CN100448503C - Pre-filled crystallization plates and methods for making and using same - Google Patents
Pre-filled crystallization plates and methods for making and using same Download PDFInfo
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- CN100448503C CN100448503C CNB2004800024104A CN200480002410A CN100448503C CN 100448503 C CN100448503 C CN 100448503C CN B2004800024104 A CNB2004800024104 A CN B2004800024104A CN 200480002410 A CN200480002410 A CN 200480002410A CN 100448503 C CN100448503 C CN 100448503C
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- 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
Abstract
The present invention generally relates to encapsulation of crystallization solutions (16) in pierceable reservoirs (14) in order to prevent evaporation of the crystallization solutions, thereby allowing safe transport/shipping, from a workstation to another workstation, and safe storage of pre-filled microplates (10) used to carry large number of vapor-diffusion crystallization experiments.
Description
Background technology
Invention field
The present invention relates to crystallography and, especially relate to transportation and processing before being used to use with the pre-microwell plate of filling of precipitation solution.
The description of prior art
Crystallography is the exceedingly useful instrument of scientist, and therefore is the research field that attracts many people.It provides the molecule three-dimensional structure accurately and the strong instrument of describing in detail and have very great help for understanding its function.The high molecular crystallography of albuminoid is widely used in academic research and industrial now.
Though the three-dimensional structure of monomeric protein can obtain by the method for crystallising that needs crystal to form, it is not easy to do obtaining crystal from macromolecule.For example, even the optimum condition of given molecular crystalline does not need thousands of tests, also need hundreds of tests to obtain.Therefore, developed the multiple instrument that can test in a large number fast relatively and method, comprised hanging drop and sit the method for dripping.All these methods all use the characteristics of benefit of vapor diffusion to obtain crystal.
According to vapor diffusion technique, the macromolecule sample of a little volume is mixed with the crystallization or the precipitation solution of approximately equal volume.The drop that produces is sealed in the chamber of the crystallization solution with a large amount of liquid storage volumes.Drop is by hanging from crystal surface or keeping separating with the reservoir of crystallization solution by the pedestal that is seated at more than the reservoir crystallization solution level.Along with the progress of time, crystallization drop and crystallization solution reach balance by the benefit of vapor diffusion of volatile chemical.Carry out balance by benefit of vapor diffusion and be present between drop and the reservoir, cause the crystallization of macromolecule sample in the drop up to realizing high molecular supersaturation.
Yet the process of growth biomacromolecule crystal is highly experimental process utilizing trial-and-error method to make in the process that different crystallization parameters change.Usually those parameters are: the pH in the crystallization drop, temperature and salinity, the concentration of high molecular concentration to be crystallized and precipitating reagent (wherein having hundreds of).Utilize trial-and-error method, people can test many crystallization conditions (screening crystallization condition) by changing aforesaid parameter, make the crystallization condition of the polymer crystal growth of good diffraction with higher probability acquisition.Just can make breadboard operating efficiency higher in order to prepare all different solutions that carry out many different tests, introduce the pre-preparation of forming by the different crystallization solutions of 10mL of compound 24 pipes usually as the company of Hampton Research and screened.Follow the tracks of the market trace of Hampton Research, the EmeraldBiostructures of other companies of similar MDL and Jena Biosciences begin to introduce the crystallization screening of pre-formulation.A problem of all that crystallization solution is need or automatically change crystallization plates over to their craft when carrying out crystallization trial.This has to carry out work tediously long and effort with regard to the qualified technician who means this area.
In addition, shifting crystallization solution in microwell plate need promptly carry out in order to avoid the evaporation of crystallized liquid usually.This evaporation will change the composition of solution, will cause the problem of reproducing the same terms thus if produce crystal growth.Usually, utilize micro-pipette to shift.The technical staff has to open the test tube that comprises solution and then solution is moved into crystallization reservoir.Quicker for the transfer in the SBS standard crystallization plates, can use little pipette of multichannel and SBS standard deep, it is filled in advance up to 2mL, sets 96 kinds of different crystallization conditions (preparing 96 microporous crystalline plates).Therefore, the researcher needs centrifugal stop block (cross pollution of micropore when preventing to break seal), unpacking stop block and begin to shift solution.In case a problem is that the stop block unlatching is just evaporated; This is that a major issue is because all processes can spend a few minutes.Another problem is if the researcher does not use all solution stop blocks to need sealing again.In order to use all solution, the researcher needs pre-minimum 10 plates of filling, even generally need 3 plates (1 of each temperature).No less important ground is to it may be noted that when handling hundreds of different solution the error of possible occurrence flag and dispersion and cross pollution.
Another method is to use by similar Tecan, the accurate automated fluid treating stations of the company of Gilson or Robodesign exploitation.When comprising the conventional molecular biology experiment that DNA handles, can use those stations, because the decreased number of pending solution and evaporation also are problem of smaller (albumen are more unstable all the more along with the rising of time and temperature than DNA).For example, the United States Patent (USP) 6,148,878 that licensed to people such as Ganz on November 21st, 2002 discloses the robot of filling a plurality of microwell plates.A problem of this system is that it is not suitable for filling compound microwell plate in advance and carries out crystallization trial, because the offhand any equipment of micropore to be used to store before using at it that is used to automatically and effectively seal microwell plate.
In a word, the micropore of filling microwell plate with crystallization solution is a difficult task, if not suitably carrying out under the standard that presets of strictness, may cause the undesirable extensive variation of result of the test, jeopardizes the repeatability of test simultaneously.
