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WO2009152520A2 - Système de cristallisation microcapillaire de type nanovolume - Google Patents

Système de cristallisation microcapillaire de type nanovolume Download PDF

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Publication number
WO2009152520A2
WO2009152520A2 PCT/US2009/047414 US2009047414W WO2009152520A2 WO 2009152520 A2 WO2009152520 A2 WO 2009152520A2 US 2009047414 W US2009047414 W US 2009047414W WO 2009152520 A2 WO2009152520 A2 WO 2009152520A2
Authority
WO
WIPO (PCT)
Prior art keywords
channel
crystal
crystal card
summand
card
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2009/047414
Other languages
English (en)
Other versions
WO2009152520A3 (fr
Inventor
Cory Gerdts
Mark Elliott
Peter Nollert-Von-Specht
Lansing J. Stewart
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Emerald Biostructures Inc
Original Assignee
Decode Biostructures Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Decode Biostructures Inc filed Critical Decode Biostructures Inc
Priority to JP2011513753A priority Critical patent/JP2011525166A/ja
Priority to EP09763807A priority patent/EP2352591A4/fr
Publication of WO2009152520A2 publication Critical patent/WO2009152520A2/fr
Publication of WO2009152520A3 publication Critical patent/WO2009152520A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/20—Jet mixers, i.e. mixers using high-speed fluid streams
    • B01F25/23—Mixing by intersecting jets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/06—Crystallising dishes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/54—Organic compounds
    • C30B29/58—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers

Definitions

  • One aspect of the discussed subject matter includes a system for protein crystallization, which comprises a pumping system and pieces of software configured to execute on the protein crystallization system to control the pumping system.
  • the system further includes one or more crystal cards coupled to the pumping system, each configured to house a mixer and a microfluidic capillary that is coupled to the mixer to facilitate storage and inspection of protein crystallization.
  • Another aspect of the subject matter includes a method for gradient screening, which comprises regulating aqueous streams by independently controlling each aqueous stream with a pumping system exercised by pieces of software.
  • the method further comprises mapping out crystallization phase space of a protein to illustrate transition from precipitation, to microcrystals, to single crystals in a protein crystallization experiment.
  • a further aspect of the subject matter includes a method for hybrid screening, which comprises pre-forming precipitant plugs and pre-forming plug spacers, each separating two precipitant plugs from each other.
  • the method further comprises forming gradients by merging precipitant plugs, plug spacers, and a protein stream.
  • the method further includes mapping out crystallization phase space of a protein to illustrate transition from precipitation, to microcrystals, to single crystals in a protein crystallization experiment.
  • a further aspect of the subject matter includes a method which comprises receiving a crystal card with capillaries, coating capillaries with a reagent to reduce the surface energy, and removing the reagent.
  • the subject matter includes a crystal card, which comprises a substrate configured to house a mixer circuit and an inspection circuit.
  • the crystal card further includes a layer bonded to the substrate and configured to peel from the substrate.
  • FIGURE 1 is a block diagram illustrating an exemplary nanovolume microcapillary crystallization system
  • FIGURE 2 is a block diagram illustrating an exemplary pumping system of the nanovolume microcapillary crystallization system
  • FIGURE 3A is a pictorial diagram illustrating an exemplary user interface for configuring the pumping system
  • FIGURE 3B is a pictorial diagram illustrating an exemplary user interface for priming fluids to a crystal card of the system
  • FIGURE 3C is a pictorial diagram illustrating an exemplary user interface for specifying the production of nanoplugs in the crystal card wherein the nanoplugs are of equal size and equal content according to one embodiment of the subject matter;
  • FIGURE 3D is a pictorial diagram illustrating an exemplary user interface for specifying the production of nanoplugs in the crystal card wherein the nanoplugs have varying concentrations of protein and precipitant according to one embodiment of the subject matter;
  • FIGURE 3E is a pictorial diagram illustrating an exemplary user interface for specifying the production of nanoplugs in the crystal card wherein the nanoplugs have varying size and concentration for multiple precipitants according to another embodiment of the subject matter;
  • FIGURE 4A is a pictorial diagram illustrating a top isometric view of one embodiment of a crystal card
  • FIGURE 4B is a pictorial diagram illustrating a bottom isometric view of one embodiment of a crystal card
  • FIGURE 4C is a pictorial diagram illustrating a top view of one embodiment of a crystal card
  • FIGURE 4D is a pictorial diagram illustrating a side view of one embodiment of a crystal card
  • FIGURE 4E is a pictorial diagram illustrating a bottom view of one embodiment of a crystal card;
  • FIGURE 5A is a pictorial diagram illustrating a top isometric view of another embodiment of a crystal card
  • FIGURE 5B is a pictorial diagram illustrating a bottom isometric view of another embodiment of a crystal card
  • FIGURE 5C is a pictorial diagram illustrating a top view of another embodiment of a crystal card
  • FIGURE 5D is a pictorial diagram illustrating a side view of another embodiment of a crystal card
  • FIGURE 5E is a pictorial diagram illustrating a bottom view of another embodiment of a crystal card
  • FIGURE 6A is a pictorial diagram illustrating a top isometric view of a third embodiment of a crystal card
  • FIGURE 6B is a pictorial diagram illustrating a bottom isometric view of a third embodiment of a crystal card
  • FIGURE 6C is a pictorial diagram illustrating a top view of a third embodiment of a crystal card
  • FIGURE 6D is a pictorial diagram illustrating a side view of a
  • FIGURE 7A is a pictorial diagram illustrating a top isometric view of a fourth embodiment of a crystal card
  • FIGURE 7B is a pictorial diagram illustrating a bottom isometric view of a fourth embodiment of a crystal card
  • FIGURE 7C is a pictorial diagram illustrating a top view of a fourth embodiment of a crystal card
