JP2014240343A - Nano-volume micro capillary crystallization system - Google Patents

Abstract

A nanovolume microcapillary crystallization system for proteins is provided. A plurality of software running on a protein crystallization system to control the pumping system, and one or more crystal cards 400 coupled to the pumping system, each comprising a mixer 404 and A crystal card 400 is provided that houses a microfluidic capillary 406 coupled to a mixer 404 to facilitate storage and protein crystallization testing. [Selection] Figure 4A

Description

This application claims the benefit of US Provisional Patent Application No. 61/061536, filed Jun. 13, 2008, which is hereby incorporated by reference.

Statement of Government License Rights The subject matter of the present invention was made, at least in part, with government support provided by NIGMS U54 GM074961 awarded by the National Institute of General Medical Sciences.

  The field of structural biology is creating technology every year that improves throughput and efficiency. Such progress motivated the development of genes to three-dimensional structures in three days. In order to improve efficiency, it is desirable to minimize the amount of protein required so that sufficient material for crystallization screening and optimization can be obtained from cell-free synthesis. With the structural goal of “3 days” in mind, it is desirable to develop several technologies that increase the efficiency of gene-to-structure pipelines.

  This summary is provided to introduce a selection of concepts in a further description that are further described below. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

  One aspect of the subject matter discussed includes a system for protein crystallization comprising a pumping system; and a plurality of software configured to execute on the protein crystallization system to control the pumping system. . The system comprises one or more crystal cards coupled to a pumping system, each comprising a mixer and a microfluidic capillary coupled to the mixer to facilitate storage and protein crystallization studies. Further included is a crystal card configured to receive.

  Another aspect of the present subject matter includes a method for gradient screening comprising adjusting water flow by independently controlling each water flow using a pumping system that operates with a plurality of software. The method further comprises mapping the crystallization phase space of the protein to show a transition from precipitation to microcrystals to single crystals in protein crystallization experiments.

  Further aspects of the present subject matter include methods for hybrid screening comprising pre-forming a precipitant plug; and pre-forming a plug spacer, each separating two precipitant plugs from each other. The method further comprises forming a gradient by combining the precipitant plug, the plug spacer, and the protein stream. The method further includes mapping the crystallization phase space of the protein to show a transition from precipitation to microcrystals to single crystals in protein crystallization experiments.

    Further aspects of the present subject matter include methods comprising receiving a crystal card with a capillary; coating a capillary with a reagent to reduce surface energy; and removing the reagent.

    In another aspect, the present subject matter includes a crystal card comprising a substrate configured to receive a mixer circuit and an inspection circuit. The crystal card further includes a layer configured to be bonded to and peeled from the substrate.

    The foregoing aspects, and many of the advantages associated with the present invention, will be better understood and will be more readily appreciated by reference to the following detailed description taken in conjunction with the accompanying drawings.

1 is a block diagram illustrating an example nanovolume microcapillary crystallization system. FIG. 1 is a block diagram illustrating a pumping system that is an example of a nanovolume microcapillary crystallization system. FIG. It is a pictorial diagram which shows the example user interface for comprising a pumping system. FIG. 2 is a pictorial diagram illustrating an example user interface for priming fluid on a crystal card of the system. FIG. 5 is a pictorial diagram illustrating an example user interface for specifying the generation of nanoplugs in a crystal card, wherein the nanoplugs are equal in size and content according to one embodiment of the present subject matter. FIG. 2 is a pictorial diagram illustrating an example user interface for specifying the generation of nanoplugs in a crystal card, wherein the nanoplugs have varying concentrations of protein and precipitant according to one embodiment of the present subject matter. FIG. 4 is a pictorial diagram illustrating an example user interface for specifying the generation of nanoplugs in a crystal card, wherein the nanoplugs have various sizes and concentrations for a plurality of precipitants according to another embodiment of the present subject matter. FIG. 5 is a pictorial diagram illustrating a top isometric view of one embodiment of a crystal card. FIG. 3 is a pictorial diagram illustrating a bottom isometric view of one embodiment of a crystal card. It is a pictorial diagram which shows the top view of one Embodiment of a crystal card. It is a pictorial diagram which shows the side view of one Embodiment of a crystal card. It is a pictorial diagram which shows the bottom view of one Embodiment of a crystal card. FIG. 6 is a pictorial diagram showing a top isometric view of another embodiment of a crystal card. FIG. 6 is a pictorial diagram showing a bottom isometric view of another embodiment of a crystal card. It is a pictorial diagram which shows the top view of another embodiment of a crystal card. It is a pictorial diagram which shows the side view of another embodiment of a crystal card. It is a pictorial diagram which shows the bottom view of another embodiment of a crystal card. It is a pictorial diagram showing a top isometric view of a third embodiment of the crystal card. It is a pictorial diagram which shows the bottom isometric view of 3rd Embodiment of a crystal card. It is a pictorial diagram which shows the upper side figure of 3rd Embodiment of a crystal card. It is a pictorial diagram which shows the side view of 3rd Embodiment of a crystal card. It is a pictorial diagram which shows the bottom view of 3rd Embodiment of a crystal card. It is a pictorial diagram which shows the upper surface isometric view of 4th Embodiment of a crystal card. It is a pictorial diagram which shows the bottom isometric view of 4th Embodiment of a crystal card. It is a pictorial diagram which shows the upper side figure of 4th Embodiment of a crystal card. It is a pictorial diagram which shows the side view of 4th Embodiment of a crystal card. It is a pictorial diagram which shows the bottom view of 4th Embodiment of a crystal card. FIG. 10 is a pictorial diagram showing a top isometric view of a fifth embodiment of a crystal card. It is an illustration which shows the bottom isometric view of 5th Embodiment of a crystal card. It is a pictorial diagram showing a top view of an embodiment of a crystal card. It is a pictorial diagram which shows the side view of 5th Embodiment of a crystal card. It is a pictorial diagram which shows the bottom view of 5th Embodiment of a crystal card. It is a pictorial diagram which shows the upper surface isometric view of 6th Embodiment of a crystal card. It is a pictorial diagram which shows the bottom isometric view of 6th Embodiment of a crystal card. It is a pictorial diagram which shows the three-dimensional exploded isometric view of 6th Embodiment of a crystal card. It is a pictorial diagram which shows the top view of 6th Embodiment of a crystal card. It is a pictorial diagram which shows the side view of 6th Embodiment of a crystal card. It is a pictorial diagram which shows the bottom view of 6th Embodiment of a crystal card. It is a pictorial diagram showing one embodiment of a 3 plus 1 mixer of one embodiment of a crystal card. It is a pictorial diagram showing another embodiment of a 3 plus 1 mixer of one embodiment of a crystal card. It is a pictorial diagram which shows 3rd Embodiment of 3 plus 1 mixer of one Embodiment of a crystal card. It is a pictorial diagram which shows 4th Embodiment of 3 plus 1 mixer of one Embodiment of a crystal card. It is a pictorial diagram which shows sectional drawing of one Embodiment of a crystal card. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 15A-15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system.