Therefore, attempted to seek the method for pre-filling microwell plate.A kind of known method comprises the top with adhesive tape or heated sealant thin foil sealing crystallization microwell plate.After the accumulating, need before carrying out crystallization experiment, remove seal so that the inlet of plate top surface to be provided, herein lubricating grease is coated in each micropore around.When unpacking, have and take away the risk that may be present in liquid on the seal, this causes that by volume or the concentration that changes surplus solution in the reservoir unwanted test changes thus owing to the transportation or the shrinkage when occurrence temperature changes of plate.In order to remove the liquid on the sealing device, the researcher needs centrifugal before use plate; This may make automation complicated, and needs optional equipment and additional step.In addition, the seal at microwell plate top and centrifugal solution only are only applicable to hanging-drop crystallization plates.In fact, be positioned under the situation that the seat of plate top surface and the micropore below the top seal device drips at crystal surface, the crystallization solution that micropore contains does not contact with crystal surface, and not with its pollution.In addition, in this case, centrifugal can not being used for removed liquid below the top seal device, because this centrifugal partially crystallizable solution that will cause contacts crystal surface, thereby produces more problems.
According to applicant's knowledge, nobody can fill before transported/handled plate or microwell plate in advance up to now, and described plate or microwell plate are used to sit a crystallization experiment.This is because crystal surface (containing the position that high molecular drop to be crystallized is sat) is being damaged/is polluting between the delivery period between processing/each site probably, fills microwell plate and carries out the crystallization drop setting in described each site.
The researcher always manages to use less drop so that the protein content that uses during the crystallization minimizes when carrying out the benefit of vapor diffusion crystallization experiment.When the droplet that uses less than 1ml, thereby some problems may take place because balance can not reach good crystallization condition too rapidly.Therefore when the solution with considerably less volume carries out the benefit of vapor diffusion test, need to seek some measure that equilibrium process is slowed down or control.Having tested several different methods control/correction/change realizes the required time of complete equipilibrium between drop and mother liquor (crystallization solution).Some method tried to use oils slow down/control evaporation process (D ' Arcy etc. (1996) J.Crystal Growth168,175-180).A shortcoming of this method is to carry out additional step oils is distributed in the reservoir on the crystallization solution.In addition, when preparing hanging-drop crystallization set-up, perhaps oils can reduce the quality of the observed image of microscopically.At last, the micropore of microwell plate may can not add enough oils too for a short time sometimes.
The growth course of biology polymer crystal remains highly experiential process in a word.By thousands of a large amount of variable conditions that influence crystal growth of crystallization trial experiment, the final crystallization condition that obtains the best.Therefore market demand the present invention produces many reproducible crystallization trials fast and easily.Therefore, need to be easy to be provided with the multiple crystallization test, and during this set, make the device of the risk minimization of sum of errors cross pollution.Another important needs are owing to filling microwell plate with crystallization solution and evaporation between the crystallization drop is being set or device/method that the liquid loss increases the reproducibility of experiment.At last, need to make the researcher to control the device of the vapour phase-diffusive equilibrium ratio of multiple crystallization test in the multiple microwell plate.
Summary of the invention
The purpose of this invention is to provide with the pre-crystallization plates of filling of crystallization solution.
Another object of the present invention is to prevent that crystal surface is polluted by the crystallization reaction thing during the seat of transportation or the pre-filling of processing drips crystallization plates.
Further aim of the present invention provide that control sit to be dripped or the hanging drop crystallization experiment in the device of benefit of vapor diffusion ratio.
Further purpose of the present invention is to make simple to operateization that vapor diffusion technique is carried out crystallization experiment of passing through that must carry out.
Further purpose of the present invention provides the method for filling microwell plate with crystallization solution in advance.
Further purpose of the present invention provides the new method of carrying out crystallization experiment.
Further purpose of the present invention provides the standard that is used for preparation experiment and sits drip apparatus and be inverted the new crystallization plates that plate obtains the hanging drop experiment.
Further purpose of the present invention provides has the new hanging-drop crystallization plates of integrating hanging-drop support.
Therefore according to the present invention, the kit that is used to carry out crystallization experiment is provided, the crystallization plates that comprises pre-filling with many micropores, each micropore of described many micropores is opened on its top and is used to accept crystallization solution, comprise that its upper end is with the primary seal spare of the independent seal of upper/lower positions in recessed each micropore, be used for temporarily crystallization solution being sealed in micropore before using, transporting and to handle the crystallization plates of pre-filling safely, thereby and the secondary seal that on the described plate more than the described primary seal spare, comprises sealing surface be used for described micropore is sealed in more than the described primary seal spare and after independent seal is broken, benefit of vapor diffusion take place.
According to another general aspect of the present invention there, the kit that is used to carry out crystallization experiment is provided, described kit comprises at least a at least a crystallization solution that is sealed in many independent capsules, and at least aly comprises that many adaptations load the crystallization plates of the micropore of above-mentioned capsule.
According to further overview of the present invention, the kit that is used for by the benefit of vapor diffusion grown crystal is provided, the crystallization plates that comprises the micropore of having fixed many pre-filling crystallization solutions, at least a seal that is used for respectively and seals described micropore separately with contained crystallization solution, but wherein said seal is made by pierceable material and is used for hole is fixed on the sealing part so that benefit of vapor diffusion to take place.
According to another general aspect of the present invention there, provide the method for making pre-crystallization plates of filling, comprised the steps: the crystallization plates that crystallization solution is provided and has many micropores, and crystallization solution has been sealed in the many capsules that hold in the described micropore.
