solution was filled. Further, by passing air under a positive pressure, excess glucose isomerase solution is removed from the first liquid supply channel 33 through the port 30, and a first weighing for weighing the first liquid (protein solution) is performed. The glucose isomerase solution was weighed at all seven locations of the channel 36.
Next, air is circulated into the first liquid supply channel 33 from the port 28 using a positive pressure higher than the pressure for removing the excessive glucose isomerase solution, and the weighed glucose isomerase solution is mixed. Drained into channel 34.
Next, 10% PEG8000 (Hampton Research HR2-535) was added to 10% MgCl in a buffer (pH 6.5) of 100 mM MES (Hampton Research HR2-587).₂(Hampton Research HR2-559) was added to the second liquid supply channel 35 and the second weigh channel 37 for weighing the second liquid from the precipitant solution adjusted to 200 mM by the positive pressure from the port 31a. And the excess precipitant solution in the second liquid supply channel 35 is removed via the port 31b by flowing air under positive pressure, and the second liquid is weighed. 2 The precipitant solution was weighed in the weighing channel 37. Next, air is circulated from the port 31a to the second liquid supply channel 35 using a positive pressure higher than the pressure for removing the excess precipitant solution, and the weighed precipitant solution is mixed with the mixing channel 34. Spilled into. As a result, the glucose isomerase solution and the precipitant solution contacted and merged in the mixing channel 34. This operation was performed at five locations to prepare five liquids in which the glucose isomerase solution and the precipitant solution were combined.
When this chip was observed with a stereomicroscope, crystals were observed in the liquid in all five liquids in which the glucose isomerase solution and the precipitant solution were combined.
[0099]
Example 4 Crystallization of glucose isomerase (contact between liquid and continuous liquid)
The experiment was performed using the same chip as in Example 3 and using the same reagents as in Example 1.
First, in order to weigh the first liquid (protein solution), glucose isomerase (Hampton) adjusted to a concentration of 20 mg / mL in water using positive pressure from the port 28 to the first liquid supply channel 33. Research HR7-102) solution was filled. Further, by passing air under a positive pressure, excess glucose isomerase solution is removed from the first liquid supply channel 33 through the port 30, and a first weighing for weighing the first liquid (protein solution) is performed. The glucose isomerase solution was weighed at all seven locations of the channel 36.
Next, 10% PEG8000, MgCl2 in MES buffer (PH6.5) at a concentration of 100 mM.₂The precipitant solution adjusted to be 200 mM was filled from the port 28 to the first liquid supply channel 33 using positive pressure. As a result, the glucose isomerase solution and the precipitant solution contacted each other at the opening of the first weighing channel 36 of the first liquid supply channel 33.
When this chip was observed with a stereomicroscope, the interface between the glucose isomerase solution and the precipitant solution was present (the opening between the first liquid supply channel 33 and the first weighing channel 36), and the glucose isomerase solution side (first Crystals were observed in the liquid on the one weighing channel 36) and the precipitant solution side (first liquid supply channel 33 side). In particular, many crystals were observed in the liquid on the precipitant side.
[0100]
As described above, the micro liquid weighing structure of the present invention and the microchip having the structure and the protein separation using the microchip, the determination of glucose by enzyme reaction, and the crystallization of glucose isomerase have been specifically described. The invention is not limited to the specifically illustrated embodiments and examples. For example, only the portion near the second channel (channel B) of the third channel (channel C) is selectively selected.Properties that are harder to wet than other parts of the third channel (channel C)If the modification can be performed so that the fourth flow path (flow path D) having a narrow width is not provided, the third flow path having a wide width is disposed in the second flow path (flow path B) having the same width. Can also be connected directly. Further, the shape of the third flow path for measuring the liquid can be any shape such as a circle, an ellipse, and a triangle in addition to a rectangle.
[0101]
【The invention's effect】
As described above, according to the present invention, it is possible to obtain a trace liquid weighing structure capable of quantitatively weighing a trace amount of liquid by a simple operation with a simple configuration. Furthermore, according to the present invention, it is possible to reduce the dead volume of the sample in various apparatuses that require quantitative liquid handling, and to realize space saving and cost reduction of the entire apparatus. It is possible to obtain a micro liquid weighing structure that can be made.
[0102]
That is, according to the method of the present invention, since the liquid can be weighed in a minute amount in the microchannel, observation, measurement, mixing, phase change, reaction, etc. can be made minute. Further, the dead volume of the sample is small, and particularly when a plurality of third flow paths are connected to the first flow path, the dead volume can be drastically reduced. In addition, there is an advantage that the area where the weighed liquid is in contact with the outside air is small, so that drying or concentration due to evaporation hardly occurs. Furthermore, since the liquid can be weighed by a simple operation with a simple operation and can be easily operated after that, it is possible to realize space saving and low cost of the entire apparatus.
