Abstract:
Technologies are described for methods and systems effective for flex plates. The flex plates may comprise a base plate. The base plate may include walls that define an insert location opening in the base plate. The insert location opening in the base plate may be in communication with a securement area. The flex plates may comprise an insert. The insert may include a reservoir region and a crystallization region separated by a wall including channels. The reservoir region and the crystallization region may include a backing. The insert may further include securement tabs. The securement tabs may be configured to secure the insert to the base plate at the securement area.

Description:
[0001]    This application claims benefit of U.S. Provisional Application No. 62/100,962 filed on Jan. 8, 2015, the contents of which are incorporated herein by reference in its entirety. 
     
    
       [0002]    The present application was made with government support under contract number DE-SC0012704 awarded by the U.S. Department of Energy. The United States government has certain rights in the invention(s). 
     
    
     FIELD OF THE INVENTION 
       [0003]    This disclosure relates generally to a tray for growing crystals such as proteins, nucleic acids, and/or carbohydrates. Crystals grown in crystallization trays may be analyzed for applications involving drug design and for controlling drug delivery. 
       BACKGROUND 
       [0004]    Molecules such as proteins, nucleic acids, and/or carbohydrates may be crystallized in order to study the crystalline structure to obtain detail about the molecules&#39; function. Crystallization may be performed by providing a preparation of a solution of a target compound and altering the environment of the dissolved target compound to make the target compound revert to its solid form as a crystal. A precipitate may be introduced into the environment to make the target compound less soluble and to induce crystallization. 
       SUMMARY 
       [0005]    In some examples, flex plates are generally described. The flex plates may comprise a base plate. The base plate may include walls that define an insert location opening in the base plate. The insert location opening in the base plate may be in communication with a securement area. The flex plates may comprise an insert. The insert may include a reservoir region and a crystallization region separated by a wall including channels. The reservoir region and the crystallization region may include a backing. The insert may further include securement tabs. The securement tabs may be configured to secure the insert to the base plate at the securement area. 
         [0006]    In some examples, methods to make samples are generally described. The methods may comprise placing a preparation within a crystallization region of an insert secured to a base plate. The methods may comprise placing a precipitant within a reservoir region of the insert. The reservoir region may be separated from the crystallization region by walls defining channels. The methods may comprise allowing vapor from the precipitant to achieve equilibrium between the reservoir region and the crystallization region. The methods may comprise removing the insert from the base plate. The methods may comprise placing the insert into a testing device to test the sample. 
         [0007]    In some examples, flex plates are generally described. The flex plates may comprise a base plate wherein the base plate includes walls that define an insert location opening in the base plate. The insert location opening may be in communication with a securement area. The flex plates may comprise an insert. The insert may include a reservoir region and a crystallization region separated by a wall including channels. The reservoir region and the crystallization region may include a backing. The insert may further include securement tabs. The securement tabs may be configured to secure the insert to the base plate at the securement area. The flex plates may comprise a cover. The cover may include an adhesive sheet configured to cover the flex plate and define holes. The holes may be configured such that preparation and precipitant may be placed in at least one of the crystallization region and the reservoir region through the cover. 
         [0008]    The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0009]    The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which: 
           [0010]      FIG. 1  illustrates an example flex plate with a removable insert removed; 
           [0011]      FIG. 2  illustrates an example base plate with a removable insert removed; 
           [0012]      FIG. 3  illustrates an example flex plate including a base plate with removable inserts with multiple reservoir regions and with a removable insert removed; 
           [0013]      FIG. 4  illustrates an example flex plate cover; 
           [0014]      FIG. 5  illustrates an example insert cover; all arranged according to at least some embodiments described herein. 
       
    
    
     DETAILED DESCRIPTION 
       [0015]    In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. 
         [0016]    As used herein, any compound, material or substance which is expressly or implicitly disclosed in the specification and/or recited in a claim as belonging to a group or structurally, compositionally and/or functionally related compounds, materials or substances, includes individual representatives of the group and all combinations thereof. 
         [0017]      FIG. 1  illustrates an example flex plate with a removable insert removed, arranged in accordance with at least some embodiments presented herein. As discussed in more detail below, a flex plate used for crystal growth may include removable inserts which may be removed from the flex plate and utilized for in situ data collection. 
