U.S. patent application number 09/822595 was filed with the patent office on 2001-10-11 for protein crystallization in microfluidic structures.
Invention is credited to Sygusch, Jurgen, Weigl, Bernhard H..
Application Number | 20010027745 09/822595 |
Document ID | / |
Family ID | 22715327 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010027745 |
Kind Code |
A1 |
Weigl, Bernhard H. ; et
al. |
October 11, 2001 |
Protein crystallization in microfluidic structures
Abstract
A device for promoting protein crystal growth (PCG) using
microfluidic channels. A protein sample and a solvent solution are
combined within a microfluidic channel having laminar flow
characteristics which forms diffusion zones, providing for a well
defined crystallization. Protein crystals can then be harvested
from the device. The device is particularly suited for microgravity
conditions.
Inventors: |
Weigl, Bernhard H.;
(Seattle, WA) ; Sygusch, Jurgen; (Montreal,
CA) |
Correspondence
Address: |
JERROLD J. LITZINGER
SENTRON MEDICAL, INC.
4445 LAKE FOREST DR.
SUITE 600
CINCINNATI
OH
45242
US
|
Family ID: |
22715327 |
Appl. No.: |
09/822595 |
Filed: |
March 30, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60193867 |
Mar 31, 2000 |
|
|
|
Current U.S.
Class: |
117/206 |
Current CPC
Class: |
Y10T 117/1024 20150115;
B01F 33/30 20220101; Y10S 117/90 20130101; B01L 2300/0867 20130101;
G01N 2035/00336 20130101; B01L 3/06 20130101; Y10T 117/10 20150115;
C30B 29/58 20130101; C30B 30/08 20130101; B01L 3/50273 20130101;
B01L 2400/0481 20130101; B01L 2400/0605 20130101; B01L 2400/0487
20130101; B01L 2200/0636 20130101; C30B 7/00 20130101; B01L
2300/0816 20130101; C30B 27/00 20130101; G01N 25/14 20130101; B01F
33/3039 20220101; B01L 2200/0684 20130101; B01L 3/502776 20130101;
B01L 7/54 20130101; B01L 3/5025 20130101; C30B 27/00 20130101; C30B
29/58 20130101; C30B 7/00 20130101; C30B 29/58 20130101 |
Class at
Publication: |
117/206 |
International
Class: |
C30B 001/00 |
Claims
What is claimed is:
1. A device for promoting protein crystallization growth from
solution, comprising: a body structure; means located within said
body structure for introduction of at least one solution containing
protein and at least one solution containing a solvent; and at
least one microfluidic channel connected to said introduction means
wherein said protein solution and said solvent solution interact to
induce formation of protein crystals within said channel.
2. The device of claim 1, wherein said protein solution and said
solvent solution flow laminarly in parallel contact within said
microfluidic channel to establish a concentration gradient within
said channel, allowing for protein crystallization.
3. The device of claim 1, wherein said protein solution
introduction means and said solvent solution introduction means are
each connected to said crystallization channel by a microfluidic
channel.
4. The device of claim 3, wherein said protein microfluidic
channel, said solvent microfluidic channel, and said
crystallization channel form a T-Sensor structure.
5. The device of claim 1, further comprising a chamber coupled to
said crystallization channel for harvesting said formed protein
crystals.
6. The device of claim 1, further comprising fluid movement
generating means coupled to said protein solution introduction
means and said solvent solution introduction means for propelling
said solutions through said crystallization channel.
7. The device of claim 6, wherein said fluid movement generating
means comprises an air bellows.
8. The device of claim 1, further comprising a mixing means,
coupled between said protein solution introduction means and said
solvent introduction means interface and said crystallization
channel for mixing said protein solution and said solvent solution
completely to form a homogeneous mixture.
9. The device of claim 8, wherein said mixing means comprises a jet
vortex mixer.
10. The device of claim 9, further comprising a solvent absorbing
means coupled to said crystallization channel for absorbing solvent
from said homogeneous mixture to increase the concentration of
protein within said mixture, thereby inducing increased protein
crystallization.
11. A device for promoting protein crystallization growth from
solution, comprising: a body structure; means located within said
body structure for introduction of at least one solution containing
protein, at least one solution containing a solvent, and at least
one solution containing a combination of protein and a buffer; a
microfluidic structure coupled to said protein solvent introduction
means and said combination protein and buffer solution introduction
means for flowing said solutions laminarly in parallel to remove
irreversible protein aggregates from said combined solutions; and
at least one microfluidic channel connected to said solvent
introduction means and the output of said microfluidic structure
wherein said solvent solution and said combined solutions interact
to induce formation of protein crystals within said channel.
12. The device of claim 11, further comprising a waste chamber
coupled to said microfluidic structure to retain said irreversible
protein aggregates.
13. The device of claim 11, further comprising a chamber coupled to
said crystallization chamber for harvesting said formed protein
crystals.
14. The device of claim 11, further comprising fluid movement
generating means coupled to said protein solution introduction
means, said solvent solution introduction means, and said combined
protein and buffer solution introduction means for propelling said
solutions through said crystallization channel.
