JP4255891B2 - Protein crystallization method - Google Patents

Description

  The present invention relates to a protein crystallization method using a trace liquid weighing structure. More specifically, the present invention relates to a micro liquid weighing structure suitable for use in performing analysis or chemical reaction using various samples, and a protein crystallization method using a microchip having the structure in a substrate. .

  Conventionally, various apparatuses for performing analysis by electrophoresis, chromatography, and the like are known, and in such an apparatus, an accurate analysis result can be obtained by quantitatively weighing a liquid such as a sample to be used. It is something that can be done.

  For this reason, various methods for quantitatively weighing a liquid such as a sample in various apparatuses used for such electrophoresis and chromatography have been proposed. For example, Patent Document 1 describes a microchip electrophoresis apparatus, and Patent Document 2 describes an apparatus and method for electrophoretic separation of a fluid substance mixture. However, in any of these conventional methods, there is a problem that a sample more than the amount necessary for the analysis is actually required, and the dead volume of the sample cannot be reduced.

  Furthermore, in a chemical reaction or analysis using a liquid such as a small amount of sample, a microchip composed of a microchip may be used. Even when such a microchip is used, an accurate result can be obtained by quantitatively weighing the liquid such as the sample to be used. However, in the analysis using the microchip, the liquid such as the sample to be handled is used. Since the volume of the liquid is extremely small, it is difficult to weigh the liquid quantitatively, and various complicated structures are required for that purpose, and there is a problem that the operation for handling the structure becomes complicated.

  On the other hand, batch methods, dialysis methods, vapor diffusion methods, gel methods, and the like have been known as protein crystallization methods. However, each of these methods has a problem that a large amount of protein is required, and particularly when searching for a crystallization condition of a protein whose crystallization condition is unknown, a problem that a huge amount of protein is required. Had.

  In addition, in order to reduce the amount of protein to be used, a protein solution is dispensed by inkjet (for example, see Non-Patent Document 1) or electrospray (for example, see Patent Document 3), and the protein crystallization conditions are searched. The attempt was known. However, this method has a problem that since the droplets are exposed to the air, the droplets evaporate and dry or concentrate.

  Furthermore, attempts to produce crystals in a laminar flow using a microchannel device (for example, see Patent Document 4) or attempts to obtain crystals by strictly controlling temperature (for example, see Non-Patent Document 2) are known. It was. However, this method has a small reaction field and excellent reaction control. However, the introduction method to the crystallization site of the protein solution is the same as the conventional method, and the ratio of dead volume is extremely large. Had.

Japanese Patent Laid-Open No. 10-148628 (see FIGS. 1 and 2) JP-A-7-198680 (see FIGS. 1 and 2) International Publication No. WO01 / 92293 Pamphlet US Pat. No. 6,409,832 “Journal of Applied Crystallography” (2002), 35, p. 278-281 “Analytical Chemistry” (2002), 74, p. 3505-3510

  Accordingly, an object of the present invention is to provide a protein crystallization method using a trace liquid weighing structure that allows a small quantity of liquid to be quantitatively weighed by a simple operation with a simple configuration. It is.

  Another object of the present invention is to reduce the dead volume of the sample in various apparatuses that require quantitative liquid handling, and to realize space saving and cost reduction of the entire apparatus. It is an object of the present invention to provide a method for crystallizing a protein using a micro liquid weighing structure.

  The above-mentioned problem is solved by the trace liquid weighing structure used in the present invention. The trace liquid weighing structure used in the present invention is made by using the capillary phenomenon (capillary repulsion) caused by the liquid with respect to the flow path, paying attention to the surface tension of the liquid.

  According to the first embodiment of the trace liquid weighing structure used in the present invention, the first flow path and the second flow path that are each extended in a predetermined direction, and the flow path wall of the first flow path The third flow path that opens to the flow path wall, and the one end of the third flow path that connects to the second flow path and opens to the flow path wall of the second flow path. The fourth channel is narrower than the other three channels, and the liquid introduced into the first channel is the channel of the first channel. 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 It is characterized in that a liquid having a volume corresponding to the volume is weighed.

  According to the second embodiment of the trace liquid weighing structure used in 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 The third flow path that opens to the flow path wall, and the one end of the third flow path that connects to the second flow path and opens to the flow path wall of the second flow path. And a fourth channel that is narrower than the other three channels, and the liquid introduced into the first channel flows in the first channel. The third channel is removed by removing the liquid remaining in the first channel after being drawn into the third channel through the opening of the third channel opening in the road wall. The system has at least two systems for weighing a liquid having a volume corresponding to the volume of the first flow path, and shares the first flow path or the second flow path.

  According to the third embodiment of the trace liquid weighing structure used in the present invention, two or more of the fourth flow paths are connected to the third flow path, or the fourth flow path is It is characterized by branching into two or more.

  According to a fourth embodiment of the trace liquid weighing structure used in the present invention, a plurality of sets of the third and fourth flow paths connected to the third flow path are formed.

  According to the fifth embodiment of the trace liquid weighing structure used in the present invention, it opens to the flow path wall of the fourth flow path, is thinner or the same thickness as the fourth flow path, and is difficult to wet ( It is further characterized by further having a fifth flow path formed of a flow path wall having a property that the capillary attraction force is relatively difficult to work.

  According to the sixth embodiment of the trace liquid weighing structure used in the present invention, the second channel has a channel width narrower than a normal channel width of the channel at a part of the channel. The narrow channel portion is characterized by being less wettable than a normal channel width portion (or relatively less susceptible to capillary attraction).

  According to the seventh embodiment of the trace liquid weighing structure used in the present invention, the liquid weighed in a volume corresponding to the volume of the third flow path is passed through the fourth flow path. It further has outflow means for flowing out into the two flow paths.

  According to the eighth embodiment of the trace liquid weighing structure used in the present invention, when the second flow path in the vicinity of the opening of the fourth flow path is filled with the liquid, It further has an outflow means for causing the liquid weighed in a volume corresponding to the volume of the flow path to flow out to the second flow path through the fourth flow path.

According to the ninth embodiment of the trace liquid weighing structure used in 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.
The present invention is a protein crystallization method using a trace liquid weighing structure having the above characteristics or a chip on which the structure is formed.

  As described above, the trace liquid weighing structure used in the present invention is a trace liquid weighing structure that can quantitatively weigh a minute quantity of liquid by a simple operation with a simple configuration. . Furthermore, the trace liquid weighing structure used in the present invention can reduce the dead volume of the sample in various apparatuses that require quantitative liquid handling, and can reduce the space and cost of the entire apparatus. This is a trace liquid weighing structure that can realize the above.

That is, the trace liquid weighing structure used in the present invention can quantitatively weigh the liquid in the microchannel, so that observation, measurement, mixing, phase change, reaction, etc. can be reduced in quantity. Further, the dead volume of the sample is small, and particularly when a plurality of third flow paths are connected to the first flow path, the dead volume can be drastically reduced. In addition, there is an advantage that the area where the weighed liquid is in contact with the outside air is small, so that drying or concentration due to evaporation hardly occurs. Furthermore, since the liquid can be weighed by a simple operation with a simple operation and can be easily operated after that, it is possible to realize space saving and low cost of the entire apparatus.
The trace liquid weighing structure having the above-described features and the microchip including the same can be particularly suitably used for protein crystallization.

  Hereinafter, embodiments of a trace liquid weighing structure used in the present invention will be described in detail with reference to the accompanying drawings.

1 (a), 1 (b) and 1 (c) are conceptual diagrams for explaining the principle of the trace liquid weighing structure used in the present invention. Four channels A (first channel), channel B (second channel), channel C (third channel), and channel D (fourth channel) are: Between A and B, the flow path C from the flow path A side toward the flow path B has a structure in which the flow path D bridges in series. A liquid is introduced into the channel C via the channel A. Since the channel D having a channel wall that is difficult to wet (or relatively hard to attract capillary attraction) is thinner than other channels, a larger force is required to introduce the channel D, so 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 FIG. 1 (a) and FIG. 1 (b)).
In the present specification, “the property of being hardly wetted (or relatively difficult to work with capillary attraction)” means that when the liquid is drawn into the channel C, it cannot pass through the channel D with the same pressure, It means the nature of the flow path where the liquid stops.

