U.S. patent application number 13/776526 was filed with the patent office on 2014-03-13 for nanovolume microcapillary crystallization system.
This patent application is currently assigned to EMERALD BIOSTRUCTURES, INC.. The applicant listed for this patent is EMERALD BIOSTRUCTURES, INC.. Invention is credited to Mark William Elliott, Cory John Gerdts, Peter Kurt Nollert-von Specht, Lansing Joseph Stewart.
Application Number | 20140073055 13/776526 |
Document ID | / |
Family ID | 42266414 |
Filed Date | 2014-03-13 |
United States Patent
Application |
20140073055 |
Kind Code |
A1 |
Gerdts; Cory John ; et
al. |
March 13, 2014 |
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.
Inventors: |
Gerdts; Cory John; (Clear
Lake, IA) ; Elliott; Mark William; (San Diego,
CA) ; Nollert-von Specht; Peter Kurt; (Bainbridge
Island, WA) ; Stewart; Lansing Joseph; (Bainbridge
Island, WA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
EMERALD BIOSTRUCTURES, INC.; |
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US |
|
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Assignee: |
EMERALD BIOSTRUCTURES, INC.
Bainbridge Island
WA
|
Family ID: |
42266414 |
Appl. No.: |
13/776526 |
Filed: |
February 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12484983 |
Jun 15, 2009 |
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13776526 |
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61061563 |
Jun 13, 2008 |
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Current U.S.
Class: |
436/4 ;
422/245.1 |
Current CPC
Class: |
B01D 9/00 20130101; Y10T
428/31855 20150401; B01D 9/0072 20130101 |
Class at
Publication: |
436/4 ;
422/245.1 |
International
Class: |
B01D 9/00 20060101
B01D009/00 |
Goverment Interests
STATEMENT OF GOVERNMENT LICENSE RIGHTS
[0002] This subject matter was made, at least in part, with
Government support as provided for by the terms of NIGMS U54
GM074961, awarded by the National Institute of General Medical
Sciences. The Government has certain rights in the subject matter.
Claims
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.RTM. 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.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
12/484,983, filed Jun. 15, 2009, which claims the benefit of U.S.
Provisional Patent Application No. 61/061,536, filed Jun. 13, 2008,
the entire disclosures of which are incorporated herein by
reference.
BACKGROUND
[0003] The field of structural biology is generating technologies
that increase throughput and efficiency each year. Such advances
have inspired progression from gene to three-dimensional structure
in three days. In an effort to improve efficiency, it is desirable
to minimize the volume of protein required such that sufficient
material for crystallization screening and optimization can be
obtained from cell-free synthesis. With the "three day" structure
goal in mind, it is desirable to develop several technologies to
increase efficiency in the gene to structure pipeline.
SUMMARY
[0004] This summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This summary is not intended to identify
key features of the claimed subject matter, nor is it intended to
be used as an aid in determining the scope of the claimed subject
matter.
[0005] One aspect of the discussed subject matter includes a system
for protein crystallization, which comprises a pumping system and
pieces of software configured to execute on the protein
crystallization system to control the pumping system. The system
further includes 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.
[0006] Another aspect of the subject matter includes a method for
gradient screening, which comprises regulating aqueous streams by
independently controlling each aqueous stream with a pumping system
exercised by pieces of software. The method further comprises
mapping out crystallization phase space of a protein to illustrate
transition from precipitation, to microcrystals, to single crystals
in a protein crystallization experiment.
[0007] A further aspect of the subject matter includes a method for
hybrid screening, which comprises pre-forming precipitant plugs and
pre-forming plug spacers, each separating two precipitant plugs
from each other. The method further comprises forming gradients by
merging precipitant plugs, plug spacers, and a protein stream. The
method further includes mapping out crystallization phase space of
a protein to illustrate transition from precipitation, to
microcrystals, to single crystals in a protein crystallization
experiment.
[0008] A further aspect of the subject matter includes a method
which comprises receiving a crystal card with capillaries, coating
capillaries with a reagent to reduce the surface energy, and
removing the reagent.
[0009] In another aspect, the subject matter includes a crystal
card, which comprises a substrate configured to house a mixer
circuit and an inspection circuit. The crystal card further
includes a layer bonded to the substrate and configured to peel
from the substrate.
DESCRIPTION OF THE DRAWINGS
[0010] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0011] FIG. 1 is a block diagram illustrating an exemplary
nanovolume microcapillary crystallization system;
[0012] FIG. 2 is a block diagram illustrating an exemplary pumping
system of the nanovolume microcapillary crystallization system;
[0013] FIG. 3A is a pictorial diagram illustrating an exemplary
user interface for configuring the pumping system;
[0014] FIG. 3B is a pictorial diagram illustrating an exemplary
user interface for priming fluids to a crystal card of the
system;
[0015] FIG. 3C is a pictorial diagram illustrating an exemplary
user interface for specifying the production of nanoplugs in the
crystal card wherein the nanoplugs are of equal size and equal
content according to one embodiment of the subject matter;
[0016] FIG. 3D is a pictorial diagram illustrating an exemplary
user interface for specifying the production of nanoplugs in the
crystal card wherein the nanoplugs have varying concentrations of
protein and precipitant according to one embodiment of the subject
matter;
[0017] FIG. 3E is a pictorial diagram illustrating an exemplary
user interface for specifying the production of nanoplugs in the
crystal card wherein the nanoplugs have varying size and
concentration for multiple precipitants according to another
embodiment of the subject matter;
[0018] FIG. 4A is a pictorial diagram illustrating a top isometric
view of one embodiment of a crystal card; FIG. 4B is a pictorial
diagram illustrating a bottom isometric view of one embodiment of a
crystal card; FIG. 4C is a pictorial diagram illustrating a top
view of one embodiment of a crystal card; FIG. 4D is a pictorial
diagram illustrating a side view of one embodiment of a crystal
card; FIG. 4E is a pictorial diagram illustrating a bottom view of
one embodiment of a crystal card;
[0019] FIG. 5A is a pictorial diagram illustrating a top isometric
view of another embodiment of a crystal card; FIG. 5B is a
pictorial diagram illustrating a bottom isometric view of another
embodiment of a crystal card; FIG. 5C is a pictorial diagram
illustrating a top view of another embodiment of a crystal card;
FIG. 5D is a pictorial diagram illustrating a side view of another
embodiment of a crystal card; FIG. 5E is a pictorial diagram
illustrating a bottom view of another embodiment of a crystal
card;
[0020] FIG. 6A is a pictorial diagram illustrating a top isometric
view of a third embodiment of a crystal card; FIG. 6B is a
pictorial diagram illustrating a bottom isometric view of a third
embodiment of a crystal card; FIG. 6C is a pictorial diagram
illustrating a top view of a third embodiment of a crystal card;
FIG. 6D is a pictorial diagram illustrating a side view of a third
embodiment of a crystal card; FIG. 6E is a pictorial diagram
illustrating a bottom view of a third embodiment of a crystal
card;
[0021] FIG. 7A is a pictorial diagram illustrating a top isometric
view of a fourth embodiment of a crystal card; FIG. 7B is a
pictorial diagram illustrating a bottom isometric view of a fourth
embodiment of a crystal card; FIG. 7C is a pictorial diagram
illustrating a top view of a fourth embodiment of a crystal card;
FIG. 7D is a pictorial diagram illustrating a side view of a fourth