15. The device of claim 14, wherein said fluid movement generating
means comprises an air pump.
16. The device of claim 11, wherein said microfluidic structure
comprises an H-filter.
17. The device of claim 1, wherein said body structure is
constructed from plastic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application takes priority from U.S. Provisional
Application Serial No. 60/193,867, filed Mar. 31, 2000, which
application is incorporated herein in its entirety by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to a device for growing
crystals, and, more particularly, to a device for promoting protein
crystal growth using microfluidic structures.
[0004] 2. Description of the Related Art
[0005] Macromolecular crystals have become keystones of molecular
biology, biochemistry, and biotechnology. Understanding how
crystals express their function depends on knowledge of the
macromolecular architecture at the atomic level.
[0006] The determination of the three dimensional atomic structure
of crystals is one of the most important areas of pure and applied
research. This field, known as X-ray crystallography, utilizes the
diffraction of X-rays from crystals in order to determine the
precise arrangement of atoms within the crystal. The result may
reveal the atomic structure of substances as varied as metal alloys
to the structure of deoxyribonucleic acid (DNA). The limiting step
in all of these areas of research involves the growth of a suitable
crystalline sample.
[0007] One important and rapidly growing field of crystallography
is protein crystallography. Proteins are polymers of amino acids
and contain thousands of atoms in each molecule. Considering that
there are 20 essential amino acids in nature, one can see that
there exists virtually an inexhaustible number of combinations of
amino acids to form protein molecules. Inherent in the amino acid
sequence or primary structure is the information necessary to
predict the three dimensional structure. Unfortunately, science has
not yet progressed to the level where this information can be
obtained quickly and easily. Although considerable advances are
being made in the area of high field nuclear magnetic resonance, at
the present time the only method capable of producing a highly
accurate three dimensional structure of a protein is by the
application of X-ray crystallography. This requires the growth of
reasonably ordered protein crystals (crystals which diffract X-rays
to at least 3.0 angstroms resolution or less), as the accuracy of
structures determined by X-ray crystallography is limited by the
disorder in the crystallized protein.
[0008] The maximum extent of a diffraction pattern is generally
considered to be a function of the inherent statistical disorder of
the molecules of protein crystals rather than the result of purely
thermal effects. Statistical disorder present in protein crystals
has two principal sources: 1) intrinsic structural or
conformational variability of protein molecules, and 2) spatial
distribution of the individual molecules about lattice sites
occupied.
[0009] In addition, other inherent limitations in the
crystallization process involve the effects of molecular
convection, thermal effects, and buoyancy, all due to the earth's
gravitational field. Therefore, it has been proposed to conduct
crystallization experiments in the microgravity ({fraction
(1/1000)} g to {fraction (1/10,000)} g) of space, on board the
space shuttle, international space station, or other similar
vehicles. Several patents disclose crystallization in microgravity
to improve the size, morphology and diffraction quality of
crystals. U.S. Pat. Nos. 5,362,325 and 4,755,363 are exemplary of
patents disclosing microgravity crystallization.
[0010] Focus of microgravity research in protein crystal growth
(PCG) has been based on the observation that PCG in a microgravity
environment yields protein crystals that are of reduced disorder.
Reduction in lattice disorder by protein crystals grown in
microgravity compared to ground controls offers enhanced resolution
of diffracted intensities and translates at the atomic level into
more precise knowledge of the protein architecture. The detailed
knowledge of how ligands interact with binding sites at the atomic
level permits insight into catalytic mechanisms and recognition in
biological systems, a prerequisite for structure-assisted drug
design. In a pharmaceutical industry setting, higher resolution
implies significant manpower reduction in synthetic chemistry to
explore the drug-binding site and results in more rapid
optimization of drug target interaction. Accelerated drug design is
extremely cost effective, allowing a pharmaceutical company to
quickly recover R&D costs and improve profitability.
[0011] Several important advances have recently accelerated the
structure determination process using even small crystals. These
include selenomethionyl proteins, cryo-crystallography, high
intensity synchrotron radiation sources, CCD detectors, and
multiwavelength anomalous diffraction (MAD) phasing. With these
advances, a protein structure can be solved by MAD phasing
literally within hours of data collection at a synchrotron
radiation source. The outstanding uncertainty faced by protein
crystallography is the growth of high quality protein crystals.
[0012] In the very near future, it is expected that the field of
structural genomics will foster a tremendous explosion in demand
for protein structure determination. Genome sequencing or genomics
is significantly impacting biological research by changing our
understanding of biological processes through identification of
novel proteins that may be involved in disease or are unique to
pathogenic organisms. Genome project results have shown that in
most organisms, more than 50% of the proteins have no assigned
function. In the human genome, this amounts to over 50,000
proteins. These uncharacterized proteins thus represent a reservoir
of untapped biological information that is widely acknowledged as
the next generation of protein therapeutics and targets for
pharmaceutical development. With large-scale genomic sequencing now
becoming routine, attention is being focused on understanding the
structure and function of these biological macromolecules. Recently
published examples where knowledge of a three-dimensional structure
of an unknown protein can provide clues to its function is expected
to open the gates to a massive need for high quality structure
determination.