         [0018]    Flex plate  102  may be a crystal growth plate used to grow crystals such as proteins, nucleic acids, and/or carbohydrates. Flex plate  102  may include a base plate  106  and multiple fragments or removable inserts  104 . Flex plate  102 , including base plate  106 , and fragments or removable inserts  104  may be made from a polymer material such as acrylonitrile-butadiene-styrene copolymer, polycarbonate, polydimethylsiloxane (PDMS), polyethylene (PE), polymethylmethacrylate (PMMA), polymethylpentene, polypropylene, polystyrene, polysulfone, polytetrafluoroethylene (PTFE), polyurethane (PU), polyvinylchloride (PVC), polyvinylidene chloride (PVDC), polyvinylidine fluoride, styrene-acryl copolymers, polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), silicones, epoxy resins, poly ether block amide, polyester, acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics, polyacetal (POM), polyacrylates (acrylic), polyacrylonitrile (PAN), polyamide (PA), polyamide-imide (PAI), polyaryletherketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polyketone (PK), polyester/polythene/polyethene, polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), and mixtures thereof. 
         [0019]    Insert  104  may include securement tabs  110 , walls defining a crystallization region  112 , walls defining a reservoir region  114 , and walls defining channels  116 . Insert  104  may include transparent backing  126  that may be adhered to both crystallization region  112  and to reservoir region  114 . Transparent backing  126  may be adhered to an underside of insert  104  and may be transparent to electromagnetic radiation such as x-rays. Securement tabs  110  may secure insert  104  to base plate  106  through a mating between securement tabs  110  and a securement area  108  defined by walls in base plate  106 . Securement tabs  110  may secure insert  104  to base plate  106  such that insert  104  may be removed or disassembled from base plate  106 . Securement of insert  104  to base plate  106  may be performed by any securement method, including, dovetailing, snap-in, friction fit, sockets, etc. Securement tabs  110  may also be compatible with adapters for goniometers. 
         [0020]    Base plate  106  may include walls which define insert location openings  120 . Insert location openings  120  may communicate with securement area  108 . Insert  104  may be secured within insert location openings  120  when securement tabs  110  mate with securement areas  108 . Flex plate  102  may include base plate  106  with a respective insert  104  secured within each insert location opening  120 . Flex plate  102 , including inserts  104  secured in flex plate base  106 , may be used to grow protein crystals for protein crystallography. Base plate  106  of flex plate  102  may include a raised area  122  above a pedestal  124 . Flex plate  102  may be about 128 mm in length (at longest point on pedestal  124 ), by about 85.5 mm in width (at widest point on pedestal  124 ), by about 11.5 mm high (total height of pedestal  124  and raised area  122 ). Flex plate  102  may be configured to have preparations and precipitants applied to crystallization region  112  and precipitant region  114  respectively, by protein crystallography automated equipment. 
         [0021]    Crystallization region  112  of insert  104  may be separated from reservoir region  114  by wall  115 . Wall  115  may prevent liquid precipitant  118  from crossing from reservoir region  114  to crystallization region  112 . Channels  116  may be defined by wall  115  along a top edge of wall  115 . Channels  116  may be grooves within the top edge of wall  115  running between reservoir region  114  and crystallization region  112  and may allow air and vapor of precipitant  118  to flow between reservoir region  114  and crystallization region  112 . Crystallization region  112  of each insert  104  may accommodate chemical screening preparations. A liquid handling system, such as acoustic droplet ejection (ADE) may be operated in connection with flex plate  102  and insert  104  to place preparations  119  within crystallization region  112 . Crystallization region  112  may accommodate over  81  distinct chemical screening preparations. Reservoir region  114  may include precipitant  118 . Precipitant  118  may be any precipitant used in protein crystallography, including but not limited to, poly ethylene glycol (PEG) 600, PEG 4K, PEG 10K, (NH 4 ) 2 SO 4 , PO 4 , and citrate. Precipitant  118  in vapor form may diffuse across channels  116  to crystallization region  112 . Precipitant  118  in vapor form may achieve equilibrium between reservoir region  114  and crystallization region  112 . Precipitant vapor equilibrium may take a time period, such as 24 hours and crystals may form in crystallization region  112 . As discussed in more detail below, a cover may be placed over insert  104  or flex plate  102 , including multiple inserts, to prevent loss of precipitate when preparation volumes are of nanoliter scale. 