embodiment of a crystal card; FIG. 7E is a pictorial diagram
illustrating a bottom view of a fourth embodiment of a crystal
card;
[0022] FIG. 8A is a pictorial diagram illustrating a top isometric
view of a fifth embodiment of a crystal card; FIG. 8B is a
pictorial diagram illustrating a bottom isometric view of a fifth
embodiment of a crystal card; FIG. 8C is a pictorial diagram
illustrating a top view of an embodiment of a crystal card; FIG. 8D
is a pictorial diagram illustrating a side view of a fifth
embodiment of a crystal card; FIG. 8E is a pictorial diagram
illustrating a bottom view of a fifth embodiment of a crystal
card;
[0023] FIG. 9A is a pictorial diagram illustrating a top isometric
view of a sixth embodiment of a crystal card; FIG. 9B is a
pictorial diagram illustrating a bottom isometric view of a sixth
embodiment of a crystal card; FIG. 9C is a pictorial diagram
illustrating an exploded isometric view of a sixth embodiment of a
crystal card; FIG. 9D is a pictorial diagram illustrating a top
view of a sixth embodiment of a crystal card; FIG. 9E is a
pictorial diagram illustrating a side view of a sixth embodiment of
a crystal card; FIG. 9F is a pictorial diagram illustrating a
bottom view of a sixth embodiment of a crystal card;
[0024] FIG. 10 is a pictorial diagram illustrating one embodiment
of a three-plus-one mixer of one embodiment of a crystal card;
[0025] FIG. 11 is a pictorial diagram illustrating another
embodiment of a three-plus-one mixer of one embodiment of a crystal
card;
[0026] FIG. 12 is a pictorial diagram illustrating a third
embodiment of a three-plus-one mixer of one embodiment of a crystal
card;
[0027] FIG. 13 is a pictorial diagram illustrating a fourth
embodiment of a three-plus-one mixer of one embodiment of a crystal
card;
[0028] FIG. 14 is a pictorial diagram illustrating a cross section
through one embodiment of a crystal card; and
[0029] FIGS. 15A-15V are process diagrams illustrating an exemplary
method for crystallizing molecules using a nanovolume
microcapillary crystallization system.
DETAILED DESCRIPTION
[0030] Various embodiments of the subject matter describe a
nanovolume microcapillary crystallization system which comprises a
pump, software configured to control the pump, and a crystal card
that houses a mixer circuit and an inspection circuit. The crystal
cards are suitably manufactured using materials that include one or
more properties selected from a group consisting of X-ray
transmission, optical clarity, moldability, chemical resistance and
surface energy. The crystal cards house macromolecular crystals in
various phases enabling either extraction of crystals from the
crystal card or in situ X-ray diffraction. The crystals are
promoted inside the crystal cards by formation of nanoplugs by the
nanovolume microcapillary crystallization system. Nanoplugs are
formed by combining streams of aqueous solutions with an immiscible
and biologically inert carrier fluid, such as fluorocarbon
solution. Streams of aqueous solutions, such as those composed of a
target molecule, buffer, and precipitant solutions, are combined at
the mixer circuit to form nanoplug crystallization experiments. The
nanoplugs are incubated and monitored for crystallization. Nanoplug
crystallization experiments can be suitably used to shed light on
scientific questions regarding protein crystal nucleation and
growth and to generate crystals for novel structure solution.
[0031] The nanovolume microcapillary crystallization system
facilitates two screening styles: gradient mode and hybrid mode. As
used herein, the term gradient mode includes any suitable screening
method that provides various crystallization phases of molecules.
The gradient mode allows a crystallographer to finely scan a
crystal card to reveal crystallization phase space of a particular
molecule. Because each stream of aqueous solution used in the
nanovolume microcapillary crystallization system can be
independently controlled using the pump via the software,
concentration gradients of desired granularity over a series of
nanoplugs are suitably formed by changing the flow rates of the
individual streams. As a precipitant stream decreases in flow rate,
the nanovolume microcapillary crystallization system increases a
flow rate of a buffer stream such that that the sum of the flow
rates remains constant. Using the gradient mode, crystallization
phase space of a particular molecule, such as a protein, can be
mapped out to show a transition from precipitation, to
microcrystals, to single crystals.
[0032] As an enhancement to gradient mode, hybrid mode combines
gradients with sparse matrix screening on one crystal card. Sparse
matrix screening of molecule crystals in nanoplugs can be achieved
by generating a pre-formed cartridge of different crystallizing
agents. As used herein, the term hybrid mode includes hybrid
screening, including any suitable screening method that includes
pre-formed cartridges. The hybrid mode extends the concept of
sparse matrix screening by pre-forming precipitant nanoplugs,
separated by a nanoplug spacer (gas bubble), and forming a
concentration gradient as they are merged with a molecule stream.
Similar to the gradient mode, the hybrid mode generates gradients
by coordinating flow rate change between the pre-formed precipitant
nanoplugs and the buffer stream. By performing sparse matrix and
gradient screening together on one crystal card, the hybrid mode is
able to sample a large area of the crystallization phase space,
generating 20-40 experiments from each pre-formed precipitant
nanoplug.
[0033] As used hereinabove and hereinbelow, the term "nanoplug"
refers to a nanoliter-volume sized drop, such as a 10-20 nL aqueous
drop, that fills a microfluidic channel of the system. Each
nanoplug comprises a distinct microcrystallization experiment. As
used hereinabove and hereinbelow, the term "mixer circuit" means
the inclusion of a circuit having three aqueous channels and one
carrier fluid channel that come together at a point upstream of the
inspection circuit of a crystal card. Additional configurations are
possible, such as mixers having four or five aqueous channels and
one carrier fluid channel. The aqueous channels come together and
suitably intersect the carrier fluid channel at a 90 degree angle.
As used herein, the term "macro-micro interface" means the
inclusion of a coupling between the syringes and a crystal card. In
some embodiments, the syringes are connected to the mixer circuit
or the inspection circuit via tubing, such as Teflon.RTM. (PTFE)
tubing. In other embodiments, the tubing is connected to the mixer
circuit or the inspection circuit using connectors configured to
fluidically connect the inlets and outlets of the circuits with the
tubing. As used hereinabove and hereinbelow, the term "inspection
circuit" refers to a capillary or channel where fluids come
together and form aqueous nanoplugs that flow inside the capillary.
The inspection circuit can also be used to inspect the nanoplugs
for crystal formation. Further, the inspection circuit can also be
used to store crystals formed by the methods of the subject matter,
and is therefore also referred to herein as a storage capillary. As
used herein, the term "main channel" refers to the area of the
inspection circuit that locates downstream of the mixer where the
aqueous solutions and carrier fluid combine to form aqueous
nanoplugs. As used hereinabove and hereinbelow, the term "molecule"
includes small molecules, such as organic compounds and/or
chemicals, and macromolecules. The term "biological molecule"
refers to a molecule that is derived from, modeled on, or
corresponds to a molecule from a biological source. The term also
includes molecules synthesized or produced in vitro, such as by
cell-free synthesis, and/or in vivo, such as recombinant proteins,
mutant proteins, and artificial proteins, natural and artificial
nucleic acid molecules, and other biological molecules that do not
occur in nature. As used herein, the term "macromolecule" includes
biopolymers such as nucleic acids, proteins, carbohydrates and
lipids. For simplicity, the terms "protein" and "protein solution"
are used herein to encompass other types of molecules in addition
to proteins.
