MXPA01008052A - Laboratory cap and well for hanging-drop crystallization methods - Google Patents

Abstract

The invention relates to a device for molecular and macromolecular crystallization. More particularly, the device comprises a well and a transparent cap for growing diffraction-quality protein crystals by conventional vapor diffusion techniques. The present device is particularly advantageous in that it allows the pre-filling of the well with a solution for transport and handling.

Description

CAVITY AND LABORATORY COVER FOR CRYSTALLIZATION METHODS BY DROP PLACEMENT Field of the Invention The present invention relates to a device for handling macromolecular and molecular crystallization. More particularly, the device comprises a cap and cavity installation for the growth of protein crystals by vapor diffusion techniques. The present device is particularly advantageous in that since A10 facilitates the pre-filling of the cavity with a solution for transport and handling before use by a technician.

BACKGROUND OF THE INVENTION Crystallography is an extremely useful tool for scientists, and therefore it is a field of research that attracts great interest. It is a powerful medium that provides the detailed and accurate description of the three-dimensional structure of the molecules, ^ fc and is very helpful in understanding its functions. The crystallography of macromolecules like proteins is used extensively nowadays, academically as well as industrially. Although the three-dimensional structures of simple proteins have been obtained through crystallization methods, it is not always easy to obtain the crystals from the macromolecules. For example, the preferred conditions for Crystallization of a given molecule can take several hundred if not thousands of trials. As a result, means and methods have been developed to perform a large number of relatively rapid tests, including drop settling and drop placement methods. All such methods use the benefit of vapor diffusion to obtain the crystals. The drop placement method is currently the most commonly used technique to explore various crystallization conditions of macromolecules, such as proteins. It comprises suspending a droplet of approximately 2 to 20 μL of solution containing the macromolecule to be crystallized and a precipitating agent, on a precipitating solution, such as 20% conventional polyethylene glycol or 40% ammonium sulfate, contained in a container or cavity. The system is sealed tightly. After a while, the vapor diffusion of the solvent or solvent mixtures between the droplet and the solution in the vessel reaches equilibrium. The final result is a reduction of water in the droplet, and an increase in the macromolecule and concentration of precipitating agent therein, thus originating the crystallization of the macromolecule under optimal conditions. The current technique for the assembly of drop settling or drop placement experiments is a long and arduous task and must be carried out by experienced and qualified technical personnel. Conventionally, a commercially available dish made of an inert thermoplastic material comprising kyL?.? & É »? Z.ii ?? z.l, .J .. 8JVtUx. "J BB * - -.i - - * z.y.t * y -". L ^^. J ^^ A _. a plurality of containers or cavities, and the precipitating solution is placed in each container or cavity manually. The macromolecule solution is thus mixed with a precipitating agent on a glass plate (coverslip) and the total is inverted over the cavities, thereby causing the macromolecule solution to overflow into the cavity. Before placing a glass plate on a cavity, the edge of each cavity is greased to ensure adequate closure. Care must be taken when placing the plate on each cavity, since the fat can easily contaminate the macromolecule solution. The crystallization process is followed with the help of a microscope. After the crystal is obtained, the glass plate is removed. Again, this must be done with great care to prevent contamination of the crystallized macromolecule with grease, and / or rupture of the glass plate. On top of that, the plates are hardly of repeated use for any experiment because the fat is difficult to remove, and some of it remains in the plates. An advantage of drop drop and drop placement methods is that they provide purification conditions for crystallization, and truly represent a microcrystallization technique. The diffusion of vapor in the settlement of drop or suspension allows the depuration of a wide range of conditions and requires a relatively small amount of macromolecules. In addition, it allows a relatively clear visualization of the results, and the eventual crystals are free, that is, they do not adhere or stick to any surface. t? Jt? ^ itt ^ Jt, Typically, several hundred experiments are required to find the proper crystallization conditions for the production of high quality crystals. According to the above, the experiments of drop settlement and drop placement are a very intense process that require experienced and qualified technical personnel. For example, multiple distribution stages and component aspirants, multiple