  • FIGURE 7D is a pictorial diagram illustrating a side view of a fourth embodiment of a crystal card
  • FIGURE 7E is a pictorial diagram illustrating a bottom view of a fourth embodiment of a crystal card
  • FIGURE 8A is a pictorial diagram illustrating a top isometric view of a fifth embodiment of a crystal card
  • FIGURE 8B is a pictorial diagram illustrating a bottom isometric view of a fifth embodiment of a crystal card
  • FIGURE 8C is a pictorial diagram illustrating a top view of an embodiment of a crystal card
  • FIGURE 8D is a pictorial diagram
  • FIGURE 9A is a pictorial diagram illustrating a top isometric view of a sixth embodiment of a crystal card
  • FIGURE 9B is a pictorial diagram illustrating a bottom isometric view of a sixth embodiment of a crystal card
  • FIGURE 9C is a pictorial diagram illustrating an exploded isometric view of a sixth embodiment of a crystal card
  • FIGURE 9D is a pictorial diagram illustrating a top view of a sixth embodiment of a crystal card
  • FIGURE 9E is a pictorial diagram illustrating a side view of a sixth embodiment of a crystal card
  • FIGURE 9F is a pictorial diagram illustrating a bottom view of a sixth embodiment of a crystal card
  • FIGURE 10 is a pictorial diagram illustrating one embodiment of a three-plus-one mixer of one embodiment of a crystal card;
  • FIGURE 11 is a pictorial diagram illustrating another embodiment of a three-plus-one mixer of one embodiment of a crystal card
  • FIGURE 12 is a pictorial diagram illustrating a third embodiment of a three-plus-one mixer of one embodiment of a crystal card
  • FIGURE 13 is a pictorial diagram illustrating a fourth embodiment of a three-plus-one mixer of one embodiment of a crystal card
  • FIGURE 14 is a pictorial diagram illustrating a cross section through one embodiment of a crystal card; and FIGURES 15A- 15V are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system;.
  • a nanovolume microcapillary crystallization system which comprises a pump, software configured to control the pump, and a crystal card that houses a mixer circuit and an inspection circuit.
  • the crystal cards are suitably manufactured using materials that include one or more properties selected from a group consisting of X-ray transmission, optical clarity, moldability, chemical resistance and surface energy.
  • the crystal cards house macro molecular crystals in various phases enabling either extraction of crystals from the crystal card or in situ X-ray diffraction.
  • the crystals are promoted inside the crystal cards by formation of nanoplugs by the nanovolume microcapillary crystallization system.
  • Nanoplugs are formed by combining streams of aqueous solutions with an immiscible and biologically inert carrier fluid, such as fluorocarbon solution. Streams of aqueous solutions, such as those composed of a target molecule, buffer, and precipitant solutions, are combined at the mixer circuit to form nanoplug crystallization experiments. The nanoplugs are incubated and monitored for crystallization. Nanoplug crystallization experiments can be suitably used to shed light on scientific questions regarding protein crystal nucleation and growth and to generate crystals for novel structure solution.
  • an immiscible and biologically inert carrier fluid such as fluorocarbon solution.
  • the nanovolume microcapillary crystallization system facilitates two screening styles: gradient mode and hybrid mode.
  • the term gradient mode includes any suitable screening method that provides various crystallization phases of molecules.
  • the gradient mode allows a crystallographer to finely scan a crystal card to reveal crystallization phase space of a particular molecule.
  • concentration gradients of desired granularity over a series of nanoplugs are suitably formed by changing the flow rates of the individual streams.
  • concentration gradients of desired granularity over a series of nanoplugs are suitably formed by changing the flow rates of the individual streams.
  • the nanovolume microcapillary crystallization system increases a flow rate of a buffer stream such that that the sum of the flow rates remains constant.
  • crystallization phase space of a particular molecule such as a protein, can be mapped out to show a transition from precipitation, to microcrystals, to single crystals.
  • hybrid mode combines gradients with sparse matrix screening on one crystal card.
  • Sparse matrix screening of molecule crystals in nanoplugs can be achieved by generating a pre-formed cartridge of different crystallizing agents.
  • the term hybrid mode includes hybrid screening, including any suitable screening method that includes pre-formed cartridges.
  • the hybrid mode extends the concept of sparse matrix screening by pre-forming precipitant nanoplugs, separated by a nanoplug spacer (gas bubble), and forming a concentration gradient as they are merged with a molecule stream. Similar to the gradient mode, the hybrid mode generates gradients by coordinating flow rate change between the pre-formed precipitant nanoplugs and the buffer stream. By performing sparse matrix and gradient screening together on one crystal card, the hybrid mode is able to sample a large area of the crystallization phase space, generating 20-40 experiments from each pre-formed precipitant nanoplug.
  • nanoplug refers to a nano liter- volume sized drop, such as a 10-20 nL aqueous drop, that fills a micro fluidic channel of the system. Each nanoplug comprises a distinct microcrystallization experiment.
  • mixer circuit means the inclusion of a circuit having three aqueous channels and one carrier fluid channel that come together at a point upstream of the inspection circuit of a crystal card. Additional configurations are possible, such as mixers having four or five aqueous channels and one carrier fluid channel. The aqueous channels come together and suitably intersect the carrier fluid channel at a 90 degree angle.
  • the term “macro-micro interface” means the inclusion of a coupling between the syringes and a crystal card.
  • the syringes are connected to the mixer circuit or the inspection circuit via tubing, such as Teflon ® (PTFE) tubing.
  • the tubing is connected to the mixer circuit or the inspection circuit using connectors configured to fluidically connect the inlets and outlets of the circuits with the tubing.
  • the term “inspection circuit” refers to a capillary or channel where fluids come together and form aqueous nanoplugs that flow inside the capillary. The inspection circuit can also be used to inspect the nanoplugs for crystal formation.