[0034] A nanovolume microcapillary crystallization system 100
useful for molecule crystallization is shown in FIG. 1. A prepared
sample 102 comprising a molecule for which the crystal structure is
desired, for example a prepared protein sample, is provided in an
aqueous solution 104. Additional aqueous solutions are also
provided; for example, a buffer solution and a precipitant
solution. The buffer solution may comprise the buffer used to
prepare the biological sample 102. A carrier fluid is also
provided. The carrier fluid is immiscible with the aqueous
solutions. Suitable examples of carrier fluids include fluorinated
oils; for example, FC-40 (3M Corp., St. Paul, Minn.). The aqueous
solutions and carrier fluid 104 are provided to one or more
syringes 106 which are connected to one or more pumps 108. The pump
108 is controlled by software executed on a nanoplug-forming
computer 110. The software executed on the nanoplug-forming
computer 110 regulates the flow of the aqueous solutions and
carrier fluid 104 in a crystal card 112. The flow of the aqueous
solutions and carrier fluid in the crystal card 112 are observed
through a magnifying device such as a microscope 114.
[0035] A pumping system 200 useful for regulating the flow of
various fluids through a crystal card is shown in FIG. 2. Pump 1
202 controls syringe 1 204 and syringe 2 208. Syringe 1 204 is
loaded with an aqueous solution such as buffer 206. Syringe 2 is
filled with an aqueous solution such as a precipitant reagent 210.
Pump 2 212 controls syringe 3 214 and syringe 4 218. Syringe 3 214
is filled with an immiscible fluid such as a carrier fluid 216.
Syringe 4 218 is filled with an aqueous solution containing a
molecule, such as a protein of interest 220. Suitable pumps include
Harvard Twin 33 syringe pumps (Harvard Apparatus, Holliston,
Mass.). In some embodiments, the syringe pumps have been modified
by the manufacturer to provide better accuracy. Suitable syringes
include Hamilton syringes, such as an 1800 series Hamilton Gas
Tight syringe. Suitable syringe volumes range from 10 ul to 100 ul.
The pumping system 200 is controlled by software executed on a
nanoplug-forming computer 110.
[0036] Suitable software is provided for controlling the pumping
system 108. FIGS. 3A-3E illustrate representative user interfaces
of the software of the system showing various modes that control
the pumping system 200. FIG. 3A shows a representative user
interface 300 of a configuration mode of the software. FIG. 3B
shows a representative user interface 302 of a prime mode of the
software. FIG. 3C shows a representative user interface 304 of a
constant mode of the software. FIG. 3D shows a representative user
interface 306 of a gradient mode of the software. FIG. 3E shows a
representative user interface 308 of a hybrid mode of the software
of the system.
[0037] Referring now to the crystal cards of the subject matter
disclosed herein, a representative example of one embodiment of a
crystal card is shown in FIGS. 4A-4E. The crystal card 400 is
configured to be about the same size as a standard microscope
slide, being about 76.20 mm long and about 25.40 mm wide (or about
3 inches long by about 1 inch wide). The crystal card 400 is about
1.0 to 1.5 mm thick. The crystal card is manufactured of
transparent polycarbonate by injection molding (Siloam Biosciences,
Inc.).
[0038] Referring now to the embodiment shown in FIGS. 4A and 4B,
the crystal card 400 has an upper surface 402 and a lower surface
414 that is parallel to the upper surface 402. The crystal card 400
further comprises a substrate configured to house a mixer circuit
404 and a storage and inspection circuit 406. The mixer circuit 404
is comprised of four microfluidic channels 421, 422, 424, and 426.
See FIG. 4C. Channels 421, 422, and 424 come together and intersect
channel 426 at a 90 degree angle. Each channel comprises an inlet
410. See FIG. 4E. The inspection circuit 406 comprises a long
microfluidic capillary channel that locates just downstream of the
mixer 404 and ends at an outlet 412. The length of the microfluidic
capillary 406 is about 67 cm. The microfluidic capillary channel
406 is also referred to as an inspection circuit, in which crystals
produced in the card may be stored in the channel 406 until
subjected to in situ X-ray diffraction analysis or extracted for
cryocooling. The microfluidic channels 421, 422, 424, 426 and the
capillary channel 406 are substantially square in cross-section and
have an inner diameter of about 200 micrometers (.mu.m).times.200
.mu.m. However, other configurations of the channels are
possible.
[0039] Referring now to FIG. 4D, the crystal card 400 further
comprises a layer 420 that is thermally bonded to the substrate and
configured to peel from the substrate. The peelable layer 420 is
thermally bonded to the substrate surface 414. In other
embodiments, the peelable layer 420 may be chemically bonded to the
substrate. The peelable layer 420 is about 0.10 to 0.14 mm thick.
The peelable layer 420 is suitably configured such that removal of
the peelable layer 420 exposes the interior space of the inspection
circuit channel 406. The crystal card 400 further comprises a
macro-micro interface that connects the syringes to the crystal
card. In one embodiment, the macro-micro interface includes
sections of rigid plastic tubing 430 (for example, tubing made of
PEEK.TM. polymer) that are connected at one end to the inlets 410
and outlet 412, and are connected at the other end to slip fit
connectors 432 made of flexible silicone tubing. The slip fit
connectors 432 are configured to accept Teflon.RTM. tubing (PTFE)
(not shown). The other end of the tubing is connected to a syringe
of the system. The Teflon.RTM. tubing has an inner diameter of 360
um and an outer diameter of 760 um (ID/OD 360/760), whereas the
connecter 432 has an inner diameter of 760 um, thereby forming a
gas and liquid tight seal when the Teflon.RTM. tubing is inserted
into the connecter 432.
[0040] In operation, the channel 421 is connected to tubing that is
filled with an aqueous solution, such as a buffer that is used in
the protein solution of interest. Channel 422 is connected to
tubing that is filled with a precipitant solution. As used herein,
it is understood that the term precipitant is interchangeable with
the term crystallant. Channel 424 is connected to tubing that is
filled with a solution containing a target molecule of interest. In
one embodiment, the target biological molecule is a protein.
Channel 426 is connected to tubing that is filled with a carrier
fluid. Suitable examples of carrier fluids include fluorinated oils
or fluorocarbons, such as FC-40, although others are possible. The
carrier fluid is immiscible with the aqueous fluids and
preferentially wets the walls of the inspection circuit
microchannel, thereby separating segments of the combined aqueous
solution into nanoplugs that span the width of the channel. In one
embodiment, the aqueous nanoplugs are about 10-20 nL in volume.
