JP2004163104A - Minute quantity liquid scaling structure and microchip having the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a minute quantity liquid scaling structure having a simple structure and quantitatively dealing with a minute quantity of a liquid in only simple operation. <P>SOLUTION: After the liquid introduced into a first flow path is drawn into a third flow path, the liquid remaining in the first flow path is removed and the liquid having volume corresponding to volume of the third flow path is scaled by using the structure having the first flow path (the flow path A) and a second flow path (a flow path B) for respectively extending in the predetermined direction, the third flow path (the flow path C) open to a flow path wall of the first flow path and a fourth flow path (a flow path D) open to a flow path wall of the second flow path, coupling one end of the third flow path to the second flow path, having a wetproof property (or a property for relatively preventing capillary force) and narrower than the other three flow paths. If the second flow path is filled with the liquid, the structure also has a fifth flow path (a flow path E) comprising a flow path wall open to a flow path wall of the fourth flow path, narrower than or the same as the fourth flow path and having a wetproof property (or a property for relatively preventing capillary force). <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a micro liquid weighing structure. More specifically, the present invention relates to a microfluidic liquid weighing structure suitable for use in performing an analysis or a chemical reaction using various samples, and a microchip having the structure in a substrate.
[0002]
[Prior art]
Conventionally, various devices for performing analysis by electrophoresis, chromatography, and the like are known. In such devices, accurate analysis results can be obtained by quantitatively weighing a liquid such as a sample to be used. Can be done.
[0003]
Therefore, various methods have been proposed for quantitatively weighing a liquid such as a sample in various devices used for such electrophoresis and chromatography. For example, Patent Literature 1 describes a microchip electrophoresis apparatus, and Patent Literature 2 describes an apparatus and a method for electrophoretic separation of a fluid substance mixture. However, any of these conventional techniques requires a sample larger than the amount actually required for analysis, and has a problem that the dead volume of the sample cannot be reduced.
[0004]
Further, in a chemical reaction or analysis using a liquid such as a small amount of a sample, a microchip composed of a minute chip may be used. Even when such a microchip is used, accurate results can be obtained by quantitatively weighing a liquid such as a sample to be used.However, in the analysis using a microchip, a liquid such as a sample to be handled is used. Since the volume of the liquid is extremely small, it is difficult to quantitatively weigh the liquid, and various complicated configurations are required, and the operation for handling the configuration is complicated.
[0005]
On the other hand, conventionally, as a method of crystallizing a protein, a batch method, a dialysis method, a vapor diffusion method, a gel method, and the like have been known. However, each of these methods has a problem that a large amount of protein is required. 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 is required. Had.
[0006]
In addition, in order to reduce the amount of protein 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 crystallization conditions of the protein are searched. The attempt was known. However, in this method, since the droplets are exposed to the air, there is a problem that the droplets evaporate and dry or concentrate.
[0007]
Furthermore, an attempt to generate a crystal in a laminar flow using a microchannel device (for example, see Patent Document 4) or an attempt to obtain a crystal by strictly controlling the temperature (for example, see Non-Patent Document 2) is known. I was However, in this method, the reaction field is minute and the reaction control is excellent, but the method of introducing the protein solution to the crystallization site is the same as the conventional method, and the ratio of dead volume is extremely large. Had.
[0008]
[Patent Document 1]
JP-A-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]
U.S. 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]
Therefore, an object of the present invention is to provide a trace liquid weighing structure which can quantitatively measure a trace amount of liquid with only a simple operation with a simple configuration.
[0010]
Further, another object of the present invention is to reduce the dead volume of a sample and realize space saving and cost reduction of the entire device in various devices that require quantitative liquid handling. It is an object of the present invention to provide a micro liquid weighing structure which can be used.
[0011]
[Means to solve the problem]
The above object is solved by the micro liquid weighing structure of the present invention. The microfluidic liquid weighing structure of the present invention focuses on the surface tension of the liquid and utilizes the capillary phenomenon (capillary repulsion) caused by the liquid in the flow path.
[0012]
According to the first embodiment of the trace liquid weighing structure of the present invention, the first flow path and the second flow path each extending in a predetermined direction, and the flow path wall of the first flow path A third flow path that is open, and one end of the third flow path that is open in the flow path wall of the second flow path and connects the second flow path, and is less likely to wet (or relatively capillary) A fourth flow path having a property of being less attractive (attracting force does not work) and being thinner than the other three flow paths, and a liquid introduced into the first flow path is formed by a flow path wall of the first flow path. After being drawn into the third flow path through the opening of the third flow path that opens at the above, the liquid remaining in the first flow path is removed, and the volume of the third flow path is removed. Is characterized in that a liquid having a volume corresponding to is measured.
[0013]
According to the second embodiment of the trace liquid weighing structure of the present invention, the first flow path and the second flow path each extending in a predetermined direction, and the flow path wall of the first flow path A third flow path that is open, and one end of the third flow path that is open in the flow path wall of the second flow path and connects the second flow path, and is less likely to wet (or relatively capillary) A fourth flow path having a property of being less attractive (attraction is unlikely to work) and being thinner than the other three flow paths, and the liquid introduced into the first flow path is used as the flow path of the first flow path. After being drawn into the third flow path through the opening of the third flow path that opens in the wall, the liquid remaining in the first flow path is removed, and the third flow path is removed. It is characterized by having at least two systems for weighing a liquid having a volume corresponding to the volume, and sharing the first flow path or the second flow path.
[0014]
According to the third embodiment of the trace liquid weighing structure of the present invention, two or more of the fourth flow paths are connected to the third flow path, or two or more of the fourth flow paths are connected to the third flow path. It is characterized by being branched 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 thereto are formed.
[0016]
According to the fifth embodiment of the trace liquid weighing structure according to the present invention, the opening is formed in the flow path wall of the fourth flow path, is thinner or the same thickness as the fourth flow path, and is less likely to be wet (or It is characterized by further having a fifth flow path composed of a flow path wall having a property that the capillary attraction does not work relatively easily.
[0017]
According to a sixth embodiment of the trace liquid weighing structure of the present invention, the second flow path has a flow path width smaller than a normal flow path width of a part of the flow path. Characterized in that the narrow channel portion has a property that it is harder to wet (or relatively hard to apply capillary attraction) than a normal channel width portion.
[0018]
According to a seventh embodiment of the trace liquid weighing structure of the present invention, the liquid weighed in a volume corresponding to the volume of the third flow path is supplied to the second flow path via the fourth flow path. Characterized by further comprising an outflow means for outflow to the flow path.
[0019]
According to an eighth embodiment of the trace liquid weighing structure of the present invention, when the second flow path near the opening of the fourth flow path is filled with the liquid, the third flow path is provided. It is characterized by further comprising an outlet means for causing the liquid weighed in a volume corresponding to the volume of the passage to flow out to the second flow passage via the fourth flow passage.
[0020]
According to a ninth embodiment of the trace liquid weighing structure of the present invention, the first flow path, the second flow path, the third flow path, the fourth flow path, and the fifth flow path. Each of the flow paths is a channel formed on the substrate.
[0021]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, an embodiment of a trace liquid weighing structure according to the present invention will be described in detail with reference to the accompanying drawings.
[0022]
1 (a), 1 (b) and 1 (c) are conceptual illustrations for explaining the principle of the present invention. The four flow paths A (first flow path), flow path B (second flow path), flow path C (third flow path), and flow path D (fourth flow path) A has a structure in which the flow path C and the flow path D are bridged in series from the flow path A to the flow path B between the flow path A and the flow path B. The liquid is introduced into the channel C via the channel A. Since the flow path D having a flow path wall that is hard to wet (or relatively hard to exert a capillary attractive force) is thinner than other flow paths and requires a larger force for introduction, an appropriate pressure is applied to the liquid. Then, the liquid can be stopped at the boundary surface c2 between the flow path C and the flow path D (see FIGS. 1A and 1B).
In the present specification, “the property that is hard to wet (or relatively hard to apply capillary attraction)” means that when the liquid is drawn into the channel C, the liquid cannot pass through the channel D with the same pressure, It means the nature of the flow path where the liquid stops.