lubrication stages, etc. must be performed. , in the experimental assembly. In addition, for each cavity, a separate coverslip must be inverted manually. The number and complexity of stages can therefore produce a wide undesirable variation in the experimental results. As stated above, fat is conventionally used to provide a seal between the cavity and the coverslip. Other ways to seal the system have been proposed. For example, the grease can be replaced with immersion oil or an adhesive tape. As with grease, these sealing means have serious drawbacks. Grease is not always easy to distribute around the upper edge of the cavity, and it is a time-consuming operation. Technicians who repeat the operation thousands of times occasionally suffer from physical pain in their hands. Other significant problems and risks arise when the glass is handled on a greasy cover slide. The cover slide sometimes breaks during the process, which can cause injury to the technician, in addition to the loss of the crystals. Immersion oil is also problematic. One has to use a certain volume of oil. Too much oil leads to contamination within the cavity, while not enough will lead to non-hermetic closure which can result in evaporation of the precipitating solution. An adhesive tape allows the simplest and fastest manipulations, but all the experiments are sealed at the end of the assembly, thus introducing the experimental variations between the 1st and 24th drop. In addition, the crystals frequently stick to the tape, making the recovery of the crystals impossible, and the operations for the recovery of the drop are also problematic. These conditions promoted the automation of the procedure. Some automated crystallization devices already exist. The well-known Cyberlab-200 ™ device distributes the solutions in the cavities, greases the upper edge of each cavity, pours the droplets on the cover slides held by a vacuum arm, and places the cover slides over the cavities. However, such an apparatus still has some drawbacks, specifically a complicated experimental setup, and the remarkable use of grease. In addition such an apparatus is extremely expensive. Relevant references in the field include 2,366,886 of E.U.; 3, 107,204 of E. U; 3,297, 184 of E.U; 3,537,956 of E.U; 3,597,326 of E.U; 3,649,464 of E.U; 3,692,498 to E. U; 3,729,382 of E.U; 3,745,091 of E.U; 3,907,505 of E. U; 4,038, 149 of E.U; 4, 154,795 of E. U; 4,495,289 of E.U; 4,917,707 of E.U; 5,271, 795 from E.U. Therefore, it would be highly desirable to develop a device for crystallizing the macromolecules that could overcome the ijt-.t-? zix xz previous deficiencies. Such a device would eliminate the requirement of external means such as grease, oil or other adhesive tape to seal the cavity and cover, and would preferably be easy to handle, either manually or automatically. Finally, the process of the experimental assembly of the device could be facilitated and accelerated greatly, while simultaneously eliminating the possible risks of contamination of the possible crystals. Finally, such a device could be useful for various crystallization processes such as drop settling or drop placement processes.

BRIEF DESCRIPTION OF THE INVENTION According to the present invention, there is provided a saucer comprising an upper surface, a lower part, a straight circumferential wall extending from the upper part to the lower part, and a plurality of cavities extending downwards from, and opening in, the upper part, to receive a precipitating solution; and a cap provided for each cavity, each cap comprising closure members for closing the cap in the cavity in a sealed manner. Such a dish is particularly advantageous for the growth of macromolecular and molecular crystals.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the perspective view of a first cavity and lid installation according to the present invention; Figure 2 illustrates a section view of a modality preferred of a cover; Figure 3 illustrates a perspective view of a tool adapted to install and remove a lid from the cavity; Figure 4 illustrates a sectional view of the tool of Figure 3; and Figures 5, 6 and 7 illustrate other embodiments of the cavity and lid installation according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION 10 It is an object of the present invention to provide a # glass forming device using the vapor diffusion method. The device comprises a cavity and a transparent cover designed to close the cavity and form a sealed volume, sealing the cavity without the need to add a sealing material such as grease, oil, adhesive tape and similar between the cavity and the lid. The lid is made of transparent material to allow the examination and monitoring of crystal growth, as well as the manipulation of the glass under a microscope. The present device therefore represents an important advance in the methods for the growth of crystals of macromolecules, especially in the field of drop settling and drop placement. Due to its simplicity, the operations of filling the cavity with the precipitating solution, placing a drop of the macromolecule solution on the lid and sealing the cavity when putting the lid on position on the cavity, can be made by any technician Ít A? Jt? L ?. I.i.