  More specifically, when the liquid 100 (see the shaded area in FIG. 1) is introduced into the flow path A, the liquid 100 passes through the opening c1 of the thin flow path C that opens at the flow path wall aa of the flow path A. Can be introduced into the channel C. When the flow path A and the flow path C have a flow path wall that is easily wetted (or is easy to work with capillary attraction), if the flow path C is made thinner than the flow path A, the liquid 100 will have a stronger capillary attraction. It is spontaneously drawn into the channel C through the opening c1 of the channel C that opens to the channel wall aa of the channel A. When the flow path A and the flow path C have a flow path wall that is difficult to get wet (or relatively hard to work with capillary attraction), the flow path can be obtained 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).

  At this time, the liquid 100 that has reached the end face c2 on the flow path C side of the flow path D (the flow path section between c2 and d1) is blocked by the capillary repulsion of the flow path D having a flow path wall that is difficult to wet. There is no entry into Road D. Even when the channel C is hard to get wet, the capillary repulsion of the channel D is stronger than the capillary repulsion of the channel C, so that the liquid 100 does not enter the channel D (FIG. 1B). )reference).

  Subsequently, the liquid 100 remaining in the flow path A is moved to a lower pressure side by causing an appropriate pressure difference at both ends of the flow path A, for example. It moves to the position which does not contact the part c1, or removes from the inside of the flow path A (refer FIG.1 (c)). At this time, the liquid 100 in the channel C does not normally enter the channel A back (see FIG. 1C). As a result, the end face 100a and the end face 100b, which are both end faces of the liquid 100 in the flow path C, are positioned on the opening c1 of the flow path C and the end face c2 on the side connected to the flow path A. It is possible to weigh a volume of liquid according to the volume of the interval between c1 and c2 (see FIG. 1C). However, the end face of the liquid is formed to the extent that it substantially matches the end face of the opening c1 or c2 due to the influence of surface tension, contact angle, etc., and may not exactly match the end face of the opening c1 or c2. Therefore, the volume of the liquid weighed in accordance with the volume of the sections c1 and c2 of the flow path C may not exactly match the volume of the sections c1 and c2 of the flow path C.

  Further, the liquid weighed in the flow path C flows by causing an appropriate pressure difference between the flow paths so that the pressure in the flow path A is slightly larger than the pressure in the flow path B. It is easy to introduce into the flow path B through the path D and its opening d1. As a result, this liquid can be used for the purpose of reaction or analysis, for example, by transferring it by air pressure.

  Of the trace liquid weighing structure used in the present invention, the first embodiment includes a first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction, A third channel (channel C) that opens to the channel wall of the first channel (channel A) and a channel wall that opens to the channel wall of the second channel (channel B) One end of the third flow path (flow path C) and the second flow path (flow path B) are connected to each other and have the property of being difficult to get wet (or relatively difficult to perform capillary attraction). The third flow path has a fourth flow path (flow path D) that is thinner than the flow path, and the liquid introduced into the first flow path opens in the flow path wall of the first flow path. After being drawn into the third flow path through the opening of the path, the liquid remaining in the first flow path is removed, and a volume of liquid corresponding to the volume of the third flow path is weighed. It ’s something that I ’m trying to take .

  Therefore, according to the first embodiment of the trace liquid weighing structure used in the present invention, the volume of the third flow path is increased by introducing the liquid from the first flow path into the third flow path. Since the liquid of the corresponding volume can be weighed, the liquid can be weighed quantitatively with a simple configuration with only a simple operation, and the dead volume of the sample can be reduced and the entire device can be saved. And low cost.

  The second embodiment of the trace liquid weighing structure used in the present invention includes a first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction. A third channel (channel C) that opens in the channel wall of the first channel (channel A), and a channel wall in the second channel (channel B). One end of the third flow path (flow path C) and the second flow path (flow path B) are connected to each other and have the property of being hard to get wet (capillary attractive force does not work) and narrower than the other three flow paths. An opening portion of the third flow path that has a fourth flow path (flow path D) and in which the liquid introduced into the first flow path opens in the flow path wall of the first flow path. A system that removes the liquid remaining in the first flow path after being drawn into the third flow path through the first flow path and weighs a volume of liquid corresponding to the volume of the third flow path. Have at least two, Without even the two systems together, it is obtained so as to share the first flow path or the second flow path (see FIG. 2 (a) and Figure 2 (b)).

  Therefore, according to the second embodiment of the trace liquid weighing structure used in the present invention, since the two systems share the first flow path or the second flow path with each other, for example, the first flow path When the same type of liquid is quantitatively weighed in each of the two systems sharing the same, the different types of liquid weighed in the two systems and the second flow path of each system are combined. Dilution by 1 and reaction by coalescence, analysis by reaction after coalescence, etc. can be performed (see FIG. 2A). On the other hand, for example, when different types of liquids are quantitatively weighed in each of the two systems sharing the second flow path, a plurality of the created plurality of liquids are created in the second flow path shared by the two systems. It is possible to perform the analysis by the coalescence, the dilution by coalescence, the reaction by coalescence, the analysis by the reaction after coalescence, etc. (see FIG. 2B).

  Further, as in the third embodiment of the trace liquid weighing structure used in the present invention, in the trace liquid weighing structure according to any one of the first embodiment or the second embodiment, the fourth flow is measured. The path (flow path D) may be divided into two or more flow paths, or may be branched into two or more (see FIG. 3A). This makes it possible to adjust the flow velocity of the fluid in the flow path D independently of the capillary repulsion.

  Further, among the trace liquid weighing structures used in the present invention, the fourth embodiment is the trace liquid weighing structure according to any one of the first embodiment, the second embodiment, or the third embodiment. Two sets or more of the third channel (channel C) and the fourth channel (channel D) are formed (see FIG. 3B). Thus, according to the fourth embodiment of the trace liquid weighing structure used in the present invention, a plurality of sets of the third flow channel and the fourth flow channel (for example, the flow channel C in FIG. 3B). A plurality of liquids of different volumes can be weighed quantitatively and in parallel by the flow path D, the flow path D, the flow path C ′ and the flow path D ′, and the flow path C ″ and the flow path D ″).

  Further, among the trace liquid weighing structures used in the present invention, the fifth embodiment is the trace liquid according to any one of the first embodiment, the second embodiment, the third embodiment, or the fourth embodiment. In the weighing structure, the flow path opens in the flow path wall of the fourth flow path (flow path D) and has the property that it is thinner than the fourth flow path or has the same thickness and is difficult to get wet (capillary attractive force does not work). A fifth channel (channel E) made of walls is further provided (see FIG. 4).

  FIG. 4 shows a conceptual explanatory diagram for explaining the principle of the trace liquid weighing structure of the fifth embodiment. As shown in FIG. 4A, the five channels A, B, C, D, and E are connected between the channels A and B. The road D is connected in series and bridges. When the liquid is introduced from the flow path A to the flow path C, the flow path D has a flow path wall that is difficult to wet (or has no capillary attraction), so 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.

  When the liquid 100 (see the shaded area in FIG. 4) is introduced into the thick channel A, the liquid 100 flows through the opening c1 of the narrow channel C that opens at the channel wall aa of the channel A. (See FIG. 4B). This phenomenon occurs when the liquid in the vicinity of the opening d1 of the flow path D in the flow path B is not filled with the first embodiment, the second embodiment, the third embodiment, and the fourth embodiment. Although it is the same structure as any one of the trace liquid weighing structures of the embodiment, when the liquid 200 (gray area in FIG. 4) is introduced into the flow path B in advance, the first embodiment In the micro liquid weighing structure according to any one of the second embodiment, the third embodiment, and the fourth embodiment, the introduction of the liquid 100 into the flow path C occurs in an incomplete form and is completed. do not do. This is because there is no escape route for the gas remaining in the channel C.