[0041] Referring now to FIGS. 5A-5E, a representative example of
another embodiment of a crystal card of the subject matter is
shown. Similar elements between different figures have similar
reference numbers, wherein the first digit increases by one and
corresponds to the figure number. For the sake of brevity, elements
that are similar between the different Figures will not be
described further. In the embodiment shown in FIGS. 5A-5E, the
inlet 510 is located in a shallow cylindrical depression 508
located in a top surface 502 of the crystal card 500. The
cylindrical depression 508 is configured for attaching a connector
(not shown) that connects tubing to the inlets 510 and outlet 512.
The dimensions of the crystal card 500 are shown in FIG. 5E. The
crystal card 500 is 76.2 mm long and 25.4 mm wide. The inlets 510
are spaced 4.5 mm apart. The parallel channels of the inspection
circuit 506 are 2.0 mm apart. However, as will be appreciated by a
person skilled in the art, other suitable configurations are
possible.
[0042] Referring now to FIGS. 6A-6E, a representative example of a
third embodiment of a crystal card is shown. For the sake of
brevity, similar elements that are described in previous figures
are not described here. In the embodiment shown in FIGS. 6A-6E, the
inlets 610 and outlet 612 are positioned below a cylindrical
projection 608 that is connected to and extends outwardly from the
surface 602. The projection 608 is configured for attaching a
connector (not shown) that connects tubing to the inlets 610 and
outlet 612. Digressing, the crystal cards illustrated in the
embodiments shown in FIGS. 4-6 are manufactured from transparent
polycarbonate plastic by injection molding (Siloam Biosciences,
Inc.).
[0043] Returning now to FIGS. 7-9, representative embodiments of a
second type of crystal card will be described. FIGS. 7A-7E
illustrate a representative example of another embodiment of a
crystal card of the subject matter. For the sake of brevity,
similar elements that are described in previous figures are not
described here. In the embodiment shown in FIGS. 7A-7E, the top
surface 702 of the crystal card 700 further comprises two rows of
ports 708. The ports are configured to receive a plastic connector
(not shown) that is suitable for connecting tubing to the inlets
710 and outlets 712 located beneath the port 708. The surface 702
comprises 28 ports 708. However, different numbers of ports are
possible depending on the design of the crystal card 700. The port
708 extends about 2.5 mm above the surface 702 of the crystal card
700. A hole is suitably drilled in the bottom center portion of the
port 708 such that it aligns with and is in fluidic connection with
the inlets 710 and outlets 712. The center of the ports are spaced
about 4.5 mm apart. The hole drilled in the bottom of the port 708
is about 0.2 mm (200 um) in diameter. It will be understood that
not every port is connected to the circuit channels such that only
desired ports to connect tubing to the inlets 710 and the outlets
712 need be drilled. In other embodiments, a laser is used to drill
holes through the peelable layer 720 before it is bonded to the
bottom surface 714. The laser-drilled holes are configured to be in
fluidic connection with the inlets 710 and the outlets 712. Tubing
is connected to the laser-drilled holes using a specially designed
crystal card holder (not shown).
[0044] Referring still to FIGS. 7A-7E, the crystal card further
comprises two separate asymmetrical microfluidic channel circuits
706A, 706B. In 706A, the inspection circuit is about 270 mm long.
In 706B, the inspection circuit is about 306 mm long. In both
circuits 706A and 706, the outlet 712 is located on the opposite
side of the circuit from the inlets 710 and the mixer circuits
704A, 704B. The embodiment shown in FIGS. 7A-7E comprises two
separate configurations of the mixer circuit 704A, 704B. As shown
in more detail in FIG. 10, the mixer circuit 704A comprises a short
neck region approximately 0.20 mm long between the aqueous channels
and the carrier fluid channel. As shown in more detail in FIG. 11,
the mixer circuit 704B lacks a neck region between the aqueous
channels and the carrier fluid channel. The mixer circuit 704A was
found to be suitable for aqueous nanoplug formation in a crystal
card.
[0045] Referring now to FIGS. 8A-8E, a representative example of
another embodiment of a crystal card of the subject matter is
shown. For the sake of brevity, elements that are similar to
previously described elements are not further described here. The
crystal card 800 comprises two separate symmetrical microfluidic
channel circuits 806. In this embodiment, the outlet 812 is located
on the same side of the circuit 806 as the mixer 804 and the inlets
810.
[0046] Referring now to FIGS. 9A-9E, a representative example of
another embodiment of a crystal card of the subject matter is
shown. For the sake of brevity, elements that are similar to
previously described elements are not further described here. A
crystal card 900 comprises a single microfluidic circuit comprising
one mixer circuit 904 and a long inspection circuit 906. The
inspection circuit 906 is about 665 mm long. FIG. 9C illustrates an
exploded view of the crystal card 900. Piece 930 comprising ports
908 is bonded to piece 940 comprising the microfluidic circuit
channels. The peelable layer 920 is thermally bonded to the bottom
surface 914 of piece 940. However, in other embodiments, the
peelable layer 920 may be chemically bonded to the substrate
surface 914. The peelable layer 920 is suitably configured such
that removal of the peelable layer 920 exposes the interior space
of the inspection circuit channel 906. Digressing, the crystal
cards illustrated in the embodiments shown in FIGS. 7-9 are
manufactured from transparent cyclic olefin copolymer (COC) or
comparable plastic (ThinXXS Microtechnology AG, Germany).
[0047] Returning now to FIGS. 10-13, representative examples of
mixer circuits will now be described. FIG. 10 shows a
representative example of one embodiment of a mixer circuit that
corresponds to the mixer 704A shown in FIG. 7. The mixer circuit
1000 comprises three aqueous channels 1021, 1022 and 1024. The
aqueous channels are separated from the carrier fluid channel 1026
by a neck region 1007. The channels are oriented such that the
three channels 1021, 1022, 1024 containing aqueous solutions come
together and intersect the channel 1026 containing the carrier
fluid at a 90 degree angle. The mixer 1000 further comprises a
portion of an inspection circuit 1006. Referring still to FIG. 10,
the dimensions of the mixer 1000 will now be described. The neck
region 1007 is about 0.2 mm long. Channel 1021 is about 0.2 mm in
diameter. Channels 1022, 1024 are about 0.141 mm in diameter.
Channels 1006, 1026 are about 0.2 mm in diameter. However, other
suitable dimensions for a mixer circuit are possible.
[0048] FIG. 11 shows a representative example of another embodiment
of a mixer circuit that corresponds to the mixer circuit 704B shown
in FIG. 7. The mixer circuit 1100 comprises three aqueous channels
1121, 1122 and 1124. The aqueous solution channels connect directly
to the carrier fluid channel 1106 in the absence of a neck region.
The mixer circuit feeds into the inspection circuit 1126. The
channels are oriented such that the three channels containing
aqueous solutions come together and intersect the channel
containing the carrier fluid at a 90 degree angle. The diameter of
channel 1121 is about 0.2 mm. The diameter of channels 1122, 1124
is about 0.141 mm. The diameter of the junction region between the
aqueous channels and the carrier fluid channel 1126 is about 0.285
mm. However, other suitable dimensions for a mixer are
possible.