[0023]
More specifically, when the liquid 100 (see the hatched area in FIG. 1) is introduced into the flow channel A, the liquid 100 is passed through the opening c1 of the thin flow channel C that opens in the flow channel wall aa of the flow channel A. Can be introduced into the flow path C. When the flow path A and the flow path C have a flow path wall having a property of being easily wetted (or easily acting on the capillary attraction), if the flow path C is made thinner than the flow path A, the liquid 100 will have a stronger capillary attraction. The channel C is spontaneously drawn into the channel C via the opening c1 of the channel C that opens in the channel wall aa of the channel A. When the flow path A and the flow path C have flow path walls having a property of being hard to wet (or relatively hard to exert a capillary attraction), by applying an appropriate pressure to the liquid 100 from the end face of the flow path A, The liquid 100 can be introduced into C (see FIG. 1B).
[0024]
At this time, the liquid 100 that has reached the end face c2 of the flow path D (the flow path section between c2 and d1) on the side of the flow path C is blocked by the capillary repulsion of the flow path D having the flow path wall that is difficult to wet, and It does not get into road D. Even in the case where the flow path C is a flow path wall that is difficult to wet, the liquid 100 does not enter the flow path D because the capillary repulsion of the flow path D is stronger than the capillary repulsion of the flow path C (FIG. 1 (b)). )reference).
[0025]
Subsequently, the liquid 100 remaining in the flow path A is moved to a lower pressure side by generating an appropriate pressure difference between both ends of the flow path A, for example, and the opening of the flow path C in the flow path A is reduced. The part is moved to a position not in contact with the part c1 or removed from the inside of the flow path A (see FIG. 1C). At this time, the liquid 100 in the flow path C does not usually return into the flow path A (see FIG. 1C). As a result, the end faces 100a and 100b, which are both end faces of the liquid 100 in the flow path C, are located at 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 measure a liquid having a volume corresponding to the volume of the section between c1 and c2 (see FIG. 1C). However, the end face of the liquid is formed so as to substantially match the end face of the opening c1 or c2 due to the influence of surface tension, contact angle, or the like, and may not exactly match the end face of the opening c1 or c2. Therefore, the volume of the liquid measured according to the volume of the section between c1 and c2 of the flow path C may not exactly match the volume of the section between c1 and c2 of the flow path C.
[0026]
Further, the liquid weighed in the channel C is caused to flow by causing an appropriate pressure difference between the two channels so that the pressure in the channel A becomes slightly larger than the pressure in the channel B. It is easy to introduce into the flow path B through the path D and the opening d1. As a result, the liquid can be used for reaction or analysis by transferring the liquid by air pressure or the like.
[0027]
According to the first aspect of the present invention, the first flow path (flow path A) and the second flow path (flow path B) each extending in a predetermined direction, and the first flow path A third flow path (flow path C) opening on the flow path wall of (flow path A) and a third flow path (flow path C) opening on the flow path wall of the second flow path (flow path B). One end of the flow path C) is connected to the second flow path (flow path B), and has a property that it is hardly wet (or relatively hard to apply capillary attraction), and is thinner than the other three flow paths. 4 (flow path D), and the liquid introduced into the first flow path has an opening in the third flow path that opens in the flow path wall of the first flow path. After being drawn into the third flow path through the third flow path, the liquid remaining in the first flow path is removed, and a liquid having a volume corresponding to the volume of the third flow path is weighed. Things.
[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, a liquid having a volume corresponding to the volume of the third flow path is obtained. With a simple configuration, the liquid can be quantitatively weighed with only a simple operation, while reducing the dead volume of the sample, saving space and reducing the cost of the entire apparatus. Can be realized.
[0029]
Further, the invention according to claim 2 of the present invention is characterized in that the first flow path (flow path A) and the second flow path (flow path B) extending in a predetermined direction, respectively, A third flow path (flow path C) opening on the flow path wall of the flow path (flow path A); and a third flow path opening on the flow path wall of the second flow path (flow path B). A fourth flow path which is connected to one end of the flow path (flow path C) and the second flow path (flow path B) and has a property of being hard to wet (capillary attraction does not work) and being thinner than the other three flow paths. (Flow path D), and the liquid introduced into the first flow path is provided through the opening of the third flow path which is opened in the flow path wall of the first flow path. After being drawn into the third flow path, the liquid remaining in the first flow path is removed, and at least two systems for measuring a liquid having a volume corresponding to the volume of the third flow path are provided. At least two systems as described above Together, in which so as to share the first flow path or the second flow path (see FIG. 2 (a) and Figure 2 (b)).
[0030]
Therefore, according to the second aspect 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 a plurality of liquids of the same type are quantitatively weighed in each of the systems, different types of liquids weighed in the two systems are combined with each other in the second flow path of each system, and dilution and unification by union are performed. One reaction, analysis after the union reaction, and the like can be performed (see FIG. 2A). Conversely, for example, when different types of liquids are quantitatively weighed in each of two systems sharing the second channel, the plurality of liquids created in the second channel shared by the two systems are , Dilution by coalescence, reaction by coalescence, analysis by reaction after coalescence, etc. (see FIG. 2 (b)).
[0031]
According to the third aspect of the present invention, in the trace liquid weighing structure according to any one of the first and second aspects, the fourth flow path (flow path D) May be divided into two or more flow paths, or may be branched into two or more flow paths (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, in the trace liquid weighing structure according to any one of the first, second, and third aspects, the third flow path (flow path) C) and two or more sets of the fourth flow path (flow path D) are formed (see FIG. 3B). Thus, according to the invention described in claim 4 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. The flow paths C ′ and D ′ and the flow paths C ″ and D ″) allow a plurality of different volumes of liquid to be weighed quantitatively and in parallel.
[0033]
According to a fifth aspect of the present invention, in the trace liquid weighing structure according to any one of the first, second, third, and fourth aspects, the fourth flow path is provided. A fifth flow path (flow path) composed of a flow path wall which is open to the flow path wall of the flow path (flow path D) and has the property of being thinner or the same thickness as the fourth flow path and less likely to wet (capillary attraction does not work). (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 flow paths A, B, C, D, and E flow between the flow path A and the flow path B and flow path C. The road D has a structure that connects in series to form a bridge. When the liquid is introduced from the flow path A to the flow path C, the flow path D has a flow path wall having a property of being hardly wet (or the capillary attraction does not work). Therefore, the liquid stops at the interface c2 between the flow path C and the flow path D. In order to introduce the liquid into the flow path D, a larger force is required.
[0035]
When the liquid 100 (see the hatched area in FIG. 4) is introduced into the thick flow channel A, the liquid 100 flows into the flow channel C through the opening c1 of the thin flow channel C that opens in the flow channel wall aa of the flow channel A. (See FIG. 4B). This phenomenon is caused by any one of claims 1, 2, 3, and 4 when the liquid is not filled in the vicinity of the opening d1 of the flow path D in the flow path B. It has the same structure as the described trace liquid weighing structure, but when the liquid 200 (the gray area in FIG. 4) has been introduced into the flow channel B in advance, claims 1, 2, and 3. In the third and fourth aspects of the present invention, 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 flow path C.
[0036]
According to the fifth aspect of the present invention, when the liquid 200 (the gray area in FIG. 4) has been introduced into the flow channel B, the flow of the liquid The gas in the inside is purged into the flow path E having an opening e1 in the flow path wall of the flow path D (the end face of the flow path E opposite to e1 is open to the atmosphere). (See FIG. 4B). Also in this case, the liquid 100 that has reached the end face c2 between the flow path C and the flow path D has a flow path D (a flow path between c2 and d1) having a flow path wall that is difficult to wet (or does not exert capillary attraction). The channel section is blocked by capillary repulsion 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 by, for example, generating an appropriate pressure difference at both ends of the flow path A and moving it to a lower pressure side. As a result, a liquid having a volume corresponding to the volume of the flow path C (the section between c1 and c2) can be weighed (see FIG. 4C).