  According to the fifth embodiment, when the liquid 200 (gray area in FIG. 4) has been introduced into the flow path B, the liquid 100 has been introduced into the flow path C until the liquid 100 has been introduced. The gas that has been purged is purged into the flow path E having the opening e1 on the flow path wall of the flow path D (the end surface of the flow path E opposite to e1 is open to the atmosphere). (See FIG. 4 (b)). Also in this case, the liquid 100 that has reached the end surface c2 between the channel C and the channel D does not get wet (or the channel between the channels C2 and d1 has a channel wall that does not act as a capillary attraction). It is blocked by the capillary repulsion of the road section) and does not enter the flow path D (see FIG. 4B).

  Subsequently, the liquid 100 remaining in the flow path A is removed from the flow path A, for example, by causing an appropriate pressure difference at both ends of the flow path A to move to a lower pressure side. As a result, it is possible to weigh a volume of liquid corresponding to the volume of the flow path C (section between c1 and c2) (see FIG. 4C).

  Further, the liquid formed in the channel C causes an appropriate pressure difference between the two channels so that the pressure in the channel A is slightly larger than the pressure in the channel B, and so on. It becomes possible to introduce into the liquid 200 in the flow path B via the. At this time, the end surface of the flow path E opposite to the end surface e1 needs to be closed (see FIG. 4D).

  Therefore, if the fifth embodiment of the trace liquid weighing structure used in the present invention is used, a practical technique for quantitatively introducing a trace amount of sample or reagent in a protein crystallization apparatus is provided. be able to.

  FIGS. 5A and 5B are plan views showing the trace liquid weighing structure of the sixth embodiment, and FIG. 5C is a cross-sectional view taken along the line cc in FIG. 5B. As shown in FIG. 5A, the second flow path (flow path B) is a narrow flow having a flow width narrower than the normal flow width of the flow path in a part of the flow path. It has a path portion (flow path b1 and flow path b2). The narrow channel portion has a property that it is less likely to get wet than a normal channel width portion (or relatively hard to attract capillary attraction). One end of the second flow path (flow path B) itself and the narrow flow path portion (flow path b) is open to the atmosphere or adjusted to an appropriate pressure. Therefore, a volume-limited section can be formed in a part of the second channel by sandwiching a part of the second channel between the two narrow channel parts. The third channel (channel C) can be communicated with the volume-limited section of the second channel via the fourth channel (channel D). For example, when the first liquid weighed in one third flow path (flow path C) is pushed into the volume-limited section of the second flow path via the fourth flow path (flow path D). The air in the limited volume section is released into the atmosphere through the narrow channel portion, and the extruded first liquid remains in the limited volume section. Further, the second liquid weighed in the other third flow path (flow path C ′) is passed through the other fourth flow path (flow path D ′) to limit the volume of the second flow path. When pushed in, the remaining air in the volume-limited section is also released into the atmosphere through the narrow channel portion, and the first liquid and the second liquid can be filled in the volume-limited section. Therefore, a volume-limited section sandwiched between two narrow channel portions can be used as a reaction chamber, and the amount of reaction solution to be used can be minimized. For this reason, the volume of the volume limited section of the second flow path can be determined in consideration of the total amount of liquid supplied in the limited section. For example, it is preferable to set a value that is substantially the same as or slightly larger than the sum of the volumes of one or more third flow paths connected in the limited section.

  FIG. 5B shows an embodiment in which one third flow path is connected to the volume-limited section of the second flow path, and the second flow path includes two narrow channels. It has a flow path part (flow path b1 and flow path b2), and the other part has a normal flow path width. For example, the first liquid is supplied from the second flow path having the normal flow path width to the volume-limited section through the first narrow flow path portion (flow path b1), and then the third flow path (flow The second liquid weighed in the path C) is supplied into the volume limited compartment through the fourth flow path (flow path D).

  Therefore, the second flow path does not necessarily mean only a flow path in which the liquid moves (flows), but is also used as a flow path for moving air. In particular, it is only necessary to form a volume-limited section sandwiched between two narrow channel portions capable of allowing the liquid weighed in the third channel to flow out. The planar shape of the limited section is not limited to the illustrated rectangular shape, and may be various shapes such as a circle, an ellipse, and a triangle.

  As shown in FIG. 5 (c1), the narrow channel portion (channel b1 and channel b2) of the second channel is formed on the lower substrate 1, and the normal width portion of the second channel is formed. It can be formed on the upper substrate 3 or, as shown in FIG. 5 (c2), the narrow channel portions (channel b1 and channel b2) of the second channel and the second channel All of the normal width portions can be formed on the upper substrate 3.

  Further, among the trace liquid weighing structures used in the present invention, the seventh embodiment is the first embodiment, the second embodiment, the third embodiment, the fourth embodiment, the fifth embodiment, or In the trace liquid weighing structure according to any one of the sixth embodiment, the liquid quantitatively weighed in the third flow path (flow path C) is further passed through the fourth flow path (flow path). From the opening of the fourth channel (channel D) that opens in the channel wall of the second channel (channel B) via the channel D), the second channel (channel B) It has an outflow means for flowing out.

  Specifically, in order to cause the liquid in the third flow path formed quantitatively to flow out to the second flow path, the pressure in the first flow path is slightly lower than the pressure in the second flow path. An appropriate pressure difference may be generated between the two flow paths or a centrifugal force may be applied in the outflow direction so as to increase. As a means for causing the pressure difference, for example, a known and commonly used pressure mechanism or pressure reduction mechanism is preferably used.

  Therefore, according to the seventh embodiment of the trace liquid weighing structure used in the present invention, the liquid in the third flow path quantitatively weighed can flow out to the second flow path. Therefore, it is possible to quantitatively introduce a small amount of sample or reagent in the protein crystallization apparatus.

  An eighth embodiment of the trace liquid weighing structure used in the present invention is the trace liquid weighing structure according to the seventh embodiment, wherein the second flow path (flow path B) is filled with liquid. The liquid quantitatively weighed in the third channel (channel C) is transferred to the second channel via the fourth channel (channel D). Outflow means for flowing out from the opening of the third flow path that opens in the flow path wall to the second flow path is provided.

  Specifically, according to the eighth embodiment of the trace liquid weighing structure used in the present invention, the third liquid flow path is formed quantitatively using the trace liquid weighing structure of the fifth embodiment. The liquid is placed between the two flow paths so that the pressure of the first flow path is slightly higher than the pressure of the second flow path with the end face opposite to e1 of the flow path E in FIG. It is possible to cause the second flow path to flow out by performing an operation such as generating an appropriate pressure difference or applying a centrifugal force in the outflow direction.

  Therefore, according to the fifth embodiment of the trace liquid weighing structure used in the present invention, the liquid in the third flow path quantitatively weighed can flow out to the second flow path. Therefore, it is possible to quantitatively introduce a small amount of sample or reagent in the protein crystallization apparatus.

  Further, among the trace liquid weighing structures used in the present invention, the ninth embodiment includes the first embodiment, the second embodiment, the third embodiment, the fourth embodiment, the fifth embodiment, In any one of the sixth embodiment, the seventh embodiment, and the eighth embodiment, the first flow path (flow path A), the second flow path (flow path B), and the first The third channel (channel C), the fourth channel (channel D), and the fifth channel (channel E) are channels formed in the substrate. is there.

  Therefore, according to the ninth embodiment of the trace liquid weighing structure used in the present invention, it is possible to quantitatively weigh a small volume of liquid with a simple configuration and with a simple operation. It is possible to reduce the dead volume of one sample and to save space and cost of the entire apparatus.

  Further, among the trace liquid weighing structures used in the present invention, the tenth embodiment includes the first embodiment, the second embodiment, the third embodiment, the fourth embodiment, the fifth embodiment, In any one of the sixth embodiment, the seventh embodiment, the eighth embodiment, or the ninth embodiment, the volume of the third flow path is formed to a size of picoliter to microliter order. It is what has been.

