EP2040809A2 - Mikrofluidische vorrichtung zur kristallisierung und kristallographischen analyse von molekülen - Google Patents
Mikrofluidische vorrichtung zur kristallisierung und kristallographischen analyse von molekülenInfo
- Publication number
- EP2040809A2 EP2040809A2 EP07823305A EP07823305A EP2040809A2 EP 2040809 A2 EP2040809 A2 EP 2040809A2 EP 07823305 A EP07823305 A EP 07823305A EP 07823305 A EP07823305 A EP 07823305A EP 2040809 A2 EP2040809 A2 EP 2040809A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- crystallization
- microfluidic device
- crystallization chamber
- equal
- chamber
- 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.)
- Withdrawn
Links
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- OCQZXMCGTAWGEQ-UHFFFAOYSA-N prop-2-enamide;n-[(prop-2-enoylamino)methyl]prop-2-enamide Chemical compound NC(=O)C=C.C=CC(=O)NCNC(=O)C=C OCQZXMCGTAWGEQ-UHFFFAOYSA-N 0.000 claims description 2
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- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 description 7
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- 241000287828 Gallus gallus Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 2
- 229920002799 BoPET Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- CGVLVOOFCGWBCS-RGDJUOJXSA-N n-octyl β-d-thioglucopyranoside Chemical compound CCCCCCCCS[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O CGVLVOOFCGWBCS-RGDJUOJXSA-N 0.000 description 2
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- 238000011282 treatment Methods 0.000 description 2
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- 108010052285 Membrane Proteins Proteins 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 238000001015 X-ray lithography Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- TXGJTWACJNYNOJ-UHFFFAOYSA-N hexane-2,4-diol Chemical compound CCC(O)CC(C)O TXGJTWACJNYNOJ-UHFFFAOYSA-N 0.000 description 1
- 238000007731 hot pressing Methods 0.000 description 1
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- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/0004—Crystallisation cooling by heat exchange
- B01D9/0013—Crystallisation cooling by heat exchange by indirect heat exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/005—Selection of auxiliary, e.g. for control of crystallisation nuclei, of crystal growth, of adherence to walls; Arrangements for introduction thereof
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/005—Selection of auxiliary, e.g. for control of crystallisation nuclei, of crystal growth, of adherence to walls; Arrangements for introduction thereof
- B01D9/0054—Use of anti-solvent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/0077—Screening for crystallisation conditions or for crystal forms
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
-
- 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
- B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
-
- 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
- B01L3/502707—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 the manufacture of the container or its components
Definitions
- the present invention relates to the field of crystallization. It particularly relates to a microfluidic device for the crystallization and crystallographic analysis of molecules, including biological.
- the crystallization of molecules is a complex multi-parametric process involving a large number of o physicochemical and biochemical variables.
- the search and optimization of conditions making it possible to obtain quality crystals may require a large quantity of substances to be crystallized, such as biomolecules or synthetic compounds, which material can be very expensive.
- miniaturized devices are already used for the crystallization of molecules, they can be expensive, insufficiently reliable, difficult to use, not be suitable both for screening and optimization of crystallization conditions, or not allow an analysis of the crystals in situ, in particular by X-ray diffraction.
- the inventors have now developed a device for solving all or part of the problems mentioned above.
- the subject of the invention is a microfluidic device comprising at least one crystallization chamber capable of comprising a solution in which at least one compound is present according to a concentration gradient and in which the geometry of said crystallization chamber allows to limit the phenomena of convection.
- the invention also relates to a crystallization chamber for receiving a solution comprising a crystallization agent. Consequently, the invention also relates to a crystallization chamber comprising or not this solution, as well as to a microfluidic device comprising at least one chamber with or without said solution.
- microfluidic device means a miniaturized apparatus using very small quantities of liquid sample, of the order of one microliter, or even less than one microliter.
- the geometry and the reduced dimensions of said crystallization chamber minimize the convection movements in the solutions, as observed for example in interferometry.