[0038]
Further, the liquid formed in the flow path C causes an appropriate pressure difference between the two flow paths so that the pressure in the flow path A becomes slightly larger than the pressure in the flow path B, and the like. Can be introduced into the liquid 200 in the flow path B through At this time, the end face of the flow path E opposite to the end face e1 needs to be closed (see FIG. 4D).
[0039]
Therefore, according to the invention of claim 5 of the present invention, it is possible to quantitatively introduce a small amount of a sample or a reaction reagent into various analyzers or reaction devices used for electrophoresis or chromatography. A practical approach can be provided.
[0040]
FIGS. 5A and 5B are plan views showing a trace liquid weighing structure of the present invention according to claim 6, and FIG. 5C is a cross-sectional view taken along line cc in FIG. is there. As shown in FIG. 5A, the second flow path (flow path B) has a narrow flow having a flow width smaller than a normal flow width of the flow path in a part of the flow path. It has a road portion (flow path b1 and flow path b2). The narrow channel portion has a property that it is harder to wet (or relatively hard to exert capillary attraction) than a normal channel width portion. 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, by sandwiching a part of the second flow path between the two narrow flow path parts, a volume-limited section can be formed in a part of the second flow path. The third flow path (flow path C) can be communicated with the volume-limited section of the second flow path via the fourth flow path (flow path D). For example, when the first liquid weighed in one third flow path (flow path C) is extruded through the fourth flow path (flow path D) into the limited volume section of the second flow path. The air in the volume-limited compartment is discharged into the atmosphere through the narrow channel portion, and the extruded first liquid remains in the volume-limited compartment. Further, the second liquid weighed in the other third flow path (flow path C ′) is subjected to the volume limitation section of the second flow path via the other fourth flow path (flow path D ′). When pushed into the volume-limited compartment, the remaining air in the volume-limited compartment 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 compartment. Therefore, the limited volume section sandwiched between the two narrow flow path portions can be used as a reaction chamber, and the amount of the reaction solution to be used can be minimized. For this reason, the volume of the limited volume section of the second flow path can be determined in consideration of the total amount of liquid supplied into the limited section. For example, it is preferable to set the value to be approximately equal to or slightly larger than the sum of the volumes of the one or more third flow paths connected in the limited section.
FIG. 5 (b) 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 is partially narrowed by two narrow paths. It has a flow path portion (flow path b1 and flow path b2) and the other parts have a normal flow path width. For example, the first liquid is supplied from the second channel having a normal channel width to the volume-limited section via the first narrow channel portion (channel b1), and then supplied to the third channel (flow). The second liquid weighed in the path C) is supplied into the limited volume section via the fourth flow path (flow path D).
Therefore, the second flow path does not necessarily mean only a flow path in which a liquid moves (flows), but is also used as a flow path for moving air. In particular, it suffices that a volume-limited section sandwiched by two narrow flow path portions capable of allowing the liquid weighed out in the third flow path to flow out is formed, and the shape of the second flow path, especially, the volume The planar shape of the limited section is not limited to the illustrated rectangular shape, but can be various shapes such as a circle, an ellipse, and a triangle.
As shown in FIG. 5 (c1), a narrow flow path portion (flow path b1 and flow path b2) of the second flow path is formed on the lower substrate 1, and a normal width portion of the second flow path is formed. It can be formed on the upper substrate 3 or, as shown in FIG. 5 (c2), the narrow flow path portions (flow path b1 and flow path b2) of the second flow path and the second flow path All of the normal width portion can be formed on the upper substrate 3.
[0041]
According to a seventh aspect of the present invention, a trace liquid weighing apparatus according to any one of the first, second, third, fourth, fifth and sixth aspects is provided. In the structure, the liquid quantitatively weighed in the third flow path (flow path C) is further supplied to the second flow path (flow path D) via the fourth flow path (flow path D). There is provided an outflow means for flowing out from the opening of the fourth flow path (flow path D), which opens in the flow path wall of the path B), to the second flow path (flow path B).
[0042]
Specifically, in order for the liquid in the third channel formed quantitatively to flow out to the second channel, the pressure in the first channel is slightly lower than the pressure in the second channel. 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 the size. As a means for generating a pressure difference, for example, a known and commonly used pressurizing mechanism or depressurizing mechanism is suitably used.
[0043]
Therefore, according to the invention described in claim 7 of the present invention, the liquid in the third flow path that is quantitatively weighed can flow out to the second flow path, so that electrophoresis or electrophoresis can be performed. It is possible to quantitatively introduce a small amount of a sample or a reaction reagent into various analyzers or reaction devices used for chromatography or the like.
[0044]
According to an eighth aspect of the present invention, in the micro liquid weighing structure according to the seventh aspect, the second flow path (flow path B) is further filled with a liquid. The liquid quantitatively weighed in the third flow path (flow path C) is opened at the flow path wall of the second flow path via the fourth flow path (flow path D). Outflow means for flowing out from the opening of the third flow path to the second flow path.
[0045]
Specifically, according to the invention of claim 8 of the present invention, the liquid in the third flow path that is quantitatively formed using the trace liquid weighing structure of claim 5 is a liquid flowing through the third flow path. An appropriate pressure difference is generated between the two flow paths so that the pressure in the first flow path becomes slightly higher than the pressure in the second flow path in a state where the end face of E opposite to e1 is closed. Alternatively, by performing an operation such as applying a centrifugal force in the outflow direction, it is possible to cause the outflow to the second flow path.
[0046]
Therefore, according to the invention described in claim 5 of the present invention, the liquid in the third flow path that is quantitatively weighed can flow out to the second flow path. It is possible to quantitatively introduce a small amount of a sample or a reaction reagent into various analyzers or reaction devices used for chromatography or the like. In addition, by applying the microfluidic liquid weighing structure of the present invention, introduction of a sample in nucleic acid and protein electrophoresis (injection), and facilitating or hardly wetting a surface or a necessary portion in a channel, Alternatively, it is possible to adapt to protein synthesis or separation, chemical substance synthesis or screening, etc. by performing each required treatment corresponding to the surface properties of proteins and DNA.
[0047]
Further, of the present invention, the invention described in claim 9 is any one of claim 1, claim 2, claim 3, claim 4, claim 5, claim 6, claim 7, or claim 8. In the invention described in the paragraph, the first flow path (flow path A), the second flow path (flow path B), the third flow path (flow path C), and the fourth flow path (flow path A). 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 ninth aspect of the present invention, it is possible to quantitatively weigh a small volume of liquid with only a simple operation by a simple configuration, and to further reduce the dead time of the sample. It is possible to reduce the volume, save the space of the entire apparatus, and reduce the cost.
[0049]
The invention described in claim 10 of the present invention is the invention of claim 1, claim 2, claim 3, claim 4, claim 5, claim 6, claim 7, claim 8, or claim 9. In the invention according to any one of the first to third aspects, the volume of the third flow path is formed in a size on the order of picoliter to microliter.
[0050]
Therefore, according to the microfluidic liquid weighing structure of the present invention, it is possible to quantitatively weigh microvolume liquids of various sizes from picoliters to microliters with a simple configuration and simple operations only. it can.
[0051]
The trace liquid weighing structure of the present invention can be formed in a chip. A plurality of sets of the trace liquid weighing structure 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 channel formed is preferably a microchannel. Here, the microchannel refers to a channel having a cross-sectional shape in which a micro effect appears when a liquid is introduced into a channel (flow path), that is, a liquid has some behavior change. The appearance of the micro effect depends on the physical properties of the liquid, but the length of the shortest interval (corresponding to the short side in the case of a rectangle and the minor axis in the case of an ellipse) of the cross-sectional shape, that is, the shape of the surface perpendicular to the main flow direction of the channel. The thickness 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, a chip means a member of a portion including a trace liquid weighing structure. Further, the microchip means a chip provided with a micro 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 a substance, biochemistry or a chemical reaction, or protein crystallization. The chip in the present invention is desirably disposable or replaced only for a limited number of uses, but may be used permanently. In this case, it may be possible to integrate the device with a device such as a dispenser or a measuring device. In such a case, a member including a flow path structure is called a chip.