  Therefore, according to the trace liquid weighing structure used in the present invention, it is possible to quantitatively weigh a small volume of liquid of various sizes on the order of picoliter to microliter with a simple configuration and simple operation. Can do.

  The trace liquid weighing structure used in the present invention can be formed in the chip. A plurality of trace liquid weighing structures used in the present invention may be formed on the same chip with the same structure or a combination of different structures. In the chip used in the present invention, each formed flow path is preferably a microchannel. Here, the microchannel means a channel having a cross-sectional shape in which a micro effect appears when a liquid is introduced into the channel (flow path), that is, some behavior change appears in the liquid. The manifestation of the micro effect varies depending on the physical properties of the liquid, but the cross-sectional shape, that is, the length of the shortest interval (corresponding to the short side for a rectangle and the short axis for an ellipse) of the shape perpendicular to the main flow direction of the channel. Is usually 5 mm or less, preferably 500 μm or less, more preferably 200 μm or less. The lower limit of this length is not particularly limited as long as it has a function as a microchannel.

  In the present invention, the chip means a part of a member including a trace liquid weighing structure. The microchip means a chip having a trace liquid weighing structure composed of the microchannel. The size and shape of the chip are not particularly limited, and can be arbitrarily set according to the application.

In the present invention, the chip is used for protein crystallization. The chip in the present invention is desirably replaced after use for a single use or a limited number of times, but may be used permanently. In this case, it may be integrated with a device such as a dispenser or a measuring device, but in this case, the part of the member including the flow path structure is called a chip.
The material of the chip in the present invention is not limited as long as the above channel structure can be realized. Examples of the material include polydimethylsiloxane (PDMS), glass, silicon, photoreactive resin and other resins, metals, ceramics, and combinations thereof.

  The method for creating the trace liquid weighing structure used in the present invention may be any method as long as the trace liquid weighing structure can be realized. For example, transfer technology represented by machining, injection molding and compression molding, dry etching (RIE, IE, IBE, plasma etching, laser etching, laser ablation, blasting, electrical discharge machining, LIGA, electron beam etching, FAB), wet Surface micro-machining, which forms fine structures by etching (chemical erosion), monolithic molding such as stereolithography and ceramics laying, and coating, vapor deposition, sputtering, deposition, and partial removal of various substances. It includes a method of forming a groove by forming an opening portion by using at least one sheet-like substance (film, tape, etc.), and a method of forming a channel by dropping and injecting a flow path constituent material by an ink jet or a dispenser.

  In order to produce a chip for use in the present invention, a mask may be used in the above method. The mask may have any design as long as the chip of the present invention can be finally produced, and a plurality of masks may be used. The mask is usually designed with a shape in which the flow path is projected onto a flat surface, but when processing on both sides of the flow path component to be bonded or when forming a flow path using multiple members, multiple masks are used. Therefore, the mask is not necessarily a projection of the final shape of the flow path because a part of the mask can be directly processed without using the mask. Examples of the electromagnetic wave shielding mask used for the photo-curing resin include those obtained by coating quartz or glass with chromium, or those obtained by baking a resin film with a laser.

  The mask can also be created by drawing at least a part of the channel structure and printing it on a transparent resin film using a suitable software, for example. The present invention also includes a computer-readable recording medium used for creating the mask or master chip drawn by the software and storing the electronic information of at least a part of the flow channel structure. Examples of suitable recording media include magnetic media such as floppy disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, CD-RW, and DVD, and semiconductor memories. .

  In producing the chip used in the present invention, the chip may be directly manufactured by the above method, or the chip may be shaped using this as a mold. Of course, it is also possible to shape the chip using this as a mold. In the present invention, a mold at each stage from a mold that is initially created to a chip that is actually used for operation may be referred to as a “master chip”. The master chip usually has a concave / convex pattern identical to that of an actual chip (positive) and is transferred twice, or has a concave / convex pattern opposite to that of an actual chip (negative). The master chip in the present invention is not necessarily limited to this, although it is used after being transferred twice.

The chip used in the present invention can be processed by bonding two or more materials. As bonding methods, adhesive bonding, resin bonding with primer, diffusion bonding, anodic bonding, eutectic bonding, thermal fusion, ultrasonic bonding, laser melting, bonding with solvent / dissolving solvent, adhesive tape, adhesive tape , Pressure bonding, bonding with a self-adsorbing agent, physical retention, and combination with unevenness.
In addition, a method of integrally forming the fluid branch portion and the independent flow path without requiring bonding is also possible. Specifically, it is possible to form a structure including a closed space by integral molding such as stereolithography.
The length, shape, and thickness of one side of the chip thus produced are not limited, and can be set to any value between 5 mm and 100 mm, for example.

  Note that when a microchip is manufactured by bonding a substrate using a mask and a master chip, the depth of the microchannel in the chip is, for example, that when the substrate is formed of two substrates, the channel is the upper substrate. Depending on whether it exists on the lower surface or the upper surface of the lower substrate, it can be partially set to one of two arbitrary values. The depth is not particularly limited, but preferably 1 to 200 μm. It can be set to any arbitrary value. Here, “the depth of the channel” corresponds to the height of a convex portion of a chip having a convex flow path serving as a channel mold, that is, a master chip. Further, the number of substrates is not limited to two, and a chip can be produced by laminating a plurality of arbitrary substrates as necessary.

The protein crystallization method of the present invention is a method characterized by using the above-described trace liquid weighing structure or a chip on which the structure is formed.
Of the present invention, the invention described in claim 1 is
(A) A protein solution or a precipitant solution is introduced into the first flow path of the flow path structure, and the third flow is made through an opening of a third flow path that opens to the first flow path. After the protein solution or precipitant solution is drawn into the path, the protein solution or precipitant solution remaining in the first flow path is moved to a position where it does not contact the opening of the third flow path, A step of causing a protein solution or a precipitant solution prepared in a volume corresponding to the volume of the third channel to flow out to the second channel through a fourth channel;
(B) A precipitant solution or a protein solution is introduced into the first channel, and the precipitant solution is introduced into the third channel through an opening of the third channel that opens into the first channel. Alternatively, after the protein solution is drawn, the precipitant solution or protein solution remaining in the first flow path is moved to a position where it does not contact the opening of the third flow path, and the third flow path The precipitant solution or the protein solution prepared in a volume corresponding to the volume of the liquid is allowed to flow out to the second flow path through the fourth flow path, and the protein solution and the precipitant solution are allowed to flow in the second flow path. A process of contacting and uniting;
(C) A protein crystallization method comprising a step of precipitating a protein crystal from a protein and a precipitant solution combined in a second flow path.
The invention according to claim 2 of the present invention is the flow path structure in which precipitation of protein crystals from the combined protein and precipitant solution is separated from the solution according to the invention of claim 1. The method is carried out in a state where the precipitant solution is disposed therein. In the present invention, the precipitant solution may be arranged, for example, by introducing it into an appropriate flow channel, for example, the second flow channel in advance, or bringing the protein solution and the precipitant solution into contact / unification. Then, the precipitant solution may be introduced into the first channel and placed in a state of being drawn into the third channel.

  In these inventions, either the protein solution or the precipitant solution may be introduced into the second channel first, or the second stream may be simultaneously formed using two sets of channel structures formed separately. It may be introduced to the road.

Moreover, invention of Claim 3 among this invention is the following.
(A) A protein solution is introduced into the first flow path of the trace liquid weighing structure, and the third flow path is opened through the opening of the third flow path that opens to the first flow path. After the protein solution is drawn, the protein solution remaining in the first flow path is moved to a position where it does not come into contact with the opening of the third flow path, and according to the volume of the third flow path. Creating a protein solution in a volume of
(B) introducing a precipitant solution into the first channel, bringing the protein solution and the precipitant solution into contact at the opening of the third channel, and bringing the protein solution and the precipitant solution together;
(C) A protein crystallization method comprising the step of precipitating protein crystals from the combined protein and precipitant solution.
In this method, since protein crystals are precipitated in the first and / or third flow paths, the combined solutions are allowed to stand for a suitable time as necessary, and then the presence or absence of the crystal precipitation is confirmed.