- the microfluidic environment thus promotes a more homogeneous crystalline growth in a medium that limits convection phenomena, or even free of convection phenomena.
- crystallization chamber means a chamber adapted to the crystallization of molecules, in particular a space that is impermeable to liquids and gases, and most particularly to water, to volatile solvents such as alcohols, with water vapor and / or air.
- the crystallization chamber is connected to at least one reservoir (R1).
- reservoir in the sense of the present invention, a sealed chamber for containing a fluid whose volume may be greater than that of the crystallization chamber.
- the crystallization chamber according to the invention may be arranged so as to allow crystallization by batch, by counter-diffusion, preferably by counter-diffusion.
- the crystallization chamber according to the invention has a section or a diameter, less than or equal to 400 microns, in particular less than or equal to 300 microns, in particular less than or equal to 200 microns, or even less than or equal to 100 microns.
- the crystallization chamber according to the invention may have a length greater than or equal to 10 mm, in particular greater than or equal to 30 mm.
- the crystallization chamber according to the invention may have a length / width ratio greater than or equal to 10, in particular greater than or equal to 100, and most preferably greater than or equal to 1000.
- the crystallization chamber according to the invention may have a square, rectangular, hemispherical, triangular or tubular section, in particular square or rectangular.
- the geometry of the crystallization chamber according to the invention comprises means making it possible to improve the crystallization, in particular to increase the number of crystals formed, in particular by grafting chemical functions, fillers, enzyme substrates and or ligands, or by particular geometrical arrangements, such as baffles, roughnesses or surface irregularities.
- the device according to the invention can in particular allow the crystallization of macromolecules such as enzymes, nucleic acids or membrane proteins.
- the crystallization chamber according to the invention can be produced by at least one lithography, micro-machining, injection molding, compression molding, hot pressing or cold casting and / or printing process.
- Lithography process means a method derived from the semiconductor industry, the principle of which general consists in creating an image on a substrate covered with a layer of sensitive material as described by Chang and Sze (1996, ULSI technology, MacGraw-Hill International Editions) and by Xia and Whitesides (1998, Annu Rev. Mater Sci., 28, 153-184).
- lithography processes include photolithography, X-ray lithography, EUV lithography, electronic lithography, ion lithography and nano-printing lithography. Such techniques can be easily identified by the skilled person using his general knowledge.
- Material-removing micromachining methods may be based on the use of a cutting tool or a laser.
- the material (s) constituting the crystallization chamber according to the invention and its surroundings may be "transparent", in particular allowing the visible spectrum to be passed. or the signal diffracted by the crystal.
- the material (s) may especially be chosen from the group comprising polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate and cycloolefin copolymer (COC). ) and the resin SU8, preferably polymethylmethacrylate.
- the device according to the invention can allow the kinetic monitoring of the growth of the crystals, for example by videomicroscopy, the formation of a concentration gradient, in particular by interferometry.
- At least a portion of the volume defined by the crystallization chamber according to the invention comprises a gel.
- gel means a two-phase medium consisting of a three-dimensional network of crosslinked polymer impregnated with a liquid such as a molecular solution to be crystallized.
- the crosslinking may be of physical origin in the case for example of an agarose gel, cellulose and their derivatives, or chemical in the case for example of a silica gel or acrylamide-bisacrylamide.
- Said gel according to the invention may be chosen from the group comprising agarose, cellulose gels and / or their derivatives, of silica and / or of acrylamide-bisacryamide.
- At least a portion of the volume defined by an end of the crystallization chamber according to the invention comprises a gel.
- the entire volume defined by the crystallization chamber according to the invention comprises a gel.
- the crystallization chamber according to the invention may have on at least a portion of its internal surface means for increasing the wettability.
- wettability in the sense of the invention means the ability of a. surface to be wetted with an aqueous solution, which results in the observation of a contact angle of less than 90 °.