-l. + T.4 you? lv., ... ~ and ** -. ^ M -? "^ ...", ",. £ .fc. " ? ^. a. ^ ..? mt < 8-i ** **. & **. " - ^ > a > -.j.it competent, and not only by experienced staff. In a preferred embodiment of the invention, a plurality of cavities are molded together, for example in a saucer comprising 4 rows of 6 cavities each, with corresponding transparent covers being provided therein. The saucer and the resulting covers can also be optionally treated with a hydrophobic agent such as a silicone agent. Due to the transparency of the lid and the lower surface of the cavity, crystallization can be followed with minimal handling, and without altering the vapor balance within each cavity. In addition, the visualization of the results under the microscope are simple because the lid is made of a translucent (clear) or transparent material. Preferably, the pan and lid material are the same, and comprise materials that can be easily molded at a reasonable cost. The material should be stable for extended periods of time to the various chemicals present in the cavity and the lid. Also the material should preferably not absorb water, and be of good optical quality to facilitate work and observation under a microscope. Exemplary suitable materials include various thermoplastics such as polystyrene, polypropylene, polycarbonate, polyacrylate, polymethacrylate, acrylonitrile-styrene copolymers, nitrile-acrylonitrile-styrene copolymers, polyphenylene oxide, phenoxy resins, etc. , polystyrene being the most preferred material. Another object of the present invention is to provide a crystal forming device that allows the manipulation of the growth of crystals under the microscope without any transfer of the cover, wherein the solutions can be added directly without any transfer of the crystals, in a non-fat environment. . Another main advantage of the device of the present invention is that once a series of experiments is complete, the saucer is easily of repeated use, simply by taking another series of caps containing a drop of a solution containing a macromolecule to be crystallized, and reinstalling the covers over the cavities. In addition, a given cap can be removed from its original cavity and closed in another containing a different precipitating solution. The invention also relates to a method for forming crystals of a macromolecule, the method comprising the steps of distributing a precipitating solution in a cavity.; forming a droplet in a cap comprising closure members for closing the cap on the cavity; and close and seal the cavity. In a preferred embodiment, a ring made of an elastomeric material such as polypropylene, an ethylene-propylene copolymer, Teflon ™ etc., is preferably provided between the cap and the cavity. In a more preferred embodiment, the cavity can be filled in advance and hermetically sealed, so that a dish is provided to a technician in a "ready to use" manner. Due to the ergonomics of the present invention, the lid easily engages in such a way that there is no need for special manual ability comparatively to the use of conventional thin, brittle, microscope coverslips. The presence of a cavity in the surface of the lid that faces the lower surface of the cavity allows the addition of liquid directly on the drop, after placing the lid upside down on a board, without the need to transfer the crystals to another cavity, thus limiting the manipulations that could ruin brittle crystals. The use of the cavity and lid installation of the present invention can be automated in a direct manner by providing the end of an automated arm with a simple clamping element having a limb provided with a structure adapted to the disengageable clamping of the cover. There is no need for the application of grease or the handling of pieces of fragile coverslips. The fastening element can also be manipulated manually by a technician, as described above. The cavity and lid installation of the present invention also finds applications in the field of cell cultures, cellular or molecular biology, etc. In a more preferred embodiment of the invention, the cavity is filled in advance and sealed with the cover. Therefore, the technician receives a "ready-to-use" installation, thereby eliminating the time-consuming operation of filling each cavity with the appropriate precipitating solution. Therefore, the buyer can order as many facilities with the same or different precipitating solutions. For shipping purposes, the cover can be replaced in the installation with a film to prevent contact of the precipitating solution with the cover. Such contact may need cleaning of the lid before its use. One can also use a lid for shipping purposes, and a different lid to carry out the experiments. It is important that the cavity is sealed to prevent evaporation and spillage of the precipitating solution, either during the loading of the pre-filled cavities, or during the experiments. Referring to the drawings illustrating the preferred embodiments of the