[0049] Referring now to FIG. 12, another view of the mixer circuit
704A described in FIG. 7 is shown. The mixer circuit 1200 comprises
three aqueous channels 1221, 1222 and 1224 that are connected by a
short neck region to the carrier fluid channel 1226. The channels
are oriented such that the three channels containing aqueous
solutions come together and intersect the channel containing the
carrier fluid at a 90 degree angle. Each channel has an inlet 1210.
Downstream of the mixer circuit 1204, the solutions feed into a
portion of an inspection circuit 1206. Referring still to FIG. 12,
the dimensions of the mixer circuit 1200 will now be described. The
inlets 1210 are located about 4.4 mm from the channels 1206, 1226.
The aqueous channels 1221, 1222, 1224 make a right angle turn about
2.9 mm from the inlet. The right angle turn has an inner radius
R0.300 and an outer radius R0.500. The portion of channels 1221,
1222, 1224 that are disposed in a plane parallel to channel 1206
are about 1.300 mm from channel 1206. The aqueous channels 1221,
1222, 1224 make a 45 degree turn before connecting with each other
upstream of the neck region. The inner diameter of channel 1206 is
about 0.200 mm (200 um). The parallel portions of channel 1206 are
about 1.2 mm apart. However, other suitable dimensions are
possible.
[0050] FIG. 13 shows a representative example of another embodiment
of a mixer that corresponds to the mixer circuits 804 and 904 shown
in FIGS. 8 and 9. The mixer circuit 1300 comprises aqueous channels
1321, 1322, and 1324. The aqueous channels are separated from the
carrier fluid channel 1306 and the inspection circuit 1326 by a
short neck region. The diameter of the neck region is about 0.200
mm. However, other suitable dimensions are possible. The channels
are oriented such that the three channels containing aqueous
solutions come together and intersect the channel containing the
carrier fluid at a 90 degree angle. Downstream of the mixer
circuit, the solutions flow into the inspection circuit 1326.
[0051] FIG. 14 shows a representative example of a cross-section
through a crystal card similar to the embodiment illustrated in
FIG. 9. The crystal card 1400 is comprised of three layers 1420,
1430 and 1440. Layer 1430 comprises the ports as shown in FIGS.
7-9. Layer 1430 is about 0.4 mm thick. Layer 1440 comprises the
microfluidic channel circuit and is about 1.5 mm thick at the edge.
Layer 1420 comprises the peelable layer attached to the bottom
surface of the crystal card 1400, and is about 0.14 mm thick.
[0052] FIGS. 15A-15V illustrate a method 5000 for crystallizing
molecules using a nanovolume microcapillary crystallization system.
From a start block, the method 5000 proceeds to a set of method
steps 5002, defined between a continuation terminal ("Terminal A")
and an exit terminal ("Terminal B"). The set of method steps 5002
describes the preparation of a crystal card and the connection of
the crystal card to a pump.
[0053] From Terminal A (FIG. 15B), the method 5000 proceeds to a
set of method steps 5008 where the crystal card is manufactured
from a suitable material, such as polydimethylsiloxane (PDMS) or
plastic by injection molding. The method then returns to a point of
invocation. The method 5000 next proceeds to a set of method steps
5010 defined by a continuation terminal ("Terminal A2"). The set of
method steps 5010 treats the microcapillary surface of the crystal
card to reduce the surface energy.
[0054] From Terminal A2 (FIG. 15C), the method 5000 proceeds to
decision block 5014 where a test is performed to determine whether
the crystal card is manufactured from plastic. If the answer to the
test is NO, the method proceeds to another continuation terminal
("Terminal A4"). If the answer to the test at decision block 5014
is YES, the method proceeds to another decision block 5016 where
another test is performed to determine whether the plastic is
polycarbonate. If the answer to the test at decision block 5016 is
NO, the method 5000 proceeds to another continuation terminal
("Terminal A5"). If the answer to the test at decision block 5016
is YES, the method 5000 proceeds to another continuation terminal
("Terminal A6").
[0055] From Terminal A4 (FIG. 15D), the method 5000 proceeds to
block 5018 where the method treats the crystal card as manufactured
from PDMS. The method proceeds to block 5020 where the
microcapillary surface is treated with a perfluorinated silane
solution for 2 hours at room temperature. The method then proceeds
to block 5022 where the perfluorinated silane solution is removed
by vacuum. At block 5024, the microcapillary surface of the crystal
card is dried using a gas such as air under pressure at 5-10 psi
for 1 hour. The method then returns to the point from which the
steps of Terminal A2 were invoked, and proceeds to another
continuation terminal ("Terminal A3"). See block 5012.
[0056] From Terminal A5 (FIG. 15E), the method 5000 proceeds to
block 5026 where the method treats the crystal card as made of a
plastic comprising cyclic olefin copolymer (COC) or comparable
plastic. At block 5028, the microcapillary surface is treated with
a reagent to reduce the surface energy (hydrophobicity) of the
plastic for 2 hours at room temperature. Suitable reagents for
reducing the surface energy include fluorinated copolymer
solutions, but other reagents are possible. Suitable fluorinated
copolymer solutions include a two percent fluorinated copolymer
solution in a fluorosolvent, such as Cytonix PFC 502AFA (Cytonix
Corp., Beltsville, Md.). Cytonix PFC 502AFA is manufactured to
adhere to polycarbonate and reduce the surface energy to 6-10
dyne/cm. To apply the fluorinated copolymer solution, the crystal
card is filled from the outlet with the Cytonix PFC 502AFA
solution. At block 5030, the fluorinated copolymer solution is
removed by vacuum. At block 5032, the microcapillary surface is
dried using a gas such as air under pressure of 5-10 psi for 1
hour. The method 5000 then proceeds to block 5034 where the crystal
card is heated to 60.degree. C. for 1 hour. The method then returns
to the point of invocation of the steps of Terminal A2. See block
5012 at Terminal A3.
[0057] From Terminal A6 (FIG. 15F), the method 5000 proceeds to
block 5036 where the crystal card is pre-chilled on ice. At block
5038, the microcapillary surface is treated with a fluorinated
copolymer solution such as Cytonix PFC 502AFA for 2 hours on ice.
The polycarbonate crystal card inlets may be prone to cracking if
incubated with the 502AFA solution at higher temperatures. The
method then proceeds to continuation terminal A5 where it skips to
block 5030 and performs the steps in blocks 5030, 5032, and 5034.
The method then returns to a point at which the steps of Terminal
A2 were invoked. See Terminal A3 at block 5012. The set of method
steps at block 5012 couples the crystal card to the pump.
[0058] From Terminal A3 (FIG. 15G), the method 5000 proceeds to
block 5040 where syringe 1 is filled with a buffer or aqueous
solution. At block 5042, syringe 2 is filled with a precipitant
solution. At block 5044, syringe 3 is filled with a carrier fluid.
