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:
FIELD OF THE INVENTION 
     The present invention relates to a device for handling molecular and macromolecular crystallization. More particularly, the device comprises a well and a cap assembly for growing protein crystals by conventional vapor diffusion techniques. The present device is particularly advantageous in that it facilitates the pre-filling of the well with a solution for transport and handling prior to utilization by a technician. 
     BACKGROUND OF THE INVENTION 
     Crystallography is an extremely useful tool for scientists, and is therefore a field of research attracting a lot of interest. It is a powerful means that provides precise and detailed description of the three-dimensional structure of the molecules, and is of great help in the understanding of their functions. Crystallography of macromolecules like proteins is extensively used today, academically as well as industrially. 
     Although three-dimensional structures of simple proteins have been obtained through crystallization methods, it is not always easy to obtain crystals from macromolecules. For example, the preferred conditions for the crystallization of a given molecule can take several hundreds if not thousands of trials. As a result, means and methods have been developed to perform a great number of trials relatively quickly, including hanging-drop and sitting-drop methods. All such methods use the benefit of vapor diffusion to obtain the crystals. 
     The hanging-drop method is currently the most commonly used technique for scanning 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, over a precipitating solution, such as conventional polyethylene glycol 20% or ammonium sulfate 40%, contained in a reservoir or well. The system is then sealed hermetically. After a while, vapor diffusion of the solvent or solvent mixtures between the droplet and the solution in the reservoir reaches equilibrium. The end result is a decrease of water in the droplet, and an increase of the macromolecule and precipitating agent concentration therein, thus causing crystallization of the macromolecule in optimized conditions. The actual technique for the set up of the hanging-drop or sitting-drop experiments is a long and arduous work and has to be performed by qualified and skilled technical personnel. 
     Conventionally, a commercially available tray made of an inert thermoplastic is material comprising a plurality of reservoirs or wells is prepared, and the precipitating solution is placed in each reservoir or well manually. The macromolecule solution is then mixed with a precipitating agent on a glass plate (coverglass) and the whole is inverted over the wells, thus making the macromolecule solution overhanging the well. Prior to placing a glass plate over a well, the rim of each well is greased to ensure a proper seal. Care must be taken when placing the plate over each well, since the grease 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 breaking of the glass plate. On top of that, the plates are hardly reusable for any experiment because the grease is hard to remove, and some of it remains on the plates. 
     An advantage of the hanging-drop and sitting-drop methods is that they provide screening conditions for crystallization, and truly represent a microcrystallization technique. The vapor diffusion in the hanging or sitting drop allows screening of a large range of conditions and necessitates a relatively small amount of macromolecules. Further, it allows a relatively clear visualization of the results, and the eventual crystals are free, i.e., they do not adhere or are stuck to any surface. 
     Typically, several hundreds of experiments are required to find appropriate crystallizing conditions for the production of high quality crystals. Accordingly, hanging-drop and sitting-drop experiments are a very labor-intensive process demanding skilled and experienced technical personnel. For example, multiple aspirating and dispensing steps of components, multiple greasing steps etc. must be performed in the experimental set up. Further, for each well, a separate coverglass must be manually inverted. The number and complexity of steps can therefore produce an undesirable wide variation in experimental results. 
     As stated above, grease is conventionally used to provide a seal between the well and the coverglass. Other ways for sealing the system have been proposed. For example, 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 dispense around the upper rim of the well, and is a time consuming operation. Technicians repeating the operation thousands of times occasionally suffer physical pain to their hands. Other significant problems and risks are present when manipulating the crystal on a greasy cover slide. The cover slide sometimes breaks during the process, which may cause injury to the technician, in addition to loosing the crystals. The immersion oil is also problematic. One has to use a determined volume of oil. Too much oil leads to contamination within the well, while not enough will lead to non-hermetic seal that may result in the evaporation of the precipitating solution. An adhesive tape allows quicker and simpler manipulations, but all experiments are sealed at the end of the set-up, thus introducing experimental variations between the 1 st  and the 24 th  drop. Further, crystals often stick to the tape, rendering impossible the recovery of the crystals, and the operations for the recovery of the drop are also problematic. 
     These conditions promoted the robotization of the procedure. Some automated crystallization devices already exist. The well-known Cyberlab-200™ apparatus dispenses solutions in wells, greases the upper rim of each well, pours droplets on cover slides held by a vacuum arm, and places the cover slides over the wells. However, such apparatus still has some drawbacks, namely a complicated experimental set-up, and the notable use of grease. Further such apparatus is extremely expensive. 