invention, Figure 1 illustrates a cavity and lid installation 10 comprising a bottom plate or plate 12 provided (a) with a plurality of cavities 14 and corresponding covers 16. The installation 10 may also include a cover 18 used for storage or shipping purposes. The preferred shape of the cover 18 is comprised of an insert (not shown) in each corner that allows the cover to be retained on the covers without touching them. The cover 18 also allows the storage of several experiment dishes one on top of the other. The saucer 12 comprises an edge 20 extending around four side walls thereof, and is provided with gripper clamping surfaces 22 such as those described in 4.038, 149 of E. U; in two opposite side walls for easy handling of the cymbal by the technician. Claw clamping surfaces 22 are provided to prevent malfunctioning, and to greatly facilitate handling of the covered and uncovered dishes. The cover 18 comprises a section 24 adapted to engage around the claw grip surfaces 22 for proper positioning on the installation 10. Figure 2 illustrates a sectional view of the lid 16. As can be seen, the lid 16 comprises a cylindrical groove 26 in which . .y.? z-z -.:. JX.zÍX,? zi., .- z,.? yz. i .ifcjt.iii.fcj. an O 28 ring element made of an elastic material is inserted. Such material, although optional, is provided to ensure adequate closure when the lid 16 is placed on the upper edge 30 of the cavity 14. The internal surface 32 of a groove 26 has a portion or flange 34 extending to the passage of the flat surface 36 thereby forming a cavity 38. The surface 36 may be concave or convex, but the planar configuration illustrated in Figure 2 is more preferred. As stated above, the material of the lid 16 is such that it is sufficiently transparent or translucent in such a way that the lid 16 can be placed directly under a microscope for the observation and / or manipulation of the crystals. Each cover 16 comprises a pair of closure elements 40 diametrically opposed to each other and comprising a slot portion 41. The lid 16 also comprises a further edge 46 provided with a series of spacer 45 below. Once the precipitating solution is poured into the cavity 14, the technician places the lid 16 upside down on a flat surface and places a drop of the solution containing the macromolecule on the surface 36. The lid 16 is thus released cautiously, each Closing element 40 is inserted into a corresponding opening 42 provided in the upper surface 44 of the saucer 42 until the boundary of the upper edge 30 of the cavity 14 with the ring element O 28 within the groove 26 is achieved. The lid 16 is thus rotated in such a way that the closure elements 40 each slide in a groove 43 having a length smaller than that of the opening 42 and extending in a part of the periphery of the cavity 14 until the surface The upper portion of the part 41 is completely under the upper surface 44, thereby effectively sealing the cavity and keeping the lid 16 in place. In a more preferred embodiment, a section 47 of the part 41 is perforated to facilitate sliding under the upper surface 44. To ensure even better closure and maintenance of the lid in position, a small pump (not shown) is provided in the section 49 which is adapted to be placed in a corresponding notch (not shown) present below the surface 44 after full insertion of the part 41 under the surface 44. To put the lid 16 in place in the cavity 14, or for the Removal thereof, a tool 48 may be used, as illustrated in Figures 3 and 4. The tool 48 comprises a body 58 divided in part 52 molded in a manner so as to facilitate attachment by the technician or an automated arm; a cylindrical portion 54 with an outer surface 57 having a circumference slightly greater than that of the edge 46, and an inner surface 59 having a slightly smaller circumference than that of the edge 46. The tool 48 further comprises two diametrically opposed lid fasteners 60. each provided with a clamping grip 62. The clamping element 60 can be provided on the internal surface 59, directly on the edge 66, or on the external surface 57. In operation, the tool 48 is placed on the lid 16 in such a way that each claw 62 is inserted into a slot 64 cut in the edge 46 until at least one part 65 of each element 60 is assembled at the edge 46. The tool 48 is thus rotated until the clamping claws 62 mesh completely under the edge 46, and the rotation is maintained until the closure members 40 are aligned with the slots 42. The cover 16 simply stops as well. To reintroduce the lid in position, the procedure is carried out in an opposite manner. The outer surface 53 of the part 52 should be flat, so that it can be placed on a table or under a microscope in a stable manner, and allow the technician to observe and / or work on the crystals. To be able to work under a microscope directly, the surface 53 must comprise an opening that preferably corresponds to the internal diameter of the cylindrical part 54 (see Figure 4). Figure 5 illustrates another embodiment of the present invention. The cavity and lid installation 100 comprising a bottom plate or plate (not shown a-) provided (a) with