A representative example of a suitable carrier fluid includes a
fluorinated carbon solution. Suitable examples of a fluorocarbon
fluid include FC-40. FC-40 has a high surface tension with the
detergents used in solubilizing membrane proteins. The surface
tension enables nanoplug formation and crystallization. In a
representative embodiment, the carrier fluid is a fluorinated oil
which is immiscible with aqueous fluids. The carrier fluid
surrounds and separates the aqueous nanoplugs as they are formed,
moving them forward through the crystal card during the method. At
block 5046, syringe 4 is filled with a protein solution containing
the protein of interest in a suitable buffer. At block 5048,
suitable tubing such as Teflon.RTM. tubing is attached to the
needle of each syringe. At block 5050, syringes 1 and 2 are
attached to pump 1, and syringes 3 and 4 are attached to pump 2. At
block 5052, the tubing is connected to the crystal card via a
macro-micro interface. Suitable connections for the macro-micro
interface are described above. The method then proceeds to exit
Terminal B.
[0059] From Terminal B, the method 5000 proceeds to a set of method
steps 5004, defined between a continuation terminal ("Terminal C")
and an exit terminal ("Terminal D"). The set of method steps 5004
receives instructions to regulate fluid flow through the crystal
card to obtain crystals. From Terminal C (FIG. 15H), the method
5000 proceeds to a set of method steps 5054, defined by a
continuation terminal ("Terminal C1"). The set of method steps 5054
configures the pump.
[0060] From Terminal C1 (FIG. 15I), the method 5000 proceeds to
block 5060 where the method receives instructions on the type of
syringe pump model to be controlled by the system. Suitable pumps
include Harvard Apparatus Twin Syringe Pump Model 33 (Harvard
Apparatus, Holliston, Mass.), which has been modified by the
manufacturer to provide better accuracy. As illustrated by FIG. 2,
each syringe pump controls two syringes. At block 5062, the method
receives instructions on the serial communication port of a
computer used to control the pump system. The communication ports
are configured such that each syringe pump receives instructions at
the same time, thereby preventing time delays and allowing the
solutions to flow through the crystal card simultaneously. The
method proceeds to block 5064 where the method receives
instructions on the volume of each syringe connected to the pumps.
At block 5066, the method determines the diameter of each syringe
connected to the pumps. The method then proceeds to return to a
point at which the steps of the Terminal C1 were invoked.
[0061] From block 5054, the method 5000 proceeds to a set of method
steps 5056 defined by a continuation terminal ("Terminal C2"). The
set of method steps primes fluids to the mixer circuit of the
crystal card. From Terminal C2 (FIG. 15J), the method 5000 proceeds
to block 5068 where the method receives instructions on which
syringe will be used to dispense fluids into the mixer of the
crystal card. At block 5070, the method receives instructions on
the flow rate from each syringe. At block 5072, the method receives
instructions on the volume of fluid to be dispensed by the syringe.
At block 5074, the method dispenses or aspirates fluid from a
fluidic channel upstream of the mixer circuit. The method then
continues to another continuation terminal ("Terminal C4").
[0062] From Terminal C4 (FIG. 15K), the method 5000 proceeds to
decision block 5076 where a test is performed to determine whether
the syringe is dispensing an aqueous fluid. If the answer to the
test at decision block 5076 is NO, the method proceeds to another
continuation terminal ("Terminal C5). If the answer to the test at
decision block 5076 is YES, the method proceeds to block 5078 where
the method receives instructions to stop the aqueous fluid at the
mixer circuit and before the fluid enters the inspection circuit.
The method then continues to Terminal C2 and repeats the above
identified process steps for the next syringe. From Terminal C5
(FIG. 15K), the method 5000 proceeds to block 5080 where the method
receives instructions to stop the carrier fluid downstream of the
mixer circuit and slightly inside the inspection circuit. The
method then proceeds to return to a point from which the steps of
Terminal C2 were invoked.
[0063] Digressing, an illustrative process for priming aqueous
solutions and the carrier fluid to the mixer of the crystal card
will now be described in detail. First, the empty crystal card
mixer circuit is positioned on the microscope stage for observation
during priming. The method receives instructions to dispense a
solution, for example buffer, from syringe 1 to the mixer. The
buffer is dispensed into the fluid channel connected to syringe 1
until the user observes that the solution has reached the region of
the mixer just upstream of the junction between the fluidic
channels. The method then receives instructions to stop dispensing
the solution. Solution may be removed from the channel by
instructing the method to aspirate the reagent. It is suitable to
refrain aqueous solutions from entering the inspection circuit of
the crystal card. The method is repeated for each of the three
fluid channels connected to syringes dispensing aqueous solutions;
for example, syringe 4 (protein solution) and syringe 2
(precipitant solution). The carrier fluid is then dispensed into
the fourth fluid channel connected to syringe 3. The carrier fluid
is dispensed into the fourth fluid channel until the fluid travels
through the mixer junction and just slightly enters the inspection
circuit (fifth channel) of the crystal card. The method then
receives instructions to stop dispensing the carrier fluid.
[0064] Returning to block 5056, the method 5000 proceeds to a set
of method steps 5058 defined by a continuation terminal ("Terminal
C3"). The set of method steps receives instructions to produce
aqueous nanoplugs in the inspection circuit of the crystal card.
From Terminal C3 (FIG. 15L), the method 5000 receives instructions
on which nanoplug formation protocol will be performed at block
5082. The method then proceeds to decision block 5084 where a test
is performed to determine whether the instruction received was to
perform the constant mode. If the answer to the test at block 5084
is NO, the method proceeds to another continuation terminal
("Terminal C6"). If the answer to the test at decision block 5084
is YES, the method proceeds to block 5086 where the method receives
instructions on the flow rate for each syringe. The method then
proceeds to block 5088 where the method receives instructions on
the total volume of fluid to pass through the mixer circuit. At
block 5090, the method produces aqueous nanoplugs inside the
inspection circuit of the crystal card wherein each nanoplug is
suitably of equal size and has the similar concentration of protein
and precipitant. The method then proceeds to return to a point of
invocation. From block 5058, the method proceeds to exit terminal
D.
[0065] From Terminal C6 (FIG. 15M), the method 5000 proceeds to
decision block 5092, where a test is performed to determine whether
the method was instructed to perform gradient mode. If the answer
to the test in block 5092 is NO, the method proceeds to another
continuation terminal ("Terminal C7"). If the answer to the test in
decision block 5092 is YES, the method proceeds to block 5094 where
the method receives instructions on the maximum flow rate for the
syringes with variable flow. In one embodiment, the variable flow
syringes contain the buffer and precipitant. In another embodiment,
syringes 1 and 2 are the variable flow syringes. However, the
method can designate any syringe to be a variable flow syringe. In
one embodiment, the combined flow rate of the variable flow
syringes equals the maximum flow rate. For example, in one
embodiment, the method provides instructions for the flow rate of
syringe 1 to equal 2 .mu.l/min, whereas the method provides
instructions for the flow rate of syringe 2 to equal 0 (zero)
.mu.l/min. In this embodiment, the maximum flow rate equals 2
ul/min (2+0 .mu.l/min). The method then proceeds to block 5096
where the method receives instructions on the constant flow rate
for the syringe controlling the carrier fluid. In one embodiment,
syringe 3 controls the carrier fluid. In one embodiment, the
carrier fluid flow rate equals the total flow rate of the aqueous
solutions (buffer, precipitant, and protein solutions). In another
embodiment, the flow rate for the carrier fluid may be selected to
be slower or faster than the total flow rate of the aqueous fluids.