[0013] The crystallization process generally involves several
distinct phases, such as nucleation and post-nucleation growth.
Nucleation is the initial formation of an ordered grouping of a few
protein molecules, while post-nucleation growth consists of the
addition of protein molecules to the growing faces of the crystal
lattice and requires lower concentrations than the nucleation
phase.
[0014] Most protein crystals nucleate at very high levels of
supersaturation, typically reaching up to 1000% in many cases. At
such supersaturation levels, post-nucleation crystal growth takes
place under very unfavorable conditions. Most macromolecules at the
concentrations needed to attain the very high levels of
supersaturation tend to form aggregates and clusters of both
ordered oligomeric species and/or random amorphous aggregates.
Depending on the half-life and concentration of such clusters,
formation of nuclei can involve incorporation of partially ordered
aggregated species. Quiescent conditions mitigate imperfect
post-nucleation growth at high supersaturation by reducing the
collision frequency of aggregate species of all kinds to form
larger clusters or nuclei. Microseeding a protein solution, that
is, introduction of freshly crushed crystallites, would provide a
succinct approach to circumvent growth from imperfect nuclei.
[0015] At higher levels of supersaturation, growth by absorption of
three-dimensional nuclei onto crystal faces has been observed in
crystallization studies of thaumatin, catalase, t-RNA, lysozyme,
lipase, STMV virus and canavalin. The three-dimensional nuclei have
observed average dimensions ranging between 1-10 .mu.m making them
colloidal in size. The origin of these nuclei is thought to be
protein clusters that originate from protein rich droplets
possessing short-range internal order and that undergo long-range
ordering upon interaction with the underlying crystal lattice.
[0016] Under quiescent conditions at low supersaturation, a protein
crystal grows by incorporation of individual protein molecules,
monomers, from the surrounding medium, which because of their low
diffusivities produce a concentration gradient or depletion zone
about the growing crystallite. For lysozyme, protein concentration
gradients measured by Mach-Zender interferometry surrounding a
large 1 mm crystal are the order of .about.10% over a 2-3 mm
distance. Larger aggregates in the bulk solution diffuse more
slowly than protein monomers, allowing the depletion zone to
kinetically discriminate against incorporation of large aggregates
into the crystal lattice. In effect, the depletion zone acts much
like a mass filter. The depletion zone not only tends to filter out
larger aggregates but also partially unfolded or denatured proteins
which also have larger hydrodynamic radii, hence lower
diffusivities than the compact globular native protein. Since mass
filtering is transient and based on differential diffusion of the
various species, protein crystal growth will eventually be
compromised by self-impurities as the system approaches
equilibrium. AFM studies in ground controls have shown that
macromolecular crystals tend to stop growing because of formation
of a dense impurity adsorption layer of protein restricting access
to crystal faces.
[0017] In microgravity, sedimentation and buoyancy convection
effects are suppressed and diffusion is the dominant mechanism of
protein transport. Hence, a depletion zone would be extended and
could more effectively exclude higher order protein aggregates from
incorporation into a growing protein crystal, thus leading to a
greater degree in crystal perfection. Recent PCG studies in
microgravity with lysozyme dimer self-impurities tend to support
this hypothesis. Non-quiescent conditions such as gravity induced
sedimentation of larger nuclei and/or crystallites adjacent to the
growing crystal would create disturbances in the depletion zone,
facilitating incorporation of higher concentrations of
self-impurities, and compromise its role of mass filtering.
Post-nucleation growth by absorption of three-dimensional nuclei,
observed at higher supersaturation levels, is particularly
susceptible to sedimentation effects and buoyancy-driven flow.
Particles such as nuclei of colloidal size are susceptible to
gravitational effects and this may be in large part the basis for
the beneficial effect of microgravity on PCG.
[0018] Frequently, prior to activation of a PCG experiment in
microgravity, purified protein is stored at high concentration for
as long as several weeks. For a protein maintained in soluble
state, protein instability or unfolding promotes production of
irreversible aggregates. Thus, given the high supersaturation
conditions required for nucleation, protein crystal nuclei may
contain significant concentrations of amorphous aggregates. Whether
presence of self-impurities is detrimental to subsequent ordered
post-nucleation growth and hence crystal quality is a function of
the ability of competent nuclei to promote post-nucleation growth
and concentration of competent monomeric species. Clearly, highly
ordered nuclei tend to be kinetically more stable than amorphous
aggregates, which is essential for sustaining post-nucleation
growth. However, under prolonged solution storage, irreversible
protein aggregation may compromise PCG success.
[0019] Several methods of protein crystallization have been
developed and successfully employed over the course of the last
century. These include vapor phase diffusion, liquid-liquid
interfacial diffusion, liquid-liquid turbulent mixing, and step
gradient methods.