According to further overview of the present invention, the method of carrying out crystallization experiment is provided, comprise the steps: to provide and have many crystallization plates with the pre-micropore of filling of crystallization solution, crystallization solution is sealed in the micropore separately by at least a seal that pierces through, the micropore of aiming at selection is at the seal acanthopore, and draw a part of crystallization solution in the selected micropore that breaks a seal, and carry out following operation in the micropore in described Kaifeng: the crystallization solution that will draw on drop support mixes the acquisition drips of solution with Polymer Solution, and described drips of solution is sealed in the micropore, described crystallization solution contained in described drips of solution and the micropore is separated, and the hole in the seal can make between drips of solution and crystallization solution benefit of vapor diffusion takes place.
According to another general aspect of the present invention there, the method of making pre-crystallization plates of filling is provided, comprise the steps: to provide crystallization plates with many micropores, and crystallization solution is dispersed in the micropore, and individually crystallization solution is sealed in the micropore by the light sheet material that can puncture on the crystallization solution in the heat seal micropore.
Can pierce through the crystallization solution that encapsulates in the reservoir and prevent the crystallization solution evaporation, can transport safely/load and transport to another work station from a work station thus, and the pre-microwell plate of filling of safety storage, be used to carry out various vapor diffusion crystallization experiments.Because pipette tip can carry out piercing through of reservoir, apparatus and method of the present invention are especially favourable to be that it has reduced to carry out the required number of steps of crystallization trial in the research laboratory.Another advantage of apparatus of the present invention/method is the vapor diffusion speed of crystallization control experiment easily.
One embodiment of the invention provide and can be used for crystalline protein and other molecule, the crystallization solution of especially high molecular encapsulation.According to embodiment preferred, the crystallization solution of each capsule contains preferably and is made of plastics, and enough I contain the reservoir in the micropore of microwell plate of crystallization solution with insertion, but the puncture through seal part that can be bonded to reservoir is to guarantee suitable seal, this reservoir that is used to store and the crystallization solution that inserted reservoir before sealing.But pierceable material need be made by the material that keeps perforate, can carry out the vapor diffusion experiment with this capsule in case pierce through.
According to a further general feature of the present invention, provide to use and to have pierced through the new reservoir of thin slice, thereby transported the crystallization reservoir of pre-filling safely by use heat seal desirable seal.Reservoir can be molded, and fills sealing (by piercing through thin slice) and insertion crystallization cavity.Reservoir can directly be molded as crystalline microporous, fills, then sealing (by piercing through thin slice).The present invention can transport the crystallization plates or the microwell plate of pre-filling safely, and the danger of before carrying out crystallization experiment, not polluting crystal surface.Control speed provided by the invention can be by changing the size generation vapor diffusion process will in hole, and described hole will be pierced in seal when the beginning crystallization experiment.
According to another aspect of the present invention, provide this be used to be suitable for sit drip or the manufacture method of the encapsulation crystallization solution of the microwell plate of hanging drop crystallization trial.Described capsule is molded, fills suitable crystallization solution, inserts then to sit with the sealing of film or thin foil and drips or the micropore of hanging-drop crystallization plates.
According to another aspect of the present invention, provide kit, comprised the crystallization solution of many encapsulation, and at least a crystalline microporous plate that preferably includes the micropore of many encapsulation crystallization solutions.
The present invention is also very interesting, because can load crystallization drop device for mechanical people, makes many crystallization solutions to open and close or before preparation drop device crystallization solution is shifted from another container.
According to further overview of the present invention, the crystalline microporous that contains many micropores plate is provided, each micropore comprises and is used to receive the precipitation solution reservoir of precipitation solution and has the drop chamber that is used to receive the crystal surface that contains high molecular drips of solution to be crystallized, described drop chamber communicates with described precipitation solution reservoir and can carry out benefit of vapor diffusion between drips of solution and precipitation solution in micropore sealing back, and the current limiter that in described precipitation solution reservoir, provides, be used for when the crystalline microporous plate is inverted, precipitation solution being retained in the precipitation solution reservoir and be used to implement the hanging drop experiment.
According to further overview of the present invention, the method of carrying out the hanging drop crystallization experiment is provided, comprise the steps: to provide the plate of the crystalline microporous with many micropores, the drop chamber that each micropore contains the precipitation solution reservoir and has crystal surface, described precipitation solution reservoir contains a large amount of precipitation solutions, to contain high molecular drips of solution to be crystallized is seated on the described crystal surface, with with micropore in the drips of solution that separates of the precipitation solution that contains drop is sealed in the micropore, and be inverted plate so that drips of solution hangs from described crystal surface, and described precipitation solution is retained in the described precipitation solution reservoir.
According to still further general aspect of the present invention, the hanging drop that is suitable for being upside down on the micropore that contains crystallization solution crystallization supports is provided, contain and limit the hanging drop crystallization supports that at least one is used to hold the lower surface that contains at least one chamber that precipitation solution and Polymer Solution to be crystallized drip, described chamber has bottom surface and size when adjusting to the Polymer Solution that holds predetermined volume and reaching the steam balance with box lunch between crystallization solution and drips of solution, and the bottom surface in chamber is still fully covered by solution.