  The confirmation means of crystal precipitation may be performed by an appropriate detection means known per se, such as visual observation or microscopic observation. The precipitated protein crystals can be collected by an appropriate method known per se, for example, by introducing an appropriate liquid and allowing it to flow out.

  Here, the physical property of the protein solution used for the protein crystallization method of the present invention is not particularly limited as long as it can be used in the trace liquid weighing structure of the present invention. Specifically, examples of the solvent include water, salt solutions, buffer solutions, alcohol or glycerol and solutions thereof, synthetic or natural polymer solutions, and the like.

  The precipitant solution used means a solution that promotes protein crystal formation, and the physical properties of this solution are not particularly limited as long as they can be used in the trace liquid weighing structure of the present invention as described above. Specific examples include water, salt solutions, buffer solutions, alcohol or glycerol and solutions thereof, synthetic or natural polymer solutions, and the like. In addition, proteins and polymer gels, porous silicon (see Chayen et al., Journal of Molecular Biology, (2001) 312, 591-595) are known as substances that promote crystal formation. Good.

  Further, in the method of the present invention, when sealing or adjustment of the vapor pressure is necessary, 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 channel between two narrow channel portions.

  Known methods for protein crystallization include, for example, a batch method, a vapor diffusion method, an interface contact method, and a gel method. Here, in order to perform crystallization by a batch method in the microchip having the flow channel structure of the present invention, for example, as in the method according to any one of claims 1 to 3, a protein solution and a precipitant solution are used. And the flow path may be sealed by the above method if necessary.

  In order to perform the vapor diffusion method, for example, as described in the method of claim 1 or 2, the protein solution and the precipitant solution are brought into contact and combined in the second flow path, and then as described above. If necessary, a liquid whose vapor pressure is adjusted may be introduced into the first channel, and the vapor diffusion may be adjusted by separating the contacted and united liquid from the third and / or fourth channel. However, in the vapor diffusion method, the liquid that has been contacted and united and the liquid that adjusts the vapor pressure (precipitating agent) need only be arranged across the gas phase, and the arrangement of the liquid is not necessarily the above-described form. There is no need. A liquid for adjusting the vapor pressure may be introduced halfway through the first flow path or at the end thereof, and the vapor pressure may be adjusted across the first, third, and fourth flow paths. A liquid for adjusting the vapor pressure may be introduced to the middle of the flow path or at the end portion, and the vapor pressure may be adjusted across the second flow path.

Hereinafter, the reference examples and examples will specifically illustrate the crystallization of protein using the micro liquid weighing structure of the present invention and the microchip having the structure.
Reference Example 1 Protein Separation by Ion Exchange Chromatography FIG. 6 (a) shows a microchip 10 having a trace liquid weighing structure used in the present invention. The microchip 10 shown in FIG. 6 (a) is a microchip for capillary ion exchange chromatography used for protein analysis, and is provided with a trace liquid weighing structure used in the present invention at the injection portion of a sample. .

  In FIG. 6A, port 11 and port 12 are ports for introducing a protein elution buffer. A buffer having a different ionic strength is introduced, a flow rate is changed, and a buffer having a different ionic strength is provided in the mixer 13. By mixing, a gradient of ionic strength can be formed. Therefore, the upper surfaces of the port 11 and the port 12 are open to the atmosphere, and the lower portion communicates with the channel.

  The microchannel 14 is a column for chromatography. From the damming unit 15 of ion exchange beads (as the ion exchange beads, for example, anion exchange beads manufactured by Pharmacia: particle size of 30 μm can be used). The ion exchange beads are filled in a section of 3 mm in the direction of the trace liquid weighing structure (b). A port 18 exists in the direction opposite to the damming portion 15.

  FIG. 6B is an enlarged view of the trace liquid weighing structure (b). The microchip 10 shown here is formed by stacking two plate-like substrates (upper: substrate 16, lower: substrate 17) formed of a polymer (polymer) material such as PDMS (polydimethylsiloxane) and bonding them. However, the length, shape, and thickness of one side of the microchip are not limited, and are set to any value between 5 mm and 100 mm, for example. be able to.

Microchannels having different depths are formed on the lower surface of the upper substrate 16 and the upper surface of the lower substrate 17. The microchannel existing on the lower surface of the substrate 16 has a depth of 100 μm, and the microchannel existing on the upper surface of the substrate 17 has a depth of 10 μm. Furthermore, as a feature of PDMS, the hardened surface is hydrophobic. Further, by subjecting this surface to irradiation treatment with O ₂ plasma, excimer UV laser, or the like, PDMS and PDMS, PDMS and glass, etc. can be easily bonded. From the above two points, PDMS is suitable for the production of this micro liquid weighing structure.

  Note that the depth of the microchannel in the microchip 10 is partially set to one of two arbitrary values depending on whether it exists on the lower surface of the upper substrate 16 or the upper surface of the lower substrate 17. The depth can be set to an arbitrary value of 1 to 200 μm.

  As shown in FIG. 6B and FIG. 6C1, the microchannel structure in the trace liquid weighing structure (b) is extended in the left-right direction in parallel with each other on the lower surface of the upper substrate 16. The first flow path 19 and the second flow path 20, the third flow path 21 extending perpendicularly from the first flow path 19 to the second flow path direction, and the upper surface of the lower substrate 17. A fourth flow path 22 that connects the second flow path 20 and the third flow path 21, and a fifth flow path 23 that is derived from the middle of the fourth flow path 22 and goes straight to the fourth flow path 22. Is formed. When a shallow and narrow channel is disposed on the upper surface of the lower substrate 17, each flow is partially overlapped with a deep and wide channel disposed on the lower surface of the upper substrate 16. Roads can communicate with each other. Alternatively, as shown in FIG. 6 (c2), when all the channels are arranged on the upper substrate 16 (or the lower substrate 17), it is not necessary to provide an overlapping portion in each channel. By appropriately selecting a synthetic resin material to be used as a substrate and / or making the surface of a specific flow path disposed in the substrate and a part or all of a necessary portion easy to wet or difficult to wet Accordingly, as shown in FIG. 6C2, all the flow paths can be provided only on one substrate.

  The width of the first flow path 19 and the second flow path 20 is 200 μm, the width of the third flow path 21 is 100 μm, the width of the fourth flow path 22, and the fourth flow in the fifth flow path 23. The width of the portion in contact with the path 22 is 20 μm. Note that the fourth flow path 22 needs to be thinner than the first flow path 19, the second flow path 20, and the third flow path 21 in order to make it difficult for liquid to enter the fourth flow path 22. In addition, the width of the fifth channel 23 needs to be equal to or less than the width of the fourth channel 22 in order to make it difficult for the liquid to enter the fifth channel 23.

  Next, the above-described microchip 10 is manufactured by the manufacturing process described below. Prior to the manufacturing process, first, the microchannels of the upper substrate 16 and the lower substrate 17 of the microchip 10 are arranged. Each layout pattern is printed on a transparent film at a high resolution (for example, 4064 dpi) in order to be used separately as a mask for photolithography.

First, a silicon (Si) wafer is cut with a diamond cutter in accordance with the size of a microchip to be manufactured, subjected to ultrasonic cleaning, dried, and subjected to O ₂ plasma treatment at 200 W for 30 seconds using a plasma reactor.

  Next, a negative photoresist SU-8 is spin-coated (when a 100 μm channel is prepared, SU-850 is used for 10 seconds at 2000 rpm, and when a 10 μm channel is prepared, SU-8 is used at 2000 rpm. 10 seconds) and then hold in an oven at 90 ° C. for 30 minutes.

  Next, using a mask aligner, the microchannel layout pattern on the upper substrate 16 and the lower substrate 17 in the microchip 10 printed on the film is photo-printed on separate silicon wafers coated with SU-8. After transferring using a lithography technique and holding in an oven at 90 ° C. for 30 minutes, the film is immersed in Developer (for example, 1-methoxy-2-propyl acetate can be used as Developer). Develop, wash with isopropyl alcohol, followed by distilled water and dry.