  • the inspection circuit can also be used to store crystals formed by the methods of the subject matter, and is therefore also referred to herein as a storage capillary.
  • the term “main channel” refers to the area of the inspection circuit that locates downstream of the mixer where the aqueous solutions and carrier fluid combine to form aqueous nanoplugs.
  • the term "molecule” includes small molecules, such as organic compounds and/or chemicals, and macro molecules.
  • biological molecule refers to a molecule that is derived from, modeled on, or corresponds to a molecule from a biological source.
  • the term also includes 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 other biological molecules that do not occur in nature.
  • macromolecule includes biopolymers such as nucleic acids, proteins, carbohydrates and lipids.
  • protein and “protein solution” are used herein to encompass other types of molecules in addition to proteins.
  • a nano volume microcapillary crystallization system 100 useful for molecule crystallization is shown in FIGURE 1.
  • a prepared sample 102 comprising a molecule for which the crystal structure is desired, for example a prepared protein sample, is provided in an aqueous solution 104. Additional aqueous solutions are also provided; for example, a buffer solution and a precipitant solution.
  • the buffer solution may comprise the buffer used to prepare the biological sample 102.
  • a carrier fluid is also provided.
  • the carrier fluid is immiscible with the aqueous solutions. Suitable examples of carrier fluids include fluorinated oils; for example, FC-40 (3M Corp., St. Paul, Minnesota).
  • the aqueous solutions and carrier fluid 104 are provided to one or more syringes 106 which are connected to one or more pumps 108.
  • the pump 108 is controlled by software executed on a nanoplug-forming computer 110.
  • the software executed on the nanoplug-forming computer 110 regulates the flow of the aqueous solutions and carrier fluid 104 in a crystal card 112.
  • the flow of the aqueous solutions and carrier fluid in the crystal card 112 are observed through a magnifying device such as a microscope 114.
  • FIGURE 2 A pumping system 200 useful for regulating the flow of various fluids through a crystal card is shown in FIGURE 2.
  • 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 a precipitant reagent 210.
  • Pump 2 212 controls syringe 3 214 and syringe 4 218.
  • Syringe 3 214 is filled with an immiscible fluid such as a carrier fluid 216.
  • Syringe 4 218 is filled with an aqueous solution containing a molecule, such as a protein of interest 220.
  • Suitable pumps include Harvard Twin 33 syringe pumps (Harvard Apparatus, Holliston, MA). In some embodiments, the syringe pumps have been modified by the manufacturer to provide better accuracy. Suitable syringes include Hamilton syringes, such as an 1800 series Hamilton Gas Tight syringe. Suitable syringe volumes range from 10 ul to 100 ul.
  • the pumping system 200 is controlled by software executed on a nanoplug-forming computer 110. Suitable software is provided for controlling the pumping system 108.
  • FIGURES 3A-3E illustrate representative user interfaces of the software of the system showing various modes that control the pumping system 200.
  • FIGURE 3 A shows a representative user interface 300 of a configuration mode of the software.
  • FIGURE 3B shows a representative user interface 302 of a prime mode of the software.
  • FIGURE 3C shows a representative user interface 304 of a constant mode of the software.
  • FIGURE 3D shows a representative user interface 306 of a gradient mode of the software.
  • FIGURE 3E shows a representative user interface 308 of a hybrid mode of the software of the system.
  • the crystal card 400 is configured to be about the same size as a standard microscope slide, being about 76.20 mm long and about 25.40 mm wide (or about 3 inches long by about 1 inch wide).
  • the crystal card 400 is about 1.0 to 1.5 mm thick.
  • the crystal card is manufactured of transparent polycarbonate by injection molding (Siloam Biosciences, Inc.).
  • the crystal card 400 has an upper surface 402 and a lower surface 414 that is parallel to the upper surface 402.
  • the crystal card 400 further comprises a substrate configured to house a mixer circuit 404 and a storage and inspection circuit 406.
  • the mixer circuit 404 is comprised of four microfluidic channels 421, 422, 424, and 426. See FIGURE 4C. Channels 421, 422, and 424 come together and intersect channel 426 at a 90 degree angle. Each channel comprises an inlet 410. See FIGURE 4E.
  • the inspection circuit 406 comprises a long microfluidic capillary channel that locates just downstream of the mixer 404 and ends at an 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, in which crystals produced in the card may be stored in the channel 406 until subjected to in situ X-ray diffraction analysis or extracted for cryocooling.
  • the microfluidic channels 421, 422, 424, 426 and the capillary channel 406 are substantially square in cross-section and have an inner diameter of about 200 micrometers ( ⁇ m) x 200 ⁇ m. However, other configurations of the channels are possible.
  • the crystal card 400 further comprises a layer 420 that is thermally bonded to the substrate and configured to peel 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 is about 0.10 to 0.14 mm thick.
  • the peelable layer 420 is suitably configured such that removal of the peelable layer 420 exposes the interior space of the inspection circuit channel 406.
  • the crystal card 400 further comprises a macro-micro interface that connects the syringes to the crystal card.
  • the macro-micro interface includes sections of rigid plastic tubing 430 (for example, tubing made of PEEKTM polymer) that are connected at one end to the inlets 410 and outlet 412, and are connected at the other end to slip fit connectors 432 made of flexible silicone tubing.
  • the slip fit connectors 432 are configured to accept Teflon® tubing (PTFE) (not shown). The other end of the tubing is connected to a syringe of the system.
  • the Teflon® tubing has an inner diameter of 360 um and an outer diameter of 760 um (ID/OD 360/760), whereas the connecter 432 has an inner diameter of 760 um, thereby forming a gas and liquid tight seal when the Teflon® tubing is inserted into the connecter 432.