Description of drawings
After having described characteristic of the present invention, be described referring now to accompanying drawing by its preferred embodiment of diagram, wherein:
Fig. 1 is according to the perspective view of the preferred embodiment of the invention with the pre-crystallization plates of filling of crystallization solution of encapsulation;
Fig. 2 is the end face view of the crystallization plates of pre-filling shown in Figure 1;
Fig. 3 is the cutaway view that obtains along Fig. 2 center line 3-3;
Fig. 4 is that in a single day micropore so that break pre-filling seal between crystallization solution and drop benefit of vapor diffusion just takes place with rubber belt sealing with the cutaway view that is dispersed in the pre-crystallization plates of filling of big molecule drop on the drop support that is incorporated into the plate micropore;
Fig. 5 is the end face view according to the hanging drop preparation of second embodiment of the invention;
Fig. 6 is the cutaway view that obtains along Fig. 5 center line 6-6;
Fig. 7 a to 7d diagram participates in making the different step of crystallization solution capsule according to the preferred embodiment of the invention;
Fig. 8 a to 8c is the microporous side view with drop support and crystallization retaining device, is used to make crystallization plates to be prepared as standard and sit drips a plate and be inverted then and be used to carry out the hanging drop crystallization experiment.
Fig. 9 a to 9c is the schematic side-view of micropore shown in Fig. 8 a to 8c, illustrates the loading operation of crystallization plates;
Figure 10 a to 10c is the schematic side-view of micropore shown in Fig. 8 a to 8c, and crystallization plates loads and is inverted crystal growth/monitoring position so that the hanging drop crystallization experiment can carry out at this moment;
Figure 11 is the plan view of the further aspect multi-cavity hanging-drop support lower surface according to the present invention; And
Figure 12 is the diagram elevation in a chamber of multi-cavity hanging-drop support shown in Figure 11.
The description of preferred embodiment
The invention provides to the porous crystalline plate, drip crystallization or the precipitation solution that microwell plate is filled desired number in advance, before in the laboratory, using, transport and handle such as hanging drop microwell plate or seat.According to this mode, the technical staff uses " ready-made can the use " plate, and the time-consuming operation in each hole is filled in cancellation with suitable precipitation solution thus.
A kind of possible application of the present invention of Fig. 1 illustration.More specifically, Fig. 1 shows that the seat that contains many micropores 12 drips crystallization plates.Each hole 12 comprises the central reservoir 13 that communicates with drop chamber 15, and described drop chamber 15 has crystal surface or drop support 17, contains high molecular solution to be crystallized thereon and keep static (referring to Fig. 4) during crystallization experiment.10 of plates are as ordinary construction and can adopt different structures, only otherwise deviate from scope of the present invention.
The capsule 14 that contains crystallization solution 16 (Fig. 3 and 4) is contained in the central reservoir 13 in each hole 12 in friction tight mode.Perhaps, capsule 14 can be loosely received in the hole 12 and stick wherein.Being appreciated that also that each hole 12 can be placed surpasses one capsule 14.As shown in figs. 1 and 3, each capsule 14 contains reservoir, and part cup 18 has the open top end that approaches seal 19, and preferred heated sealant is to cup 18.Part cup 18 is preferably by the injection molten thermoplastic material, carries out in the chamber mould (not shown) of injector capsule form molded such as polypropylene.Certainly the material that forms cup 18 must be with respect to the crystallization solution that wherein contains inertia chemically.Equally, contain high molecular drop to be crystallized directly situation be suspended under the situation of the hanging-drop applications above the crystallization solution, cup 18 must be enough clear or transparent, can make light by so that can examine under a microscope and monitor crystal growth on drop support.In graphic embodiment, cup 18 is generally conical butt, but very clear, and it can be an Any shape, as long as the micropore of coupling plate wherein to be placed.In graphic embodiment, each glass 18 has the semi-circular indentation 20 of axial expansion, is used for being contained in the protrusion 22 of the corresponding axial expansion that each central reservoir 13 forms.Protruding 22 is engaged in the recess 20 cup 18 spin lockings in hole 12.
Preferably can pierce through light sheet material or film, the form that is coated with the aluminium foil of shop such as polypropylene provides seal 19, its can be easily by being used to draw crystallization solution in case with its with contain the pipette top that high molecular solution to be crystallized mixes and pierce through.Also can use other perforator puncture through seal part 19.But the material that is used to form puncture through seal part 19 must be pierce through instrument leave hole 12 after such as not recovering its shape.Instrument must stay the big or small permanent hole 24 (Fig. 4) of expectation at seal 19 dresses can carry out continuous benefit of vapor diffusion between drop 26 (Fig. 4) and crystallization solution 16.By changing the size in hole 24, the benefit of vapor diffusion ratio between drop 26 and the crystallization solution 16 can be effectively controlled.But use the puncture through seal part favourable too, because seal needn't utilize extra step to remove.In addition as mentioned above, it is undesirable removing seal because when some liquid is pulled away along with seal in the reservoir change of the volume of solution or concentration can introduce unwanted experiment and change.Therefore exempted needs to centrifugal plate before opening micropore, thus the seal of guaranteeing not have liquid to remain in each hole below.But the use of puncture through seal part makes that also the independent sealing micropore that highly needs becomes possibility, and it is subjected to may needing to remove respectively the restriction of each independent seal processing up to now.But, needn't physically remove seal and even can break seal in pipette operating period of crystallization solution 16 and thus two steps were combined into a step by means of the puncture through seal part.