Slower carrier fluid rates generate larger aqueous nanoplugs with
smaller segments comprising carrier fluid between nanoplugs. Faster
carrier fluid rates generate smaller aqueous nanoplugs with larger
carrier fluid segments between the nanoplugs. The method then
proceeds to block 5098 where the method receives instructions on
the constant flow rate for the syringe controlling the protein
solution. In one embodiment, syringe 4 controls the carrier fluid.
In one embodiment, the protein flow rate equals the sum of the flow
rate of the other aqueous solutions (buffer and precipitant).
Changing the flow rate of the protein solution changes the ratio of
protein-to-crystallization conditions in each nanoplug. The method
then proceeds to block 6000 where the method receives instructions
on the total aqueous volume to be dispensed during a single
iteration or cycle of the method. The method then proceeds to
another continuation terminal ("Terminal C8").
[0066] From Terminal C8 (FIG. 15N), the method 5000 proceeds to
block 6002 where the method receives instructions on the volume of
each aqueous nanoplug that will be dispensed into the inspection
circuit. At block 6004, the method receives instructions on the
total number of iterations or cycles to be performed (i.e., the
number of times the gradient screening steps are repeated). In one
embodiment, if the method receives instructions to run zero
iterations, the pumps will stop when the total aqueous volume
selected at block 6000 is dispensed. In another embodiment, if the
method receives instructions to run one or more iterations, the
pumps will stop when the process steps described above have been
repeated the desired number of times. At block 6006, the method
reciprocally varies the flow rate of the buffer and precipitant
solutions such that the sum of the buffer and precipitant solution
flow rates equals the maximum flow rate selected at block 5094. For
example, in one embodiment, at block 5094 the method provides
instructions for the flow rate of syringe 1 to equal 2 .mu.l/min
and provides instructions for the flow rate of syringe 2 to equal 0
.mu.l/min, such that the maximum flow rate equals 2 .mu.l/min. When
the method starts, the flow rate from syringe 1 will begin at 2
.mu.l/min and ramp down to 0 .mu.l/min, while the flow rate from
syringe 2 will simultaneously ramp up from 0 .mu.l/min to 2
.mu.l/min. At block 6008, the method produces a series of aqueous
nanoplugs inside the inspection circuit wherein each drop is of
equal size but varies in the concentrations of protein and
precipitant in each drop. At block 6010, the method terminates
after the desired number of iterations or cycles has been
performed. The method then returns to block 5058 where the method
proceeds to exit terminal D.
[0067] From Terminal C7 (FIG. 15O), the method 5000 proceeds to
decision block 6012 where a test is performed to determine whether
the method was instructed to perform hybrid mode. If the answer to
the test at block 6012 is NO, the method proceeds to another
continuation terminal ("Terminal C9"). If the answer to the test at
block 6012 is YES, the method proceeds to another decision block
6014 where a test is performed to determine whether a precipitant
cartridge has been prepared. If the answer to the test at decision
block 6014 is NO, the method proceeds to another continuation
terminal ("Terminal C10"). If the answer to the test at block 6014
is YES, the method proceeds to another continuation terminal
("Terminal C11").
[0068] From Terminal C9 (FIG. 15P), the method 5000 proceeds to
decision block 6016 where a test is performed to determine whether
the method was instructed to perform the pulsatile mode. If the
answer to the test at decision block 6016 is NO, the method returns
to Terminal C3 where the above identified steps are repeated. If
the answer to the test at decision block 6016 is YES, the method
proceeds to block 6018 where the method receives instructions on
performing the pulsatile mode. The method then returns to block
5058. From block 5058, the method exits to Terminal D.
[0069] From Terminal C10 (FIG. 15Q), the method 5000 proceeds to
block 6020 where a syringe is connected to tubing, such as
Teflon.RTM. tubing, containing carrier fluid. The method then
proceeds to block 6022 where the syringe is connected to a syringe
pump. At block 6024, the method receives instructions to enter a
defined volume, for example, about 40 nL, and aspirates an air
bubble of about 40 nL into the tubing. At block 6026, the method
aspirates a defined volume, for example, about 120 nL, of a
precipitant solution into the tubing. At block 6028, the method
repeats the above two steps until a suitable number of precipitants
are loaded into the tubing. For example, a suitable number of
precipitants can range from 1-24 or more. At block 6030, the method
aspirates carrier fluid, about 1 .mu.L, into the open tip of the
tubing. At block 6032, the tubing is connected to the precipitant
inlet of the crystal card. The method then proceeds to continuation
Terminal C11.
[0070] At Terminal C11 (FIG. 15R), the method 5000 proceeds to
block 6034 where the method receives instructions on the starting
flow rate of the buffer solution (syringe 1). At block 6036, the
method receives instructions on the change in the flow rate (step
size) of the buffer solution. The step size is the change in the
rate of flow that will be applied at each ramp up or down of the
method. At block 6038, the method receives instructions on the
starting flow rate of the precipitant cartridge (syringe 2). At
block 6040, the method calculates the change in the flow rate (step
size) of the precipitant solution. In one embodiment, the step size
for the buffer equals the step size for the precipitant. At block
6042, the method sums the buffer and precipitant flow rates to
determine the total flow rate. At block 6044, the method receives
instructions on the starting flow rate for the carrier fluid
(syringe 3). At block 6046, the method receives instructions on the
change in the flow rate (step size) of the carrier fluid. The
method then proceeds to another continuation terminal ("Terminal
C12").
[0071] From Terminal C12 (FIG. 15S), the method 5000 proceeds to
block 6048 where the method receives instructions on the constant
flow rate of the protein solution (syringe 4). The method then
proceeds to block 6050 where the method receives instructions on
the number of ramp up steps (rate of flow changes) for each
precipitant. At block 6052, the method sets the number of ramp down
steps to equal the number of ramp up steps for each iteration or
cycle of the method. At block 6054, the method receives
instructions on the number of iterations or cycles to be performed.
In one embodiment, one iteration or cycle corresponds to a single
precipitant loaded in the precipitant cartridge. At block 6056, the
method receives instructions on the duration of each ramp step. For
example, in one embodiment, the duration of each ramp step is 1.5
seconds. At block 6058, the method reciprocally varies the buffer
and precipitant flow rates such that the sum equals the starting
rates. The method then proceeds to another continuation terminal
("Terminal C13").
[0072] From Terminal C13 (FIG. 15T), the method 5000 proceeds to
block 6060 where the method varies the flow rate of the carrier
fluid. The method then proceeds to block 6062 where the method
produces a series of nanoplugs inside the inspection circuit
wherein each drop has equal amounts of protein and varying amounts
of precipitant and buffer. In one embodiment, the method provides a
varied amount of precipitant with a constant amount of protein for
each cycle. Table 1 illustrates one embodiment of the method
described above for the hybrid mode. The method then proceeds to
Terminal D.