    In various embodiments of the present subject matter, a nanovolume microcapillary crystallization system is described that includes a pump; software configured to control the pump; and a crystal card containing a mixer circuit and an inspection circuit. The crystal card is suitably manufactured using a material that includes one or more properties selected from the group consisting of x-ray transmission, optical clarity, moldability, chemical resistance and surface energy. The Crystal cards contain various phases of macromolecular crystals that allow for the collection of crystals from the crystal card or in situ X-ray diffraction. The crystal grows in the crystal card through nanoplug formation by a nanovolume microcapillary crystallization system. Nanoplugs are formed by mixing an immiscible biologically inert carrier fluid, such as a fluorocarbon solution, with an aqueous stream. To configure a nanoplug crystallization experiment, an aqueous stream consisting of, for example, a target molecule, a buffer and a precipitant solution is mixed in a mixer circuit. Nanoplugs are incubated and monitored for crystallization. Nanoplug crystallization experiments can be used accordingly to elucidate scientific questions regarding nucleation and growth of protein crystals and to generate crystals in solutions of novel structure.

    The nanovolume microcapillary crystallization system facilitates two screening modalities: gradient mode and hybrid mode. As used herein, the term gradient mode includes any suitable screening method that provides various crystallization phases of the molecule. The gradient mode allows the crystallologist to scan the crystal card finely to reveal the crystallization phase space of a particular molecule. Each aqueous solution flow used in the nanovolume microcapillary crystallization system can be independently controlled by software using a pump, thus varying the flow rate of the individual streams to create a concentration gradient of the desired particle size across a series of nanoplugs. It is formed accordingly. As the precipitant stream flow rate decreases, the nanovolume microcapillary crystallization system increases the buffer stream flow rate so that the total flow rate remains constant. Gradient mode can be used to map the crystallization phase space of specific molecules, such as proteins, to show the transition from precipitation to microcrystals to single crystals.

    As an enhancement of the gradient mode, the hybrid mode combines gradient and sparse matrix screening on a single crystal card. By creating cartridges of various preformed crystallization agents, sparse matrix screening for molecular crystals in nanoplugs can be achieved. As used herein, the term hybrid mode includes hybrid screening, including any suitable screening method involving preformed cartridges. The hybrid mode expands the concept of sparse matrix screening by pre-forming precipitant nanoplugs separated by nanoplug spacers (bubbles) and forming a concentration gradient when they are combined with molecular flow. Similar to the gradient mode, the hybrid mode generates a gradient by coordinating flow changes between the preformed precipitant nanoplug and buffer flow. By performing sparse matrix and gradient screening together on one crystal card, in hybrid mode 20-40 experiments are generated from each pre-formed precipitant nanoplug, sampling a large region of the crystallization phase space Is possible.

    As described above and below, the term “nano plug” refers to a nanoliter volume sized droplet, such as a 10-20 nL droplet, that fills the microfluidic channel of the system. Each nanoplug has a separate microcrystallization experiment. As described above and below, the term “mixer circuit” is meant to include a circuit having three water channels and one carrier fluid channel that come together upstream of the inspection circuit of the crystal card. Further configurations are possible, such as a mixer with 4 or 5 water channels and one carrier fluid channel. The water channels come together and intersect the carrier fluid channel accordingly at an angle of 90 degrees. As used herein, the term “macro-micro interface” is meant to include a connection between a syringe and a crystal card. In some embodiments, the syringe is connected to a mixer circuit or test circuit through tubing such as Teflon (PTFE) tubing. In other embodiments, the tubing is connected to a mixer circuit or a test circuit using connectors configured to fluidly connect circuit inlets and outlets to the tubing. As described above and below, the term “test circuit” refers to a capillary or channel in which fluids together form an aqueous nanoplug that flows through the capillary. The inspection circuit can also be used to inspect the nanoplug for crystal formation. Furthermore, the test circuit can also be used to store crystals formed by the subject method and is therefore also referred to herein as a storage capillary. As used herein, the term “main channel” refers to the region of the test circuit that is located downstream of the mixer and where the aqueous solution and carrier fluid mix to form an aqueous nanoplug. As described above and below, the term “molecule” includes small molecules such as organic compounds and / or chemicals, as well as macromolecules. The term “biological molecule” refers to a molecule that is derived from, corresponds to, or models a biological origin. This term refers to molecules synthesized or produced in vitro, such as by cell-free synthesis, and / or in vivo, such as recombinant proteins; mutant proteins, and artificial proteins, natural and artificial nucleic acid molecules, and others that do not occur in nature As well as other biological molecules. As used herein, the term “macromolecule” includes biopolymers such as nucleic acids, proteins, carbohydrates and lipids. For clarity, the terms “protein” and “protein solution” as used herein encompass other types of molecules in addition to proteins.

    A nanovolume microcapillary crystallization system 100 useful for molecular crystallization is shown in FIG. A prepared sample 102 comprising a molecule for which a crystal structure is desired, such as a prepared protein sample, is provided in the aqueous solution 104. Additional aqueous solutions such as buffer solutions and precipitant solutions are also provided. The buffer solution can comprise a buffer used to prepare the biological sample 102. A carrier fluid is also provided. The carrier fluid is immiscible with the aqueous solution. Suitable examples of carrier fluids include fluorinated oils; for example, FC-40 (3M Corp., St. Paul, Minnesota). The aqueous solution and carrier fluid 104 are supplied to one or more syringes 106 connected to one or more pumps 108. The pump 108 is controlled by software executed on the nanoplug forming computer 110. Software running on the nanoplug forming computer 110 regulates the flow of the aqueous solution and carrier fluid 104 in the crystal card 112. The flow of the aqueous solution and carrier fluid in the crystal card 112 is observed using a magnifying device such as the microscope 114.

    A pumping system 200 that helps regulate the flow of various fluids through the crystal card is shown in FIG. Pump 1 202 controls syringe 1 204 and syringe 2 208. Syringe 1 204 is loaded with an aqueous solution such as buffer 206. Syringe 2 is filled with an aqueous solution such as precipitant reagent 210. Pump 2 212 controls syringe 3214 and syringe 4 218. Syringe 3 214 is filled with an immiscible fluid, such as carrier fluid 216. Syringe 4 218 is filled with an aqueous solution containing molecules such as protein 220 of interest. Suitable pumps include a Harvard Twin 33 syringe pump (Harvar Apparatus, Holliston, Mass.). In some embodiments, the syringe pump has been modified by the manufacturer to provide better accuracy. Suitable syringes include Hamilton syringes such as the 1800 series Hamilton Gas Tight syringe. Suitable syringe volumes range from 10 μl to 100 μl. The pumping system 200 is controlled by software executed on the nanoplug forming computer 110.

    Appropriate software is provided to control the pumping system 108. 3A-3E show exemplary user interfaces of the system software that display various modes of controlling the pumping system 200. FIG. FIG. 3A shows an exemplary user interface 300 in software configuration mode. FIG. 3B shows an exemplary user interface 302 for the main mode of software. FIG. 3C shows an exemplary user interface 304 for certain modes of software. FIG. 3D shows an exemplary user interface 306 in software gradient mode. FIG. 3E shows an exemplary user interface 308 in the hybrid mode of the system software.

    Referring now to the subject crystal card disclosed herein, a representative example of one embodiment of a crystal card is shown in FIGS. The crystal card 400 is configured to be approximately the same size as a standard microscope slide and is approximately 76.20 mm long and approximately 25.40 mm wide (or approximately 3 inches long x approximately 1 inch wide). The crystal card 400 has a thickness of about 1.0 to 1.5 mm. The crystal card is produced from transparent polycarbonate by injection molding (Siloam Bioscience).