[0020] Approximately 90% of protein crystallization experiments in
microgravity (and on the ground) in the past decade have used the
vapor diffusion or hanging drop method, in which water is
transported through the vapor phase from a drop of protein and
precipitant solution to a concentrated precipitant solution. This
method has several advantages, especially at 1.times.g, including
the relative absence of container surfaces, slow approach to
supersaturation, low volume requirement, and ease of observation of
crystal nucleation and growth, and it is fairly viscosity
independent. It also has a number of disadvantages, including
limited volume in the case of hanging (but not sessile) drops,
limited control over saturation rate, and a potential for the
establishment of convection currents at the liquid - air interface.
The sessile-hanging drop, like the hanging-drop method, removes
water only from the crystal-growth solutions. Unlike hanging drop
in the sessile drop method, buoyancy-driven fluid upwelling often
occurs, and the rate of water removal depends on vapor pressure.
Examples of devices which use the vapor-diffusion method include
U.S. Pat. Nos. 4,886,646; 5,103,531; 5,096,676; and 5,130,105.
[0021] Interfacial diffusion or liquid-liquid interfacial diffusion
as a technique for protein crystal growth involves superposition of
protein and precipitant solutions across an interface. PCG then
depends on mutual self-diffusion of protein and precipitant across
the resultant interface to grow protein crystals. Due to convection
effects, such interfaces are not stable on earth but can be
reproducibly generated in microgravity. The transient concentration
gradient affords control over nucleation events by spatially
reducing the number of nucleation sites. Protein dilution by the
precipitant solution as system equilibration takes place diminishes
the potential for protein aggregate incorporation into nuclei and
crystallites. In this method, a depletion zone will only be
established once the system has approached equilibrium. Mixing of
highly viscous fluids by interfacial diffusion occurs very slowly
and can correspond to a time scale incompatible with the duration
of a shuttle mission but is compatible with the ISS mission.
[0022] Turbulent mixing will result essentially in the system being
brought to its equilibrium value at the onset of the PCG experiment
and maintained at equilibrium throughout the experiment. This is
useful in allowing comparisons to be made where it is important to
know the final end point of a system and is akin to batch
crystallization. Turbulent mixing also overcomes difficulties
associated with mixing of viscous precipitants.
[0023] In the step gradient approach, homogeneous nucleation and
crystal growth are treated as separate steps. Homogeneous
nucleation is induced by bringing, carefully, a near saturated
protein solution into contact with a highly supersaturating
solution of precipitant (1.2-3.0 times saturating concentration,
for example). This exposure lasts just long enough to cause
nucleation, then the crystals are transferred to a slightly
saturating concentration of precipitant for quiescent crystal
growth. This method has been successfully used to grow protein
crystals in space.
[0024] An essential difference between vapor phase diffusion and
liquid-liquid interfacial diffusion is in their mutually orthogonal
approach to equilibrium in the protein solubility phase. Vapor
diffusion starts from a dilute protein solution that becomes
concentrated at equilibrium, while liquid-liquid interfacial
diffusion dilutes the protein starting condition.
[0025] All of the methods discussed above have gravity-dependent
components. Crystals more dense than the mother liquor sediment
away from the zone of crystallization, while those less dense float
away from this zone. Sedimentation against a vessel wall modifies
the habit of the crystal. Rapid nucleation on a dialysis membrane
or vessel wall sometimes leads to large numbers of small crystals.
Ideally, motionless, contactless crystal growth is desired, and the
microgravity environment of space flight comes very close to
providing these conditions.
[0026] Modern protein crystallography data collection techniques
make use of protein crystals flash frozen in liquid nitrogen to
minimize radiation damage. Crystals of large dimensions (0.5-1 mm)
are more readily damaged during flash freezing while smaller
crystals (.about.0.2 mm or less in average dimension) can be cooled
rapidly enough to prevent ice formation. Using 2.sup.nd and
3.sup.rd generation synchrotron radiation sources and CCD
detectors, even smaller crystals have been successfully exploited.
The device of the present invention thus targets growth of high
quality small and medium size crystals. The presence of
self-impurities is more likely to compromise growth of larger
crystals than smaller crystals largely in part to the longer time
scale involved for growth of large crystals, making them more
susceptible to protein denaturation phenomena.
[0027] Protein denaturation, if it does occur prior to PCG
activation in microgravity, can compromise PCG success by formation
of irreversible aggregates, self-impurities, in the protein
solution. If irreversible aggregation does take place in ground
experiments and compromises PCG success, the facility should be
able to mitigate against the protein aggregate population at the
time of PCG activation.
[0028] Protein crystals can be stressed or even damaged during
harvesting and/or in subsequent manipulations and therefore become
unsuitable for data collection. The present device should allow
facile harvesting of protein crystals for flash freezing. In
particular, potential crystal entrapment in corners should be
avoided.
[0029] The device should afford facile integration and dispersement
of large number of PCG experiments by a PCG mission integration
center as well as allow ready documentation of post-flight
results.