     Relevant references in the field include U.S. Pat. No. 2,366,886; U.S. Pat. No. 3,107,204; U.S. Pat. No. 3,297,184; U.S. Pat. No. 3,537,956; U.S. Pat. No. 3,597,326; U.S. Pat. No. 3,649,464; U.S. Pat. No. 3,692,498; U.S. Pat. No. 3,729,382; U.S. Pat. No. 3,745,091; U.S. Pat. No. 3,907,505; U.S. Pat. No. 4,038,149; U.S. Pat. No. 4,154,795; U.S. Pat. No. 4,495,289; U.S. Pat. No. 4,917,707; U.S. Pat. No. 5,271,795; 
     It would therefore be highly desirable to develop a device for crystallizing macromolecules that would overcome the above deficiencies. Such device would eliminate the requirement of external means like grease, oil or an adhesive tape to seal the well and the cover, and would preferably be easy to manipulate, either manually or automatically. Ultimately, the process of experimental set up of the device would be greatly facilitated and accelerated, while simultaneously eliminating possible risks of contamination of the eventual crystals. Finally, such device should be usable for various crystallization processes such as hanging-drop or sitting-drop processes. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, there is provided a tray comprising a top surface, a bottom, an upright circumferential wall extending from the top surface to the bottom, and a plurality of wells extending downwardly from, and being open at, the top surface, for receiving a precipitating solution; and a cap provided for each well, each cap comprising locking members for locking the cap onto the well in a sealed manner. Such tray is particularly advantageous for growing molecular and macromolecular crystals. 
    
    
     
       IN THE DRAWINGS 
         FIG. 1  illustrates perspective view of a first cap and well assembly in accordance with the present invention; 
         FIG. 2  illustrates a section view of a preferred embodiment of a cap; 
         FIG. 3  illustrates a perspective view of a tool adapted to install and remove a cap from the well; 
         FIG. 4  illustrates a section view of the tool of  FIG. 3 ; and 
         FIGS. 5 ,  6  and  7  illustrate other embodiments of the cap and well assembly according to the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     It is an object of the present invention to provide a crystal forming device using vapor diffusion method. The device comprises a well and a transparent cap designed to close the well and form a sealed volume, the well being sealed without the need to add a sealing material like grease, oil, adhesive tape and the like between the well and the cap. The cap is made of transparent material to allow examination and monitoring of crystal growth, as well as manipulation of the crystal under a microscope. The present device therefore represents an important advance in methods for growing crystals of macromolecules, especially in the field of hanging-drop and sitting-drop. 
     Because of its simplicity, the operations of filling the well with the precipitating solution, placing a drop of the macromolecule solution onto the cap and sealing the well by putting the cap in position over the well can be accomplished by any competent technician, and not only skilled personnel. 
     In a preferred embodiment of the invention, a plurality of wells are molded together, for example in a tray comprising 4 rows of 6 wells each, with corresponding transparent caps are provided thereon. The resulting tray and caps may also be optionally treated with a hydrophobic agent such as a siliconing agent. 
     Because of the transparency of the cap and of the bottom surface of the well, crystallization can be followed with minimal handling, and without disturbing the vapor equilibrium within each well. Further, visualization of the results under the microscope are simple because the cap is made of a transparent or translucent (clear) material. 
     Preferably, the material of the tray and the cap are the same, and comprise materials that can be molded easily at a reasonable cost. The material should be stable for extended periods of time towards the various chemical products present in the well and onto the cap. The material should also preferably not absorb water, and be good optical quality to facilitate work and observation under a microscope. Example suitable materials include various thermoplastics such as polystyrene, polypropylene, polycarbonate, polyacrylate, polymethacrylate, acrylonitrile-styrene copolymers, nitrile-acrylonitrile-styrene copolymers, polyphenyleneoxide, plienoxy resins, etc., the most preferred material being polystyrene. 
     It is another object of the present invention to provide a crystal-forming device that allows the manipulation of the growth crystals under the microscope without any transfer from the cap, where solutions can be added directly without any transfer of the crystals, in a greaseless environment. 
     Another major advantage of the device of the present invention is that once a series of experiments is completed, the tray is readily reusable, simply by taking another series of caps containing a drop of a solution containing a macromolecule to be crystallized, and reinstalling the caps over the wells. Further, a given cap may be removed from its original well and locked onto another one containing a different precipitating solution. 