a plurality of cavities 1 12 and corresponding layer 1 14, comprising a cylindrical groove 1 16 in which inserts an O 18 ring element made of an elastic material. As for the previously illustrated embodiment, the O-ring, although optional, is provided to ensure adequate closure. Each cover 1 14 comprises a pair of closure element 120 diametrically opposed to each other and comprising a flange portion 122. The cover 1 14 closes in position on the pan by inserting each closure element 120 into a corresponding aperture 124 provided in FIG. the upper surface 126 of the pan until the lower surface 128 of the lid 1 14 lies flat on the upper surface 126 of the pan. The cover 1 14 is thus rotated in such a way that the elements of closure 120 slides in a slot 130 having a length smaller than that of the aperture 124 and extending into a portion of the periphery of the cavity 12 until the top surface of the part • the flange 122 is completely under the upper surface 5 126, thereby effectively sealing the cavity and keeping the lid 1 14 in place. Figure 6 illustrates another simple variation of the present invention, wherein the upper surface 152 of a cavity 150 comprises a groove 154 together with its circumference and is adapted for receiving a ring element O 1 56 coupled to a cover 158. The section # of the groove 154 is such that it is slightly smaller than that of the element 156, such that in the insertion in the groove, a seal is formed by the closure of the lid 158 in the cavity 150 without the need of any adhesive or grease. Figure 7 illustrates still another embodiment of the invention, wherein the cap 170 is screwed into the cavity 172. This construction as well as the other illustrated in Figures 1, 2 and 5 is provided for the gradual engagement of the mechanism of closing the lid with the cavity for movement between a position of engagement and a closing position. The present cavity and lid installation is particularly suitable for both drop settlement or drop placement crystallization methods. With respect to the method of droplet settling, although not specifically illustrated in the drawings, any person skilled in the art will readily appreciate that any Conventional bead support can be inserted or molded into the cavity. ÁÁít? Ti 4-4 * MAA? .iz-á.3aÉií * á **. - ~ _ * »» > .,. ,,, "«, _, ^^ x ^ -. ^. z ^^ j? éMr ^? x ^ s &? ^^^^ aí ^? ma Examples of such droplet settlement support include vibrio drop settling rollers or Micro-Bridges ™ developed and sold by Hampton Research (Laguna Hills, California). Each cavity is filled carefully with a selected equilibrating solution. Subsequently, a drop of selected protein is deposited on the cover. The shape and texture of the lower surface can be varied to obtain optimal results for a particular protein solution to be crystallized, for example, when lower surface tension solutions, including solutions protein-containing detergents are used. The addition of • Balancing solution and the protein drops in the device can be carried out either manually or through a commercial automated pipetting apparatus, and the sealing of the lid on the solution can also be carried out manually or in an automated way. While the invention has been described in relation to the specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any of the variations, uses or adaptations of the invention by following, in general, the principles of the invention and including such tendencies of the present how they come within the usual or known practice within the subject matter to which the invention pertains, and how it can be applied in the essential features set forth above, and as follows within the scope of the appended claims. k ^ xt?. ?? x.?z Jj? .- z, .ix- ^ xzXzm ^ _x ^ z ^ tia,? S? Mz? y, .. ^, ,, ¿- ^ »^ t, tt * A? A? Au? Kab Í6xtto < ß ?? ßtH íi ^^

Claims (10)

1. CLAIMS 1. A device for forming crystals by vapor diffusion, comprising a saucer having a lower part and a straight peripheral wall extending from the lower part, said device also being provided with a number of cavities located within said saucer and opening at an upper extremity of the same to receive a precipitating solution, providing individual removable covers to cover separately and independently each of a plurality of said cavities, each of said individual removable layers having a concealed part defining a support surface for suspending a solution droplet that contains a molecule or a macromolecule to be crystallized on the precipitating solution contained in an associated of said plurality of cavities, said support surface being at least partially transparent to allow the examination and monitoring of the crystal growth, each of said tap Individuals are further provided with a first structurally engagable integral closure member with a corresponding second closure member associated with each of said plurality of cavities for releasably closing said individual removable covers in said plurality of cavities in a sealing manner without need for an operator to apply an external sealing material between the removable covers and the plurality of cavities.
