Nanovolume microcapillary crystallization system

Abstract

A nanovolume microcapillary crystallization system allows nanoliter-volume screening of crystallization conditions in a crystal card that allows crystals to either be removed for traditional cryoprotection or in situ X-ray diffraction studies on protein crystals that grow within. The system integrates formulation of crystallization cocktails with preparation of the crystallization experiments. The system allows the researcher to select either gradient screening in crystallization experiments for efficient exploration of crystallization phase space or a combination of sparse matrix with gradient screening to execute one comprehensive hybrid crystallization trial.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 
     
         1 . A protein crystallization system, comprising:
 a pumping system;   pieces of software configured to execute on the protein crystallization system to control the pumping system; and   one or more crystal cards coupled to the pumping system, each configured to house a mixer and a microfluidic capillary that is coupled to the mixer to facilitate storage and inspection of protein crystallization.   
     
     
         2 . The protein crystallization system of  claim 1 , wherein the pumping system includes a syringe pumping system or a pressure pumping system, wherein the syringe pumping system includes four-channel syringe pumps to regulate aqueous solutions being conveyed into the one or more crystal cards through second, third, and fourth microfluidic channels, and fluorous solutions being conveyed into a fifth microfluidic channel. 
     
     
         3 . The protein crystallization system of  claim 1 , wherein the pieces of software facilitate control of each pump of the four-channel syringe pump and control of each channel of second, third, fourth, and fifth microfluidic channels to generate granular gradients of flow of aqueous solutions. 
     
     
         4 . The protein crystallization system of  claim 1 , wherein the one or more crystal cards are formed from materials having properties that are selected from a group consisting of X-ray transmissive, optical clarity, modable, chemical resistive, suitable surface energy, and a combination of two or more of the foregoing recited properties. 
     
     
         5 . The protein crystallization system of  claim 1 , wherein the mixer includes a junction of second, third, fourth, and fifth microfluidic channels where aqueous plugs are formed, the second, third, fourth, and fifth microfluidic channels being formed from microfluidic channels that are approximately 200 by 200 micrometers. 
     
     
         6 . The protein crystallization system of  claim 5 , wherein the junction defines a hydrophobic surface that supports formation of aqueous plugs, which are approximately in a range of 10 nanoliters to 20 nanoliters, and wherein the microfluidic capillary transports the aqueous plugs away from the junction. 
     
     
         7 . The protein crystallization system of  claim 2 , wherein the one or more crystal cards are formed from plastic configured for fine gradient screening and are alternatively formed from PDMS/Teflon® configured for hybrid screening and membrane proteins. 
     
     
         8 . The protein crystallization system of  claim 1 , further comprising syringes with needles coupled to the pumping system, and yet further comprising tubings having distal ends and proximal ends configured to act as macro-micro interface between the needles of the syringes and the one or more crystal cards, each tubing having an inner diameter of about 360 micrometers and an outer diameter of about 760 micrometers, the distal end of a tubing configured to slide onto a needle and the proximal end of the tubing configured to coupled to the one or more crystal cards. 
     
     
         9 . A method for gradient screening, comprising:
 regulating aqueous streams by independently controlling each aqueous stream with a pumping system exercised by pieces of software; and   mapping out crystallization phase space of a protein to illustrate transition from precipitation, to microcrystals, to single crystals in a protein crystallization experiment.   
     
     
         10 . The method of  claim 9 , wherein the act of regulating includes forming concentration gradients over a series of aqueous plugs by changing flow rates of each aqueous stream. 
     
     
         11 . The method of  claim 10 , wherein the act of regulating includes regulating aqueous streams selected from proteins, crystallization agents, fluorocarbons, precipitants, ligands, protein partners, DNA complexes, buffers, and cryoprotectants. 
     
     
         12 . The method of  claim 11 , wherein the act of regulating includes increasing a flow rate of an aqueous stream of a buffer when a flow rate of an aqueous stream of a precipitant decreases so that a sum of flow rates remains constant. 
     
     
         13 . A method for hybrid screening, comprising:
 pre-forming precipitant plugs;   pre-forming plug spacers, each separating two precipitant plugs from each other;   forming gradients by merging precipitant plugs, plug spacers, and a protein stream;   mapping out crystallization phase space of a protein to illustrate transition from precipitation, to microcrystals, to single crystals in a protein crystallization experiment.   
     
     
         14 . The method of  claim 13 , wherein pre-forming plug spacers includes pre-forming using gas bubbles. 
     
     
         15 . The method of  claim 13 , wherein forming gradients includes coordinating flow rate change between a stream formed from the precipitant plugs, plug spacers, and a buffer stream. 
     
     
         16 . The method of  claim 13 , wherein each precipitant plug is about 100 nanoliters. 
     
     
         17 . A method comprising:
 receiving a crystal card with capillaries;   coating capillaries with a reagent to reduce a surface energy; and   removing the reagent.   
     
     
         18 . The method of  claim 17 , further comprising incubating the crystal card on ice for a predetermined number of hours. 
     
     
         19 . The method of  claim 17 , wherein the capillaries include inside surfaces and the act of coating capillaries includes coating the inside surfaces of the capillaries to reduce surface energy to about six to ten dynes per centimeter. 
     