TABLE-US-00001 TABLE 1 Values specified by the method in hybrid
mode. One Carrier Total cycle Buffer Precipitant Fluid Protein Sum
Duration time up Step (ul/min) (ul/min) (ul/min) (ul/min) Aqueous
(sec) (sec) 0 0.2 0.6 2.2 0.6 1.4 1.5 Ramp 1 0.3 0.5 2.0 0.6 1.4
1.5 up 2 0.4 0.4 1.8 0.6 1.4 1.5 3 0.5 0.3 1.6 0.6 1.4 1.5 4 0.6
0.2 1.4 0.6 1.4 1.5 6.0 Ramp 1 0.5 0.3 1.6 0.6 1.4 1.5 down 2 0.4
0.4 1.8 0.6 1.4 1.5 3 0.3 0.5 2.0 0.6 1.4 1.5 4 0.2 0.6 2.2 0.6 1.4
1.5 6.0 Step 0.1 0.1 0.2 stable size
[0073] From Terminal D at block 5004, the method 5000 proceeds to a
set of method steps 5006, defined between a continuation terminal
("Terminal E") and an exit terminal ("Terminal F"). The set of
method steps 5006 performs diffraction experiments on the crystals
obtained from the crystal card. From Terminal E (FIG. 15U), the
method 5000 proceeds to decision block 6064 where a test is
performed to determine whether crystals were extracted from the
inspection circuit of the crystal card prior to diffraction. If the
answer to the test at block 6064 is NO, the method proceeds to
another continuation terminal ("Terminal E1"). If the answer to the
test at block 6064 is YES, the method proceeds to block 6066 where
a peelable layer is removed from the bottom surface of the crystal
card. In one embodiment, the peelable layer is bonded to the
plastic part of the crystal card that contains the microfluidic
channels. The bond is designed to be strong enough to prevent fluid
from leaking out of the microfluidic circuit but weak enough to be
manually peeled off. In one embodiment, the bond is a thermal bond.
In another embodiment, the bond is a chemical bond. Removal of the
peelable layer exposes the interior of the microfluidic channels of
the crystal card, allowing access to the aqueous nanoplugs. In
another embodiment, the aqueous nanoplugs that contain crystals are
retained in the microfluidic channels of the crystal card after the
peelable layer is removed. At block 6068, the crystal formed in the
inspection circuit is extracted from the crystal card using a
cryoloop. In one embodiment, the cryoloop is a nylon cryoloop. At
block 6070, the crystal is cryocooled, and diffraction data is
obtained. The method then proceeds to exit Terminal F where the
method terminates execution.
[0074] From Terminal E1 (FIG. 15V), the method 5000 proceeds to
block 6072 where the crystal card containing crystals is mounted
onto the goniometer of an X-ray source. At block 6074, the method
obtains diffraction data from crystals located in situ inside the
inspection circuit. The method then proceeds to block 5006 and exit
Terminal F. The method then terminates execution.
[0075] The above described crystal extraction steps can be used in
combination with the gradient screening of various embodiments of
the subject matter to generate crystals of methionine-R-sulfoxide
reductase. Crystals were removed from the crystal card using a
cryoloop and then cryocooled for diffraction experiments. As an
example, a 1.7 .ANG. data set was collected at SBC-CAT beamline
19BM located at the Advanced Photon Source at Argonne National
Laboratories and the structure was subsequently solved and refined.
The final coordinates and structure factors were deposited to the
Protein Data Bank (accession code 3CXK).
[0076] The crystal card of various embodiments of the subject
matter is also suitable for in situ diffraction. In situ
diffraction allows the crystallographer to assess the quality of a
crystal before being altered by the cryoprotection process. For
robust crystals, it can allow complete diffraction data to be
collected. The crystal card is sufficiently X-ray transparent to be
mounted onto the goniometer of an X-ray source for diffraction data
collection at room temperature. For example, a simple test was
conducted to analyze the absorption of the X-Rays by the crystal
card. The beam current in the ion chamber normalized to the APS
ring current (I/I.sub.0) was measured with and without the crystal
card inserted at a wavelength of 0.979261 A (12.66099 keV).
I/I.sub.0 without the crystal card measured 1.91671 E-6 and
I/I.sub.0 with the crystal card measured 1.5511 E-6. This
constitutes a 19% X-ray absorbance by the crystal card. Further,
the crystal card can be translated along its X and Y axis to
collect data from multiple crystals to be combined for a complete
data set. To demonstrate this technique, a crystal card containing
Lysozyme crystals was mounted on the goniometer head at NE-CAT
beamline 24ID-C located at the Advanced Photon Source at Argonne
National Laboratories. Data were collected at room temperature from
three crystals in the crystal card. Crystallographic data are
provided in Appendix A.
[0077] Regarding structure determination, data sets were collected
at the Advanced Photon Source: beamline 19BM at 100K for
methionine-R-sulfoxide reductase and beamline 24-IDC at room
temperature for lysozyme. Data were integrated and scaled with
HKL2000. For the lysozyme structure, intensities were integrated
separately for each of the three data sets using the mosflm
package. The structures of lysozome and methionine-R-sulfoxide
reductase were solved by molecular replacement using Molrep and PDB
entries HEE and 3CEZ as the search models, respectively. Structures
were refined with Refmac5 and model building was performed with
Coot.
[0078] While illustrative embodiments have been illustrated and
described, it will be appreciated that various changes can be made
therein without departing from the spirit and scope of the
invention.
TABLE-US-00002 APPENDIX A Crystallographic Data: methionine-R-
Lysozyme sulfoxide reductase Data Collection Unit Cell (.ANG.) a =
79.18, a = 42.00, b = 45.17 c = 38.38 c = 45.40, .alpha. = 88.4
.beta. = 83.7, .gamma. = 69.1 Space group P4.sub.32.sub.12 P1 (No.
1) (No. 96) Resolution (.ANG.) 50-1.90 50-1.70 Wavelength (.ANG.)
0.97950 0.97932 Total Reflections 54,338 118,181 Unique Reflections
10,151 32,539 I/(sigI)* 11.4 (2.9) 23.1 (2.2) R.sub.merge (%)* 13.7
(58.4) 6.8 (42.3) Completeness (%)* 98.8 (98.5) 95.3 (87.4)
Redundancy 5.4 (5.0) 3.6 (3.3) Wilson B factor (.ANG..sup.2) 24.1
22.1 Refinement Resolution (.ANG.) 50-1.90 50-1.70 Reflections
(working/test) 9,412/480 30,828/1,650 R.sub.working/R.sub.free (%)
19.6/23.0 16.6/19.9 Number of atoms (protein/water) 1001/45
2082/180 r.m.s. deviation bond length (.ANG.) 0.016 0.015 r.m.s.
deviation bond angle 1.607 1.408 (degrees) Average B factor
(.ANG..sup.2) (All atoms) 28.8 28.3 Average B factor (.ANG..sup.2)
(Protein) 28.5 27.5 Average B factor (.ANG..sup.2) (Water) 35.9
37.0 Coordinate error (.ANG.) Based on R.sub.free 0.149 0.095
Ramachandran Analysis (%) Most Favored (chain A/B) 89.4 91.7/90.8
Additionally Allowed (chain A/B) 10.6 7.3/8.3 *Parenthesis
indicates values for the 2.00 .ANG. to 1.90 .ANG. resolution shell
for lysozyme and 1.76 .ANG. to 1.70 .ANG. shell for
methionine-R-sulfoxide reductase.
* * * * *