The material of the chip in the present invention is not particularly limited as long as the above-mentioned flow path structure can be realized. Examples of the material include polydimethylsiloxane (PDMS), glass, silicon, photoreactive resin and other resins, metal, ceramic, and a combination 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, mechanical processing, transfer technology represented by injection molding and compression molding, dry etching (RIE, IE, IBE, plasma etching, laser etching, laser ablation, blast processing, electric discharge machining, LIGA, electron beam etching, FAB), wet Surface micro-machining, which forms a fine structure by etching (chemical erosion), solid molding such as stereolithography, filling of ceramics, etc., coating, vapor deposition, sputtering, depositing and partially removing various substances in layers The method includes a method of forming a groove by forming an opening portion by using at least one sheet-like substance (film, tape, or the like), and a method of forming a flow path forming material by dropping and injecting the material using an ink jet or a dispenser.
A mask may be utilized in the above method to make a chip of the present invention. The mask may have any design or a plurality of masks as long as the chip of the present invention can be finally produced. The mask is usually designed in a shape in which the flow path is projected onto a plane, but when processing is performed on both sides of the flow path component to be bonded, or when the flow path is formed using a plurality of members, a plurality of masks are used. Can be directly processed without using a mask, and a mask is not always a projection of the shape of the final channel. Examples of a mask for shielding electromagnetic waves used for a photocurable resin include a mask obtained by coating chromium on quartz or glass, or a mask obtained by baking a resin film with a laser.
The mask can also be created by, for example, drawing at least a part of the flow channel structure using a computer and appropriate software, and printing the drawn channel structure on a transparent resin film. The present invention also includes a computer-readable recording medium used to create the mask or the master chip in which at least a part of the electronic information of the flow path structure, which is drawn by the software, is stored. Here, examples of suitable recording media include magnetic media such as flexible disks, hard disks, and magnetic tapes; optical disks such as CD-ROMs, MOs, CD-Rs, CD-RWs, and DVDs; and semiconductor memories. .
In manufacturing the chip of the present invention, the chip may be directly manufactured by the above method, or the chip may be shaped as a mold. Of course, it is possible to further shape the chip using this as a mold. In the present invention, the mold at each stage from the mold created first to the front of the chip actually used for the operation may be referred to as a “master chip”. The master chip usually has the same concavo-convex pattern as the actual chip (positive) and is transferred twice, or the master chip has the concavo-convex pattern opposite to the actual chip (negative). The master chip in the present invention is not necessarily limited to this, although it is used after repeated transfer.
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. Bonding methods include bonding with an adhesive, resin bonding with a primer, diffusion bonding, anodic bonding, eutectic bonding, heat fusion, ultrasonic bonding, laser melting, lamination with a solvent or dissolving solvent, adhesive tape, adhesive tape. , Compression, bonding with a self-adsorbent, physical holding, and combination by unevenness.
In addition, a method in which the fluid branch portion and the independent flow path are integrally formed without the need for lamination is also possible. Specifically, it is possible to form a structure including a closed space by integral molding such as an optical molding method.
The length, shape, and thickness of one side of the chip thus produced are not limited, and may be set to an arbitrary value of, for example, 5 mm to 100 mm per side.
[0052]
Note that when a microchip is manufactured by attaching a substrate using a mask and a master chip, the depth of a microchannel in the chip is, for example, when the microchannel is formed using two substrates, the channel is formed on the upper substrate. Depending on which of the lower surface and the upper surface of the lower substrate is present, it can be partially set to one of two arbitrary values, and the depth thereof is not particularly limited, but is preferably 1 to 200 μm. Can be set to any value. Here, the “depth of the channel” corresponds to a chip having a channel having a convex structure serving as a channel mold, that is, a height of a convex portion of a master chip. Further, the number of substrates is not limited to two, and a chip may be formed by laminating an arbitrary number of substrates as needed.
[0053]
Of the present invention, the invention according to claim 20 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 passed through an opening of a third flow path that opens to the first flow path; After the protein solution or the precipitant solution is drawn into the channel, the protein solution or the precipitant solution remaining in the first channel is moved to a position where it does not contact the opening of the third channel, Allowing the protein solution or the precipitant solution prepared in a volume corresponding to the volume of the third flow path to flow out to the second flow path via a fourth flow path;
(B) introducing a precipitant solution or a protein solution into the first flow path, and passing the precipitant solution into the third flow path through the opening of the third flow path that opens to the first flow path; Alternatively, after the protein solution is drawn in, the precipitant solution or the protein solution remaining in the first flow path is moved to a position where the precipitant solution or the protein solution does not come into contact with the opening of the third flow path. The precipitant solution or the protein solution prepared in a volume corresponding to the volume of the solution is allowed to flow out through the fourth flow path to the second flow path, and the protein solution and the precipitant solution are passed through the second flow path. Contacting and uniting;
(C) A method for crystallizing a protein, comprising a step of precipitating protein and a protein crystal from a precipitant solution in the second channel.
According to a twenty-first aspect of the present invention, in the twentieth aspect, the precipitation of the protein crystals from the combined protein and precipitant solution is separated from the solution by a flow channel structure. The method is performed in a state where a precipitant solution is disposed therein. In the present invention, the precipitant solution may be disposed, for example, in advance by introducing it into an appropriate flow path, for example, a second flow path, or by contacting / coalescing the protein solution with the precipitant solution. After that, the precipitant solution may be introduced into the first flow path and arranged in a state of being drawn into the third flow path.
In these inventions, either of the introduction of the protein solution and the precipitant solution into the second channel may be performed first, or the second channel may be simultaneously formed using two separately formed channel structures. It may be introduced to the road.
Further, of the present invention, the invention described in claim 22 is:
(A) A protein solution is introduced into the first flow path of the trace liquid weighing structure, and the protein solution is introduced into the third flow path through an opening of a third flow path that opens to the first flow path. After the protein solution is drawn in, 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 is moved according to the volume of the third flow path. Creating a protein solution with the adjusted volume,
(B) introducing a precipitant solution into the first channel, bringing the protein solution and the precipitant solution into contact with each other at the opening of the third channel, and combining the protein solution and the precipitant solution;
(C) A method for crystallizing a protein, comprising a step of precipitating protein crystals from the combined protein and precipitant solution.
In this method, protein crystals are precipitated in the first and / or third channels, and if necessary, the combined solution is allowed to stand for an appropriate time, and then the presence or absence of crystal precipitation is confirmed.
The means for confirming the crystal precipitation may be determined by a suitable detection means known per se, for example, visual observation, microscopic observation and the like. The precipitated protein crystals can also be collected by an appropriate method known per se, for example, by introducing and flowing out an appropriate liquid.
Here, the physical properties of the protein solution used in the protein crystallization method of the present invention are not particularly limited as long as they can be used in the trace liquid weighing structure of the present invention. Specifically, examples of the solvent include water, salt solution, buffer solution, alcohol or glycerol and its solution, synthetic or natural polymer solution, and the like.
The precipitant solution used refers to a solution that promotes the formation of protein crystals, 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 their solutions, and synthetic or natural polymer solutions. Gels of proteins and macromolecules, porous silicon (see Chayen et al., Journal of Molecular Biology, (2001) 312, 591-595) and the like are known as substances that promote crystal formation. Good.
In the method of the present invention, when sealing or adjustment of vapor pressure is required, it is preferable that the volume of the gas phase in contact with the solution is 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 flow path between two narrow flow path portions.
As a method for crystallizing a protein, for example, a batch method, a vapor diffusion method, an interface contact method, a gel method and the like are known. Here, in order to perform crystallization by a batch method in the microchip having the channel structure of the present invention, for example, as in the method according to any one of claims 20 to 22, a protein solution and a precipitant solution are used. May be brought into contact with and united with each other, and the flow channel may be sealed by the above-described method as necessary.