The microchip having the above-described characteristics of the flow channel structure according to the present invention can be suitably used for, for example, separation / analysis of various substances, biochemistry or chemical reaction, protein crystallization, and the like.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a conceptual explanatory diagram for explaining the principle of the present invention, (a) showing its structure, and (b) and (c) explaining a liquid weighing method.
FIG. 2 is an explanatory view of a trace liquid weighing structure having two liquid production systems according to claim 2, wherein (a) shares the flow path A, and (b) shares the flow path B. This is the case.
FIG. 3 is an explanatory view of the trace liquid weighing structure according to claim 3 and claim 4, wherein (a) shows a case where the flow path D is divided into three flow paths, and (b) It is explanatory drawing in case there exist three sets of the flow paths C and the flow paths D from which thickness differs.
FIG. 4 is an explanatory view of a micro liquid weighing structure according to claim 5, wherein (a) shows the structure, and (b), (c) and (d) are filled in the flow path B with liquid. It is explanatory drawing of the liquid introduction method in the case of being carried out.
5 (a) and 5 (b) are plan views showing a trace liquid weighing structure according to the present invention as set forth in claim 6, and FIG. 5 (c) is a cross section taken along the line cc in FIG. 5 (b). FIG.
6 is an explanatory diagram of a microchip 10 having a trace liquid weighing structure according to the present invention used for protein separation by ion exchange chromatography in Example 1, wherein (a) is a layout of the entire chip; (B) is a quantification of a small amount of sample and an enlarged explanatory view of an introduction part, and (c1) and (c2) are cross-sectional views along the line cc in FIG.
7 (a) is a plan view of a microchip 24 according to another embodiment of a microfluidic weighing structure according to the present invention, which is used for glucose determination by enzymatic reaction of Example 2, and FIG. (A) It is the elements on larger scale of the b section in a figure, (c) is the elements on larger scale of the c section in (b) figure.
8 is a plan view of a microchip 41 used for crystallization of glucose isomerase in Examples 3 and 4, and FIG. 8 (b) is a partially enlarged plan view of a portion b in FIG. 8 (a).
[Explanation of symbols]
10 Microchip for capillary chromatography
11 Elution buffer introduction port
12 Elution buffer introduction port
13 Mixer
14 microchannels
15 Damping part
16 PDMS flat plate (top)
17 PDMS flat plate (bottom)
18 ports
19 First flow path
20 Second flow path
21 Third flow path
22 Fourth channel
23 Fifth flow path
24 Microchips for 50 kinds of two-component mixed reaction
25 Upper substrate
26 Lower board
27 channels
28 First liquid inlet port
29 channels
30 First liquid outlet port
31a Second liquid inlet port
31b Second liquid outlet port
32a port
32b port
33 First liquid supply channel
34 mixing channels
35 Second liquid supply channel
36 First Weighing Channel
37 Second weighing channel
38 1st narrow channel
39 Second narrow channel
100 liquid (sample, etc.)
100a Lower end of liquid in channel C (channel A side)
100b Upper end of liquid in channel C (channel D side)
200 liquid (liquid chromatographic mobile phase, etc.)
aa, aa ′ Channel wall of channel A
bb, bb 'Channel walls of channel B
c1 Opening of channel C (channel A side)
c2 Opening of channel C (channel D side)
d1 Opening of channel D (channel B side)
e1 Opening of channel E (channel D side)

Claims (18)

A first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction, and a first flow path opening in the flow path wall of the first flow path (flow path A). 3 channel (channel C) and one end of the third channel (channel C) opened to the channel wall of the second channel (channel B) and the second channel And a fourth channel (channel D) having a property that the capillary attraction is relatively less likely to work than the third channel (channel C), and is introduced into the first channel. The drawn liquid is drawn into the third flow path through the opening of the third flow path that opens in the flow path wall of the first flow path, and then into the first flow path. A trace liquid weighing structure characterized by removing the remaining liquid and weighing a volume of liquid corresponding to the volume of the third flow path. A first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction, and a first flow path opening in the flow path wall of the first flow path (flow path A). 3 channel (channel C) and one end of the third channel (channel C) opened to the channel wall of the second channel (channel B) and the second channel And a fourth channel (channel D) having a property that the capillary attractive force is relatively less likely to work than the third channel (channel C) , and the first channel After the introduced liquid is drawn into the third flow path through the opening of the third flow path that opens in the flow path wall of the first flow path, the first flow path At least two systems that remove the liquid remaining in the liquid and weigh a volume of liquid corresponding to the volume of the third flow path, and share the first flow path or the second flow path Trace liquid characterized by Intake structure.   Two or more fourth channels (channel D) are connected to the third channel (channel C), or two or more fourth channels (channel D) are connected. The trace liquid weighing structure according to claim 1, wherein the structure is branched.   