As shown in Figure 3, the seal of crystallization solution can be isolated crystal surface 17 and crystallization solution 16 easily, thereby avoids the pollution of crystal surface 17 during the pre-plate 10 of filling of transportation and processing before its use.By Fig. 3, can easily understand the end face of reservoir 18, promptly seal 19 is at the recessed micropore 12 in a segment distance place of plate 10 end faces.Thus, the end face of plate and lid, all adhesive tape 30 effective sealed engagement as shown in Figure 4 provide the secondary seal that is independent of the first order seal that is formed by seal 19 sealings.Second level seal be used for sealing gland crystallization solution 16 and from the drop 26 of atmosphere so that between it benefit of vapor diffusion can take place after first order seal is broken.According to Fig. 6 as can be seen, the spacing of seal 19 and plate top surface provides and the drop of solution need be suspended on the space more than the crystallization solution in the hanging-drop crystallization set-up.This is essential for the situation that pierced holes does not meet the size of holding drop.Yet, can easily seal be applied to the top in hole in some cases and limit the enough hole dimensions that allow the suspension drop to run through the pierced holes that limits in the seal.This will guarantee that the observation of drop is not subjected to the influence that seal exists.
In the application, the technical staff uses with crystallization solution 16 pre-crystallization plates 10 of filling, as shown in Figure 3.Subsequently, the biology macromolecule drop of selecting 26 (Fig. 4) is dispersed on the crystal surface 17 of micropore 12 of desired number.Contained crystallization solution 16 and is down into the crystallization solution of drawing aequums in the micropore 12 from each hole 12 with pipette by pipetting with pipette top pierced holes 24 usefulness pipettes in these micropores 12 in the seal 19 of micropore 12 then.At this moment wait, pipette can also be used to add a given substance into crystallization solution, thereby can utilize the different tests of different crystallization solutions on the pre-same plate 10 of filling with single crystallization solution.Different additives can be introduced in each hole.The solution drawn with macromolecule drop 26 each crystal surface 17 on mixed thereafter.At last, by on the micropore 12 that independent adhesive tape 30 is placed on preparation like this so that it is closed to make finishes preparation, as shown in Figure 4.The adhesive tape that it should be noted that single length can cover simultaneously above a hole.The type that also is appreciated that the lid that uses be not the lid of emphasis of the present invention and various types be used in macromolecule drop poured into wherein after sealing gland micropore 12.For example, also can use the independent lid of describing such as among the PCT application PCT/CA00/00119, its content is incorporated herein by reference herein.
Drawing crystallization solution in the dispersion of macromolecule drop and the micropore can manually or via the business automation suction means carry out.The secondary seal micropore also can be manually or the automation mode carry out.
The encapsulation of crystallization solution is illustrated among Fig. 7 a to 7d.Part cup 18 preferably obtains in the chamber mould (not shown) that forms capsule by the injection thermoplastic.Be injected into after the mould, make the thermoplastic cooling produce cup 18.Then, cup 18 is filled crystallization solution shown in Fig. 7 b.Thereafter, but be shown in preferably heated sealant pierceable material thin foil 19 of each cup 18 top as Fig. 7 c and 7d.Shown in Fig. 7 a, the top of each cup 18 is the mating surfaces that produce between the top edge with graphic thin foil 19 and cup 18 among raising Fig. 7 d of inclination.In heat seal operating period, the angled end on cup top will melt and form the flat edge shown in Fig. 7 d.At last, cup 18 is inserted micropore 12.
It should be noted that cup 18 is whole by its direct mold entering plate 10 micropores 12 and micropore 12 are formed, thereby exempt the additional step that cup 18 is inserted the micropore 12 of pre-infill panel.Yet have following shortcoming when seal 19 is heat seal: plate 10 must be fit to material by the heat seal that must not have best optical characteristics and make.
Fig. 5 and 6 illustrates the possible application of another kind of the present invention, and wherein Feng Zhuan crystallization solution is applied in the hanging drop test equipment.Similarly reference number is used in reference to similar part.
Under the situation of hanging drop experiment, but importantly be limited to hole 24 ' and macromolecule drop 26 vertical alignments that hang in the puncture through seal part 19 ', and therefore observe crystal growth via drop support 17 ' so that light is passed through.For this purpose, can provide the visible indication for the treatment of pierced holes 24 with reference to the end face that thing 32 is provided at seal 19 '.Same adhesive tape is substituted by glass plate 30 ' and provides lubricating grease to provide sealing between glass plate 30 ' and micropore around each micropore.
It should be noted that but capsule 14 can also take pouch or any other to be adapted to hold the form of the puncture through seal container of crystallization solution.In addition, according to the further embodiment of not graphic the present invention, but crystallization solution can be dispersed directly into micropore and the puncture through seal part can be applied on the micropore or in the micropore with crystallization solution sealing wherein.Internal end surface must be limited in each micropore with the thin foil heat seal in micropore.If but the puncture through seal part is heat sealed to the end face of crystallization plates, micropore can also be opened separately from one another by hole independent in the seal on the micropore that pierces through selection.Then, but lubricating grease etc. can be applied directly on the puncture through seal part around each the independent hole that is defined in wherein, the suitable sealing of opening between micropore and the inverted cover plate (hanging-drop support) thereof is provided.Perhaps, lubricating grease can be substituted by the adhesive on the cover plate.In this case, but cover plate will be simply placed in the top of crystallization plates directly contacts with the puncture through seal part.
As mentioned above, but the encapsulation of crystallization solution 16 or drop support 17 is provided and sit drips in the main reservoir 13 of plate 10 micropores 12 the puncture through seal part between the contained mass crystallization solution 16 and have equally to make to sit and drip plate and be inverted characteristics of carrying out the hanging drop crystallization experiment.This shows, can utilize seat to drip a plate and carry out the hanging drop crystallization trial.This is favourable, because no longer need to be dispersed in separate support with comprising high molecular solution to be crystallized, on cover plate, it undoubtedly is upside down on the corresponding micropore of microwell plate subsequently then with its sealing.Needing this separate support that is used for the hanging drop experiment up to now is to be inverted because means known can not place solution droplets 26 on the crystallization plates and with whole plate.Precipitation solution will drip from reservoir 13, thereby cause the pollution of crystal surface or drop support 17 when operation panel.