  The master thus fabricated has a convex structure that serves as a microchannel template for the upper substrate 16 and the lower substrate 17.

  Next, in order to facilitate the removal of the PDMS replica after molding, the surface treatment is performed by holding in a 3% dimethyloctadecylchlorosilane / toluene solution for 2 hours before pouring the PDMS prepolymer.

  Next, the PDMS prepolymer and a curing reagent (for example, “Sylgard 184: Dow Corning Co., MI” can be used as a curing reagent) are mixed at a ratio of 10: 1 and sufficiently stirred. Then, in order to install the master, it is poured into a frame made of acrylic and kept at 90 ° C. for 30 minutes for curing.

After the curing, the PDMS replica is peeled off from the master to obtain the upper substrate 16 and the lower substrate 17 of the microchip 10, respectively. Further, the lower surface of the upper substrate 16 and the upper surface of the lower substrate 17 are treated with O ₂ plasma and bonded to obtain the microchip 10.

  A micro liquid weighing operation for sample injection actually performed using the microchip 10 obtained by the above method will be described below.

  First, the sample is filled into the first channel 19 and the third channel 21 by flowing the sample from one side of the first channel 19 by a pressure flow. At this time, the fourth flow path 22 has the property that it is difficult to wet or that the capillary attraction is relatively difficult to operate, and more than the first flow path 19, the second flow path 20, and the third flow path 21. The sample does not enter the fourth flow path 22 because it is thin and difficult to wet with a hydrophilic liquid.

  In a state where the second channel 20 is filled with liquid, if the fifth channel 23 does not exist, the sample in the third channel 21 is introduced when the sample is introduced into the third channel 21. Air leaks into the second flow path 20.

  However, the presence of the fifth flow path 23 causes air in the third flow path 21 to escape to the fifth flow path 23 even when the second flow path 20 is filled with liquid. Therefore, the sample can be introduced into the third channel 21 without air leaking into the second channel 20.

  Next, by introducing air into the first channel 19 by pressure flow, the sample in the first channel is pushed out, the sample remains only in the third channel 21, and the third channel The sample remaining in the flow path 21 does not flow back into the first flow path 19 again. In the example of the chip shown here, the amount of sample remaining in the third flow path 21 is designed to be 5 nl.

  Subsequently, by increasing the pressure in the first flow path 19, the sample in the third flow path 21 leaks into the fourth flow path 22, and the sample reaches the second flow path 20. A sample is introduced into the second flow path 20. At this time, since the outlet side of the fifth flow path 23 is closed, the sample does not leak into the fifth flow path 23.

  In order to reduce the possibility that the sample leaks into the fifth channel 23, the channel width of the fifth channel 23 is desirably equal to or less than the channel width of the fourth channel 22, Both are set to 20 μm.

In order to prevent adsorption of a sample (protein or the like) for separation by chromatography on PDMS, only the first channel 19, the second channel 20, and the third channel 21 are used using HCl. Treatment to make it hydrophilic. However, the hydrophilization treatment is not limited to HCl, and a method using O ₂ plasma or a method using albumin is also possible. PDMS itself is a hydrophobic substance.

  Actually, in the chromatography using sample injection by the above-mentioned micro liquid control operation, a mixed solution of albumin and IgG labeled with FITC is introduced, and Tris / HCl Buffer (pH 8.0) is flowed at a flow rate of 1.0 μl / min. Then, using anion exchange beads (Pharmacia: particle size: 30 μm), after adsorption, they were successfully separated within 1 minute using a 0-1 M gradient buffer with sodium chloride.

  In the above Reference Example 1, this micro liquid weighing structure was fabricated by PDMS of silicon polymer. However, by making part or all of the inner surface and / or end face of the flow path easy to wet or difficult to wet, The structure can be applied to any material (for example, silicon, polymer, glass, ceramics, metal, etc.) as long as the wetness in the road can be adjusted and the structure can be manufactured. In addition, this micro liquid weighing structure can be used by a composite use of these different materials, or a material mixed with temperature, pH responsive substance PIPAAm (N-isopropylacrylamide) and the like.

Reference Example 2 Quantification of Glucose by Enzymatic Reaction FIG. 7A is a plan view of a microchip 24 according to another reference example of the trace liquid weighing structure used in the present invention. The microchip 24 shown in FIG. 7A is a microchip capable of individually performing up to 50 kinds of reactions or analyzes by mixing two liquids quantitatively weighed. Adopts a micro liquid weighing structure used in the present invention. The microchip 24 shown in FIG. 7A processes up to 50 types of reactions or analyses, but microchips that process a larger number of reactions or analyzes can naturally be produced.

  By using the microchip 24 as shown in FIG. 7 (a), for example, reaction or analysis by mixing one kind of reagent individually with respect to 50 kinds of samples, and very simple operation, and Can be done at the same time. Alternatively, for example, reaction or analysis by mixing 50 kinds of reagents can be performed individually and simultaneously with respect to one kind of sample.

  The microchip 24 as shown in FIG. 7A is similar to the microchip 10 shown in FIG. 5 in that two flat plates formed of a polymer material, for example, PDMS (polydimethylsiloxane). The substrate is formed by overlapping and bonding the substrate, and has a disk shape with a diameter of 90 mm and a thickness of 3 mm. Of course, other diameters and thicknesses can be used. The shape of the microchip 24 is not limited to the illustrated disk shape. It can also be formed using a rectangular or polygonal substrate.

  In the microchip 24 shown in FIG. 7A, microchannels having different depths are formed on the lower surface of the upper substrate 25 and the upper surface of the lower substrate 26, and exist on the lower surface of the substrate 25. For example, the depth of the microchannel is 100 μm, and the depth of the microchannel existing on the upper surface of the substrate 26 is 10 μm, for example. Depths other than these can of course be used.

  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.

  In FIG. 7B, the first liquid supply channel 33 having a substantially annular shape in the center is cut at one place in the middle thereof, and one cut end is ported through a channel 27 extending radially outward. 28 is connected to and communicated with the port 30 via a channel 29 extending radially outwardly. Port 28 is the inlet for the first liquid to be mixed and port 30 is the outlet for the liquid. The liquid introduced from the port 28 enters the first liquid supply channel 33 through the channel 27, flows through the first liquid supply channel 33, and flows out from the port 30 through the channel 29. Alternatively, port 28 can be the outlet and port 30 can be the inlet.

  7B is an inlet for the second liquid to be mixed, and communicates with the outlet port 31b through the second liquid supply channel 35. Since the inlet port 31a and the outlet port 31b communicate with each other via the second liquid supply channel 35, the liquid injected from the inlet port 31a can be easily introduced into the second liquid supply channel 35 by an appropriate means such as air pressure. Can be satisfied. Adjacent to the port 31a and the port 31b, a port 32a and a port 32b are disposed through the mixing channel 34, respectively. The mixing channel 34 also serves as a reaction chamber for both mixed liquids. Since the port 32a and the port 32b communicate with each other through the mixing channel 34, air pressure is applied to the mixing channel 34 through the port 32a and / or the port 32b to promote mixing of the liquid in the channel. Alternatively, the reaction product in the mixing channel 34 can be used to take out.

  Thirty structures for measuring and mixing a trace amount of liquid comprising ports 31a and 31b, a second liquid supply channel 35, ports 32a and 32b, and a mixing channel 34 are disposed radially outside the first liquid supply channel 33. Is arranged. On the other hand, 20 structures for weighing and mixing the same trace liquid are arranged on the radially inner side. The number of arrangement is not limited to the illustrated embodiment.