  • the channel 421 is connected to tubing that is filled with an aqueous solution, such as a buffer that is used in the protein solution of interest.
  • Channel 422 is connected to tubing that is filled with a precipitant solution.
  • precipitant is interchangeable with the term crystallant.
  • Channel 424 is connected to tubing that is filled with a solution containing a target molecule of interest.
  • the target biological molecule is a protein.
  • Channel 426 is connected to tubing that is filled with a 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 fluids and preferentially wets the walls of the inspection circuit microchannel, thereby separating segments of the combined aqueous solution into nanoplugs that span the width of the channel.
  • the aqueous nanoplugs are about 10-20 nL in volume.
  • the inlet 510 is located in a shallow cylindrical depression 508 located in a top surface 502 of the crystal card 500.
  • the cylindrical depression 508 is configured for attaching a connector (not shown) that connects tubing to the inlets 510 and outlet 512.
  • the dimensions of the crystal card 500 are shown in FIGURE 5E.
  • the crystal card 500 is 76.2 mm long and 25.4 mm wide.
  • the inlets 510 are spaced 4.5 mm apart.
  • the parallel channels of the inspection circuit 506 are 2.0 mm apart.
  • other suitable configurations are possible.
  • FIGURES 6A-6E a representative example of a third embodiment of a crystal card is shown.
  • the inlets 610 and outlet 612 are positioned below a cylindrical projection 608 that is connected to and extends outwardly from the surface 602.
  • the projection 608 is configured for attaching a connector (not shown) that connects tubing to the inlets 610 and outlet 612.
  • the crystal cards illustrated in the embodiments shown in FIGURES 4-6 are manufactured from transparent polycarbonate plastic by injection molding (Siloam Biosciences, Inc.).
  • FIGURES 7A-7E illustrate a representative example of another embodiment of a crystal card of the subject matter.
  • the top surface 702 of the crystal card 700 further comprises two rows of ports 708.
  • the ports are configured to receive a plastic connector (not shown) that is suitable for connecting tubing to the inlets 710 and outlets 712 located beneath the port 708.
  • the surface 702 comprises 28 ports 708.
  • the port 708 extends about 2.5 mm above the surface 702 of the crystal card 700.
  • a hole is suitably drilled in the bottom center portion of the port 708 such that it aligns with and is in fluidic connection with the inlets 710 and outlets 712.
  • the center of the ports are spaced about 4.5 mm apart.
  • the hole drilled in the bottom of the port 708 is about 0.2 mm (200 um) in diameter. It will be understood that not every port is connected to the circuit channels such that only desired ports to connect tubing to the inlets 710 and the outlets 712 need be drilled.
  • a laser is used to drill holes through the peelable layer 720 before it is bonded to the bottom surface 714.
  • the laser-drilled holes are configured to be in fluidic connection with the inlets 710 and the outlets 712.
  • the crystal card further comprises two separate asymmetrical micro fluidic channel circuits 706A, 706B.
  • the inspection circuit is about 270 mm long.
  • the inspection circuit is about 306 mm long.
  • the outlet 712 is located on the opposite side of the circuit from the inlets 710 and the mixer circuits 704A, 704B.
  • the embodiment shown in FIGURES 7A-7E comprises two separate configurations of the mixer circuit 704A, 704B.
  • the mixer circuit 704A comprises a short neck region approximately 0.20 mm long between the aqueous channels and the carrier fluid channel.
  • the mixer circuit 704B lacks a neck region between the aqueous channels and the carrier fluid channel.
  • the mixer circuit 704A was found to be suitable for aqueous nanoplug formation in a crystal card.
  • the crystal card 800 comprises two separate symmetrical micro fluidic channel circuits 806.
  • the outlet 812 is located on the same side of the circuit 806 as the mixer 804 and the inlets 810.
  • a crystal card 900 comprises a single microfluidic circuit comprising one mixer circuit 904 and a long inspection circuit 906.
  • the inspection circuit 906 is about 665 mm long.
  • FIGURE 9C illustrates an exploded view of the crystal card 900.
  • Piece 930 comprising ports 908 is bonded to piece 940 comprising the microfluidic circuit channels.
  • the peelable layer 920 is thermally bonded to the bottom surface 914 of piece 940. However, in other embodiments, the peelable layer 920 may be chemically bonded to the substrate surface 914.
  • the peelable layer 920 is suitably configured such that removal of the peelable layer 920 exposes the interior space of the inspection circuit channel 906. Digressing, the crystal cards illustrated in the embodiments shown in FIGURES 7-9 are manufactured from transparent cyclic olefin copolymer (COC) or comparable plastic (ThinXXS Micro techno logy AG, Germany).
  • FIGURE 10 shows a representative example of one embodiment of a mixer circuit that corresponds to the mixer 704A shown in FIGURE 7.
  • the mixer circuit 1000 comprises three aqueous channels 1021, 1022 and 1024.
  • the aqueous channels are separated from the carrier fluid channel 1026 by a neck region 1007.
  • the channels are oriented such that the three channels 1021, 1022, 1024 containing aqueous solutions come together and intersect the channel 1026 containing the carrier fluid at a 90 degree angle.
  • the mixer 1000 further comprises a portion of an inspection circuit 1006. Referring still to FIGURE 10, the dimensions of the mixer 1000 will now be described.
  • the neck region 1007 is about 0.2 mm long.
  • Channel 1021 is about 0.2 mm in diameter.
  • Channels 1022, 1024 are about 0.141 mm in diameter.
  • Channels 1006, 1026 are about 0.2 mm in diameter.
  • other suitable dimensions for a mixer circuit are possible.
  • FIGURE 11 shows a representative example of another embodiment of a mixer circuit that corresponds to the mixer circuit 704B shown in FIGURE 7.
  • the 1100 comprises three aqueous channels 1121, 1122 and 1124.