    Referring now to the embodiment shown in FIGS. 4A and 4B, the crystal card 400 has a top surface 402 and a bottom surface 414 parallel to the top surface 402. Crystal card 400 includes a substrate configured to accommodate mixer circuit 404 and storage and inspection circuit 406. The mixer circuit 404 is composed of four microfluidic channels 421, 422, 424, and 426. See Figure 4C. Channels 421, 422, and 424 come together and intersect channel 426 at an angle of 90 degrees. Each channel includes an inlet 410. See Figure 4E. The test circuit 406 comprises a long microfluidic capillary channel located just downstream of the mixer 404 and ending at the outlet 412. The length of the microfluidic capillary 406 is about 67 cm. The microfluidic capillary channel 406 is also referred to as an inspection circuit, and crystals generated with this card can be subjected to in situ X-ray diffraction analysis or stored in the channel 406 until harvested for freezing. . Microfluidic channels 421, 422, 424, 426, and capillary channel 406 are substantially square in cross section and have an inner diameter of approximately 200 micrometers (μm) × 200 μm. However, other channel configurations are possible.

    Referring next to FIG. 4D, the crystal card 400 further comprises a layer 420 configured to be thermally bonded to the substrate and peeled from the substrate. The peelable layer 420 is thermally bonded to the substrate surface 414. In other embodiments, the peelable layer 420 may be chemically bonded to the substrate. The peelable layer 420 has a thickness of about 0.10 to 0.14 mm. The peelable layer 420 is appropriately configured so that removal of the peelable layer 420 exposes the interior space of the test circuit channel 406. The crystal card 400 further includes a macro / micro junction that connects a syringe to the crystal card. In one embodiment, the macro / micro junction is connected at one end to the inlet 410 and outlet 412 and at the other end to a slip-fit connector 432 made of flexible silicon tubing (eg, PEEK ™). Including a cross-section of rigid plastic tubing 430)). Slip-fit connector 432 is configured to receive Teflon (PTFE) tubing (not shown). The other end of the pipe is connected to the syringe of the present system. The Teflon (registered trademark) pipe has an inner diameter of 360 μm and an outer diameter of 760 μm (ID / OD 360/760), while the connector 432 has an inner diameter of 760 μm. As a result, the Teflon (registered trademark) pipe has a connector 432. A seal is formed that is free of gas and fluid leaks when inserted into the.

    In operation, channel 421 is connected to tubing filled with an aqueous solution, such as a buffer used for the protein solution of interest. The channel 422 is connected to a pipe filled with a precipitant solution. As used herein, the term precipitant is understood to be synonymous with the term crystallant. The channel 424 is connected to a pipe filled with a solution containing the target molecule of interest. In one embodiment, the biological target molecule is a protein. Channel 426 is connected to tubing filled with carrier fluid. Suitable examples of carrier fluids include fluorinated oils or fluorocarbons such as FC-40, although others are possible. The carrier fluid is immiscible with the aqueous fluid and selectively wets the walls of the test circuit microchannel. As a result, the mixed aqueous solution portion is divided into nanoplugs across the channel width. In one embodiment, the aqueous nanoplug has a volume of about 10-20 nL.

    Referring now to FIGS. 5A-5E, a representative example of another embodiment for the subject crystal card is shown. Similar elements between different drawings have similar reference numbers, with the most significant digit incremented by 1 corresponding to the drawing number. For the sake of brevity, similar elements between the different figures are not further described. In the embodiment shown in FIGS. 5A-5E, the inlet 510 is located in a shallow cylindrical depression 508 located on the top surface 502 of the crystal card 500. Cylindrical indentation 508 is configured to attach a connector (not shown) that connects the piping to inlet 510 and outlet 512. The dimensions of the crystal card 500 are shown in FIG. 5E. The crystal card 500 has a length of 76.2 mm and a width of 25.4 mm. The inlets 510 are arranged with an interval of 4.5 mm. The parallel channels of the inspection circuit 506 are arranged with an interval of 2.0 mm. However, other suitable configurations are possible as will be appreciated by those skilled in the art.

    With reference now to FIGS. 6A-6E, a representative example of a third embodiment of a crystal card is shown. For the sake of brevity, similar elements described in the previous figures are not described here. In the embodiment shown in FIGS. 6A-6E, the inlet 610 and the outlet 612 are connected to the surface 602 and are positioned below a cylindrical protrusion 608 extending out therefrom. The protrusion 608 is configured to attach a connector (not shown) that connects piping to the inlet 610 and the outlet 612. Deviating from the subject matter, the crystal card illustrated in the embodiment shown in FIGS. 4-6 is made from transparent polycarbonate plastic by injection molding (Siloam Biosciences).

    Returning now to FIGS. 7-9, an exemplary embodiment of a second type of crystal card will be described. Figures 7A-7E show representative examples of another embodiment for the subject crystal card. For the sake of brevity, similar elements described in the previous figures are not described here. In the embodiment shown in FIGS. 7A-7E, the top surface 702 of the crystal card 700 further comprises two rows of ports 708. The port is configured to receive a plastic connector (not shown) suitable for connecting tubing to inlet 710 and outlet 712 located below port 708. Surface 702 includes 28 ports 708. However, depending on the design of the crystal card 700, various numbers of ports are possible. Port 708 extends approximately 2.5 mm above the surface 702 of crystal card 700. A hole is drilled in the center of the bottom of the port 708 to align with the inlet 710 and outlet 712 and to be fluidly connected thereto. The center of the port is about 4.5 mm apart. The hole drilled in the bottom of the port 708 has a diameter of about 0.2 mm (200 μm). Of course, not all ports are connected to the circuit channel, and only the desired ports to which piping should be connected to the inlet 710 and outlet 712 need to be drilled. In other embodiments, before the peelable layer 720 is bonded to the bottom surface 714, a hole is drilled in the layer using a laser. The laser drill hole is configured to fluidly connect with the inlet 710 and the outlet 712. Pipes are connected to the laser drill holes using specially designed crystal card holders (not shown).

    Still referring to FIGS. 7A-7E, the crystal card further comprises two asymmetric microfluidic channel circuits 706A and 706B. In 706A, the inspection circuit is approximately 270 mm long. In 706B, the inspection circuit is about 306 mm long. In both circuits 706A and 706B, the outlet 712 is located on the opposite side of the circuit from the inlet 710 and the mixer circuits 704A, 704B. The embodiment shown in FIGS. 7A to 7E has a two-part configuration including mixer circuits 704A and 704B. As shown in more detail in FIG. 10, the mixer circuit 704A includes a short neck region approximately 0.20 mm in length between the water channel and the carrier fluid channel. As shown in more detail in FIG. 11, the mixer circuit 704B has no neck region between the water channel and the carrier fluid channel. It has been found that the mixer circuit 704A is suitable for forming the aqueous nanoplug in the crystal card.

    With reference to FIGS. 8A-8E, a representative example of another embodiment for the subject crystal card is shown. For the sake of brevity, elements similar to those described above will not be further described here. The crystal card 800 includes a symmetric microfluidic channel circuit 806 divided into two. In this embodiment, the outlet 812 is located on the same side of the circuit 806 as the mixer 804 and inlet 810.