- the invention is remarkable in that in the case where the crystallization chamber has a hydrophobic surface, which is the case with elastomers or plastics, the addition of wetting agent such as surfactants makes it possible to spontaneously penetrate the aqueous solutions, including those comprising proteins, in the crystallization chamber, especially when it is in the form of channels.
- wetting agent such as surfactants
- An alternative according to the invention consists in chemically modifying the surface of the chamber so as to render it more hydrophilic.
- surface modifications by physical or chemical treatments or a combination of the two, for example plasmas, in particular oxygen, ozone and ultraviolet treatments , by ions, the adsorption of surfactants, the grafting of hydrophilic groups;
- the crystallization chamber is capable of being filled by capillarity.
- capillarity means a phenomenon resulting in the rise of a fluid in a small diameter tube.
- solution in the sense of the present invention, a homogeneous liquid comprising at least one solvent and a solute, said solute being dissolved in the solvent.
- the term "compound” means a chemical substance.
- said compound is a crystallization agent.
- crystallization agent in the sense of the present invention means an organic or inorganic compound, natural or synthetic, promoting the crystallization of molecules.
- crystallizing agents examples include salts such as sodium chloride, ammonium sulphate, alcohols such as methyl-2,4-pentanediol, ethanol, polymers such as polyethylene glycols and their derivatives and polyamines.
- concentration gradient in the sense of the present invention, the variation of the concentration of a compound of the most concentrated medium to the least concentrated medium.
- the microfluidic device makes it possible to obtain a concentration gradient of compound, and especially of crystallization agent, ranging from a concentration of less than or equal to 25%, in particular 20% or even 15%, in particular 10%, particularly 5%, or even 0%, at a concentration greater than or equal to 50%, even 75%, especially 85%, in particular 95% and especially 100% of the saturation concentration of compound and in particular of crystallization agent.
- said concentration gradient is established on at least 20% of the length, in particular at least 40% of the length, in particular at least 60% of the length, especially at least 80% of the length.
- the device according to the invention makes it possible to obtain a very large variety, a continuum, of crystallization conditions.
- This device makes it possible particularly to obtain a continuous or almost continuous variation of the conditions.
- the device according to the invention comprises several crystallization chambers
- said compound, in particular the crystallization agent is present at a different concentration in each crystallization chamber.
- this device when a crystallization condition is identified, this device can be used to optimize it.
- the device according to the invention by limiting the convection phenomena, can make it possible to obtain quality crystals with very small quantities of material to be crystallized.
- the device according to the invention can allow the crystallization of molecules in a medium free of air and / or gas leading to the degradation of compounds, and in particular molecules to be crystallized. This can thus allow the crystallization of sensitive molecules, especially oxidation.
- the device according to the invention may comprise at least one solution comprising a surfactant substance, in particular chosen from the group comprising nonionic and zwitterionic surfactants, in particular making it possible to solubilize the molecules to be crystallized.
- a surfactant substance in particular chosen from the group comprising nonionic and zwitterionic surfactants, in particular making it possible to solubilize the molecules to be crystallized.
- surfactant substance means a chemical compound having surfactant properties.
- surfactants examples include octylglucoside, octylthioglucoside, nonylglucoside, LDAO (lauryl-diamine oxide), Triton X-100® (polyoxyethylene octyl phenyl ether), CHAPS (acid 3 ((3-cholamidopropyl) dimethylammonio) propanesulfonic acid) and their derivatives, in particular octylglucoside.
- the concentration of surfactant substance can vary depending on the product chosen, especially from 1 to 100% or more of the critical micelle concentration (CMC).
- the CMC in water of octylglucoside is 20 mM
- that of octylthioglucoside is 6.5 mM
- nonylglucoside is 9.5 mM
- LDAO is 2 mM
- Triton X-100® is 0.9 mM
- CHAPS is 8 mM.
- the device according to the invention is devoid of:
- the device according to the invention can be easy to use, have improved reliability and / or have a reduced production cost.