  • the aqueous solution channels connect directly to the carrier fluid channel 1106 in the absence of a neck region.
  • the mixer circuit feeds into the inspection circuit 1126.
  • the channels are oriented such that the three channels containing aqueous solutions come together and intersect the channel containing the carrier fluid at a 90 degree angle.
  • the diameter of channel 1121 is about 0.2 mm.
  • the diameter of channels 1122, 1124 is about 0.141 mm.
  • the diameter of the junction region between the aqueous channels and the carrier fluid channel 1126 is about 0.285 mm.
  • other suitable dimensions for a mixer are possible.
  • the mixer circuit 1200 comprises three aqueous channels 1221, 1222 and 1224 that are connected by a short neck region to the carrier fluid channel 1226.
  • the channels are oriented such that the three channels containing aqueous solutions come together and intersect the channel containing the carrier fluid at a 90 degree angle.
  • Each channel has an inlet 1210. Downstream of the mixer circuit 1204, the solutions feed into a portion of an inspection circuit 1206.
  • the inlets 1210 are located about 4.4 mm from the channels 1206, 1226.
  • the aqueous channels 1221, 1222, 1224 make a right angle turn about 2.9 mm from the inlet.
  • the right angle turn has an inner radius RO.300 and an outer radius RO.500.
  • the portion of channels 1221, 1222, 1224 that are disposed in a plane parallel to channel 1206 are about 1.300 mm from channel 1206.
  • the aqueous channels 1221, 1222, 1224 make a 45 degree turn before connecting with each other upstream of the neck region.
  • the inner diameter of channel 1206 is about 0.200 mm (200 um).
  • the parallel portions of channel 1206 are about 1.2 mm apart. However, other suitable dimensions are possible.
  • FIGURE 13 shows a representative example of another embodiment of a mixer that corresponds to the mixer circuits 804 and 904 shown in FIGURES 8 and 9.
  • the mixer circuit 1300 comprises aqueous channels 1321, 1322, and 1324.
  • the aqueous channels are separated from the carrier fluid channel 1306 and the inspection circuit 1326 by a short neck region.
  • the diameter of the neck region is about 0.200 mm. However, other suitable dimensions are possible.
  • the channels are oriented such that the three channels containing aqueous solutions come together and intersect the channel containing the carrier fluid at a 90 degree angle. Downstream of the mixer circuit, the solutions flow into the inspection circuit 1326.
  • FIGURE 14 shows a representative example of a cross-section through a crystal card similar to the embodiment illustrated in FIGURE 9.
  • the crystal card 1400 is comprised of three layers 1420, 1430 and 1440.
  • Layer 1430 comprises the ports as shown in FIGURES 7-9.
  • Layer 1430 is about 0.4 mm thick.
  • Layer 1440 comprises the microfluidic channel circuit and is about 1.5 mm thick at the edge.
  • Layer 1420 comprises the peelable layer attached to the bottom surface of the crystal card 1400, and is about 0.14 mm thick.
  • FIGURES 15A- 15V illustrate a method 5000 for crystallizing molecules using a nanovolume microcapillary crystallization system. From a 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"). The set of method steps 5002 describes the preparation of a crystal card and the connection of the crystal card to a pump.
  • the method 5000 proceeds to a set of method steps 5008 where the crystal card is manufactured from a suitable material, such as polydimethylsiloxane (PDMS) or plastic by injection molding. The method then returns to a point of invocation. The method 5000 next proceeds to a set of method steps 5010 defined by a continuation terminal ("Terminal A2"). The set of method steps 5010 treats the microcapillary surface of the crystal card to reduce the surface energy. From Terminal A2 (FIGURE 15C), the method 5000 proceeds to decision block 5014 where a test is performed to determine whether the crystal card is manufactured from plastic. If the answer to the test is NO, the method proceeds to another continuation terminal ("Terminal A4").
  • a suitable material such as polydimethylsiloxane (PDMS) or plastic by injection molding.
  • the method then returns to a point of invocation.
  • the method 5000 next proceeds to a set of method steps 5010 defined by a continuation terminal ("Terminal A2").
  • the set of method steps 5010 treats the micro
  • the method 5000 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 (FIGURE 15D), the method 5000 proceeds to block 5018 where the method treats the 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.
  • the microcapillary surface of the crystal card is dried using a gas such as air under pressure at 5-10 psi for 1 hour. The method then returns to the point from which the steps of Terminal A2 were invoked, and proceeds to another continuation terminal ("Terminal A3"). See block 5012.
  • a gas such as air under pressure at 5-10 psi for 1 hour.
  • the method 5000 proceeds to block 5026 where the method treats the crystal card as made of a plastic comprising cyclic olefin copolymer (COC) or comparable plastic.
  • the microcapillary surface is treated with a reagent to reduce the surface energy (hydrophobicity) of the plastic for 2 hours at room temperature.
  • Suitable reagents for reducing the surface energy include fluorinated copolymer solutions, but other reagents are possible.
  • Suitable fluorinated copolymer solutions include a two percent fluorinated copolymer solution in a fluoro solvent, such as Cytonix PFC 502AFA (Cytonix Corp., Beltsville, MD).
  • Cytonix PFC 502AFA is manufactured to adhere to polycarbonate and reduce the surface energy to 6-10 dyne/cm.
  • the crystal card is filled from the outlet with the Cytonix PFC 502AFA solution.
  • the fluorinated copolymer solution is removed by vacuum.
  • the microcapillary surface is dried using a gas such as air under pressure of 5-10 psi for 1 hour.
  • the method 5000 then proceeds to block 5034 where the crystal card is heated to 6O 0 C for 1 hour. The method then returns to the point of invocation of the steps of Terminal A2. See block 5012 at Terminal A3.
  • the method 5000 proceeds to block 5036 where the crystal card is pre-chilled on ice.