    Referring now to FIGS. 9A-9E, a representative example of another embodiment for the subject crystal card is shown. For the sake of brevity, elements similar to those described above will not be further described here. The crystal card 900 comprises a single microfluidic circuit with one mixer circuit 904 and a long test circuit 906. The inspection circuit 906 has a length of about 665 mm. FIG. 9C shows an exploded view of the crystal card 900. Portion 930 with port 908 is joined to portion 940 with a microfluidic circuit channel. The peelable layer 920 is thermally bonded to the bottom surface 914 of the portion 940. However, in other embodiments, the peelable layer 920 may be chemically bonded to the substrate surface 914. The peelable layer 920 is accordingly configured so that removal of the peelable layer 920 exposes the interior space of the test circuit channel 906. Deviating from the subject matter, the crystal cards described in the embodiments shown in FIGS. 7-9 are made from a transparent cyclic olefin copolymer (COC) or equivalent plastic (ThinXS Microtechnology AG, Germany).

    Returning now to FIGS. 10-13, a representative example of a mixer circuit will now be described. FIG. 10 shows a representative example of one embodiment of a mixer circuit corresponding to the mixer 704A shown in FIG. The mixer circuit 1000 includes three water channels 1021, 1022, and 1024. The water channel is separated from the carrier fluid channel 1026 by a neck region 1007. The channel is oriented so that the three channels 1021, 1022, 1024 containing the aqueous solution come together and intersect the channel 1026 containing the carrier fluid at an angle of 90 degrees. The mixer 1000 further includes a part of the inspection circuit 1006. Still referring to FIG. 10, the dimensions of the mixer 1000 will now be described. The neck region 1007 is about 0.2 mm in length. The channel 1021 has a diameter of about 0.2 mm. Channels 1022, 1024 are about 0.141 mm in diameter. Channels 1006 and 1026 are about 0.2 mm in diameter. However, other suitable dimensions for the mixer circuit are possible.

    FIG. 11 shows a representative example of another embodiment of a mixer circuit corresponding to the mixer circuit 704B shown in FIG. The mixer circuit 1100 includes three water channels 1121, 1122, and 1124. The aqueous solution channel is connected directly to the carrier fluid channel 1106 without a neck region. The mixer circuit flows into the inspection circuit 1126. The channel is oriented so that the three channels containing the aqueous solution come together and intersect the channel containing the carrier fluid at an angle of 90 degrees. The diameter of the channel 1121 is about 0.2 mm. The diameter of the channels 1122, 1124 is about 0.141 mm. The diameter of the junction area between the water channel and the carrier fluid channel 1126 is about 0.285 mm. However, other suitable dimensions for the mixer are possible.

    Referring now to FIG. 12, another view of the mixer circuit 704A described in FIG. 7 is shown. The mixer circuit 1200 includes three water channels 1221, 1222 and 1224 connected to the carrier fluid channel 1226 by a short neck region. The channels are oriented so that the three channels containing the aqueous solution come together and intersect the channel containing the carrier fluid at an angle of 90 degrees. Each channel has an inlet 1210. Downstream of the mixer circuit 1204, the solution flows into a portion of the inspection circuit 1206. Still referring to FIG. 12, the dimensions of the mixer circuit 1200 will now be described. Inlet 1210 is located about 4.4 mm from channels 1206, 1226. The water channels 1221, 1222, 1224 bend at a right angle about 2.9 mm from the inlet. The right angle bend has an inner diameter R0.300 and an outer diameter R0.500. Channel portions 1221, 1222, 1224, which are arranged in a plane parallel to channel 1206, are approximately 1.300 mm from channel 1206. The water channels 1221, 1222, and 1224 bend 45 degrees before connecting to each other upstream of the neck region. The inner diameter of the channel 1206 is about 0.200 mm (200 μm). The parallel portions of channel 1206 are about 1.2 mm apart. However, other suitable dimensions are possible.

    FIG. 13 shows a representative example of another embodiment of a mixer corresponding to the mixer circuits 804 and 904 shown in FIGS. The mixer circuit 1300 includes water channels 1321, 1322, and 1324. The water channel is separated from the carrier fluid channel 1306 and the test circuit 1326 by a short neck region. The neck region has a diameter of about 0.200 mm. However, other suitable dimensions are possible. The channels are oriented so that the three channels containing the aqueous solution come together and intersect the channel containing the carrier fluid at an angle of 90 degrees. Downstream of the mixer circuit, the solution flows into the inspection circuit 1326.

    FIG. 14 shows a typical example of a cross section of a crystal card similar to the embodiment shown in FIG. Crystal card 1400 consists of three layers 1420, 1430 and 1440. Layer 1430 comprises ports as shown in FIGS. Layer 1430 has a thickness of about 0.4 mm. Layer 1440 comprises a microfluidic channel circuit and is about 1.5 mm thick at the ends. Layer 1420 comprises a peelable layer affixed to the bottom of crystal card 1400 and has a thickness of about 0.14 mm.

    15A-15V illustrate a method 5000 for crystallizing molecules using a nanovolume microcapillary crystallization system. From the start block, the method 5000 proceeds to a set of method steps 5002 defined between a continuation terminal (“Terminal A”) and an exit terminal (“Terminal B”). A set of method steps 5002 describes the preparation of the crystal card and the connection of the crystal card to the pump.

    From Terminal A (FIG. 15B), the method 5000 proceeds to a set of method steps 5008 in which the crystal card is manufactured from a suitable material such as polydimethylsiloxane (PDMS) or plastic by injection molding. The method then returns to the launch point. The method 5000 then proceeds to a set of method steps 5010 defined by a continuation terminal ("Terminal A2"). A set of method steps 5010 treats the microcapillary surface of the crystal card to reduce surface energy.

    From Terminal A2 (FIG. 15C), the method 5000 proceeds to decision block 5014 where a test is performed to determine whether the crystal card has been manufactured from plastic. If the answer to the test is no, the method proceeds to another continuation terminal ("Terminal A4"). If the answer to the test at decision block 5014 is YES, the method proceeds to another decision block 5016 where another test is performed to determine whether the plastic is polycarbonate. If the answer to the test at decision block 5016 is NO, the method 5000 proceeds to another continuation terminal (“Terminal A5”). If the answer to the test at decision block 5016 is YES, the method 5000 proceeds to another continuation terminal (“Terminal A6”).

    From Terminal A4 (FIG. 15D), the method 5000 proceeds to block 5018 where the method processes a crystal card as manufactured from PDMS. The method proceeds to block 5020 where the microcapillary surface is treated with a perfluorinated silane solution for 2 hours at room temperature. The method then proceeds to block 5022 where the perfluorinated silane solution is removed by vacuum. At block 5024, the microcapillary surface of the crystal card is dried for 1 hour using a gas such as air under a pressure of 5-10 psi. The method then returns to the point where the terminal A2 step was activated and proceeds to another continuation terminal ("Terminal A3"). See block 5012.

    From Terminal A5 (FIG. 15E), the method 5000 proceeds to block 5026 where the method treats the crystal card as being made of a plastic comprising cyclic olefin copolymer (COC) or equivalent plastic. In block 5028, the microcapillary surface is treated with a reagent for 2 hours at room temperature to reduce the surface energy (hydrophobicity) of the plastic. Suitable reagents for reducing the surface energy include fluorinated copolymer solutions, but other reagents are possible. Suitable fluorinated copolymer solutions include 2% fluorinated copolymer solutions in fluoro solvents, such as Cytonix PFC 502AFA (Cytonix, Beltsville, MD). Cytonix PFC 502AFA is manufactured for adhesion to polycarbonate and reduces the surface energy to 6-10 dynes / cm. Cytonix PFC 502 AFA solution is filled from the outlet to apply the fluorinated copolymer solution to the crystal card. At block 5030, the fluorinated copolymer solution is removed by vacuum. In block 5032, the microcapillary surface is dried for 1 hour using a gas such as air under a pressure of 5-10 psi. The method 5000 then proceeds to block 5034 where the crystal card is heated to 60 ° C. for 1 hour. The method then returns to the starting point of the terminal A2 step. See block 5012 at terminal A3.