         [0022]    Upon formation of crystals  128 , or at an end of crystal preparation, if no crystal forms, the flex plate cover may be removed from flex plate  102  and insert  104 . Flex plate  102  may be covered with an x-ray transparent adhesive plastic plate cover. The adhesive plastic plate cover may be cut with a razor blade such that each insert  104  is separated from other inserts  104 . Each insert  104  may be removed or disassembled from base plate  106 . Insert  104  may provide for in situ data collecting, as securement tabs  110  of insert  104  may be placed within standard mounts for a goniometer  130 . Goniometer  130  and x-ray source  132  may thus perform x-ray analysis of samples from within crystalline region  112  of insert  104  while samples are within insert  104 . 
         [0023]      FIG. 2  illustrates an example base plate with a removable insert removed, arranged in accordance with at least some embodiments presented herein. Those components in  FIG. 2  that are labeled identically to components of  FIG. 1  will not be described again for the purposes of clarity. 
         [0024]    As previously discussed, base plate  106  may include walls defining insert location openings  120  and securement areas  108 . A size of insert  104 , as well as location and patterning of inserts  104  within base plate  106  may be varied based on chemical libraries to be used as preparation. In an example, 96 chemicals may be used in 8×12 layouts. Base plates  106  may be configured to accommodate from 1 to 64 inserts  104  in any configuration of 1-16 rows and 1-16 columns, for example, 5 rows by 2 columns, 5 rows by 4 columns, 6 rows by 4 columns, 8 rows by 4 columns, etc. 
         [0025]      FIG. 3  illustrates an example flex plate including a base plate with removable inserts with multiple reservoir regions and with a removable insert removed, arranged in accordance with at least some embodiments presented herein. Those components in  FIG. 3  that are labeled identically to components of  FIGS. 1-2  will not be described again for the purposes of clarity. 
         [0026]    Flex plate  102  may include base plate  106  with inserts  140  secured throughout base plate  106  such that an insert  140  is secured within all insert location openings  120  in base plate  106 . Flex plate  102  may be prepared with preparations and precipitants in crystallization region  112  and reservoir region  114  by hand or by automated measures such as ADE. As described in more detail below, inserts  140  may be configured for more than one precipitant  118 . Flex plate  102  may include insert  140  with different configurations within base plate  106 , depending upon the criteria to be tested. For example, an insert  140  configured with one precipitate reservoir may be secured in base plate  106  next to an insert  140  configured with four precipitate reservoirs. 
         [0027]    In an example, a protein crystallization experiment may desirably use more than one precipitant  118 . Insert  140  may be configured to include more than one reservoir region  114 , for example reservoir regions  114   a,    114   b,    114   c,  and  114   d.  Precipitants  118   a,    118   b,    118   c,  and  118   d  may be prepared and placed in each reservoir region respectively. Precipitants  118   a,    118   b,    118   c,  and  118   d,  in vapor form may diffuse across channels  116   a,    116   b,    116   c,  and  116   d  to crystallization regions  112   a,    112   b,    112   c,  and  112   d  respectively. Crystallization regions  112   a,    112   b,    112   c,  and  112   d  may each be prepared with preparations  119   a,    119   b,    119   c,  and  119   d  respectively. Preparations  119   a,    119   b,    119   c,  and  119   d  may be identical or different preparations. Preparations  119   a,    119   b,    119   c,  and  119   d  may grow crystals with precipitants  118   a,    118   b,    118   c,  and  118   d  respectively. A configuration of insert  140  may be determined based on preparation criteria and testing to be completed. 
         [0028]      FIG. 4  illustrates an example flex plate cover, arranged in accordance with at least some embodiments herein. Those components in  FIG. 4  that are labeled identically to components of  FIGS. 1-3  will not be described again for the purposes of clarity. 