     The invention is also concerned with a method for forming crystals of a macromolecule, the method comprising the steps of dispensing a precipitating solution in a well; forming a droplet in a cap comprising locking members to lock the cap over the well; and locking and sealing the well. In a preferred embodiment, a ring made of an elastomeric material like polypropylene, an ethylene-propylene copolymer, Teflon™ etc., is preferably provided between the cap and the well. In a further preferred embodiment, the well can be filled in advance and tightly sealed, so that they a tray is provided to a technician in a “ready-to-use” manner. 
     Because of the ergonomics of the present invention, the cap is engaged easily so that there is no need for special manual dexterity comparatively to the use of conventional thin, fragile, microscope coverglasses. The presence of a cavity in the surface of the cap facing the bottom surface of the well allows the addition of liquid directly over the drop, after placing the cap upside down on a table, without the need to transfer the crystals to another well, thus limiting the manipulations that might ruin the fragile crystals. 
     The use of the cap and well assembly of the present invention can be automated in a straightforward manner by providing the extremity of an automated arm with a simple grip element having an end provided with a structure adapted to releasably grip the cap. There is no need for the application of grease or the manipulation of fragile coverglass pieces. The grip element may also be manipulated manually by a technician, as described hereinbelow. 
     The cap and well assembly of the present invention also finds applications in the field of cell cultures, molecular or cellular biology etc. 
     In a most preferred embodiment of the invention, the well is filled beforehand and sealed with the cap. The technician therefore receives a “ready-to-use” assembly, thus eliminating the time-consuming operation of filling each well with the appropriate precipitating solution. The buyer may therefore order as many assemblies as desired with the same or different precipitating solutions. For shipping purposes, the cap may be replaced on the assembly with a film to prevent contact of the precipitating solution with the cap. Such contact would necessitate the cleaning of the cap prior to its use. One may also use a cap for shipping purposes, and a different cap to carry out the experiments. It is important that the well be sealed to avoid evaporation and spilling of the precipitating solution, either during shipment of the pre-filled wells, or during the experiments. 
     Referring to the drawings which illustrate preferred embodiments of the invention,  FIG. 1  illustrates a cap and well assembly  10  which comprises a tray or base plate  12  provided with a plurality of wells  14  and corresponding caps  16 . Assembly  10  may also include a cover  18  used for shipping or storage purposes. The preferred form of cover  18  comprises with an insertion (not shown) at each corner that allows retention of the cover over the caps without touching them. Cover  18  further allows the storage of several trays of experiments one on top of the other. Tray  12  comprises a rim  20  extending about the four side walls thereof, and is provided with finger grip surfaces  22  such as those described in U.S. Pat. No. 4,038,149, on two opposed side walls for easier handling of the tray by the technician. Finger grip surfaces  22  are provided to avoid mishaps, and greatly facilitate handling of covered and uncovered trays. Cover  18  comprises a section  24  adapted to engage around finger grip surfaces  22  for proper fitting over assembly  10 . 
       FIG. 2  illustrates a section view of cap  16 . As it can be seen, cap  16  comprises a cylindrical slot  26  into which is inserted an O-ring element  28  made of a resilient material. Such material, although optional, is provided to ensure an appropriate seal when cap  16  is fitted over upper rim  30  of well  14 . The inner surface  32  of slot  26  has a portion or ridge  34  extending passed the planar surface  36  thereby forming a cavity  38 . Surface  36  may be concave or convex, but the planar configuration illustrated on  FIG. 2  is much preferred. As stated above, the material of cap  16  is such that it is sufficiently transparent or translucent so that cap  16  can be placed directly under a microscope for observation and/or manipulation of the crystals. 
     Each cap  16  comprises a pair of locking element  40  diametrically opposed to each other and comprising a ridge portion  41 . Cap  16  also comprises a further rim  46  provided with a series of spacer  45  underneath. Once the precipitating solution is poured into well  14 , the technician puts cap  16  upside down on a flat surface and places a drop of the macromolecule-containing solution onto surface  36 . Cap  16  is then flipped over cautiously, and each locking element  40  is inserted into a corresponding opening  42  provided onto the upper surface  44  of tray  12  until the abutment of upper rim  30  of well  14  with the O-ring element  28  inside slot  26  is achieved. Cap  16  is then rotated so that locking elements  40  slide each into a slot  43  having a width smaller than that of opening  42  and extending on a portion of the periphery of well  14  until the upper surface of portion  41  is entirely under upper surface  44 , thereby efficiently sealing well and maintaining cap  16  in place. In a most preferred embodiment, a section  47  of portion  41  is tapered to facilitate sliding under upper surface  44 . To ensure even better locking and maintenance of the cap in position, a small bump (not shown) is provided onto section  49  that is adapted to fit into a corresponding recess (not shown) present under surface  44  after complete insertion of portion  41  under surface  44 . 