According to characteristic of the present invention, can prepare experimental rig and drip crystallization experiment just as sitting, then whole seat being dripped crystallization plates is inverted (that is upset fully) and will comprises high molecular solution droplets 26 to be crystallized thus and hang on their corresponding drop support 17 (Figure 10 a to 10c).In case plate is inverted,, can prevent that the crystallization solution 16 of main reservoir 13 from flowing out owing in reservoir 13, there is seal 19.The hole 24 of piercing through in seal 19 must be large enough to and can between drop 26 and crystallization solution 16 vapor diffusion take place, yet but must be small enough to assurance crystallization solution 16 will rely on the surface tension of solution 16 to be retained in the reservoir 13.
Shown in Fig. 8 a to 8c, can also be undertaken by the type of flow that different other current limiter is provided in the main reservoir 13 of crystallization plates.For example, each micropore 13 can utilize the flange 34 of inner annular integrally to make, and described flange 34 inwardly stretches into from the outlet of reservoir 13 it is partly sealed, shown in Fig. 8 a.The hole 36 that annular lip 34 limits guarantees that the surface tension of solution 16 prevents to pour out reservoir when solution from working as the plate inversion, allows simultaneously between solution 16 and drop 26 vapor diffusion to take place shown in Figure 10 a.The size of opening 36 preferably can be accepted the micro-pipette end of rule and the dispensing heads of fluid operated robot.The structure of opening must be able to make the crystallization solution precipitation and keep solution when being inverted.Can add extra shutoff device after filling in reservoir is retained in liquid in the reservoir when turning around fully.
Perhaps, current limiter can adopt the form of the openend pore filtration membrane that is provided at reservoir 16.Described film may impenetrable liquid but steam thoroughly.
According to the embodiment shown in Fig. 8 b and the 8c, reservoir 13 ' and 13 " can have enough little internal diameter when being inverted with convenient crystallization plates solution 16 remain on reservoir 13 ' and 13 by capillary effect " inside, shown in Figure 10 b and 10c.Usually, the internal diameter of this capillary reservoir is less than 3.5mm.
As Fig. 9 a, in the application shown in 9b and the 9c, each crystallization plates is filled by dispersed crystalline solution at reservoir 13,13 ' and 13 " seat that only has rule in drips plate; draw reservoir 13; 13 ' and 13 " in a part of crystallization solution, and the crystallization solution that will draw and crystal surface 17,17 ' and 17 " on Polymer Solution mix the drop of acquisition solution 26.End face at each plate applies seal 38 sealing micropores 12,12 ' and 12 then ".Then, the crystallization plates of being inverted preparation like this is extremely as Figure 10 a, and the crystal growth/monitoring position shown in 10b and the 10c is so that carry out the hanging drop crystallization experiment.
Add and to have the reservoir of the current limiter of integral drop support, drip the current limiter of finding on the plate, drip or the universal crystallization plates of hanging drop experiment is prepared for forming to select to be used for sitting such as sitting in standard to crystallization plates.
The present invention can carry out easily that the experiment of multiple hanging drop must manually/automatically be inverted and index test carry out during crystallization supports on the wells/reservoirs.According to the present invention, the user can be inverted the whole crystallization plates of upset simply from as Fig. 9 a, 9b, and the load/unload position shown in the 9c arrives crystal growth/monitoring position.
The invention provides hanging-drop plates, get rid of thus separate crystallization supports is upside down in needs on the plate micropore with integral crystallization surface.According to the hanging drop preparation of routine, the micropore of microwell plate row can not cover in single operation with corresponding hanging-drop support, because hanging-drop support must be inverted single file at every turn, because the mirror effect when a kind of product is upside down on the another kind of product.According to the present invention, the precipitation solution reservoir and the drop support of each micropore are side by side, and therefore the macromolecule drop of plate by just being in suspension status in the once-through operation of simple inversion plate.Therefore simple greatly processing.
It should be noted that drop support 17 may not be the plane.They can be different structures so that promote the location of macromolecule drop and the extension subsequently that prevents drop.Can be undertaken by barrier or the drop retainer that recess or other type are provided in drop support.
Figure 11 has shown the multi-cavity hanging-drop support 40 that is adapted to be upside down on the crystallization plates micropore and its sealing is used for carrying out a large amount of crystallization experiments in identical micro pores.
Hanging-drop support 40 can or be adjacent in micropore on the pore openings end plate, and having the lid form that is adapted to the device (not shown) of corresponding lid geared assembly good engagement provides.As shown in Figure 9, hanging-drop support is characterised in that being limited to its lower surface 44 is used to receive a large amount of annular chamber 42a, 42b...42f or the recesses that comprise high molecular solution droplets to be crystallized accordingly.Chamber 42a, 42b...42f prevent the diffusion of drop, thereby prevent that drop from contacting with each other.Chamber 42a, 42b...42f can be used for the observation that is positioned at drop on the hanging-drop support and helps lend some impetus to crystal growth equally.
The size of determining each chamber is to accept predetermined molecular solution with volume.More particularly, the size in given chamber and the association between the liquor capacity must be satisfied and be dispersed in that the initial volume of solution in the chamber is no more than the liquor capacity of chamber capacity and in case reach balance between crystallization solution in micropore and the Polymer Solution that hangs from drop support 40, the bottom surface in chamber will be covered fully by keeping a large amount of Polymer Solutions.This will provide the better visualization that forms crystal easily, improve the reproducibility of test simultaneously.Usually the volume in chamber is equivalent to it and wants the drop that comprises.Thereby in a single day solution droplets is poured into the chamber and may contacts and avoid spreading of drop with the sidewall around the chamber.