In order to carry out the vapor diffusion method, for example, as in the method according to claim 20 or 21, after the protein solution and the precipitant solution are brought into contact and combined in the second flow path, If necessary, a liquid for adjusting the vapor pressure may be introduced into the first flow path, and the diffusion of the vapor may be adjusted by separating the contacted / unified liquid from the third and / or fourth flow paths. However, in the vapor diffusion method, a liquid (precipitant) for adjusting a vapor pressure and a liquid that has been brought into contact with and coalesced may be disposed with a gas phase therebetween, and the liquid is not necessarily arranged as described above. No need. A liquid for adjusting the vapor pressure may be introduced to the middle of the first flow path or at the end thereof to adjust the vapor pressure 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 to the end, and the vapor pressure may be adjusted through the second flow path.
[0054]
【Example】
Hereinafter, specific examples of the trace liquid weighing structure of the present invention, the microchip having the structure, and the qualitative / quantitative reaction using the microchip will be described by way of examples.
Example 1 Separation of proteins by ion exchange chromatography
FIG. 6A shows a microchip 10 having a microfluidic liquid weighing structure according to the present invention. The microchip 10 shown in FIG. 6 (a) is a microchip for capillary ion exchange chromatography used for protein analysis, and the sample injection portion is provided with the microfluid weighing structure according to the present invention.
[0055]
In FIG. 6A, ports 11 and 12 are ports for introducing a buffer for eluting a protein. Buffers having different ionic strengths are introduced, and the flow rates are respectively changed. By mixing, a gradient of ionic strength can be formed. Accordingly, the upper surfaces of the ports 11 and 12 are open to the atmosphere, and the lower portions communicate with the channels.
[0056]
The microchannel 14 is a column for chromatography. The microchannel 14 extends from the damming portion 15 of ion exchange beads (for example, an anion exchange bead manufactured by Pharmacia: particle size of 30 μm can be used as the ion exchange beads). The section of 3 mm in the direction of the trace liquid weighing structure (b) is filled with ion exchange beads. A port 18 is provided in a direction opposite to the blocking unit 15.
[0057]
FIG. 6B is an enlarged view of the micro liquid weighing structure (b). In the microchip 10 shown here, two flat substrates (upper: substrate 16, lower: substrate 17) formed of a polymer material, for example, PDMS (polydimethylsiloxane) are stacked and bonded. It is a square having a side of 20 mm and a thickness of 5 mm, but there is no limitation on the length, shape, and thickness of one side of the microchip, and is set to an arbitrary value of, for example, 5 mm to 100 mm on a side. 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, respectively. 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 characteristic of PDMS, the cured surface is hydrophobic. In addition, this surface is ₂ Irradiation treatment with plasma or excimer UV laser can easily bond PDMS to PDMS, PDMS to glass, and the like. From the above two points, PDMS is suitable for the production of this trace 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 is present on the lower surface of the upper substrate 16 or on 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 FIGS. 6 (b) and 6 (c1), the microchannel structure in the microfluidic weighing structure (b) extends on the lower surface of the upper substrate 16 in the left-right direction in parallel with each other. The first flow path 19 and the second flow path 20, the third flow path 21 extending perpendicularly to the second flow direction from the first flow path 19, 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 is orthogonal to the fourth flow path 22. Is formed. When a shallow, narrow channel is provided on the upper surface of the lower substrate 17, each channel is partially overlapped with a deeper, wide channel provided on the lower surface of the upper substrate 16, so that each channel is formed. The roads can communicate with each other. Alternatively, as shown in FIG. 6 (c2), when all the flow paths are provided on the upper substrate 16 (or the lower substrate 17), it is not necessary to provide an overlapping portion for each flow path. By appropriately selecting the synthetic resin material used as the substrate and / or to make the surface of a specific flow path provided in the substrate and a part or all of a necessary portion easy or difficult to wet. Thus, 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 path 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 the liquid to enter the fourth flow path 22. In addition, the width of the fifth flow path 23 needs to be equal to or less than the width of the fourth flow path 22 in order to make it difficult for the liquid to enter the fifth flow path 23.
[0062]
Next, the above-described microchip 10 is manufactured by the following manufacturing process. Prior to the manufacturing process, first, the microchannels of the upper substrate 16 and the lower substrate 17 of the microchip 10 are formed. The layout patterns are separately printed on a transparent film at a high resolution (for example, 4064 dpi) in order to use them as masks for photolithography.
[0063]
First, a silicon (Si) wafer is cut with a diamond cutter in accordance with the size of a microchip to be produced, washed with ultrasonic waves, dried, and dried using a plasma reactor. ₂ Plasma treatment is performed at 200 W for 30 seconds.
[0064]
Next, a negative photoresist SU-8 is applied by spin coating (in the case of producing a 100 μm flow path, use SU-850 at 2000 rpm for 10 seconds and in the case of producing a 10 μm flow path, using SU-8, 2000 rpm) For 10 seconds) and then kept in an oven at 90 ° C. for 30 minutes.
[0065]
Next, using a mask aligner, the pattern of the layout of the microchannels on the upper substrate 16 and the lower substrate 17 of the microchip 10 printed on the film was photo-printed on separate silicon wafers coated with SU-8. The image is transferred using a lithography technique, further kept in an oven at 90 ° C. for 30 minutes, and then immersed in Developer (for example, 1-methoxy-2-propyl acetate or the like can be used as Developer). Develop, wash with isopropyl alcohol followed by distilled water and dry.
[0066]
The master thus manufactured has a convex structure that serves as a mold for the microchannel of the upper substrate 16 and the lower substrate 17.
[0067]
Next, in order to smoothly remove the PDMS replica after the molding, before pouring the prepolymer of PDMS, it is kept in a 3% dimethyloctadecylchlorosilane / toluene solution for 2 hours to perform a surface treatment.
[0068]
Next, a prepolymer of PDMS and a curing reagent (for example, “Sylgard 184: Dow Corning Co., MI” can be used as the curing reagent) are mixed at a ratio of 10: 1 and sufficiently stirred. After that, the mixture is poured into a frame made of acrylic for installing a master, and is held at 90 ° C. for 30 minutes to perform curing.
[0069]
After the curing, the PDMS replica is peeled off from the master, whereby the upper substrate 16 and the lower substrate 17 of the microchip 10 are obtained. Further, the lower surface of the upper substrate 16 and the upper surface of the lower substrate 17 are ₂ The microchip 10 is obtained by processing with plasma and bonding.
[0070]
Using the microchip 10 obtained by the above-described method, a trace liquid weighing operation for sample injection actually performed will be described below.
[0071]
First, the sample is filled in the first flow path 19 and the third flow path 21 by flowing the sample from one side of the first flow path 19 by a pressure flow. At this time, the fourth flow path 22 has a property that it is hard to wet or relatively hard to exert a capillary attraction, and is higher than the first flow path 19, the second flow path 20, and the third flow path 21. The sample does not enter the fourth channel 22 because it is so thin that it is hard to wet with a hydrophilic liquid.
[0072]
In the state where the second flow path 20 is filled with the liquid, if the fifth flow path 23 does not exist, when the sample is introduced into the third flow path 21, Air leaks into the second flow path 20.
[0073]
However, due to the presence of the fifth flow path 23, even when the second flow path 20 is filled with liquid, the air in the third flow path 21 flows out to the fifth flow path 23. Therefore, the sample can be introduced into the third channel 21 without air leaking into the second channel 20.
[0074]
Next, by continuously introducing air into the first flow path 19 by a pressure flow, the sample in the first flow path is pushed out, the sample remains only in the third flow path 21, and the third The sample remaining in the channel 21 does not flow back into the first channel 19 again. In the example of the chip shown here, the sample amount 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 to the fourth flow path 22, and the sample reaches the second flow path 20. The sample is introduced into the second channel 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 smaller than the channel width of the fourth channel 22. Both are set to 20 μm.
[0077]
In order to prevent adsorption of a sample (such as a protein) for separation by chromatography on PDMS, only the first flow path 19, the second flow path 20, and the third flow path 21 were formed using HCl. The treatment to make it hydrophilic is performed. However, the hydrophilization treatment is not limited to HCl, ₂ A method using plasma, a method using albumin, and the like are also possible. PDMS itself is a hydrophobic substance.