- the device according to the invention can allow an in situ analysis of the crystals present in the crystallization chamber by X-ray diffraction.
- the device according to the invention can be transparent or translucent to light, in particular to enable observation of the crystals with the naked eye, in optical magnification, especially in optical magnification.
- said solution according to the invention also comprises at least one molecule of interest, of chemical, biological, medical and / or pharmaceutical origin, in particular an inorganic or organic molecule, a natural or synthetic macromolecule, in particular chosen in the group comprising nucleic acids, proteins, supramolecular complexes and viruses.
- the microfluidic device according to the invention may comprise means making it possible to obtain a given temperature throughout the device or in at least one crystallization chamber.
- the microfluidic device according to the invention may comprise means for obtaining a temperature gradient in at least a portion of at least one crystallization chamber, in particular over the entire length of at least one crystallization chamber and particularly in the entire device according to the invention.
- Peltier effect is meant a heat displacement effect in the presence of electric current in conductive materials of different natures linked by junctions. One of the junctions then cools slightly while the other junction heats up.
- the microfluidic device according to the invention may comprise means for obtaining a temperature gradient in at least a portion of at least one crystallization chamber, or even over the entire length of at least one crystallization chamber.
- thermoelectric elements As examples of means for obtaining a temperature gradient in at least a portion of at least one crystallization chamber, or even over the entire length of at least one crystallization chamber, mention may be made of the Peltier elements.
- the invention also relates to the use of the device according to the invention for one of the following applications:
- the device according to the invention can be used to screen and optimize the crystallization conditions of molecules. . . •
- the subject of the invention is also the use of the device according to the invention in a device allowing X-ray diffraction analysis of the crystals present in the crystallization chamber.
- the device according to the invention can be used to analyze crystals in situ without handling which can deteriorate their quality.
- the invention also relates to a crystallization process comprising at least the steps of:
- said crystallization chamber is included in a device according to the invention.
- the crystallization process according to the invention further comprises the step of:
- Figure 1 illustrates PDMS devices according to the invention.
- Figure IA illustrates in the form of a drawing a mask having 3 types of crystallization chamber geometries in the form of isolated channels, comb and tree.
- Figure 1B depicts a PDMS substrate having four tree-shaped devices molded by the casting method.
- FIG. 2 illustrates in the form of diagrams three types of devices according to the invention.
- FIG. 2A illustrates a device with a total thickness of 4-5 mm, formed by a layer of PDMS (represented by stripes) in which the crystallization chambers in the form of channels are molded and which are closed by bonding a second layer of PDMS.
- Figure 2B illustrates a device formed by a layer of PDMS 0.5-1 mm thick, in which are molded the crystallization chambers in the form of channels and which are closed by a transparent plastic film (shown in dark gray) .
- the PDMS layer is stiffened by a PMMA support layer (shown in light gray).
- FIG. 2C illustrates a device of thickness 0.25 mm, formed by a PMMA layer in which the crystallization chambers are molded in the form of channels and which are closed by a transparent plastic film.
- Figure 3 illustrates the filling of a device according to the invention in PDMS.
- Figure 4 illustrates in the form of photos the formation of turnip yellow mosaic virus (VMJN), thaumatin, hen and turkey lysozyme crystals in devices. according to the invention.
- FIG. 5 illustrates • positioning the device according to the invention on a synchrotron beam line for an X-ray analysis
- the device was fixed on a standard microplate (NUNC 96-well plate to removable rows), the set being placed in the X-ray beam at 200 mm from the MAR CCD detector and held by the gripper of the manipulator arm of a robot (Staubli, France).
- FIG. 6 (A to D) illustrates the in situ analysis of hen lysozyme crystals by X-ray diffraction.
- FIG. 6A shows a device according to the invention in PMMA whose crystallization chambers are arranged in a tree structure. The device is fixed on a microplate and held by a clamp.