[0030] The PCG experiment should allow for crystallization in small
volumes comparable to volumes (.mu.L) used in routine laboratory
PCG screening, thus consuming as little protein as possible.
[0031] Technically, the facility should provide efficient
separation of protein and precipitant prior to orbit activation
with no absorption and leakage of fluids over the course of the
microgravity mission.
[0032] Microfluidic devices have been recognized to have great
potential in such areas as DNA sequencing and medical diagnostics.
Beyond this, they have the potential to allow separations, chemical
reactions, and calibration-free analytical measurements to be
performed directly on very small quantities of complex samples such
as whole blood and contaminated environmental samples. Therefore,
use of disposable microfluidic devices should be investigated as
means for growing protein crystals in microgravity.
[0033] The embodiment of the technology uses microfluidic
integrated circuits. These devices are thin transparent plastic or
glass structures, roughly credit card in size. Laminar flow
structures in these chips afford crystal growth by free
liquid-liquid interface diffusion, batch methods, or vapor
diffusion, depending on circuit design. The chips are readily
loaded with fluid samples, which, manufactured from transparent
material, allow facile documentation of PCG results and also permit
facile unloading and harvesting of protein crystals grown.
[0034] Most fluids show laminar behavior in miniature flow
structures with channel cross sections below 0.5 mm. Two or more
distinct fluid streams moving in such flow structures do not
develop turbulence at the interface between them or at the
interface with the capillary walls. Different layers of miscible
fluids and particles can thus flow next to each other in a
microchannel without any interaction, other than by diffusion of
their constituent molecular and particulate components.
Microfluidic channels typically have either width or height less
than .about.500 .mu.m. Liquids with viscosities comparable to water
or that flow slower than several cm/sec follow predictable laminar
paths. These conditions correspond to values of the non-dimensional
Reynolds numbers of .about.1 or less. The Reynolds number
characterizes the tendency of a flowing liquid to develop
turbulence; values greater than 2000 indicate turbulent flow.
Values between 1 and 2000 allow for so-called laminar
recirculation, which is frequently used in microfluidic mixing
structures.
[0035] Recent advances in device miniaturization have led to the
development of integrated microfluidic devices, so-called
labs-on-a-chip. In these tiny microchips etched with grooves and
chambers, a multitude of chemical and physical processes for both
chemical analysis and synthesis can occur. These devices, also
known as micro-total analysis systems (.mu.TAS), can be mass
produced in silicon by techniques similar to those used in the
semiconductor industry, or, for even lower cost, made out of
plastics by using casting, cutting, and stamping techniques. Recent
advances in microfabrication have extended the production of these
devices to include a wide range of materials. They offer many
advantages over traditional analytical devices: they consume
extremely low volumes of both samples and reagents. Each chip is
inexpensive and small. The sampling-to-result time is extremely
short. In addition, because of the unique characteristics exhibited
by fluids flowing in microchannels ("microfluidics"), it is
possible that these designs of analytical devices and assay formats
would not function on a macroscale. For PCG, microfluidic
structures offer a novel, innovative and modular concept different
from the current available PCG hardware. There are a number of ways
in which these microfluidic structures are relevant to PCG.
[0036] Several microfluidic structures have been recently developed
which can be useful as "building blocks" for a variety of different
disposable crystallization chips. These devices make it possible to
deliver small volumes (tens of nanoliters to tens of microliters)
of sample and reagents at flow rates down to nanoliters per
second.
[0037] Due to the low Reynolds Number conditions in microfluidic
systems, mixing is usually limited to laminar diffusion mixing or
laminar recirculating mixing. However, it is possible to introduce
turbulence into microfluidic systems. Devices have been developed
which allow quasi-turbulent mixing of both two or more single-phase
liquids or liquids containing solid particles. It consists of a
series of chambers, connected by small-diameter channels. Once the
mixer is filled, the fluid contained in the mixer can be subjected
to a series of reversals of direction. Each time the fluid is
pulsed in the forward or reverse direction, each tangential channel
produces a laminar jet in each chamber. Because each laminar jet
causes the fluid in each chamber to rotate as a vortex in the same
direction, the rotational shear field induces mixing. Fluid mixing
can also be achieved by separately dividing each fluid channel into
narrow finger channels and then recombining the all finger channels
into one channel.
[0038] U.S. Pat. Nos. 5,716,852 and 5,932,100 are directed to
microfluidic structures which operate on the principle of laminar
flow within a microscale channel wherein separate input streams are
placed in laminar contact within a single flow channel such that
desired particles can be detected or extracted by virtue of
diffusion. U.S. Pat. No. 5,716,852, which patent is hereby
incorporated by reference, discloses a device, known as a T-Sensor,
which can be used to analyze the presence and concentration of
small particles in streams containing both small particles and
larger ones by diffusion principles. The speed of the diffusion
mixing is a function of the size of the diffusion particles. U.S.
Pat. No. 5,932,100, which also is hereby incorporated by reference,
discloses a device known as an H-Filter, which, by laminar flow,
allows separation of particles based on diffusion coefficients on a
continuous basis without the need for semipermeable membranes. The
H-Filter can also be used as a dilution tool, or, by using several
H-Filters in series, a highly accurate serial dilution
structure.