    From Terminal A6 (FIG. 15F), the method 5000 proceeds to block 5036 where the crystal card is pre-cooled on ice. At block 5038, the microcapillary surface is treated on ice for 2 hours with a fluorinated copolymer solution such as Cytonix PFC 502AFA. The inlet of the polycarbonate crystal card may be fragile when incubated with a 502 AFA solution at higher temperatures. The method then proceeds to terminal A5 and jumps to block 5030 to perform the steps at blocks 5030, 5032, and 5034. The method then returns to the point where the terminal A2 step was activated. See terminal A3 at block 5012. One set of method steps at block 5012 couples the crystal card to the pump.

    From Terminal A3 (FIG. 15G), the method 5000 proceeds to block 5040 where the syringe 1 is filled with a buffer or aqueous solution. At block 5042, syringe 2 is filled with a precipitant solution. At block 5044, the syringe 3 is filled with a carrier fluid. A representative example of a suitable carrier fluid includes a fluorinated carbon solution. A suitable example of a fluorocarbon fluid includes FC-40. FC-40 has a high surface tension against detergents used to solubilize membrane proteins. The surface tension allows nanoplug formation and crystallization. In an exemplary embodiment, the carrier fluid is a fluorinated oil that is immiscible with an aqueous fluid. As aqueous nanoplugs are formed, the carrier fluid surrounds and separates them and is advanced through the crystal card during the method. In block 5046, the syringe 4 is filled with a protein solution containing the protein of interest in an appropriate buffer. At block 5048, appropriate tubing, such as Teflon tubing, is attached to the needle of each syringe. In block 5050, syringes 1 and 2 are attached to pump 1 and syringes 3 and 4 are attached to pump 2. In block 5052, the piping is connected to the crystal card via a macro / micro junction. Suitable connections for the macro / micro junction are as described above. The method then proceeds to exit terminal B.

    From Terminal B, method 5000 proceeds to a set of method steps 5004 defined between a continuation terminal (“Terminal C”) and an exit terminal (“Terminal D”). A set of method steps 5004 receives instructions to regulate the flow of fluid in the crystal card to obtain crystals. From Terminal C (FIG. 15H), the method 5000 proceeds to a set of method steps 5054 defined in the continuation terminal (“Terminal C1”). One set of method steps 5054 constitutes a pump.

    From terminal C1 (FIG. 15I), the method 5000 proceeds to block 5060 where the method receives instructions regarding the type of syringe pump model that the system controls. Suitable pumps include the Harvard Apparatus Twin Syringe Pump Model 33 (Harvar Apparatus, Holliston, Mass.) Modified by the manufacturer to provide better accuracy. As shown in FIG. 2, each syringe pump controls two syringes. At block 5062, the method receives instructions regarding the serial communication port of the computer used to control the pump system. The communication port is configured such that each syringe pump receives instructions simultaneously. As a result, time delay is prevented and the solution can flow through the crystal card simultaneously. The method proceeds to block 5064 where the method receives instructions regarding the volume of each syringe connected to the pump. In block 5066, the method determines the diameter of each syringe connected to the pump. The method then proceeds to return to the point where the terminal C1 step was activated.

    From block 5054, the method 5000 proceeds to a set of method steps 5056 defined in the continuation terminal ("Terminal C2"). The set of method steps primes the fluid into the mixer circuit of the crystal card. From Terminal C2 (FIG. 15J), the method 5000 proceeds to block 5068 where the method receives instructions on which syringe to use to dispense fluid into the crystal card mixer. At block 5070, the method receives instructions regarding the flow rate from each syringe. At block 5072, the method receives instructions regarding the volume of fluid dispensed by the syringe. At block 5074, the method dispenses or aspirates fluid from the fluid channel upstream of the mixer circuit. The method then continues to another continuation terminal ("Terminal C4").

    From Terminal C4 (FIG. 15K), the method 5000 proceeds to decision block 5076 where a test is performed to determine whether the syringe is dispensing an aqueous fluid. If the answer to the test at decision block 5076 is NO, the method proceeds to another continuation terminal (“Terminal C5”). If the answer to the test at decision block 5076 is YES, the method proceeds to block 5078. The method receives instructions to stop the fluid in the mixer circuit before the aqueous fluid enters the test circuit. The method then continues to terminal C2 and repeats the process steps shown above for the next syringe. From terminal C5 (FIG. 15K), the method 5000 proceeds to block 5080 where the method receives instructions to stop the carrier fluid downstream of the mixer circuit and slightly within the test circuit. The method then proceeds to return to the point where the terminal C2 step was activated.

    Deviating from the subject matter, an exemplary process for priming an aqueous solution and carrier fluid into a crystal card mixer is described in detail. Initially, an empty crystal card mixer circuit is placed on the microscope stage for observation during priming. The method receives instructions from a syringe 1 to dispense a solution, for example a buffer, into a mixer. Buffer is dispensed into the fluid channel connected to the syringe 1 until the user observes that the solution has reached the mixer region immediately upstream of the confluence of the fluid channels. The method then receives instructions to stop dispensing the solution. The solution can also be removed from the channel by providing the method with instructions for aspirating the reagent. Suitably the aqueous solution refrains from entering the inspection circuit of the crystal card. The method is repeated for each of the three fluid channels connected to syringes dispensing aqueous solutions; for example, syringe 4 (protein solution) and syringe 2 (precipitant solution). The carrier fluid is then dispensed into a fourth fluid channel connected to the syringe 3. The carrier fluid is dispensed into the fourth fluid channel until it passes through the confluence of the mixer and slightly enters the inspection circuit of the crystal card (fifth channel). The method then receives instructions to stop dispensing carrier fluid.

    Returning to block 5056, the method 5000 proceeds to a set of method steps 5058 defined in the continuation terminal ("Terminal C3"). One set of method steps receives instructions for generating aqueous nanoplugs in the crystal card inspection circuit. From terminal C3 (FIG. 15L), the method 5000 receives instructions at block 5082 regarding which nanoplug formation protocol to perform. The method then proceeds to decision block 5084 where a test is performed to determine if the received instruction was to execute a constant mode. If the answer to the test at block 5084 is NO, the method proceeds to another continuation terminal ("Terminal C6). If the answer to the test at decision block 5084 is YES, the method Proceed to block 5086 where the method receives instructions regarding the flow rate to the syringe, which proceeds to block 5088 where the method then receives instructions regarding the total volume of fluid passing through the mixer circuit. Each nanoplug is reasonably equal in size, and an aqueous nanoplug with similar protein and precipitant concentration is generated in the crystal card inspection circuit, and the method then proceeds back to the activation point. The method proceeds to exit terminal D.