2. 2. A device according to claim 1, characterized in that the cavities and the covers are cylindrical.
3. 3. A device according to claim 1, characterized in that one edge of each cavity extends above a top surface of the pan. A device according to claim 3, characterized in that the lid is tubular and said first closure member comprises closing elements equally spaced apart, each locking element being attachable to a corresponding opening in a surface of the dish adjacent to the edge of the wall. The cavity. A device according to claim 4, characterized in that each cover comprises a tubular section around an external surface thereof, thereby defining a slot therebetween which closes in an upper part thereof, the slot being adapted to receive in the same the edge of an associated cavity, the tubular section comprising the closing elements coupled thereto. A device according to claim 4 or 5, characterized in that each closure element comprises a needle extending from the top to the bottom of the lid, the needle comprising a part extending perpendicularly to the exterior of the external surface of the needle. the lid. A device according to claim 1, characterized in that an elastomeric material is inserted between each cap and the upper surface of the pan to seal the cavities. A device according to claim 5, characterized in that an elastomeric material is inserted in the groove between the edge of the cavity and the top of the slot. 9. A device according to claim 1, characterized in that each cover comprises a cavity in a surface that is? in front of the lower surface of the associated cavity and adapting to receive a drop of a solution containing macromolecule or molecule. A device according to claim 9, characterized in that the cavity has a flat bottom surface. eleven . A device according to claim 10, characterized in that the cavity comprises a circumferential wall extending perpendicularly at the flat bottom surface. 12. A device according to claim 2, characterized in that each cover comprises an edge on an external surface thereof, the edge comprising equally spaced apart slots. 13. A device according to claim 1, characterized in that it further comprises a cover placed on and around the saucer. 1
4. 4. A device according to claim 1, characterized in that it also comprises claw clamping surfaces equally spaced apart in the peripheral cavity. 1
5. 5. A device according to claim 1, characterized in that the plate and the lid are made of a translucent or transparent material. 1
6. 6. A device according to claim 1, characterized in that the material is selected from a group of materials consisting of: polystyrene, polypropylene, polycarbonate, polyacrylate, polymethacrylate, l? it & u? í? *, tx, .B * A- - y. ~ * t? al í. ~ ~ z. xx¿ * m zz .. * z. _,,. . ^ *. ^ «^ T *?» ^ And ~ x ?. » ia¡ ?? iti? x & * ~ ** j * ~ x Acrylonitrile-styrene copolymers, nitrile-acrylonitrile-styrene copolymers, polyphenylene oxide, phenoxy resins, and mixtures thereof. A device according to any of claims 1 to 16, characterized in that said first closing member and said second closing member are gradually closable between a released position and a closed position. 18. A device according to claim 17, characterized in that said closing members, first and second, are rotatably closable with another between said released position and said closed position. 19. A device according to claim 18, characterized in that said members, first and second, includes tapering the threads respectively in each of said covers and each of said cavities. A method for forming macromolecular or molecular crystals, comprising the steps of: distributing a precipitating solution in a cavity, providing a droplet of a solution containing a molecule or macromolecule in a droplet support having a first clutch closure means with a second corresponding closing means of the cavity, inverting the drop support on the cavity in such a way that the droplet is suspended on the precipitating solution contained in the cavity, and engaging said first, first and second closing means, to close simultaneously said drop holder in said cavity and sealing the cavity. twenty-one . A method according to claim 20, characterized because the gear stage includes rotating the closing means, first and second, relative to another. 22. A method according to claim 1, comprising the step of pre-filling the cavity with the precipitating solution and sealing the cavity before shipping to a comparator. •