  The structure shown in FIG. 7 (c) is formed for the purpose similar to the structure shown in FIG. 2 (b), and includes a microchannel structure for weighing a small amount of liquid and two types. It consists of channels for mixing liquids. A first liquid supply channel 33 (flow path A in FIG. 2B) that supplies one liquid with a mixing channel 34 (corresponding to the flow path B in FIG. 2B) functioning as a reaction chamber interposed therebetween. And a second liquid supply channel 35 for supplying the other liquid (corresponding to the flow path A ′ in FIG. 2B). One liquid supplied from the first liquid supply channel 33 is weighed by a certain amount by the first weigh channel 36 (corresponding to the flow path C in FIG. 2B). Similarly, the other liquid supplied from the second liquid supply channel 35 is weighed by a certain amount by the second weighing channel 37 (corresponding to the flow path C ′ in FIG. 2B). The liquid in the first weigh channel 36 and the second weigh channel 37 passes through the first narrow channel 38 and the second narrow channel 39 which are narrower than these channels, respectively, using conventional means such as a pressure difference. It can be taken into the mixing channel 34 and mixed in the channel.

  In FIG. 7, the channel width of the first liquid supply channel 33 for supplying one liquid, the second liquid supply channel 35 for supplying the other liquid, and the mixing channel 34 is, for example, 200 μm, and the first weighing channel 36 and The channel width of the second weighing channel 37 is, for example, 100 μm, the length is, for example, 600 μm, and the channel width of the first narrow channel 38 and the second narrow channel 39 is, for example, 20 μm. Other channel widths and channel lengths may be used as a matter of course. For example, in FIG. 7C, the volume of the two liquids to be mixed is 6 nl, but this volume is equal to the volume of the first weighing channel 36 and the second weighing channel 37. Therefore, it can be arbitrarily adjusted within the range of 1 pl to 1 μl by changing the volume of each weighing channel.

  The microchip 24 of FIG. 7 can be manufactured by the same manufacturing process as the microchip 10 shown in FIG.

  The microchip 24 shown in FIG. 7 can be used, for example, for micro liquid weighing and mixing operations for quantitative analysis of glucose by the glucose oxidase / peroxidase method.

  The micro liquid weighing and mixing operation for quantitative analysis of glucose by the glucose oxidase / peroxidase method will be described below. In the glucose oxidase / peroxidase method, a phosphate buffer mixed with appropriate amounts of glucose oxidase, peroxidase, mutarotase, 4-aminoantipyrine and phenol is used as a reagent, and an aqueous glucose solution is used as a sample.

  First, about 1 μl of the reagent is introduced from the port 28 to the port 30 in FIG. 7A by pressure flow, and then air is introduced in order to allow excess reagent to flow. The introduced reagent and air flow in the first liquid supply channel 33 in the clockwise direction. In FIG. 7C, when the reagent is introduced into the first weighing channel 36, the first narrow channel 38 is hydrophobic and difficult to wet with the liquid, and the first liquid supply channel 33 and Since the channel width is narrower than the first weighing channel 36, the reagent stays in the first weighing channel 36 and does not enter the first narrow channel 38.

  By this operation, a reagent liquid having a volume equal to the volume of the first weighing channel 36 can be weighed in the first weighing channel 36. Also, a predetermined amount of reagent liquid is weighed at once in the 50 first weighing channels 36 by performing only one operation of introducing the reagent from the port 28 to the port 30 and subsequent air introduction. It is possible. By such an extremely simple operation, it is possible to further finely distribute a minute amount of liquid and simultaneously form a large number of extremely small amounts of liquid. Moreover, in such an operation, it is expected that the amount of residual liquid that is not distributed after the operation will be extremely small.

  Subsequently, similarly to the introduction of the reagent, the sample is introduced from the port 31a toward the port 31b, and a predetermined amount of the sample liquid is weighed into the second weighing channel 37 through the second liquid supply channel 35. Can do.

  Subsequently, the reagent in the first weighing channel 36 and the sample in the second weighing channel 37 are respectively increased by increasing the pressure of air in the first liquid supply channel 33 and the second liquid supply channel 35. It is pushed into the mixing channel 34 and mixed in the channel. Since the two mixed solutions exhibit a red color by a chemical reaction, quantitative analysis of glucose can be performed by evaluating the red value. Needless to say, the microchip 24 shown in FIG. 7 can also be used for other qualitative and / or quantitative analysis.

Example 1 Crystallization of glucose isomerase (unification of liquids)
Protein (glucose isomerase) was crystallized using the microchip 41 shown in FIG. FIG. 8B is an enlarged view of the micro liquid weighing structure structure part (b) of the microchip 41 used. The microchip 41 is basically substantially the same as the microchip 24 shown in FIG. 7A except for the shape and the number of arranged micro liquid weighing structures.

  A microchip 41 shown in FIG. 8A is a microchip for protein crystallization, and a micro liquid weighing structure used in the present invention is provided in an injection portion of a solution (protein solution and precipitant solution). ing. PDMS was used as a material, and a method according to Reference Example 1 was used. The size of the chip is 24 × 46 mm and the thickness is 60 μm. Each of the first liquid supply channel 33 and the second liquid supply channel 35 has a width of 200 μm, and the mixing channel 34 has a width of 300 μm. The width of the first weighing channel 36 and the second weighing channel 37 is 100 μm, and the length is 1000 μm. The first narrow channel 38 and the second narrow channel 39 each have a width of 20 μm and a length of 400 μm.

In FIG. 8A, ports 28 and 30 are ports for introducing a protein solution, and ports 31a and 31b are ports for introducing a precipitant solution.
In the micro liquid weighing structure formed on the microchip, the first liquid supply channel (first flow path) 33 is provided with a first weighing channel (weighing the first liquid (protein solution)). Seven (third flow paths) 36 are connected, and a first narrow channel (fourth flow path) 38 is connected to each of the seven locations, and the first narrow channel 38 is a mixing channel (second flow path). ) 34. The mixing channel (second flow path) 34 formed at each of the seven locations has a second liquid supply channel 35 and a second weighing channel 37 for weighing the second liquid (precipitant solution). And the flow path structure which consists of the 2nd narrow channel 39 is connected.

First, in order to weigh the first liquid (protein solution), glucose isomerase (Hampton) adjusted to a concentration of 20 mg / mL in water using positive pressure from the port 28 to the first liquid supply channel 33. Research HR7-102) solution was filled. Further, by passing air under a positive pressure, excess glucose isomerase solution is removed from the first liquid supply channel 33 through the port 30, and a first weighing for weighing the first liquid (protein solution) is performed. The glucose isomerase solution was weighed at all seven locations of the channel 36.
Next, air is circulated into the first liquid supply channel 33 from the port 28 using a positive pressure higher than the pressure for removing the excessive glucose isomerase solution, and the weighed glucose isomerase solution is mixed. Drained into channel 34.

Then, in a buffer (pH 6.5) of 100mM of MES (Hampton Research Corporation HR2-587), PEG8000 (Hampton Research Corporation HR2-535) is 10%, MgCl ₂ (Hampton Research Corporation HR2-559) within 200mM The precipitant solution adjusted so as to be filled into the second liquid supply channel 35 and the second weigh channel 37 for weighing the second liquid using the positive pressure from the port 31a, and the air is further under the positive pressure , The excess precipitant solution in the second liquid supply channel 35 is removed via the port 31b, and the precipitant solution is weighed in the second weigh channel 37 for weighing the second liquid. I took it. Next, air is circulated from the port 31a to the second liquid supply channel 35 using a positive pressure higher than the pressure for removing the excess precipitant solution, and the weighed precipitant solution is mixed with the mixing channel 34. Spilled into. As a result, the glucose isomerase solution and the precipitant solution contacted and merged in the mixing channel 34. This operation was performed at five locations to prepare five liquids in which the glucose isomerase solution and the precipitant solution were combined.

  When this chip was observed with a stereomicroscope, crystals were observed in the liquid in all five liquids in which the glucose isomerase solution and the precipitant solution were combined.