    From terminal C6 (FIG. 15M), the method 5000 proceeds to decision block 5092 where a test is performed to determine if the method has received instructions to perform gradient mode. If the answer to the test at block 5092 is NO, the method proceeds to another continuation terminal (“Terminal C 7”). If the answer to the test at decision block 5092 is YES, the method proceeds to block 5094 where the method receives instructions on the maximum flow rate for the variable flow syringe. In one embodiment, the variable flow syringe includes a buffer and a precipitant. In another embodiment, syringes 1 and 2 are variable flow syringes. However, the method can designate any syringe as a variable flow syringe. In one embodiment, the combined flow rate of the variable flow syringe is equal to the maximum flow rate. For example, in one embodiment, the method provides instructions where the flow rate of syringe 1 is equal to 2 μl / min, while the flow rate of syringe 2 is equal to 0 (zero) μl / min. In this embodiment, the maximum flow rate is equal to 2 μl / min (2 + 0 μl / min). The method then proceeds to block 5096 where the method receives instructions on a constant flow rate for the syringe that controls the carrier fluid. In one embodiment, the syringe 3 controls the carrier fluid. In one embodiment, the flow rate of the carrier fluid is equal to the total flow rate of the aqueous solution (buffering agent, precipitant and protein solution). In another embodiment, the flow rate for the carrier fluid can be selected to be less than or greater than the total flow rate of the aqueous fluid. The smaller the carrier fluid velocity, the smaller the portion with the carrier fluid between the nanoplugs, producing a larger aqueous nanoplug. The greater the carrier fluid velocity, the greater the carrier fluid portion between the nanoplugs, producing a smaller aqueous nanoplug. The method then proceeds to block 5098 where the method receives instructions on a constant flow rate for the syringe controlling the protein solution. In one embodiment, the syringe 4 controls the carrier fluid. In one embodiment, the protein flow rate is equal to the sum of the flow rates of the other aqueous solutions (buffer and precipitant). Changing the flow rate of the protein solution changes the ratio of protein to crystallization conditions in each nanoplug. The method then proceeds to block 6000 where the method receives instructions regarding the total volume of water dispensed during one iteration or cycle of the method. The method then proceeds to another continuation terminal ("Terminal C8").

    From Terminal C8 (FIG. 15N), the method 5000 proceeds to block 6002 where the method receives instructions regarding the volume of each aqueous nanoplug dispensed into the test circuit. At block 6004, the method receives instructions regarding the total number of iterations or cycles to perform (ie, the number of times the gradient screening step is repeated). In one embodiment, if the method receives instructions to perform zero iterations, the pump stops when the entire volume of water selected at block 6000 has been dispensed. In another embodiment, if the method receives instructions to perform one or more iterations, the pump stops when the above-described process steps are repeated a desired number of times. In block 6006, the method changes the flow rates of the buffer and precipitant solution relative to each other such that the sum of the flow rates of buffer and precipitant solution is equal to the maximum flow rate selected in block 5094. For example, in one embodiment, the method provides instructions where the flow rate of Syringe 1 is equal to 2 μl / min and the flow rate of Syringe 2 is equal to 0 μl / min so that the maximum flow rate is equal to 2 μl / min at block 5094. Provide instructions. When the method starts, the flow rate from syringe 1 starts at 2 μl / min and ramps down to 0 μl / min, while the flow rate from syringe 2 ramps up from 0 μl / min to 2 μl / min simultaneously. At block 6008, the method generates a series of aqueous nanoplugs in the test circuit that have the same size for each drop but different protein and precipitant concentrations in each drop. At block 6010, the method ends after performing the desired number of iterations or cycles. The method then returns to block 5058 where the method proceeds to exit terminal D.

    From terminal C7 (FIG. 15O), the method 5000 proceeds to decision block 6012 where a test is performed to determine if the method has received instructions for performing the hybrid mode. If the answer to the test at block 6012 is NO, the method proceeds to another continuation terminal (“Terminal C9”). If the answer to the test at block 6012 is YES, the method proceeds to another decision block 6014 where a test is performed to determine whether the precipitant cartridge has been prepared. If the answer to the test at decision block 6014 is NO, the method proceeds to another continuation terminal (“Terminal C 10”). If the answer to the test at block 6014 is YES, the method proceeds to another continuation terminal (“Terminal C11”).

    From terminal C9 (FIG. 15P), the method 5000 proceeds to decision block 6016 where a test is performed to determine if the method has received instructions for performing the pulsation mode. If the answer to the test at decision block 6016 is NO, the method returns to terminal C3 where the steps shown above are repeated. If the answer to the test at decision block 6016 is YES, the method proceeds to block 6018 where the method receives instructions regarding performing a beat mode. The method then returns to block 5058. From block 5058, the method exits to terminal D.

    From terminal C10 (FIG. 15Q), the method 5000 proceeds to block 6020 where the syringe is connected to tubing such as Teflon tubing containing carrier fluid. The method then proceeds to block 6022 where the syringe is connected to a syringe pump. At block 6024, the method receives an instruction that enters a defined volume, eg, about 40 nL, and draws about 40 nL of air bubbles into the tubing. At block 6026, the method draws a defined volume, eg, about 120 nL of precipitant solution into the tubing. At block 6028, the method repeats the above two steps until the appropriate number of precipitants is loaded into the piping. For example, a suitable number of precipitating agents can range from 1 to 24 or more. In block 6030, the method draws about 1 μl of carrier fluid into the open end of the tubing. At block 6032, the tubing is connected to the crystal card precipitation agent inlet. The method then proceeds to continuation terminal C11.

    At terminal C11 (FIG. 15R), the method 5000 proceeds to block 6034 where the method receives instructions regarding the initial flow rate of the buffer solution (syringe 1). At block 6036, the method receives instructions regarding the flow rate change (step size) of the buffer solution. The step size is the change in flow applied to each ramp up or ramp down of the method. At block 6038, the method receives instructions regarding the initial flow rate of the precipitant cartridge (syringe 2). In block 6040, the method calculates the flow rate change (step size) of the precipitant solution. In one embodiment, the step size for the buffer is equal to the step size for the precipitant. In block 6042, the method sums the buffer and precipitant flow rates to determine the total flow rate. At block 6044, the method receives instructions for an initial flow rate for the carrier fluid (syringe 3). At block 6046, the method receives instructions regarding carrier fluid flow rate change (step size). The method then proceeds to another continuation terminal ("Terminal C12").

  From terminal C12 (FIG. 15S), the method 5000 proceeds to block 6048 where the method receives instructions regarding a constant flow rate of the protein solution (syringe 4). The method then proceeds to block 6050 where the method receives instructions on the number of ramp-up steps (flow rate of change) for each precipitant. At block 6052, the method sets the number of ramp down steps equal to the number of ramp up steps for each iteration or cycle of the method. At block 6054, the method receives instructions regarding the number of iterations or cycles to perform. In one embodiment, one iteration or cycle corresponds to a single precipitant loaded into the precipitant cartridge. At block 6056, the method receives instructions regarding the duration of each ramp step. For example, in one embodiment, the duration of each ramp step is 1.5 seconds. In block 6058, the method changes the buffer and precipitation flow rates relative to one another so that the sum is equal to the initial flow rate. The method then proceeds to another continuation terminal ("Terminal C13").

  From terminal C13 (FIG. 15T), the method 5000 proceeds to block 6060 where the method changes the flow rate of the carrier fluid. The method then proceeds to block 6062 where the method generates a series of nanoplugs in a test circuit where each droplet has an equal amount of protein and various amounts of precipitant and buffer. In one embodiment, the method provides varying amounts of precipitant with a fixed amount of protein for each cycle. Table 1 shows one embodiment of the above-described method for the hybrid mode. The method then proceeds to terminal D.