Multi-cavity hanging-drop support 40 is preferred by the plastic material manufacturing with superior optical characteristics and preferably obtain by injection molding.Thereby drop support 40 is observed crystal growth at the location transparency in chamber at least.Each chamber 42a, the bottom surface 41 of 42b...42f is preferably the plane.Equally, as shown in figure 12, bottom surface 41 merges so that promote the regeneration of crystal via the angled wall fragment 43 that limits about miter angle θ with sidewall.
The chamber provides forming the better visualization of crystallization on the hanging-drop support.Described chamber can make the liquid level balance be used for better visualization.
Know that very hanging-drop support 40 can be equipped with single recess or chamber at its lower surface.This will be superior to the hanging-drop support of prior art, and the hanging-drop support of prior art lacks improvement usually to forming any equipment that crystal is estimated on it.
Claims (34)
1. be used to carry out the kit of crystallization experiment, the crystallization plates that comprises pre-filling with many micropores, each micropore of described many micropores is used to accept crystallization solution at its top end opening, contain the primary seal spare of interior its top of recessed each micropore with the independent seal of upper/lower positions, be used for temporarily the crystallization solution heat seal at micropore to transport and to handle the crystallization plates of pre-filling before using safely, and the secondary seal that is included in the sealing surface on the described plate on the described primary seal spare, described secondary seal is used to be sealed in the described micropore on the described primary seal spare, thereby makes and after being broken benefit of vapor diffusion can take place at independent seal.
2. the kit that limits of claim 1, wherein said secondary seal further comprises the cover means with described sealing surface sealed engagement.
3. the kit that limits of claim 2, wherein said cover means comprise that many independent cover components are used for sealing respectively and cover described micropore.
4. the kit that limits of claim 1 wherein apply independent seal respectively so that the many thermosealed capsule that comprises crystallization solution to be provided on various piece cup (18), and wherein said capsule is placed on respectively in the described micropore.
5. the kit that limits of claim 4, wherein said part cup is transparent, makes to check and to monitor crystal growth.
6. the kit that limits of claim 4, but wherein said independent seal by can be therein fixedly the pierceable material of permanent hole make.
7. the kit that limits of claim 1, wherein each described independent seal comprises the thin foil that is connected in its bottom one segment distance with described many micropore heat seals mutually.
8. the kit that limits of claim 7, but wherein said thin foil by can be therein fixedly the pierceable material of the permanent hole of different size make the speed that benefit of vapor diffusion takes place with control.
9. the kit that limits of claim 1, the crystallization plates of wherein said pre-filling drips crystallization plates for sitting, described micropore comprises corresponding crystal surface at a segment distance place of its bottom, and described crystal surface and crystallization solution are during transportation isolated by described independent seal.
10. the kit of claim 8 qualification further comprises the visible indication reference of piercing through described independent seal inspection and monitoring crystal growth is provided.
11. the kit of claim 1, it is used to carry out crystallization experiment, described kit comprises at least a crystallization solution that is encapsulated in many independent capsules, and the crystallization plates with many micropores that comprises of the kit of at least a claim 1, and described micropore is used to load above-mentioned capsule.
12. the kit that claim 11 limits, wherein said capsule can be pierced the hole of different sizes is permanently affixed in the capsule, the speed of benefit of vapor diffusion takes place in the size decision in each hole when beginning crystallization experiment.
13. the kit that claim 11 limits, but wherein each described capsule comprises the part cup that is filled with described crystallization solution and is sealed by the puncture through seal part.
14. the kit that claim 13 limits, wherein said seal provides with the form that is heat-sealing to described reservoir and seals the light sheet material of its open top end.
15. the kit of claim 1, it is used for by the benefit of vapor diffusion grown crystal, the crystallization plates of the many micropores of qualification that comprises the kit of claim 1, described micropore is filled in advance with crystallization solution, at least a seal is used for wherein contained described crystallization solution and seals described micropore, but wherein said seal by independent hole being limited to stab material dress so that but the pierceable material of benefit of vapor diffusion takes place separately in each described micropore makes.
16. many visible indication references that provide seal to be pierced in the position with respect to micropore further are provided the kit that claim 15 limits.
17. the kit that claim 15 limits, but wherein said at least one puncture through seal part is heat-sealing to the end face of described the above plate of micropore.
18. the kit that claim 15 limits, wherein said seal provides with the thin foil form that can pierce through separately of recessed described corresponding micropore.
19. the kit that claim 18 limits, the wherein said thin foil that pierces through is heat-sealing to the corresponding part cup (18) of filling with crystallization solution and is engaged in the micropore.
20. make the method for the crystallization plates of the pre-filling in the kit of claim 1, comprise the steps: the crystallization plates that crystallization solution is provided and has many micropores, and crystallization solution is encapsulated in the many capsules that hold in the described micropore.
21. the method that claim 20 limits, the step that wherein encapsulates crystallization solution comprises molded capsule, seals each capsule with piercing through light sheet material then with the crystallization solution filled capsules.
22. the method that claim 21 limits further is included in the step of inserting capsule in the micropore.
23. the method that claim 21 limits comprises the directly step of molded capsule in the micropore of crystallization plates.
24. the method that claim 23 limits, wherein crystallization plates and capsule carry out the molded encapsulation that is used for simultaneously by the chamber mould that the thermoplastic with fusing injects the formation microwell plate with binding cavity.