  • the microcapillary surface is treated with a fluorinated copolymer solution such as Cytonix PFC 502AFA for 2 hours on ice.
  • the polycarbonate crystal card inlets may be prone to cracking if incubated with the 502AFA solution at higher temperatures.
  • the method then proceeds to continuation terminal A5 where it skips to block 5030 and performs the steps in blocks 5030, 5032, and 5034.
  • the method then returns to a point at which the steps of Terminal A2 were invoked. See Terminal A3 at block 5012.
  • the set of method steps at block 5012 couples the crystal card to the pump.
  • syringe 1 is filled with a buffer or aqueous solution.
  • syringe 2 is filled with a precipitant solution.
  • syringe 3 is filled with a carrier fluid.
  • a representative example of a suitable carrier fluid includes a fluorinated carbon solution.
  • Suitable examples of a fluorocarbon fluid include FC-40.
  • FC-40 has a high surface tension with the detergents used in solubilizing membrane proteins. The surface tension enables nanoplug formation and crystallization.
  • the carrier fluid is a fluorinated oil which is immiscible with aqueous fluids.
  • the carrier fluid surrounds and separates the aqueous nanoplugs as they are formed, moving them forward through the crystal card during the method.
  • syringe 4 is filled with a protein solution containing the protein of interest in a suitable buffer.
  • suitable tubing such as Teflon® tubing is attached to the needle of each syringe.
  • syringes 1 and 2 are attached to pump 1, and syringes 3 and 4 are attached to pump 2.
  • the tubing is connected to the crystal card via a macro-micro interface. Suitable connections for the macro-micro interface are described above. The method then proceeds to exit Terminal B.
  • the method 5000 proceeds to a set of method steps 5004, defined between a continuation terminal ("Terminal C") and an exit terminal ("Terminal D").
  • the set of method steps 5004 receives instructions to regulate fluid flow through the crystal card to obtain crystals.
  • Terminal C (FIGURE 15H)
  • the method 5000 proceeds to a set of method steps 5054, defined by a continuation terminal ("Terminal Cl ").
  • the set of method steps 5054 configures the pump.
  • the method 5000 proceeds to block 5060 where the method receives instructions on the type of syringe pump model to be controlled by the system.
  • Suitable pumps include Harvard Apparatus Twin Syringe Pump Model 33 (Harvard Apparatus, Holliston, MA), which has been modified by the manufacturer to provide better accuracy.
  • each syringe pump controls two syringes.
  • the method receives instructions on the serial communication port of a computer used to control the pump system.
  • the communication ports are configured such that each syringe pump receives instructions at the same time, thereby preventing time delays and allowing the solutions to flow through the crystal card simultaneously.
  • the method proceeds to block 5064 where the method receives instructions on the volume of each syringe connected to the pumps.
  • the method determines the diameter of each syringe connected to the pumps. The method then proceeds to return to a point at which the steps of the Terminal Cl were invoked.
  • the method 5000 proceeds to a set of method steps 5056 defined by a continuation terminal ("Terminal C2").
  • the set of method steps primes fluids to the mixer circuit of the crystal card.
  • Terminal C2 (FIGURE 15J)
  • the method 5000 proceeds to block 5068 where the method receives instructions on which syringe will be used to dispense fluids into the mixer of the crystal card.
  • the method receives instructions on the flow rate from each syringe.
  • the method receives instructions on the volume of fluid to be dispensed by the syringe.
  • the method dispenses or aspirates fluid from a fluidic channel upstream of the mixer circuit. The method then continues to another continuation terminal ("Terminal CA").
  • 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 where the method receives instructions to stop the aqueous fluid at the mixer circuit and before the fluid enters the inspection circuit. The method then continues to Terminal C2 and repeats the above identified process steps for the next syringe.
  • the method 5000 proceeds to block 5080 where the method receives instructions to stop the carrier fluid downstream of the mixer circuit and slightly inside the inspection circuit. The method then proceeds to return to a point from which the steps of Terminal C2 were invoked.
  • the empty crystal card mixer circuit is positioned on the microscope stage for observation during priming.
  • the method receives instructions to dispense a solution, for example buffer, from syringe 1 to the mixer.
  • the buffer is dispensed into the fluid channel connected to syringe 1 until the user observes that the solution has reached the region of the mixer just upstream of the junction between the fluidic channels.
  • the method then receives instructions to stop dispensing the solution. Solution may be removed from the channel by instructing the method to aspirate the reagent. It is suitable to refrain aqueous solutions 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 the fourth fluid channel connected to syringe 3.
  • the carrier fluid is dispensed into the fourth fluid channel until the fluid travels through the mixer junction and just slightly enters the inspection circuit (fifth channel) of the crystal card.
  • the method receives instructions to stop dispensing the carrier fluid.
  • the method 5000 proceeds to a set of method steps 5058 defined by a continuation terminal ("Terminal C3").
  • the set of method steps receives instructions to produce aqueous nanoplugs in the inspection circuit of the crystal card.
  • Terminal C3 (FIGURE 15L)
  • the method 5000 receives instructions on which nanoplug formation protocol will be performed at block 5082.
  • the method then proceeds to decision block 5084 where a test is performed to determine whether the instruction received was to perform the 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 proceeds to block 5086 where the method receives instructions on the flow rate for each syringe.
  • the method then proceeds to block 5088 where the method receives instructions on the total volume of fluid to pass through the mixer circuit.
  • the method produces aqueous nanoplugs inside the inspection circuit of the crystal card wherein each nanoplug is suitably of equal size and has the similar concentration of protein and precipitant.
  • the method then proceeds to return to a point of invocation. From block 5058, the method proceeds to exit terminal D. From Terminal C6 (FIGURE 15M), the method 5000 proceeds to decision block 5092, where a test is performed to determine whether the method was instructed to perform gradient mode. If the answer to the test in block 5092 is NO, the method proceeds to another continuation terminal ("Terminal C7").