ブロック５００４におけるターミナルＤから、方法５０００は、継続ターミナル（「ターミナルＥ」）および出口ターミナル（「ターミナルＦ」）間で定義された方法ステップの一組５００６へ進む。方法ステップの一組５００６は、結晶カードから得られた結晶に関する回折実験を行う。ターミナルＥ（図１５Ｕ）から、方法５０００は、回折の前に結晶カードの検査回路から結晶を採取したかどうか判断するためにテストを行う決定ブロック６０６４へ進む。もしブロック６０６４におけるテストへの答えがＮＯであれば、本方法は、別の継続ターミナル（「ターミナルＥ１」）へ進む。もしブロック６０６４におけるテストへの答えがＹＥＳであれば、本方法は、剥離可能な層を結晶カードの底面から除去するブロック６０６６へ進む。一実施形態において、剥離可能な層は、マイクロ流体チャンネルを含んだ結晶カードのプラスチック部分に接合されている。該接合は、マイクロ流体回路から流体が漏れ出すのを防ぐには十分強いが、手作業で剥がれるように十分弱く設計される。一実施形態において、該接合は熱接合である。別の実施形態において、該接合は化学結合である。剥離可能な層の除去は、結晶カードのマイクロ流体チャンネル内部を暴露し、水性ナノプラグにアクセスすることを可能にする。別の実施形態では、剥離可能な層が除去された後に、結晶を含んだ水性ナノプラグは結晶カードのマイクロ流体チャンネル中に保持される。ブロック６０６８では、検査回路中に形成された結晶がクライオループを用いて結晶カードから採取される。一実施形態において、クライオループは、ナイロンのクライオループである。ブロック６０７０では、結晶が凍結され、回折データが得られる。本方法は、次に本方法が実行を終了する出口ターミナルＦへ進む。

From Terminal D at block 5004, the method 5000 proceeds to a set of method steps 5006 defined between a continuation terminal ("Terminal E") and an exit terminal ("Terminal F"). A set of method steps 5006 performs diffraction experiments on crystals obtained from crystal cards. From Terminal E (FIG. 15U), the method 5000 proceeds to decision block 6064 where a test is performed to determine if a crystal has been taken from the inspection circuit of the crystal card prior to diffraction. If the answer to the test at block 6064 is NO, the method proceeds to another continuation terminal (“Terminal E1”). If the answer to the test at block 6064 is YES, the method proceeds to block 6066 where the peelable layer is removed from the bottom surface of the crystal card. In one embodiment, the peelable layer is bonded to the plastic portion of the crystal card that contains the microfluidic channel. The joint is designed to be strong enough to prevent fluid from leaking out of the microfluidic circuit, but weak enough to be peeled off manually. In one embodiment, the bond is a thermal bond. In another embodiment, the junction is a chemical bond. Removal of the peelable layer exposes the inside of the microfluidic channel of the crystal card and allows access to the aqueous nanoplug. In another embodiment, the aqueous nanoplug containing crystals is retained in the microfluidic channel of the crystal card after the peelable layer is removed. At block 6068, a crystal formed in the inspection circuit is collected from the crystal card using a cryoloop. In one embodiment, the cryoloop is a nylon cryoloop. At block 6070, the crystal is frozen and diffraction data is obtained. The method then proceeds to exit terminal F where the method ends execution.

  From terminal E1 (FIG. 15V), the method 5000 proceeds to block 6072 where the crystal card containing the crystal is mounted on the goniometer of the X-ray source. In block 6074, the method obtains diffraction data from the crystal in its original position in the inspection circuit. The method then proceeds to block 5006 and further to end terminal F. The method then ends execution.

  In order to produce crystals of methionine-R-sulfoxide reductase, the crystal harvesting step described above can be used in combination with the gradient screening of various embodiments in the present subject matter. Crystals were removed from the crystal card with a cryoloop and then frozen for diffraction experiments. As an example, a 1.7Å data set was collected at the SBC-CAT beamline 19BM at the Advanced Photon Source at Argonne National Laboratory, followed by elucidation and refinement of the structure. Final coordinates and structure factors have been deposited with the Protein Data Bank (accession number 3CXK).

  The crystal cards of various embodiments of the present subject matter are also suitable for in situ diffraction. In situ diffraction allows the crystallographer to assess its quality before the crystals are changed by the cryoprotection process. With strong crystals, complete diffraction data can be obtained. The crystal card has sufficient X-ray transparency to be mounted on a goniometer of an X-ray source for collecting diffraction data at room temperature. For example, a simple test was performed to analyze X-ray absorption by a crystal card. The ion chamber beam current (I / I0) normalized with the APS ring current was measured with and without the insertion of a crystal card for a wavelength of 0.979261 A (12.66099 keV). I / I0 without crystal card was measured as 1.91671E-6, and I / I0 with crystal card was measured as 1.5511E-6. As a result, the X-ray absorbance by the crystal card is 19%. Furthermore, the crystal card can be translated along the X and Y axes, and data from multiple crystals can be collected and integrated into a complete data set. To demonstrate this technology, a crystal card containing lysozyme crystals was mounted on the goniometer head of NE-CAT beamline 24ID-C at Advanced Photon Source, Argonne National Laboratory. Data was collected at room temperature from three crystals in a crystal card. Crystallographic data is presented in Appendix A.

  For structure determination, data sets were collected at Advanced Photon Source: methionine-R-sulfoxide reductase beamline 19BM, 100K; and lysozyme beamline 24-IDC, room temperature. Data was integrated and scaled using HKL2000. Using the mosflm package for the lysozyme structure, the intensity was integrated separately for each of the three data sets. Using Molrep and PDB entries 1IEE and 3CEZ as search models respectively, the structure of lysosomes and methionine-R-sulfoxide reductase was elucidated by molecular replacement. The structure was refined using Refmac5 and model building was performed using Coot.

  While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

  Appendix A

独占的な所有権または特権が請求される本発明の実施形態が、以下のように明示される。

Embodiments of the invention in which an exclusive property or privilege is claimed are set forth as follows.

Claims (40)