     
         20 . The method of  claim 17 , wherein the fluorinated copolymer solutions include two percent fluorinated copolymer solutions in fluorosolvent. 
     
     
         21 . The method of  claim 17 , wherein removing the fluorosolvent includes vacuuming the crystal card. 
     
     
         22 . The method of  claim 17 , further comprising an act of forcing clean, dry air through the crystal card, which is executed at five psi for about one hour. 
     
     
         23 . The method of  claim 17 , further comprising an act of baking the crystal card, which is executed at about sixty degree Celsius for about one hour. 
     
     
         24 . The method of  claim 17 , further comprising:
 peeling a thin layer bonded to a substrate of a crystal card;   extracting crystals by a cryoloop from microfluidic circuitry housed on the substrate;   cryocooling the crystals; and   performing diffraction experiments on the crystals to obtain diffraction data.   
     
     
         25 . The method of  claim 17 , further comprising:
 mounting a crystal card with microfluidic circuitry to a goniometer;   radiating the crystal card with X ray; and   collecting diffraction data.   
     
     
         26 . The method of  claim 25 , further comprising translating the crystal card along x and y axes to collect the diffraction data from multiple crystals stored by the microfluidic circuitry. 
     
     
         27 . A crystal card, comprising:
 a substrate configured to house a mixer circuit and an inspection circuit; and   a layer bonded to the substrate and configured to peel from the substrate.   
     
     
         28 . The crystal card of  claim 27 , wherein the layer is either thermally bonded to the substrate or chemically bonded to the substrate. 
     
     
         29 . The crystal card of  claim 27 , wherein the substrate and the layer are formed from a group consisting of an amorphous polymer, Cyclic Olefin Copolymer, a thermalplastic polymer, and Polycarbonate. 
     
     
         30 . The crystal card of  claim 27 , wherein the substrate includes a thickness of about one millimeter and the layer includes a thickness in a range of about 100 to 150 micrometers. 
     
     
         31 . The crystal card of  claim 27 , wherein the mixer circuit includes first, second, third, and fourth summand channels, each summand channel including a distal end and a proximal end, the distal end of each summand channel defining an opening configured to fluidly receive solutions, the proximal end of each summand channel defining an opening configured to fluidly communicate aqueous plugs or plug spacers, each summand channel having a first part being coupled to the distal end and a second part of the first, second, and third summand channels being coupled to the proximal end, the first part of each summand channel being spaced apart and oriented in parallel with another summand channel, the second parts of the first and third summand channels angled so that their proximal ends intersect, the second parts of the second and fourth summand channels continuing in parallel until the proximal end of the second summand channel intersects with the proximal ends of the first and third summand channels to form a vertex, a third part of the fourth summand channel continuing from the second part of the fourth summand channel at a ninety degree angle where its proximal end intersects with the vertex at another ninety degree angle. 
     
     
         32 . The crystal card of  claim 31 , wherein the first part of each summand channel is spaced apart from the first part of another summand channel by about 4.50 millimeters. 
     
     
         33 . The crystal card of  claim 31 , wherein the substrate includes a first side, a second side, a third side, and a fourth side, the second side of the substrate being spaced apart from the distal end of the third summand channel by about 3.70 millimeters, a length of the first and third side being approximately 25.40 millimeters, a length of the second and fourth side being about 76.20 millimeters, the first side of the substrate being spaced apart from the distal ends of the summand channels by about 6.00 millimeters. 
     
     
         34 . The crystal card of  claim 33 , wherein the second side of the substrate is spaced apart from the distal end of the third summand channel by about 3.70 millimeters, the first side of the substrate being spaced apart from the distal ends of the summand channels by about 6.00 millimeters, the third side of the substrate being spaced apart from the inspection circuit by approximately 6.00 millimeters. 
     
     
         35 . The crystal card of  claim 31 , wherein the inspection circuit includes a summation channel, a serpentine body, and a tail channel which terminates in an opening configured to fluidly communicate, the summation channel being coupled to the vertex and continued in a direction that is collinear with the proximal end of the fourth summand channel until the summand channel reaches an axis that is collinear with the first part of the third summand channel at which the summation channel makes a ninety degree turn to join with the serpentine body of the inspection circuit. 
     
     
         36 . The crystal card of  claim 35 , wherein the serpentine body of the inspection circuit is formed from a compound curve having multiple convex turnings coupled to each other by a serpentine channel to facilitate fluid communication, one convex turning being spaced apart from a subsequent convex turning by about 53.31 millimeters, each convex turning having a length of about 2.00 millimeters. 
     
     
         37 . The crystal card of  claim 36 , wherein a last convex turning of the serpentine body is coupled to the tail channel. 
     
     
         38 . The crystal card of  claim 37 , wherein a length of the summation channel, the serpentine body, and the tail channel is collectively about 67 centimeters, wherein cross-sectional dimensions of the summation channel, the serpentine body, and the tail channel are about 200 by 200 micrometers. 
     
     
         39 . The crystal card of  claim 27 , wherein the substrate is configured to house two mixer circuits and two inspection circuits. 
     
     
         40 . The crystal card of  claim 39 , wherein the substrate houses multiple annular ports that project upwardly, some of which multiple annular ports are adapted to fluidly receive solutions or fluidly communicate aqueous plugs or plug spacers.