  • the method proceeds to block 5094 where the method receives instructions on the maximum flow rate for the syringes with variable flow.
  • the variable flow syringes contain the buffer and precipitant.
  • syringes 1 and 2 are the variable flow syringes.
  • the method can designate any syringe to be a variable flow syringe.
  • the combined flow rate of the variable flow syringes equals the maximum flow rate.
  • the method provides instructions for the flow rate of syringe 1 to equal 2 ⁇ l/min, whereas the method provides instructions for the flow rate of syringe 2 to equal 0 (zero) ⁇ l/min.
  • the maximum flow rate equals 2 ul/min (2 + 0 ⁇ l/min).
  • the method then proceeds to block 5096 where the method receives instructions on the constant flow rate for the syringe controlling the carrier fluid.
  • syringe 3 controls the carrier fluid.
  • the carrier fluid flow rate equals the total flow rate of the aqueous solutions (buffer, precipitant, and protein solutions).
  • the flow rate for the carrier fluid may be selected to be slower or faster than the total flow rate of the aqueous fluids. Slower carrier fluid rates generate larger aqueous nanoplugs with smaller segments comprising carrier fluid between nanoplugs. Faster carrier fluid rates generate smaller aqueous nanoplugs with larger carrier fluid segments between the nanoplugs.
  • the method then proceeds to block 5098 where the method receives instructions on the constant flow rate for the syringe controlling the protein solution.
  • syringe 4 controls the carrier fluid.
  • the protein flow rate equals the sum of the flow rate 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 on the total aqueous volume to be dispensed during a single iteration or cycle of the method.
  • the method then proceeds to another continuation terminal ("Terminal C8").
  • the method 5000 proceeds to block 6002 where the method receives instructions on the volume of each aqueous nanoplug that will be dispensed into the inspection circuit.
  • the method receives instructions on the total number of iterations or cycles to be performed (i.e., the number of times the gradient screening steps are repeated).
  • the pumps will stop when the total aqueous volume selected at block 6000 is dispensed.
  • the pumps will stop when the process steps described above have been repeated the desired number of times.
  • the method reciprocally varies the flow rate of the buffer and precipitant solutions such that the sum of the buffer and precipitant solution flow rates equals the maximum flow rate selected at block 5094.
  • the method provides instructions for the flow rate of syringe 1 to equal 2 ⁇ l/min and provides instructions for the flow rate of syringe 2 to equal 0 ⁇ l/min, such that the maximum flow rate equals 2 ⁇ l/min.
  • the flow rate from syringe 1 will begin at 2 ⁇ l/min and ramp down to 0 ⁇ l/min, while the flow rate from syringe 2 will simultaneously ramp up from 0 ⁇ l/min to 2 ⁇ l/min.
  • the method produces a series of aqueous nanoplugs inside the inspection circuit wherein each drop is of equal size but varies in the concentrations of protein and precipitant in each drop.
  • the method terminates after the desired number of iterations or cycles has been performed. The method then returns to block 5058 where the method proceeds to exit terminal D.
  • the method 5000 proceeds to decision block 6012 where a test is performed to determine whether the method was instructed to perform 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 a 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 ClO"). If the answer to the test at block 6014 is YES, the method proceeds to another continuation terminal ("Terminal CH").
  • the method 5000 proceeds to decision block 6016 where a test is performed to determine whether the method was instructed to perform the pulsatile mode. If the answer to the test at decision block 6016 is NO, the method returns to Terminal C3 where the above identified steps 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 on performing the pulsatile mode. The method then returns to block 5058. From block 5058, the method exits to Terminal D.
  • the method 5000 proceeds to block 6020 where a 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 instructions to enter a defined volume, for example, about 40 nL, and aspirates an air bubble of about 40 nL into the tubing. At block 6026, the method aspirates a defined volume, for example, about 120 nL, of a precipitant solution into the tubing. At block 6028, the method repeats the above two steps until a suitable number of precipitants are loaded into the tubing.
  • tubing such as Teflon® tubing
  • a suitable number of precipitants can range from 1-24 or more.
  • the method aspirates carrier fluid, about 1 ⁇ L, into the open tip of the tubing.
  • the tubing is connected to the precipitant inlet of the crystal card. The method then proceeds to continuation Terminal CI l.
  • the method 5000 proceeds to block 6034 where the method receives instructions on the starting flow rate of the buffer solution (syringe 1).
  • the method receives instructions on the change in the flow rate (step size) of the buffer solution.
  • the step size is the change in the rate of flow that will be applied at each ramp up or down of the method.
  • the method receives instructions on the starting flow rate of the precipitant cartridge (syringe 2).
  • the method calculates the change in the flow rate (step size) of the precipitant solution. In one embodiment, the step size for the buffer equals the step size for the precipitant.
  • the method sums the buffer and precipitant flow rates to determine the total flow rate.
  • the method receives instructions on the starting flow rate for the carrier fluid (syringe 3).
  • the method receives instructions on the change in the flow rate (step size) of the carrier fluid. The method then proceeds to another continuation terminal ("Terminal C12").
  • the method 5000 proceeds to block 6048 where the method receives instructions on the 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 (rate of flow changes) for each precipitant. At block 6052, the method sets the number of ramp down steps to equal the number of ramp up steps for each iteration or cycle of the method. At block 6054, the method receives instructions on the number of iterations or cycles to be performed. In one embodiment, one iteration or cycle corresponds to a single precipitant loaded in the precipitant cartridge. At block 6056, the method receives instructions on the duration of each ramp step. For example, in one embodiment, the duration of each ramp step is 1.5 seconds. At block 6058, the method reciprocally varies the buffer and precipitant flow rates such that the sum equals the starting rates. The method then proceeds to another continuation terminal ("Terminal C13").