[0039] Although not directly related to the concept study,
understanding of a T-Sensor operation is necessary for appreciation
of the PCG concept design. A T-Sensor is a micro-total analysis
system (.mu.TAS) component that combines the separation features of
the H-Filter with detection. A T-Sensor system is demonstrated in
which a sample solution, an indicator solution, and a reference
solution are introduced in a common channel. The fluids interact
during parallel flow until they exit the microstructure. Large
particles such as blood cells would not diffuse significantly
within the time the flow streams are in contact. Small atoms such
as H.sup.+, Na.sup.+, and small molecules diffuse rapidly between
streams, whereas larger polymers diffuse more slowly and
equilibrate between streams further from the point of entry to the
device. As interdiffusion proceeds, interaction zones are formed in
which sample and reagents may bind and react. T-Sensors can be used
to let components from two different, but miscible streams diffuse
into one another and react with each other. For example, antigens
contained in one stream can diffuse into a parallel stream
containing antigens, and react with them, while the two original
streams remain largely separate.
[0040] T-Sensor-like structures can be used to induce precipitation
or crystallization of sample components. For example, components
from one stream can diffuse into a parallel stream and react with a
component there to form a precipitate. Alternatively, solvent
molecules from one stream can diffuse into a parallel stream
containing a different solvent and particles. The change in solvent
properties along the diffusion interface zone can then induce
crystallization or precipitation. Obviously, it is also possible to
apply a temperature gradient to a microchannel, either across the
channel or along its flow direction, and affect the precipitation
characteristics this way. Microseeding would be another possibility
with this device.
[0041] It should be noted that it is possible to mitigate against
protein instability using microfluidic technology. Protein
denaturation results in polydisperse protein populations that
contain higher order protein aggregates. The concentration of these
aggregates can be minimized or even eliminated through use of an
H-filter structure because of the difference in diffusion
coefficients between native protein and protein aggregates. An
H-filter set up, would preferentially concentrate the monodisperse
native protein in the filter output.
[0042] Another microfluidic device which may be useful with respect
to PCG is described in U.S. Pat. No. 5,726,751, which patent is
hereby incorporated by reference. This patent discloses a device,
known as a microcytometer, which is based on a sheath flow
cytometer design, and has at its heart a disposable laminate
cartridge technology developed specifically for microfluidic
devices. It may be possible using this technology to focus
precipitating crystals using a combination of microfluidic
hydrodynamic and geometric focusing structures. This would line up
the particles in a single file as they flow past a detector, or
allow them to settle out on the bottom of the structure in a very
controlled and precise way.
[0043] Finally, several other devices which were developed in view
of microfluidic technology are taught in U.S. Pat. Nos. 5,474,349;
5,726,404; 5,971,158; 5,974,867; 6,007,775; 5,948,684; and
5,922,210; these patents are also hereby incorporated by reference
into the present application.
SUMMARY OF THE INVENTION
[0044] Accordingly, it is an object of the present invention to
provide a device for growing protein crystals using microfluidic
structures.
[0045] It is also an object of the present invention to provide a
device in which multiple assays can be performed
simultaneously.
[0046] It is a further object of the present invention to provide a
device in which small volumes of liquids can be used to perform
protein crystal growth (PCG) experimentation.
[0047] It is a still further object of the present invention to
provide a device which is easy to use under microgravity
conditions.
[0048] These and other objects and advantages of the present
invention will be readily apparent in the description that
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 is a graphic representation of a T-Sensor which may
be used in the present invention;
[0050] FIG. 2 is a graphic representation of the T-Sensor of FIG. 1
in which the two input fluids are premixed;
[0051] FIG. 3 is a graphic representation of the T-Sensor of FIG. 2
which simulates the vapor phase or hanging drop diffusion method of
protein crystallization;
[0052] FIGS. 4A and B show graphic representations of several
molecules which have been mixed in a diffusion mixer after an
elapsed time period.
[0053] FIG. 5 is a top view of a microfluidic cartridge for use in
the present invention shown in the loading mode;
[0054] FIG. 6 is a top view of the cartridge of FIG. 5 shown in the
activation mode;
[0055] FIG. 7 is a top view showing the loading mode of a
microfluidic cartridge showing another embodiment for carrying out
the present invention.
[0056] FIG. 8 is a top view showing the loading mode of a
microfluidic cartridge showing another embodiment for carrying out
the present invention;
[0057] FIG. 9 is a top view showing the loading mode of a
microfluidic cartridge showing another embodiment for carrying out
the present invention; and
[0058] FIG. 10 is a top view showing the loading mode of a
microfluidic cartridge showing another embodiment for carrying out
the present invention.