- Figure 6B shows a chicken lysozyme crystal observed via an axial view camera.
- Figure 6C is a diffraction pattern with ranges of resolution are indicated by circles at 2.1A, 2.8A, 4.3
- Figure 6D shows an electron density map (at 2.15 ⁇ resolution) with the atomic model of the protein.
- Microfluidic devices have been made of polydimethylsiloxane (PDMS) in four successive steps:
- a mask on transparent film was obtained by laser printing.
- a thick SU8 resin mold was then made by photolitography from said mask (FIG. 1B).
- the crystallization chambers are then sealed by gluing a second layer of PDMS or a clear plastic film such as ViewSeal®, ClearSeal®, Mylar®.
- Crystallization chambers in the form of channels either isolated, comb-shaped or tree-shaped.
- Figure 1B illustrates the molding of four PDMS devices whose crystallization chambers have a tree geometry.
- PMMA polymethylmethacrylate
- Devices according to the invention were made of polymethyl methacrylate (PMMA) by laser ablation (etching of crystallization chambers in a 250 ⁇ m layer of PMMA). The crystallization chambers were then closed with a transparent plastic film (FIG. 2 C).
- PMMA polymethyl methacrylate
- the devices according to the invention manufactured in PMMA have proved particularly well suited to crystallographic analysis, in particular by X-ray diffraction, and offer numerous advantages over PDMS devices.
- the crystallization chambers of the device according to the invention have been filled by capillarity, in particular according to two techniques:
- a solution comprising a crystallizing agent was then deposited at another end of the crystallization chamber.
- a drop of solution comprising a molecule to be crystallized and a surfactant such as octylglucoside (0.5% w / v) was deposited at the end a crystallization chamber which has filled by capillarity.
- a solution comprising a gelling agent such as agarose (2% w / v) was then deposited at another end of said crystallization chamber.
- the surfactant substance made it possible to stabilize the molecules, especially the macromolecules.
- the gelling agent made it possible to immobilize the solutions and the crystals in the crystallization chambers. In addition, it has further reduced the convection phenomenon and thus promote the growth of quality crystals.
- a layer of PDMS comprising channel crystallization chambers was deposited on a thin PMMA plate (C) placed on the opposite side of the channels. These are closed with a clear plastic film (D) such as ViewSeal®, ClearSeal® and Mylar®. This assembly was then screwed onto a 5 mm thick PMMA support (B ').
- a drop of solution comprising a molecule to crystallize, a gelling agent such as agarose (0.2% - 0.5% w / v) and a detergent such as octylglucoside (0.5% w / v) a was deposited at the end of the tree of channels that have filled by capillarity (F).
- the assembly (A) was then sandwiched between the two PMMA screwable plates (B and B ') and a solution comprising a crystallizing agent was then deposited in tanks connected to the crystallization chambers.
- the assembly was then sealed with a transparent film to seal it.
- Thaumatin crystals (22kDa) are shown in Figures 4A and 4C.
- the concentration gradient of the crystallizing agent was established by diffusion from right to left.
- the crystal size increases and their number decreases as the concentration of crystallizing agent decreases "along the crystallization chamber in channel-shaped.
- Figure 4C is a close up view crystals thaumatin as bipyramids obtained 3 days in a crystallization chamber according to the invention, in the form of a channel having a section of 100 microns.
- Virus crystals of VMJN, turnip yellow mosaic virus (5.10 6 kDa) are shown in Figure 4B.
- Quadratic crystals of chicken lysozyme obtained in a PDMS device comprising crystallization chambers in the form of isolated channels are shown in Figure 4D. The crystals are clearly visible in polarized light.
- Hexagonal crystals of turkey lysozyme obtained in a PDMS device comprising crystallization chambers in the form of tree channels are shown in Figure 4E.
- the crystals are clearly visible when polarizer and analyzer are crossed (photo in the inset).