  • the method 5000 proceeds to block 6060 where the method varies the flow rate of the carrier fluid. The method then proceeds to block 6062 where the method produces a series of nanoplugs inside the inspection circuit wherein each drop has equal amounts of protein and varying amounts of precipitant and buffer. In one embodiment, the method provides a varied amount of precipitant with a constant amount of protein for each cycle. Table 1 illustrates one embodiment of the method described above for the hybrid mode. The method then proceeds to Terminal D.
  • the method 5000 proceeds to a set of method steps 5006, defined between a continuation terminal ("Terminal E") and an exit terminal ("Terminal F").
  • the set of method steps 5006 performs diffraction experiments on the crystals obtained from the crystal card.
  • Terminal E (FIGURE 15U)
  • the method 5000 proceeds to decision block 6064 where a test is performed to determine whether crystals were extracted 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 El"). If the answer to the test at block 6064 is YES, the method proceeds to block 6066 where a peelable layer is removed from the bottom surface of the crystal card.
  • the peelable layer is bonded to the plastic part of the crystal card that contains the microfluidic channels.
  • the bond is designed to be strong enough to prevent fluid from leaking out of the microfluidic circuit but weak enough to be manually peeled off.
  • the bond is a thermal bond.
  • the bond is a chemical bond. Removal of the peelable layer exposes the interior of the microfluidic channels of the crystal card, allowing access to the aqueous nanoplugs.
  • the aqueous nanoplugs that contain crystals are retained in the microfluidic channels of the crystal card after the peelable layer is removed.
  • the crystal formed in the inspection circuit is extracted from the crystal card using a cryoloop.
  • the cryoloop is a nylon cryoloop.
  • the crystal is cryocooled, and diffraction data is obtained. The method then proceeds to exit Terminal F where the method terminates execution.
  • the method 5000 proceeds to block 6072 where the crystal card containing crystals is mounted onto the goniometer of an X-ray source.
  • the method obtains diffraction data from crystals located in situ inside the inspection circuit.
  • the method then proceeds to block 5006 and exit Terminal F. The method then terminates execution.
  • Crystal extraction steps can be used in combination with the gradient screening of various embodiments of the subject matter to generate crystals of methionine-R-sulfoxide reductase.
  • Crystals were removed from the crystal card using a cryoloop and then cryocooled for diffraction experiments.
  • a 1.7 A data set was collected at SBC-CAT beamline 19BM located at the Advanced Photon Source at Argonne National Laboratories and the structure was subsequently solved and refined.
  • the final coordinates and structure factors were deposited to the Protein Data Bank (accession code 3CXK).
  • the crystal card of various embodiments of the subject matter is also suitable for in situ diffraction.
  • In situ diffraction allows the crystallographer to assess the quality of a crystal before being altered by the cryoprotection process. For robust crystals, it can allow complete diffraction data to be collected.
  • the crystal card is sufficiently X-ray transparent to be mounted onto the goniometer of an X-ray source for diffraction data collection at room temperature. For example, a simple test was conducted to analyze the absorption of the X-Rays by the crystal card. The beam current in the ion chamber normalized to the APS ring current (1/I 0 ) was measured with and without the crystal card inserted at a wavelength of 0.979261A (12.66099 keV).
  • the crystal card can be translated along its X and Y axis to collect data from multiple crystals to be combined for a complete data set.
  • a crystal card containing Lysozyme crystals was mounted on the goniometer head at NE-CAT beamline 24ID-C located at the Advanced Photon Source at Argonne National Laboratories. Data were collected at room temperature from three crystals in the crystal card. Crystallo graphic data are provided in Appendix A. Regarding structure determination, data sets were collected at the Advanced
  • Photon Source beamline 19BM at IOOK for methionine-R-sulfoxide reductase and beamline 24-IDC at room temperature for lysozyme.
  • Data were integrated and scaled with HKL2000.
  • intensities were integrated separately for each of the three data sets using the mosflm package.
  • the structures of lysozome and methionine-R-sulfoxide reductase were solved by molecular replacement using Molrep and PDB entries HEE and 3CEZ as the search models, respectively. Structures were refined with Refmac5 and model building was performed with Coot.
  • Parenthesis indicates values for the 2.00 A to 1.90 A resolution shell for lysozyme and 1.76 A to 1.70 A shell for methionine-R-sulf oxide reductase.

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Abstract

L'invention porte sur un système de cristallisation microcapillaire de type nanovolume qui permet le criblage de conditions de cristallisation de type volume en nanolitre dans une carte de cristaux soit qui permet à des cristaux d'être retirés pour une cryoprotection traditionnelle, soit qui permet des études de diffraction des rayons X in situ sur des cristaux de protéine qui se développent à l'intérieur. Le système intègre une formulation de cocktails de cristallisation avec préparation des expériences de cristallisation. Le système permet au chercheur de sélectionner soit un criblage à gradient dans des expériences de cristallisation pour une exploration efficace d'espace de phase de cristallisation, soit une combinaison de matrice creuse avec criblage à gradient pour exécuter un essai de cristallisation hybride complet.
PCT/US2009/047414 2008-06-13 2009-06-15 Système de cristallisation microcapillaire de type nanovolume Ceased WO2009152520A2 (fr)

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JP2011513753A JP2011525166A (ja) 2008-06-13 2009-06-15 ナノ体積マイクロキャピラリー結晶化システム
EP09763807A EP2352591A4 (fr) 2008-06-13 2009-06-15 Système de cristallisation microcapillaire de type nanovolume

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JP2014240343A (ja) 2014-12-25
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JP2011525166A (ja) 2011-09-15
WO2009152520A3 (fr) 2010-02-18

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