[0078]
Actually, in chromatography using sample injection by the above-mentioned microfluidic control operation, a mixture of FITC-labeled albumin and IgG was introduced, and Tris / HCl Buffer (pH 8.0) was flowed at a flow rate of 1.0 μl / min. After adsorption, using anion exchange beads (Pharmacia: particle size: 30 μm), the two were successfully separated within 1 minute using a 0 to 1 M gradient buffer with sodium chloride.
[0079]
In the above embodiment, the trace liquid weighing structure is manufactured by PDMS of silicon polymer. However, by making the inner surface and / or the end surface of the flow channel partially or entirely wettable or hard to wet, the flow channel is measured. The structure can be adapted to any material (for example, silicon, polymer, glass, ceramics, metal, etc.) if the degree of wetting inside can be adjusted and the structure can be manufactured. Also, the trace liquid weighing structure can be used by a composite use of these different materials or a material in which a temperature and pH responsive substance PIPAAm (N-isopropylacrylamide) is mixed.
[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 that can individually perform up to 50 types of reactions or analyzes by mixing two liquids quantitatively weighed, and is provided in a liquid weighing portion. Employs a micro liquid weighing structure according to the present invention. Although the microchip 24 shown in FIG. 7A processes up to 50 types of reactions or analyzes, a microchip for processing a larger number of reactions or analyzes can naturally be produced.
[0081]
By using a microchip 24 as shown in FIG. 7 (a), for example, the reaction or analysis by mixing one type of reagent for 50 types of samples can be performed individually and extremely easily. , Can be done simultaneously. Alternatively, for example, a reaction or analysis by mixing 50 types of reagents can be performed individually and simultaneously on one type of sample.
[0082]
A microchip 24 as shown in FIG. 7A has two flat plates formed of a polymer material, for example, PDMS (polydimethylsiloxane), similarly to the microchip 10 shown in FIG. It is configured by stacking and bonding substrates in a shape of a disk, having a disk shape with a diameter of 90 mm and a thickness of 3 mm. Other diameters and thicknesses can of course 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, respectively. The depth of the microchannel to be formed is, for example, 100 μm, and the depth of the microchannel existing on the upper surface of the substrate 26 is, for example, 10 μm. Naturally, other depths can 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. 7 (b), the central substantially annular first liquid supply channel 33 is cut at one point in the middle thereof, and one cut end is ported through the channel 27 extending radially outward. The other cut end is connected and connected to a port 30 via a channel 29 extending radially outward. Port 28 is the inlet for the first liquid to mix, and port 30 is the outlet for that liquid. The liquid introduced from the port 28 enters the first liquid supply channel 33 through the channel 27, flows in the first liquid supply channel 33, and flows out of the port 30 through the channel 29. Alternatively, port 28 may be an outlet and port 30 may be an inlet.
[0086]
The port 31a in FIG. 7B is an inlet for the second liquid to be mixed, and communicates with the outlet port 31b via 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 charged. The ports 32a and 32b are provided adjacent to the ports 31a and 31b via the mixing channel 34, respectively. The mixing channel 34 also functions as a reaction chamber for both mixed liquids. Since the ports 32a and 32b are in communication with each other via the mixing channel 34, air pressure can be applied to the mixing channel 34 via the port 32a and / or port 32b to promote mixing of the liquid in the channel, Alternatively, it can be used to take out the reaction product in the mixing channel 34 to the outside.
[0087]
On the radially outer side of the first liquid supply channel 33, there are 30 structures for weighing and mixing a small amount of liquid including the ports 31a and 31b and the second liquid supply channel 35 and the ports 32a and 32b and the mixing channel 34. Are located. On the other hand, on the inner side in the radial direction, 20 similar structures for weighing and mixing the minute amount of liquid are arranged. The number of arrangements is not limited to the illustrated embodiment.
[0088]
The structure shown in FIG. 7C is formed for the same purpose as the structure shown in FIG. 2B, and includes a microchannel structure for measuring a small amount of liquid, and two types. Channel for mixing the liquids. A first liquid supply channel 33 (flow path A in FIG. 2B) for supplying one of the liquids 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 (corresponding to the flow path A ′ in FIG. 2B) for supplying the other liquid. One of the liquids supplied from the first liquid supply channel 33 is weighed by a first weighing channel 36 (corresponding to the flow path C in FIG. 2B) by a certain amount. Similarly, the other liquid supplied from the second liquid supply channel 35 is weighed by the second weighing channel 37 (corresponding to the flow path C ′ in FIG. 2B) by a certain amount. The liquid in the first weighing channel 36 and the second weighing channel 37 is passed through the first narrowing channel 38 and the second narrowing channel 39, which are smaller than these channels, respectively, by using a conventional means such as a pressure difference. It can be taken into the mixing channel 34 and mixed therein.
[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. The channel width of each of the second weighing channels 37 is, for example, 100 μm, and the length is, for example, 600 μm. The channel width of each of the first narrow channel 38 and the second narrow channel 39 is, for example, 20 μm. Other channel widths and channel lengths can of course be used. For example, in FIG. 7C, the volumes of the two liquids to be mixed are each 6 nl, and this volume is equal to the volumes of the first weighing channel 36 and the second weighing channel 37. Therefore, by changing the volume of each of these weighing channels, the volume can be arbitrarily adjusted within the range of 1 pl to 1 μl.
[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, for example, a trace liquid weighing and mixing operation for quantitative analysis of glucose by a glucose oxidase / peroxidase method.
[0092]
The operation of weighing and mixing a small amount of liquid for quantitative analysis of glucose by the glucose oxidase / peroxidase method will be described below. In the glucose oxidase / peroxidase method, a phosphate buffer obtained by appropriately mixing glucose oxidase, peroxidase, mutarotase, 4-aminoantipyrine and phenol is used as a reagent, and a glucose aqueous solution is used as a sample.
[0093]
First, about 1 μl of a reagent is introduced by a pressure flow from the port 28 to the port 30 in FIG. 7A, and then air is introduced in order to allow excess reagent to flow. The introduced reagent and air flow clockwise in the first liquid supply channel 33. In FIG. 7 (c), when a reagent is introduced into the first weighing channel 36, the first narrow channel 38 is hydrophobic, hardly wets with a liquid, and the first liquid supply channel 33 and Because the channel width is narrower than the first weighing channel 36, the reagent remains 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 into the first weighing channel 36. Also, a single operation of introducing a reagent from the port 28 to the port 30 and subsequently introducing air is performed once, and a predetermined amount of the reagent liquid is weighed into the 50 first weighing channels 36 at a time. It is possible. Such an extremely simple operation makes it possible to distribute a very small amount of liquid more finely and to simultaneously form a very small amount of many liquids. In such an operation, it is expected that the amount of the residual liquid that is not distributed after the operation is 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 via the second liquid supply channel 35. Can be.
[0096]
Next, subsequently, by increasing the pressure of the air in the first liquid supply channel 33 and the second liquid supply channel 35, the reagent in the first weighing channel 36 and the sample in the second weighing channel 37 are respectively increased. Extruded into the mixing channel 34 and mixed therein. Since the two mixed solutions exhibit a red color due to a chemical reaction, a quantitative analysis of glucose can be performed by evaluating the red value. Needless to say, the microchip 24 shown in FIG. 7 can be used for other qualitative and / or quantitative analysis.
[0097]
Example 3 Crystallization of glucose isomerase (union of liquids)
The protein (glucose isomerase) was crystallized using the microchip 41 shown in FIG. FIG. 8B is an enlarged view of a micro liquid weighing structure (b) of the microchip 41 used. The microchip 41 is basically the same as the microchip 24 shown in FIG. 7A except for the shape and the number of the micro liquid weighing structures provided.