- the devices according to the invention in PDMS and PMMA are sufficiently transparent to allow observation of the crystals with the naked eye or microscope, including polarized light.
- the crystals obtained in the devices according to the invention have sizes greater than 50 ⁇ m and therefore compatible with a direct analysis by X-ray diffraction.
- agent X 100 mM acetate-Na pH 4.6, I M NaCl, 30% PEG 3350
- a set of thirty successive images collected on one of the crystals made it possible to calculate an electronic density map at a resolution of 2, 15A and determine the three-dimensional structure of the protein (Figure 6D).
- the device according to the invention makes it possible to obtain quality crystals with a small amount of sample to be crystallized.
- the device according to the invention allows both the screening and the optimization of the crystallization conditions, the monitoring by video-microscopy and the in-situ analysis of the crystals by X-ray diffraction.
- the simplicity of operation and the geometry devices should facilitate the automation of all stages, especially in the context of high throughput applications for structural genomics.
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- Enzymes And Modification Thereof (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
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Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR0606583A FR2903916B1 (fr) | 2006-07-19 | 2006-07-19 | Dispositif microfluidique pour la cristallisation et l'analyse cristallographique de molecules |
PCT/FR2007/001243 WO2008009823A2 (fr) | 2006-07-19 | 2007-07-19 | Dispositif microfluidique pour la cristallisation et l'analyse cristallographique de molecules |
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EP2040809A2 true EP2040809A2 (de) | 2009-04-01 |
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EP07823305A Withdrawn EP2040809A2 (de) | 2006-07-19 | 2007-07-19 | Mikrofluidische vorrichtung zur kristallisierung und kristallographischen analyse von molekülen |
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US (1) | US20090311666A1 (de) |
EP (1) | EP2040809A2 (de) |
JP (1) | JP2009543573A (de) |
CA (1) | CA2658403A1 (de) |
FR (1) | FR2903916B1 (de) |
WO (1) | WO2008009823A2 (de) |
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US20110195515A1 (en) * | 2008-06-20 | 2011-08-11 | Universitat Zurich | Capillary Plate and Method for Growth and Analysis of Macromolecular Crystals |
CN109634062A (zh) * | 2018-07-18 | 2019-04-16 | 上海应用技术大学 | 基于整形x射线移动曝光的微台阶加工装置及方法 |
CN115138402A (zh) * | 2021-03-31 | 2022-10-04 | 中国科学院深圳先进技术研究院 | 一种能够设置化学浓度梯度的微流控芯片及其制备方法和应用 |
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DE3941098C2 (de) * | 1989-12-13 | 1994-02-03 | Dornier Gmbh | Vorrichtung zur Herstellung von Kristallen |
US7195670B2 (en) * | 2000-06-27 | 2007-03-27 | California Institute Of Technology | High throughput screening of crystallization of materials |
US7244402B2 (en) * | 2001-04-06 | 2007-07-17 | California Institute Of Technology | Microfluidic protein crystallography |
WO2002012597A1 (es) * | 2000-07-28 | 2002-02-14 | Consejo Superior De Investigaciones Cientificas | Dispositivo y procedimiento para el crecimiento de cristales en contradifusion |
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- 2007-07-19 US US12/374,201 patent/US20090311666A1/en not_active Abandoned
- 2007-07-19 JP JP2009520013A patent/JP2009543573A/ja not_active Withdrawn
- 2007-07-19 WO PCT/FR2007/001243 patent/WO2008009823A2/fr active Application Filing
- 2007-07-19 CA CA002658403A patent/CA2658403A1/fr not_active Abandoned
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US20090311666A1 (en) | 2009-12-17 |
CA2658403A1 (fr) | 2008-01-24 |
WO2008009823A3 (fr) | 2008-03-27 |
FR2903916A1 (fr) | 2008-01-25 |
FR2903916B1 (fr) | 2009-04-03 |
WO2008009823A2 (fr) | 2008-01-24 |
JP2009543573A (ja) | 2009-12-10 |
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