Pumping system;
A plurality of software configured to run on a protein crystallization system to control the pumping system; and one or more crystal cards coupled to the pumping system, each comprising a mixer and The protein crystallization system comprising a crystal card configured to receive a microfluidic capillary coupled to the mixer to facilitate storage and protein crystallization testing.   The pumping system includes a syringe pumping system or a pressure pumping system, and the syringe pumping system is carried through the second, third, and fourth microfluidic channels into the one or more crystal cards. The protein crystallization system of claim 1 comprising a four channel syringe pump for regulating the aqueous solution and the fluorous solution carried in the fifth microfluidic channel.   The plurality of software controls each of the four-channel syringe pumps, and each of the second, third, fourth, and fifth microfluidic channels to generate a particle gradient of the aqueous solution flow. The protein crystallization system of claim 1 that facilitates control of the channel.   The one or more crystal cards are a group consisting of two or more combinations of X-ray transparency, optical transparency, modality, chemical resistance, suitable surface energy, and properties listed above. 2. The protein crystallization system of claim 1 formed from a material having properties selected from:   The mixer includes a confluence of second, third, fourth, and fifth microfluidic channels in which an aqueous plug is formed, wherein the second, third, fourth, and fifth microfluidic channels are The protein crystallization system of claim 1, formed from a microfluidic channel that is approximately 200 × 200 micrometers.   The junction point defines a hydrophobic surface that assists in forming an aqueous plug in a range of approximately 10 nanoliters to 20 nanoliters, and the microfluidic capillary transports the aqueous plug from the junction point. 5. The protein crystallization system according to 5.   The one or more crystal cards are formed from a plastic configured for fine gradient screening, or formed from PDMS / Teflon® configured for hybrid screening and membrane proteins. Item 2. The protein crystallization system of Item 2.   The protein crystallization system further comprises a syringe having a needle coupled to the pumping system and serves as a macro-micro junction between the needle of the syringe and the one or more crystal cards. And further comprising a tubing having a distal end and a proximal end configured such that each tubing has an inner diameter of about 360 micrometers and an outer diameter of about 760 micrometers, wherein the distal end of the tubing is a needle The protein crystallization system of claim 1, configured to slide up and the proximal end of the tubing configured to couple to the one or more crystal cards. Regulating water flow by independently controlling each water flow using a pumping system that works with multiple software; and in protein crystallization experiments, to show the transition from precipitation to microcrystals to single crystals A method for gradient screening comprising mapping a crystallization phase space.   The method of claim 9, wherein the act of adjusting includes creating a concentration gradient across a series of aqueous plugs by varying the flow rate of each water stream.   11. The method of claim 10, wherein the act of regulating comprises regulating a water flow selected from proteins, crystallization agents, fluorocarbons, precipitants, ligands, protein partners, DNA complexes, buffers and cryoprotectants. The method described.   12. The method of claim 11, wherein the act of adjusting includes increasing the flow rate of the buffer water stream so that the total flow rate remains constant when the flow rate of the precipitant water stream decreases. Preforming a precipitant plug;
Preforming a plug spacer, each separating the two precipitant plugs from each other;
Forming a gradient by converging precipitant plugs, plug spacers, and protein streams; and a protein crystallization phase to show a transition from precipitation to microcrystals to single crystals in protein crystallization experiments A method for hybrid screening comprising mapping a space.   The method of claim 13, wherein preforming the plug spacer comprises preforming with air bubbles.   14. The method of claim 13, wherein forming a gradient comprises coordinating flow changes between a flow formed from the precipitant plug, plug spacer, and a buffer flow.   14. The method of claim 13, wherein each precipitant plug is about 100 nanoliters. Receiving a crystal card with a capillary;
Coating the capillary with a reagent to reduce surface energy; and removing the reagent.   18. The method of claim 17, further comprising incubating the crystal card on ice for a predetermined number of hours.   18. The method of claim 17, wherein the capillary includes an inner surface and the act of coating the capillary comprises coating the inner surface of the capillary to reduce surface energy to about 6-10 dynes per centimeter.   The method of claim 17, wherein the fluorinated copolymer solution comprises 2 percent fluorinated copolymer solution in a fluorosolvent.   The method of claim 17, wherein removing the fluorosolvent comprises vacuum processing the crystal card.   18. The method of claim 17, further comprising the act of forcing clean dry air through the crystal card performed at 5 psi for about 1 hour.   The method of claim 17, further comprising the act of baking the crystal card performed at about 60 degrees Celsius for about 1 hour. Peeling off the thin layer bonded to the substrate of the crystal card;
Collecting crystals by a cryoloop from a microfluidic circuit housed on the substrate;
18. The method of claim 17, further comprising freezing the crystal; and performing a diffraction experiment on the crystal to obtain diffraction data. Mounting a crystal card with a microfluidic circuit on the goniometer;
18. The method of claim 17, further comprising irradiating the crystal card with X-rays; and collecting diffraction data.   26. The method of claim 25, further comprising translating the crystal card along x and y axes to collect the diffraction data from a plurality of crystals stored by the microfluidic circuit. A crystal card comprising: a substrate configured to receive a mixer circuit and an inspection circuit; and a layer configured to be bonded to the substrate and peeled from the substrate.   28. The crystal card of claim 27, wherein the layer is thermally bonded to the substrate or chemically bonded to the substrate.   28. The crystal card of claim 27, wherein the substrate and the layer are formed from the group consisting of an amorphous polymer, a cyclic olefin copolymer, a thermoplastic polymer, and a polycarbonate.   28. The crystal card of claim 27, wherein the substrate comprises a thickness of about 1 millimeter and the layer comprises a thickness in the range of about 100 to 150 micrometers.   The mixer circuit includes first, second, third, and fourth addend channels, each addend channel includes a distal end and a proximal end, and the distal end of each addend channel is Defining an opening configured to fluidly receive the solution, wherein the proximal end of each addend channel defines an opening configured to fluidly communicate an aqueous plug or plug spacer And each addend channel has a first portion coupled to the distal end and a second portion of the first, second, and third addend channels coupled to the proximal end. And the first part of each addend channel is spaced in parallel with another addend channel and the second part of the first and third addend channels is Are angled so that their proximal ends intersect, said second and fourth The second portion of the addend channel is parallel until the proximal end of the second addend channel intersects the proximal ends of the first and third addend channels to form a vertex. The third part of the fourth addend channel extends at an angle of 90 degrees from the second part of the fourth addend channel, and its proximal end is at another angle of 90 degrees. 28. The crystal card of claim 27, intersecting the apex.   32. The crystal card of claim 31, wherein the first portion of each addend channel is spaced about 4.50 millimeters from the first portion of another addend channel.   The substrate includes a first side, a second side, a third side, and a fourth side, wherein the second side of the substrate is from the distal end of the third addend channel. Spaced about 3.70 millimeters apart, the first and third side lengths are approximately 25.40 millimeters, and the second and fourth side lengths are about 76. 32. The crystal card of claim 31, wherein the crystal card is 20 millimeters and the first side of the substrate is spaced about 6.00 millimeters from the distal end of the addend channel.   The second side of the substrate is spaced about 3.70 millimeters from the distal end of the third addend channel, and the first side of the substrate is the addend channel 34. Spaced about 6.00 millimeters from the distal end of the substrate, and the third side of the substrate is spaced approximately 6.00 millimeters from the inspection circuit. The crystal card described in 1.   The test circuit includes a summing channel, a meandering portion, and a tail channel terminating in an opening configured to be in fluid communication, the summing channel connected to the apex, and the addend channel being Extending in a direction that is collinear with the proximal end of the fourth addend channel until an axis that is collinear with the first portion of the third addend channel is reached, the summing channel is 32. The crystal card according to claim 31, wherein the crystal card is bent by 90 degrees and connected to the meandering portion of the inspection circuit.   The meandering portion of the test circuit is formed from a compound curve having a plurality of convex bends connected to each other by meandering channels to facilitate fluid communication, wherein one convex bend is a subsequent convex bend. 36. The crystal card of claim 35, spaced about 53.31 millimeters apart, each convex bend having a length of about 2.00 millimeters.   38. The crystal card of claim 36, wherein the last convex bend of the serpentine is coupled to the tail channel.   The sum of the summing channel, the meandering portion, and the tail channel is about 67 centimeters in total, and the summing channel, the meandering portion, and the tail channel have a cross-sectional dimension of about 200 × 200 micrometers. 38. The crystal card of claim 37, wherein:   28. The crystal card of claim 27, wherein the substrate is configured to accommodate two mixer circuits and two inspection circuits.   The substrate contains a plurality of annular ports protruding upwards, some of the plurality of annular ports adapted to fluidly receive a solution or to fluidly communicate an aqueous plug or plug spacer 40. The crystal card of claim 39.

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