The microchip 41 shown in FIG. 8A is a microchip for crystallization of a protein, in which a microfluidic liquid weighing structure according to the present invention is provided at an injection portion of a solution (protein solution and precipitant solution). I have. Using PDMS as a material, it was prepared in the same manner as in Example 1. The size of the chip is 24 × 46 mm and the thickness is 60 μm. The width of each of the first liquid supply channel 33 and the second liquid supply channel 35 is 200 μm, and the width of the mixing channel 34 is 300 μm. Each of the first weighing channel 36 and the second weighing channel 37 has a width of 100 μm and a length of 1000 μm. Each of the first narrow channel 38 and the second narrow channel 39 has a width of 20 μm and a length of 400 μm each.
[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 microfluid weighing structure channel structure formed on the microchip, the first liquid supply channel (first flow channel) 33 has a first weigher for weighing the first liquid (protein solution). Seven intake channels (third flow paths) 36 are connected, and a first narrow channel (fourth flow path) 38 is connected to each of the seven locations. The first narrow channel 38 is a mixing channel (second flow path). (A flow path) 34. A 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 a second liquid (precipitant solution). And a channel structure including the second narrow channel 39 is connected.
First, in order to weigh out the first liquid (protein solution), glucose isomerase (Hampton) adjusted to a concentration of 20 mg / mL in water using positive pressure from port 28 to first liquid supply channel 33. Research HR7-102). Further, by flowing air under a positive pressure, excess glucose isomerase solution is removed from the first liquid supply channel 33 through the port 30, and the first weighing for weighing the first liquid (protein solution) is performed. The glucose isomerase solution was weighed at all seven points of the channel 36.
Next, air is passed through the first liquid supply channel 33 from the port 28 using a positive pressure higher than the pressure at which the excess glucose isomerase solution is removed, and the weighed glucose isomerase solution is mixed. Drained into channel 34.
Next, 10% of PEG8000 (Hampton Research HR2-535) and MgCl 2 were added in a buffer (pH 6.5) of 100 mM MES (Hampton Research HR2-587). ₂ (Hampton Research HR2-559), a precipitant solution adjusted to 200 mM was applied to a second liquid supply channel 35 and a second weighing channel 37 for weighing a second liquid, and a positive pressure was applied from a port 31a. To remove excess precipitant solution in the second liquid supply channel 35 via the port 31b and to weigh the second liquid by flowing air under positive pressure. The precipitant solution was weighed into two weighing channels 37. Next, using a positive pressure higher than the pressure at which the excess precipitant solution is removed, air is passed from the port 31a to the second liquid supply channel 35, and the weighed precipitant solution is mixed with the mixing channel 34. Drained. As a result, the glucose isomerase solution and the precipitant solution contacted and coalesced in the mixing channel 34. This operation was performed at five points to prepare five combined liquids of the glucose isomerase solution and the precipitant solution.
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)
An experiment was performed using the same chip as in Example 3 and using all the same reagents as in Example 1.
First, in order to weigh out the first liquid (protein solution), glucose isomerase (Hampton) adjusted to a concentration of 20 mg / mL in water using positive pressure from port 28 to first liquid supply channel 33. Research HR7-102). Further, by flowing air under a positive pressure, excess glucose isomerase solution is removed from the first liquid supply channel 33 through the port 30, and the first weighing for weighing the first liquid (protein solution) is performed. The glucose isomerase solution was weighed at all seven points of the channel 36.
Next, 10% of PEG8000 and MgCl 2 were added to a 100 mM MES buffer (PH 6.5). ₂ The precipitant solution adjusted to 200 mM was filled into the first liquid supply channel 33 from the port 28 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 stereoscopic microscope, it was confirmed that 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 (the Crystals were observed in the liquid on the first weighing channel 36) and the liquid on the precipitant solution side (the first liquid supply channel 33 side). In particular, many crystals were observed in the liquid on the precipitant side.
[0100]
As described above, the microfluidic weighing structure of the present invention and the microchip having the structure and separation of proteins using the microchip, quantification of glucose by enzymatic reaction and 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 of the third flow path (flow path C) closer to the second flow path (flow path B) is modified so as to have a property that it is hard to selectively wet (or relatively hard to exert capillary attraction). If possible, it is also possible to directly connect the wide third flow path to the same width second flow path (flow path B) without providing the narrow fourth flow path (flow path D). it can. Further, the shape of the third flow path for weighing the liquid may be any shape such as a circle, an ellipse, and a triangle in addition to the rectangular shape.
[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 a sample and realize space saving and cost reduction of the entire device in various devices that require quantitative liquid handling. It is possible to obtain a trace liquid weighing structure that can be used.
[0102]
That is, according to the method of the present invention, a small amount of liquid can be quantitatively weighed in the microchannel, so that observation, measurement, mixing, phase change, reaction, and the like can be reduced. In addition, when the dead volume of the sample is small, especially when a plurality of third flow paths are connected to the first flow path, the dead volume can be significantly reduced. In addition, there is an advantage that the area where the weighed liquid is in contact with the outside air is small, and it is difficult for evaporation to dryness or concentration to occur. Furthermore, since the liquid can be weighed out only by a simple operation with a simple configuration and the liquid operation can be easily performed thereafter, space saving and low cost of the entire apparatus can be realized.
The microchip having the above-described flow channel structure of the present invention having the above features can be suitably used for, for example, separation / analysis of various substances, biochemical or chemical reactions, crystallization of proteins, and the like.
[Brief description of the drawings]
FIG. 1 is a conceptual explanatory view for explaining the principle of the present invention, in which (a) shows its structure, and (b) and (c) explain a liquid weighing method.
FIGS. 2A and 2B are explanatory diagrams of a trace liquid weighing structure having two liquid preparation systems according to claim 2; FIG. 2A illustrates a case where the flow path A is shared, and FIG. This is the case.
FIGS. 3A and 3B are explanatory diagrams of a trace liquid weighing structure according to claims 3 and 4, wherein FIG. 3A shows a case where a flow path D is divided into three flow paths, and FIG. FIG. 5 is an explanatory diagram in a case where there are three sets of flow paths C and D having different thicknesses.
FIGS. 4A and 4B are explanatory diagrams of a trace liquid weighing structure according to claim 5, wherein FIG. 4A shows the structure, and FIGS. FIG. 5 is an explanatory diagram of a liquid introduction method in the case where a liquid is introduced.
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 cross section taken along line cc in FIG. 5 (b). FIG.
FIGS. 6A and 6B are explanatory diagrams of a microchip 10 having a microfluidic weighing structure according to the present invention, which is used for separating proteins by ion exchange chromatography in Example 1, wherein FIG. (B) is an enlarged explanatory view of the quantification of a small amount of sample and an introduction part, and (c1) and (c2) are cross-sectional views taken along line cc in FIG. (B).
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, which is used for the quantification of glucose by the enzymatic reaction of Example 2, and FIG. FIG. 3A is a partially enlarged plan view of a portion b in FIG. 4C, and FIG. 4C is a partially enlarged plan view of a portion c in FIG.
8 is a plan view of a microchip 41 used for crystallization of glucose isomerase in Examples 3 and 4, and FIG. 8B is a partially enlarged plan view of a portion b in FIG.
[Explanation of symbols]
10 Microchip for capillary chromatography
11 Port for elution buffer introduction
12 Port for introducing elution buffer
13 Mixer
14 Micro Channel
15 Damming part
16 PDMS plate (top)
17 PDMS flat plate (bottom)
18 ports
19 First channel
20 Second flow path
21 Third flow path
22 Fourth flow path
23 Fifth channel
24 Microchips for 50 kinds of two-liquid mixed reaction
25 Upper substrate
26 Lower substrate
27 channels
28 1st liquid inlet port
29 channels
30 first liquid outlet port
31a Inlet port for second liquid
31b Outlet port for second liquid
32a port
32b port
33 1st liquid supply channel
34 mixed channels
35 Second liquid supply channel
36 1st weighing channel
37 2nd weighing channel
38 1st narrow channel
39 2nd narrow channel
100 liquids (samples, etc.)