     To put cap  16  in place onto well  14 , or for removal therefrom, a tool  48  may be used, as illustrated in  FIGS. 3 and 4 . Tool  48  comprises a body  50  divided in a portion  52  shaped in a manner such as to facilitate holding by the technician or an automated arm; a cylindrical portion  54  with an external surface  57  having a circumference slightly bigger than that of rim  46 , and an internal surface  59  having a circumference slightly smaller than that of rim  46 . Tool  48  further comprises two diametrically opposed cap gripping elements  60  each provided with a gripping finger  62 . The gripping element  60  can be provided onto internal surface  59 , directly on rim  66 , or onto external surface  57 . In operation, tool  48  is placed over cap  16  so that each finger  62  is inserted into a slot  64  cut into rim  46  until at least a portion  65  of each element  60  is abutted onto rim  46 . Tool  48  is then rotated until gripping fingers  62  are completely engaged under rim  46 , and the rotation is maintained until the locking members  40  are aligned with slots  42 . Cap  16  is then simply pulled up. To reintroduce the cap in position, the procedure is carried out in an opposite manner. The external surface  53  of portion  52  should be planar, so that it can be laid 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, surface  53  must comprises an opening preferably corresponding to the internal diameter of cylindrical portion  54  (see  FIG. 4 ). 
       FIG. 5  illustrates another embodiment of the present invention. The cap and well assembly  100  which comprises a tray or base plate (not shown) provided with a plurality of wells  112  and corresponding cap  114 , which comprises a cylindrical slot  116  into which is inserted an O-ring element  118  made of a resilient material. As for the previous embodiment illustrated, the O-ring, although optional, is provided to ensure an appropriate seal. 
     Each cap  114  comprises a pair of locking element  120  diametrically opposed to each other and comprising a ridge portion  122 . Cap  114  is locked into position on the tray by inserting each locking element  120  into a corresponding opening  124  provided onto the upper surface  126  of the tray until the lower surface  128  of cap  114  lies flat onto the tray upper surface  126 . Cap  114  is then rotated so that locking elements  120  slide into a slot  130  having a width smaller than that of opening  124  and extending on a portion of the periphery of well  112  until the upper surface of ridge portion  122  is entirely under upper surface  126 , thereby efficiently sealing well and maintaining cap  114  in place. 
       FIG. 6  illustrates another simple variation of the present invention, wherein the upper surface  152  of a well  150  comprises a slot  154  along its circumference and adapted to receive an O-rig element  156  coupled to a cap  158 . The section of slot  154  is such that is slightly smaller than that of element  156 , so that upon insertion into the slot, a tight seal is formed by the locking of cap  158  to well  150  without the need of any adhesive or grease. 
       FIG. 7  illustrates yet another embodiment of the invention, wherein the cap  170  is screwed on the well  172 . This construction as well as the one illustrated in  FIGS. 1 ,  2  and  5  provides for gradual engagement of the locking mechanism of the cap with the well for movement between a released position and a locked position. 
     The present cap and well assembly is particularly suitable for both hanging-drop or sitting-drop crystallization methods. With respect to the sitting-drop method, although not specifically illustrated in the drawings, anyone skilled in the art will readily appreciate that any conventional drop support can be inserted or molded into the well. Examples of such sitting-drop support include the Micro-Bridges or the glass sitting drop rods manufactured and sold by Hampton Research (Laguna Hills, Calif.). 
     Each well is carefully filled with a selected equilibrating solution. Subsequently, a selected protein drop is deposited on the cap. The shape and the texture of the lower surface can be varied to obtain optimum results for a particular protein solution being crystallized, for example, when lower surface tension solutions, including protein solutions containing detergents, are used. The addition of the equilibrating solution and the protein drops to the device may be carried out either manually or through commercial automated pipetting apparatus, and the sealing of the cap over the solution may also be carried out manually or in an automated manner. 
     While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present description as come win known or customary practice within the art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.