25. the method that claim 21 limits, the wherein said light sheet material that pierces through is heat-sealing to each described capsule.
26. the method that claim 25 limits, but wherein said pierceable material provide with independent thin foil form, thin foil can forever be pierced through pore-forming.
27. carry out the method for crystallization experiment, comprise the steps: to provide and have many crystallization plates with the pre-micropore of filling of crystallization solution, crystallization solution is sealed in the micropore separately by at least a seal that pierces through, aim at the micropore of selecting and pierce through perforate, and draw a part of crystallization solution in the selected micropore, and carry out following operation in described micropore: the crystallization solution that will draw on drop support mixes the acquisition drips of solution with Polymer Solution, and described drips of solution is sealed in the selected micropore, described crystallization solution contained in described drips of solution and the micropore is isolated, and benefit of vapor diffusion can take place in the hole in the seal between drips of solution and crystallization solution.
28. the method that claim 27 limits wherein is sealed in described drips of solution step in the micropore of selection and comprises lubricating grease is applied directly to the step on the described seal around the described hole.
29. make the method for the crystallization plates of pre-filling used in the method for claim 27, comprise the steps: to provide crystallization plates with many micropores, and crystallization solution is distributed in the micropore, and individually crystallization solution is sealed in the micropore by the thin foil that can puncture on the crystallization solution in the heat seal micropore.
30. be used for the crystalline microporous plate that contains many micropores of the method for claim 27, each micropore comprises and is used to receive the precipitation solution reservoir of precipitation solution and has the drop chamber that is used to receive the crystal surface that contains high molecular drips of solution to be crystallized, described drop chamber communicates with described precipitation solution reservoir and can carry out benefit of vapor diffusion between drips of solution and precipitation solution in micropore sealing back, and the current limiter that in described precipitation solution reservoir, provides, be used for when the crystalline microporous plate is inverted, precipitation solution being remained on the precipitation solution reservoir and be used to implement the hanging drop experiment.
31. carry out the method for hanging drop crystallization experiment, comprise the steps: to provide the crystalline microporous plate with many micropores of claim 30, the drop chamber that each micropore contains the precipitation solution reservoir and has crystal surface, described precipitation solution reservoir contains a large amount of precipitation solutions, to contain high molecular drips of solution to be crystallized is seated on the described crystal surface, with with micropore in the drips of solution that separates of the precipitation solution that contains drop is sealed in the micropore, and be inverted plate so that drips of solution is suspended from described crystal surface, and described precipitation solution is retained in the described precipitation solution reservoir.
32. the hanging drop crystallization supports that claim 31 limits, wherein said bottom surface is made by transparent material.
33. the hanging drop crystallization supports that claim 31 limits, wherein said holder is made by plastic material.
34. the hanging drop crystallization supports that claim 33 limits, wherein said holder is made by the injection plastic material.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US44063503P | 2003-01-17 | 2003-01-17 | |
| US60/440,635 | 2003-01-17 | ||
| US60/466,074 | 2003-04-29 | ||
| US60/485,400 | 2003-07-09 |
Publications (2)
| Publication Number | Publication Date |
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| CN1852755A CN1852755A (en) | 2006-10-25 |
| CN100448503C true CN100448503C (en) | 2009-01-07 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004800024104A Expired - Fee Related CN100448503C (en) | 2003-01-17 | 2004-01-16 | Pre-filled crystallization plates and methods for making and using same |
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| CN (1) | CN100448503C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109562377A (en) * | 2016-06-30 | 2019-04-02 | 通用自动化实验技术公司 | The high resolution system, kit, device and method using Combined culture base strategy for high-flux microorganism application |
| CN106441974B (en) * | 2016-08-12 | 2023-06-02 | 福建工程学院 | Automatic sampling instrument for agricultural products |
| CN107099846B (en) * | 2017-05-25 | 2019-05-10 | 西北工业大学 | A kind of pre- cloth is micro-, nanoscale seed crystal protein crystallization board preparation method |
| CN115056555B (en) * | 2022-06-17 | 2023-09-01 | 贵州省种畜禽种质测定中心 | Special acid-proof pad for digestion furnace tube rack |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5221410A (en) * | 1991-10-09 | 1993-06-22 | Schering Corporation | Crystal forming device |
| DE19843655A1 (en) * | 1998-09-23 | 2000-03-30 | Bayer Ag | Hanging drop process and assembly for the crystallisation of biological macro-molecules minimizes the incidence of false results |
| WO2000047323A1 (en) * | 1999-02-09 | 2000-08-17 | Nextal Biotechnologie Inc. | Laboratory cap and well for hanging-drop crystallization methods |
| US20030010278A1 (en) * | 2001-07-10 | 2003-01-16 | Structural Genomix, Inc. | Tray for macromolecule crystallization and method of using the same |
-
2004
- 2004-01-16 CN CNB2004800024104A patent/CN100448503C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5221410A (en) * | 1991-10-09 | 1993-06-22 | Schering Corporation | Crystal forming device |
| DE19843655A1 (en) * | 1998-09-23 | 2000-03-30 | Bayer Ag | Hanging drop process and assembly for the crystallisation of biological macro-molecules minimizes the incidence of false results |
| WO2000047323A1 (en) * | 1999-02-09 | 2000-08-17 | Nextal Biotechnologie Inc. | Laboratory cap and well for hanging-drop crystallization methods |
| US20030010278A1 (en) * | 2001-07-10 | 2003-01-16 | Structural Genomix, Inc. | Tray for macromolecule crystallization and method of using the same |
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