100a Lower end of liquid in channel C (side of channel A)
100b Upper end of liquid in channel C (side of channel D)
200 liquids (mobile phase of liquid chromatography)
aa, aa 'Flow path wall of flow path A
bb, bb 'Flow path wall of flow path B
c1 Opening of channel C (side of channel A)
c2 Opening of channel C (side of channel D)
d1 Opening of channel D (side of channel B)
e1 Opening of flow path E (side of flow path D)

Claims (22)

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 (flow path A) opening in a flow path wall of the first flow path (flow path A). 3 (flow path C) and one end of the third flow path (flow path C) opened in the flow path wall of the second flow path (flow path B) and the second flow path And a fourth flow path (flow path D) which is less wettable (or relatively hard to exert capillary attraction) and is thinner than the other three flow paths. 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 Wherein the liquid remaining in the third channel is removed, and a liquid having a volume corresponding to the volume of the third flow path is weighed. 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 (flow path A) opening in a flow path wall of the first flow path (flow path A). 3 (flow path C) and one end of the third flow path (flow path C) opened in the flow path wall of the second flow path (flow path B) and the second flow path And a fourth flow path (flow path D) having a property of being hard to wet (or relatively hard to apply capillary attraction) and being thinner than the other three flow paths. After the liquid introduced into the first flow path is drawn into the third flow path through the opening of the third flow path opening in the flow path wall of the first flow path, the first flow The system has at least two systems for removing the liquid remaining in the path and weighing a liquid having a volume corresponding to the volume of the third flow path, and sharing the first flow path or the second flow path. Characterized by Liquid weighed structure. Two or more fourth flow paths (flow path D) are connected to the third flow path (flow path C), or two or more fourth flow paths (flow path D) 3. The structure for weighing a trace amount of liquid according to claim 1, wherein the structure is branched. The plurality of sets of the third flow path (flow path C) and the fourth flow path (flow path D) connected to the third flow path (flow path D) are formed. 2. A trace liquid weighing structure according to 1. A flow path wall that is open to the flow path wall of the fourth flow path (flow path D) and has a property of being thinner or the same thickness as the fourth flow path and less likely to be wet (or relatively hard to exert capillary attraction). The microfluid weighing structure according to any one of claims 1 to 4, further comprising a fifth flow path (flow path E) comprising: The second flow path has, in a part of the flow path, a narrow flow path part having a flow path width narrower than a normal flow path width of the flow path, and the narrow flow path part has a normal flow path part. The trace liquid weighing structure according to any one of claims 1 to 5, characterized in that it has a property of being less wet than a path width portion (or relatively hard to exert a capillary attraction). The liquid weighed at a volume corresponding to the volume of the third flow path (flow path C) is passed through the fourth flow path (flow path D) to the second flow path (flow path B). 7. The microfluidic liquid weighing structure according to claim 1, further comprising an outflow means for allowing the liquid to flow out. When the second flow path near the opening of the fourth flow path (flow path D) is filled with the liquid, the second flow path has a volume corresponding to the volume of the third flow path (flow path C). The trace liquid weighing apparatus according to claim 7, further comprising an outflow unit that causes the weighed liquid to flow out to the second flow path via the fourth flow path (flow path D). Construction. 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 (flow path D), and the 8. The trace liquid weighing structure according to claim 1, wherein each of the five flow paths (the flow path E) is a channel formed in the substrate. 9. The volume of the third flow path (flow path C) is formed to have a size on the order of picoliter to microliter. 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. The substrate has a plurality of the trace liquid weighing structures according to any one of claims 1 to 10, 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. In a substrate having a two-layer structure in which an upper substrate (16) and a lower substrate (17) are bonded, a microchannel (14) for ion exchange chromatography is provided, and elution buffer introduction ports (11, 12). And a mixer (13) communicating with the port is connected in the middle of the microchannel (14), and is connected to the ion-exchange bead blocking part (15) of the microchannel (14) and the mixer (13). A) a microfluidic weighing structure between the connecting portion and the microfluidic weighing structure, wherein the microfluidic weighing structure is provided on a first flow path (19) and a flow path wall of the first flow path (19); A third flow channel for liquid weighing that is open, and a second flow channel communicating with the third flow channel and the second flow channel that constitutes the microchannel. The width is narrower than each of the first, second and third flow paths, And a width of the fourth flow path (22), which has a property of being difficult (or relatively hard to exert capillary attraction), and a width of the fourth flow path (22) intersecting the fourth flow path (22). 5. A microchip for capillary ion exchange chromatography, comprising: a fifth channel (23) having the same width or a smaller width. 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 hardening process. 15. The microchip according to 14. A substantially annular first port having a liquid inlet port (28) at one end and an outlet port (30) at the other end in a substrate having a two-layer structure in which an upper substrate (25) and a lower substrate (26) are bonded to each other. A liquid supply channel (33), said first liquid supply channel (33) having a plurality of first weighing channels (36) opening into its channel wall, each first weighing channel (36); In contrast, a mixing channel (34) and a second liquid supply channel (35) are provided as a set, the first weighing channel (36) being wetted by the mixing channel (34). The first liquid supply channel (35) communicates through a first narrow channel (38) having a property of being difficult (or relatively hard to exert a capillary attraction), and the second liquid supply channel (35) is open to a flow path wall thereof. A second take-up channel (37); The channel (37) communicates with the mixing channel (34) through a second narrow channel (39) having a property of being hard to wet (or relatively hard to exert a capillary attraction), and the second liquid supply channel. (35) and the mixing channel (34) each have an inlet port (31a, 32a) and an outlet port (31b, 32b), and each port is provided through the upper substrate. Microchip for ultra-trace analysis and synthesis / separation. The substrate is disc-shaped, and the first weighing channel (36), the first narrow channel (38), the mixing channel (34), and the radially inner side of the substantially annular first liquid supply channel (33). Twenty sets of a second narrow channel (39), a second weighing channel (37) and a second liquid supply channel (35) are provided, and a radially outer side of the substantially annular first liquid supply channel (33). The first weighing channel (36), the first narrowing channel (38), the mixing channel (34), the second narrowing channel (39), the second weighing channel (37) and the second liquid supply channel (35). 17. 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. 18. The microchip according to 16 or 17. A mask or a master chip used for manufacturing the microchip according to any one of claims 11 to 18. A method for crystallizing a protein using the trace liquid collecting structure according to any one of claims 1 to 10, wherein the method comprises:
(A) a protein solution or a precipitant solution is introduced into the first flow path, and the protein solution or the precipitant solution is introduced into the third flow path through the opening of the third flow path that opens into the first flow path; After the precipitant solution is drawn in, the protein solution or the precipitant 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 the third flow path Flowing a protein solution or a precipitant solution prepared in a volume corresponding to the volume of the second channel through a fourth channel;
(B) introducing a precipitant solution or a protein solution into the first flow path, and passing the precipitant solution into the third flow path through the opening of the third flow path that opens to the first flow path; Alternatively, after the protein solution is drawn in, the precipitant solution or the protein solution remaining in the first flow path is moved to a position where the precipitant solution or the protein solution does not come into contact with the opening of the third flow path. The precipitant solution or the protein solution prepared in a volume corresponding to the volume of the solution is allowed to flow out through the fourth flow path to the second flow path, and the protein solution and the precipitant solution are passed through the second flow path. Contacting and uniting;
(C) a step of precipitating protein crystals from the combined protein and precipitant solution in the second channel. 21. The method according to claim 20, wherein the precipitation of the protein crystals from the combined protein and precipitant solution is performed in a state where the precipitant solution is disposed in a channel structure separated from the solution. the method of. A method for crystallizing a protein using the trace liquid collecting structure according to any one of claims 1 to 10, wherein the method comprises:
(A) After the protein solution is introduced into the first flow path and the protein solution is drawn into the third flow path through the opening of the third flow path that opens to the first flow path Moving the protein solution remaining in the first flow path to a position not in contact with the opening of the third flow path to create a protein solution with a volume corresponding to the volume of the third flow path. Process and
(B) introducing a precipitant solution into the first channel, bringing the protein solution and the precipitant solution into contact with each other at the opening of the third channel, and combining the protein solution and the precipitant solution;
(C) a step of precipitating protein crystals from the combined protein and precipitant solution.

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