[0059] FIG. 11 is a top view showing the loading mode of a
microfluidic cartridge for performing high density screening
crystallization.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0060] Solution conditions that promote ordered protein aggregation
are favorable for protein crystallization. Aggregation involves
protein interactions mediated through specific forces that are
sensitive to protein surface topology and chemical identity of the
surface groups. The complexity of these interactions represent the
difficulty encountered in obtaining X-ray diffraction quality
crystals. While developed for very simple particle interactions,
statistical mechanical models of order/disorder phase transitions
have offered insights into how to characterize the effect of
solution conditions on solubility. Attempts to characterize
proteins as simple fluids suggest that, under most crystallization
conditions, proteins experience attractions, which have a range
much shorter than their size. This has important consequences on
protein solution phase behavior. First, the solubility at a given
strength of attraction becomes weakly dependent on the extent of
the attraction. Consequently, large classes of proteins can display
a narrow range of solubility at a given level of attraction and on
which protein crystallization is dependent.
[0061] One method of characterizing the strength of the attraction
is to measure the protein 2.sup.nd virial coefficient. A second
consequence of the short-range nature of the interaction potential
is that protein solutions will show through density fluctuations a
metastable fluid/fluid phase transition. This transition appears as
a phase separation into two solutions: one rich in protein and one
dilute in protein. The critical point for this phase transition
lies at stronger attractions than the fluid/crystal phase boundary.
Therefore, crystals will ultimately grow from the protein rich
phase-separated state. The proximity of the critical point to the
fluid/crystal phase boundary plays an important role in crystal
nucleation and which is linked to the narrow range of the protein
2.sup.nd virial coefficient values consistent with protein
crystallization. The role of additives or different starting
conditions is to modify fluid/fluid phase boundaries and create
solution conditions favorable for protein crystallization.
[0062] Interaction between protein molecules is concentration
dependent and can be assayed from light scattering measurements.
The dependence of a light scattering on protein concentration in
dilute protein solutions is directly informative as to the extent
of protein interaction and the constant characterizing this
dependence is the 2.sup.nd virial coefficient, B.sub.2. Positive
values for B.sub.2 are qualitatively representative of repulsion
between protein molecules while negative values indicate attractive
interactions between protein molecules. Large negative values of
B.sub.2 imply strong attractions between protein molecules that
result in gel formation or amorphous precipitation. George and
Wilson observed that there was a commonality to the solution
conditions that are favorable for protein crystallization, and that
commonality could be expressed by the 2.sup.nd virial coefficient,
B.sub.2. The measured values for B.sub.2 using many different
protein-solvent pairs all, unambiguously, fall into a fairly narrow
range referred to as the crystallization slot. This slot is an
empirical representation of solution conditions for which PCG was
successful. The B.sub.2 values comprising the slot are slightly to
moderately negative (.apprxeq.-1 to -8.times.10.sup.-4 mol ml
g.sup.-2) and represent slightly to moderately net attractive
forces between protein molecules.
[0063] Static light scattering (SLS) is the analytical method used
to determine the 2.sup.nd virial coefficient, B.sub.2. This method
requires the intensity of light scattered by a protein solution in
excess of background due to solvent and stray light to be measured
as a function of the protein concentration. The working
relationship used to analyze the SLS data is given by the following
equation: 1 K C R = 1 M + 2 B 2 c +
[0064] where K is an optical constant dependent on refractive
index, Avogadro number, wavelength of incident light and change of
refractive index with protein concentration c. The excess Rayleigh
ratio, R.sub..theta., measured at a scattering angle of .theta. is
determined as a function of protein concentration c. M represents
the molecular weight. By plotting K.sub.c/R.sub..theta. versus c,
the 2.sup.nd virial coefficient, B.sub.2, can be obtained from the
limiting slope. B.sub.2 is a dilute solution parameter and the
protein concentration used for the SLS data depends on detection
sensitivity, ranges are typically 0.05 mg/ml for proteins of large
molecular weight to 1 mg/ml for lysozyme. In comparison to protein
concentrations used for PCG, the 2.sup.nd virial coefficient can be
determined using small quantities of protein.
[0065] Surfactants are required to solubilize membrane proteins.
Therefore, in order to crystallize a membrane protein one must
crystallize the complex of protein bound to the surfactant used.
Most membrane protein crystals to date have been observed to form
near the cloud point of the surfactant used. This cloud point is
the surfactant phase separation boundary corresponding to the
aggregation of surfactant micelles; as a solution approaches the
cloudpoint, intermicellar potentials switch from repulsive to
attractive. As static light scattering is sensitive to micellar
structures, determination of the second osmotic virial coefficient
(B.sub.2) for the protein-surfactant complex must take into account
the interactions between scattering micelles. A T-sensor detection
structure described below allows measurement of the second osmotic
virial coefficient for the protein-surfactant complex in presence
of the surfactant micelles.
[0066] Although the value of the 2.sup.nd virial coefficient is
predictive of crystallization conditions, not all starting
condition consistent with a crystallization slot value for the
2.sup.nd virial coefficient guarantee diffraction quality crystals.
Hence the value of 2.sup.nd virial coefficient will be used to
filter starting conditions, conducive for PCG trials, and all
conditions will be screened that correspond to weakly attractive
protein interactions that bracket the B.sub.2 crystallization slot
value.