         [0029]    Flex plate cover  150  may be an adhesive sheet and may cover over the entirety of flex plate  102 . Flex plate cover  150  may be transparent. Flex plate cover  150  may define holes  152 . Holes  152  may be configured to be located over reservoir regions  114  or crystallization regions  112  of any configuration of flex plate  102  such that preparation  119  and precipitant  118  may be placed within flex plate  102 . Flex plate cover  150  may prevent evaporation or loss of vapor precipitant during preparation and growing of crystals when preparation volumes are of nanoliter volumes. Flex plate cover  150  may cover about 96% of flex plate  102 . Flex plate cover  150  may be made from a polymer material such as acrylonitrile-butadiene-styrene copolymer, polycarbonate, polydimethylsiloxane (PDMS), polyethylene (PE), polymethylmethacrylate (PMMA), polymethylpentene, polypropylene, polystyrene, polysulfone, polytetrafluoroethylene (PTFE), polyurethane (PU), polyvinylchloride (PVC), polyvinylidene chloride (PVDC), polyvinylidine fluoride, styrene-acryl copolymers polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), silicones, epoxy resins, poly ether block amide, polyester, acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics, polyacetal (POM), polyacrylates (acrylic), polyacrylonitrile (PAN) polyamide (PA), polyamide-imide (PAI), polyaryletherketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polyketone (PK), polyester/polythene/polyethene, polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), and mixtures thereof. 
         [0030]      FIG. 5  illustrates an example insert cover, arranged in accordance with at least some embodiments herein. Insert cover  154  may be an adhesive sheet and may cover over the entirety of insert  104 . Insert cover  154  may be transparent. Insert cover  154  may define holes  156 . Holes  156  may be configured to be located over reservoir regions  114  or crystallization regions  112  of any configuration of insert  104  such that preparation and precipitant may be placed within insert  104 . Insert cover  154  may prevent evaporation or loss of vapor precipitant during preparation and growing of crystals within insert  104  when preparation volumes are of nanoliter volumes. 
         [0031]    In an example, flex plate  102  may be prepared with an ADE system. A chemical library may be placed by the ADE system at  81  locations in crystallization region  112  of one or more inserts  104  of flex plate  102 . Precipitant  118  may be placed in reservoir region  114  of one or more inserts  104  of flex plate  102 . Precipitant  118  may be placed with agar or allowed to soak into agar that has hardened in reservoir region  114 . Flex plate cover  150  may be placed over the flex plate  102  and equilibrium of precipitant vapor may be reached between reservoir region  114  and crystallization region  112 . Flex plate  102  may be placed in a container moistened with precipitant  118  to aid in reaching equilibrium. 
         [0032]    Protein and precipitant  118  may be placed by ADE system in crystallization region  112  of one or more inserts  104  through holes  152  in flex plate cover  150 . Preparation  119  may include a chemical library, protein and precipitant  118  placed in crystallization region  112 . Equilibrium of precipitant  118  vapor may be reached between reservoir region  114  and crystallization region  112 . Flex plate  102  may be placed in a container moistened with precipitant  118  to aid in reaching equilibrium. Flex plate  102  may include about 800 or more co-crystallization experiments. 
         [0033]    Cover  150  may be removed from flex plate  102 . Flex plate  102  may be covered with an x-ray transparent adhesive plastic plate cover. The adhesive plastic plate cover may be cut with a razor blade around each insert such that each insert  104  is separated from other inserts  104 . Inserts  104  may then be removed individually from base plate  106 . Each insert  104  may be placed onto a magnetic cap and x-ray data may be obtained with a goniometer. 
         [0034]    In another example preparation  119  may include first growing a protein sample and then adding a chemical from a chemical library. In addition to a protein sample, precipitant, and a chemical from a chemical library, preparation  119  may also include a chemical additive or other precisely targeted substance (“magic bullet”) which may improve diffraction, a cryo protectant, or may include a heavy atom additive. 
         [0035]    Among other possible benefits, a system in accordance with the present disclosure may provide for in situ data collecting. Inserts may be removed from a flex plate and the inserts may be placed within standard mounts for a goniometer. The goniometer may thus perform x-ray analysis of crystals formed within the insert while crystals are still within the insert. A user may not have to endure the laborious activity of gathering crystals in order to analyze the crystals with a goniometer. A user may not have to purchase and operate complicated and unreliable robotics to analyze crystals formed in flex plates that may not have removable inserts. A system in accordance with the present disclosure may provide high throughput by eliminating sample collection steps. The inserts may be directly placed into a goniometer as the inserts may be compatible with the centimeter sized throw of x-ray beams. Small amounts of preparations and precipitants may be used as the inserts may be used with nanoliter volumes. Waste may be reduced as the base plates may be reused. 
         [0036]    While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.