Example 2 Crystallization of glucose isomerase (contact between liquid and continuous liquid)
Experiments were performed using the same chip as in Example 1 and using the same reagents as in Example 1.
First, in order to weigh the first liquid (protein solution), glucose isomerase (Hampton) adjusted to a concentration of 20 mg / mL in water using positive pressure from the port 28 to the first liquid supply channel 33. Research HR7-102) solution was filled. Further, by passing air under a positive pressure, excess glucose isomerase solution is removed from the first liquid supply channel 33 through the port 30, and a first weighing for weighing the first liquid (protein solution) is performed. The glucose isomerase solution was weighed at all seven locations of the channel 36.
Next, a positive pressure is applied from the port 28 to the first liquid supply channel 33 with a precipitant solution adjusted to 10% PEG 8000 and 200 mM MgCl _{2 in} a MES buffer (PH 6.5) having a concentration of 100 mM. Used to fill. As a result, the glucose isomerase solution and the precipitant solution contacted each other at the opening of the first weighing channel 36 of the first liquid supply channel 33.
When this chip was observed with a stereomicroscope, the interface between the glucose isomerase solution and the precipitant solution was present (the opening between the first liquid supply channel 33 and the first weighing channel 36), and the glucose isomerase solution side (first Crystals were observed in the liquid on the one weighing channel 36) and the precipitant solution side (first liquid supply channel 33 side). In particular, many crystals were observed in the liquid on the precipitant side.

  As described above, the protein crystallization using the micro liquid weighing structure of the present invention or the microchip having the structure has been specifically described. However, the present invention is limited to the specifically exemplified embodiments and examples. It is not something. For example, only the portion close to the second channel (channel B) of the third channel (channel C) is selectively modified so as to have the property of being difficult to wet selectively (or relatively difficult to work with capillary attraction). If possible, the wide third channel can be directly connected to the second channel (channel B) having the same width without providing the fourth narrow channel (channel D). it can. Further, the shape of the third flow path for measuring the liquid can be any shape such as a circle, an ellipse, and a triangle in addition to a rectangle.

It is a conceptual explanatory drawing for demonstrating the principle of the trace liquid weighing structure used by this invention, (a) shows the structure, (b) and (c) are explaining the liquid weighing method. It is explanatory drawing of the trace liquid weighing structure which has two liquid preparation systems of a 2nd embodiment, (a) is a case where the flow path A is shared, (b) is a case where the flow path B is shared. . It is explanatory drawing of the trace liquid balance structure of a 3rd embodiment and a 4th embodiment, (a) is when the flow path D is divided into three flow paths, (b) is thickness It is explanatory drawing in case there are three sets of different flow paths C and D. It is explanatory drawing of the trace liquid balance structure of 5th Embodiment, (a) shows the structure, (b), (c), and (d) are the cases where the flow path B is filled with the liquid It is explanatory drawing of the liquid introduction method. (A) And (b) is a top view which shows the trace liquid balance structure of a 6th embodiment, (c) is sectional drawing along the cc line in (b). It is explanatory drawing of the microchip 10 provided with the trace liquid weighing structure used by this invention used for isolation | separation of the protein by the ion exchange chromatography of the reference example 1, (a) is a layout of the whole chip | tip, (b). Is an enlarged explanatory view of a quantification of a trace sample and an introduction part, and (c1) and (c2) are cross-sectional views taken along the line cc in FIG. (A) is a top view of the microchip 24 by another embodiment of the trace liquid balance structure used by this invention used for determination of glucose by the enzyme reaction of Reference Example 2, (b) is (a) It is the elements on larger scale of the b section in a figure, (c) is the elements on larger scale of the c section in (b) figure. It is a top view of the microchip 41 used by crystallization of the glucose isomerase of Example 1 and 2, (b) is the elements on larger scale of the b section in (a) figure.

Explanation of symbols

10 Microchip for capillary chromatography 11 Elution buffer introduction port 12 Elution buffer introduction port 13 Mixer 14 Microchannel 15 Damping part 16 PDMS flat plate (upper part)
17 PDMS flat plate (bottom)
18 Port 19 1st flow path 20 2nd flow path 21 3rd flow path 22 4th flow path 23 5th flow path 24 Microchip 25 for 50 kinds of minute two-liquid mixing reaction Upper substrate 26 Lower Substrate 27 Channel 28 First liquid inlet port 29 Channel 30 First liquid outlet port 31a Second liquid inlet port 31b Second liquid outlet port
32a port 32b port 33 first liquid supply channel 34 mixing channel 35 second liquid supply channel 36 first weighing channel 37 second weighing channel 38 first narrow channel 39 second narrow channel 41 microchip 100 for protein crystallization Liquid (sample, etc.)
100a Lower end of liquid in channel C (channel A side)
100b Upper end of liquid in channel C (channel D side)
200 liquid (liquid chromatographic mobile phase)
aa, aa ′ Channel wall bb of channel A, bb ′ Channel wall c1 of channel B Opening part of channel C (channel A side)
c2 Opening of channel C (channel D side)
d1 Opening of channel D (channel B side)
e1 Opening of channel E (channel D side)

Claims (3)

A protein crystallization method using a micro liquid weighing structure,
The trace liquid weighing structure includes a first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction, and the first flow path (flow path A). A third flow path (flow path C) that opens to the flow path wall of the second flow path and a flow path wall of the second flow path (flow path B) that opens to the third flow path (flow path C). One end is connected to the second flow path, and the fourth flow is narrower than the other three flow paths and has a property that the capillary attraction is less likely to work than the third flow path (flow path C). The liquid introduced into the first flow path has a path (flow path D) through the opening of the third flow path that opens in the flow path wall of the first flow path. The liquid remaining in the first flow path is removed after being drawn into the flow path, and the liquid having a volume corresponding to the volume of the third flow path is weighed, and
The method
(A) A protein solution or a precipitant solution is introduced into the first channel, and the protein solution or the precipitant solution is introduced into the third channel via an opening of the third channel that opens into the first channel. After the precipitant solution is drawn, the protein solution or precipitant solution remaining in the first flow path is moved to a position where it does not contact the opening of the third flow path, and the third flow path A step of causing a protein solution or a precipitant solution prepared in a volume corresponding to the volume of the liquid to flow out to the second flow path through a fourth flow path;
(B) A precipitant solution or a protein solution is introduced into the first channel, and the precipitant solution is introduced into the third channel through an opening of the third channel that opens into the first channel. Alternatively, after the protein solution is drawn, the precipitant solution or protein solution remaining in the first flow path is moved to a position where it does not contact the opening of the third flow path, and the third flow path The precipitant solution or the protein solution prepared in a volume corresponding to the volume of the liquid is allowed to flow out to the second flow path through the fourth flow path, and the protein solution and the precipitant solution are allowed to flow in the second flow path. A process of contacting and uniting;
(C) including a step of precipitating protein crystals from the protein and the precipitant solution combined in the second flow path. 2. The protein crystal precipitation from the combined protein and precipitant solution is performed in a state where the precipitant solution is disposed in a flow channel structure separated from the solution. the method of. A protein crystallization method using a micro liquid weighing structure,
The trace liquid weighing structure includes a first flow path (flow path A) and a second flow path (flow path B) each extending in a predetermined direction, and the first flow path (flow path A). A third flow path (flow path C) that opens to the flow path wall of the second flow path and a flow path wall of the second flow path (flow path B) that opens to the third flow path (flow path C). One end is connected to the second flow path, and the fourth flow is narrower than the other three flow paths and has a property that the capillary attraction is less likely to work than the third flow path (flow path C). The liquid introduced into the first flow path has a path (flow path D) through the opening of the third flow path that opens in the flow path wall of the first flow path. The liquid remaining in the first flow path is removed after being drawn into the flow path, and the liquid having a volume corresponding to the volume of the third flow path is weighed, and
The method
(A) After the protein solution is introduced into the first channel, and the protein solution is drawn into the third channel through the opening of the third channel that opens to the first channel. The protein solution remaining in the first flow path is moved to a position where it does not contact the opening of the third flow path, and a protein solution is created with a volume corresponding to the volume of the third flow path. Process,
(B) introducing a precipitant solution into the first channel, bringing the protein solution and the precipitant solution into contact at the opening of the third channel, and bringing the protein solution and the precipitant solution together;
(C) including a step of precipitating protein crystals from the combined protein and precipitant solution.

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