The third flow path (flow path C) and a plurality of fourth flow paths (flow path D) connected to the third flow path (flow path C) are formed in any one of claims 1 to 3. The trace liquid weighing structure as described in 1. A fifth channel (channel E) that opens to the channel wall of the fourth channel (channel D) and has a property that capillary attraction is less likely to work than the fourth channel is further provided. The trace liquid weighing structure according to any one of claims 1 to 4, characterized by comprising: The second channel has a narrow channel part having a channel width narrower than a normal channel width of the channel at a part of the channel, and the narrow channel part is a normal channel part. The micro liquid weighing structure according to any one of claims 1 to 5, wherein a capillary attractive force is relatively less likely to work than a road width portion .   The liquid weighed in a volume corresponding to the volume of the third channel (channel C) is transferred to the second channel (channel B) via the fourth channel (channel D). The trace liquid weighing structure according to any one of claims 1 to 6, further comprising an outflow means for causing the liquid to flow out into the apparatus.   When the second flow path in the vicinity of the opening of the fourth flow path (flow path D) is filled with liquid, the volume according to the volume of the third flow path (flow path C) The trace liquid weigher according to claim 7, further comprising outflow means for flowing out the weighed liquid to the second channel through the fourth channel (channel D). Construction.   The first channel (channel A), the second channel (channel B), the third channel (channel C), the fourth channel (channel D), and the first channel 8. The micro liquid weighing structure according to claim 1, wherein each of the five flow paths (flow path E) is a channel formed in the substrate.   The volume of the said 3rd flow path (flow path C) is formed in the magnitude | size of the order of picoliter to microliter, The any one of Claims 1-9 characterized by the above-mentioned. Trace liquid weighing structure.   A microchip comprising at least one trace liquid weighing structure according to any one of claims 1 to 10 in a substrate.   A plurality of the trace liquid weighing structures according to any one of claims 1 to 10 are provided in a substrate, and each trace liquid weighing structure is the same or different. A microchip according to claim 1.   13. The microchip according to claim 11, wherein the substrate has a two-layer structure in which an upper substrate and a lower substrate are bonded to each other. A substrate having a two-layer structure in which an upper substrate (16) and a lower substrate (17) are bonded to each other has a microchannel (14) for ion exchange chromatography, and an elution buffer introduction port (11, 12). And a mixer (13) communicating with the port is connected in the middle of the microchannel (14), and the ion exchange bead damming portion (15) of the microchannel (14) and the mixer (13 ) A trace liquid weighing structure portion is provided between the connecting portion and the trace liquid weighing structure portion on the first flow path (19) and the flow path wall of the first flow path (19). third flow path for preparative liquid weigher opening (21), and communicating with the flow path of said third (21) second flow passage constituting the microchannel (14) (20), said first Capillary attractive force works more than the flow path (21) of 3 A fourth channel having piles properties (22), the flow path of the fourth intersecting the flow path of said fourth (22) a width substantially the same as or which narrower than the (22) A microchip for capillary ion exchange chromatography, characterized by comprising a fifth flow path (23).   The upper substrate (16) and the lower substrate (17) are made of polydimethylsiloxane (PDMS), and the surface of the lower substrate (17) is made hydrophobic by a curing process. 14. The microchip according to 14. In a substrate having a two-layer structure in which an upper substrate (25) and a lower substrate (26) are bonded together, a substantially annular first having a liquid inlet port (28) at one end and an outlet port (30) at the other end. The first liquid supply channel (33) has a plurality of first weighing channels (36) that open to the flow path wall, and each first weighing channel (36) has a liquid supply channel (33). respect, mixing channel (34) is disposed as a second liquid feed channel (35) and a set of the set, the first weighed channel (36) wherein the mixing channel (34), wherein The second liquid supply channel (35) is connected to the flow path wall through a first narrow channel (38) having a property that capillary pulling force is relatively less likely to work than the first weighing channel (36). One second weighing channel (37) opening in And, in the second weighed channel (37) wherein the mixing channel (34), a second narrow channel having a small extent of relative capillary attraction acts than the second weighed channel (37) and (39) The second liquid supply channel (35) and the mixing channel (34) have an inlet port (31a, 32a) and an outlet port (31b, 32b), respectively, and each port has the upper part. A microchip for microanalysis and synthesis / separation characterized by being provided through a substrate.   The substrate has a disc shape, and the first weigh channel (36), the first narrow channel (38), the mixing channel (34), radially inward of the substantially annular first liquid supply channel (33), 20 sets each including a second narrow channel (39), a second weighing channel (37), and a second liquid supply channel (35) are provided, and the substantially annular first liquid supply channel (33) is radially outward. The first weigh channel (36), the first narrow channel (38), the mixing channel (34), the second narrow channel (39), the second weigh channel (37) and the second liquid supply channel (35) The microchip according to claim 16, wherein 30 sets are provided.   The upper substrate (25) and the lower substrate (26) are made of polydimethylsiloxane (PDMS), and the surface of the lower substrate (26) is made hydrophobic by a curing process. The microchip according to 16 or 17.

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