[0067] Two general types of "gravity-driven" microfluidic
structures have been manufactured: the "vertical" (GVT and GVH)
types, which have integrated sample and reagent reservoirs, and
which are operated vertically or at an incline, and the
"horizontal" (GHT and GHH) types, which have tubes attached to them
for sample and reagent filling. The letter code stands for
Gravity-driven Horizontal (or Vertical) H-Filter (or T-Sensor).
H-Filters have two inlets and two outlets, are designed to separate
components of a sample solution, and allow the collection of the
output solutions. T-Sensors have two or three inlets, and only one
waste outlet. They allow the detection of analytes directly in
complex sample solutions (such as whole blood). They are filled
with a sample solution, a indicator solution, and, for three
inlet-T-Sensors, an additional reference solution with a known
concentration of analyte.
[0068] In both GH- and GV- type structures, the flow rate depends
on the hydrostatic height of the flow column in each of the inlets,
and each of the outlets. This means that the flow speed as well as
the relative position of the centerline between the two streams can
be adjusted by changing the height of the fluid column in each
inlet and outlet. Some of the T-Sensor types are less sensitive to
differences in the fluid column height; others are more sensitive,
but these also allow to adjust the centerline very accurately.
[0069] Both GVT and GHT types can be filled with the "filling
syringes". For GV-types, place the blunt needle inside the hole of
the reservoirs on top of each cartridge. It is easiest if the
needle is placed somewhat to the side of the hole, and the
cartridge is held at a slight downward angle; fill slowly and
carefully to avoid air bubbles. The reservoir does not need to be
filled completely; however, the area close to the junction with the
inlet channels must be covered with fluid.
[0070] Sometimes the flow starts as soon as the liquid is placed in
the tube; if it is required that the fluids do not mix at all
before they enter the main T-sensor channel, then all reservoirs
should be filled while the GV cartridge lies flat. Filling GHT-type
cartridges is somewhat easier; just fill sample and indicator into
both inlet tubes at the same time and to the same level using two
syringes or pipettes.
[0071] For both GH and GV types, frequently the flow does not start
by itself when the fluids are in the tube or the inlet reservoirs.
In this case, place the "aspiration syringe" with the silicon tip
(enclosed) over the outlet channel opening and aspirate slightly
until the fluids start flowing from all inlets. Keep aspirating
until all air bubbles that may have formed are removed from the
channels. Fluids should now flow unaided as a function of
hydrostatic pressure alone.
[0072] The flow speed can be adjusted by adding or removing fluid
from the inlet tubes (GH types), or by adjusting the incline of the
cartridges (GV-types). The higher the height difference between
inlet and outlet fluid levels, the faster the fluids will flow for
a given structure. Alternatively, the flow can be increased by
placing a Q-Tip on the outlet opening (once the fluid has reached
the outlet), which increases the flow dramatically through
absorptive action.
[0073] The following presents a description of certain specific
embodiments of the present invention. However, the present
invention can be embodied in a multitude of different ways as
defined and covered by the claims. Throughout the drawings, like
parts are designated with like index numerals throughout.
[0074] A T-Sensor-like structure, generally indicated at 10, is
shown in FIG. 1 to demonstrate the principles of diffusion-based
crystallization. A sample 12 containing dissolved protein, and a
reagent 14 containing a variety of different solvents and salts,
flow together in parallel within a channel 15 of T-Sensor 10. After
establishing a laminar flow profile, the flow is significantly
slowed or stopped. The various components of both streams 12, 14
will now diffuse into each other at a certain rate, depending on
the size of the molecules within these streams, forming diffusion
interface zones 16, 18 within channel 15 of device 10. This action
establishes a concentration gradient in device 10, which allows for
a very well defined crystallization. Solvent molecules from one
stream can diffuse into a parallel stream containing a different
solvent and particles. The change in solvent properties along
diffusion interface zones 16, 18 can then induce crystallization or
precipitation. Obviously, it is also possible to apply a
temperature gradient to a microchannel, either across the channel
or along its flow direction, and affect the precipitation
characteristics this way. Microseeding would be another possibility
with this device.
[0075] Referring now to FIG. 2, a microfluidic rapid mixing
structure 20, such as a laminar jet vortex mixer which is described
in U.S. patent Ser. No. 60/206,878, a split-channel diffusion
mixer, or any other mixer that rapidly mixes fluids in the low
Reynolds-number regime can be placed upstream of crystallization
channel 15. The protein sample and the reagent are mixed at a
certain ratio, and then flow into crystallization channel 15, where
a homogeneously mixed solution 22 is slowed or stopped.
Crystallization will then occur inside channel 15. Again,
microseeding or temperature gradients can also be applied.
[0076] FIGS. 4A and 4B show the behavior of two different molecules
when mixed using a diffusion mixer. The figures demonstrate that,
within about 2 minutes, even large molecules are completely
equilibrated across 100-micrometer wide channels that make up the
split-channel diffusion mixer. FIG. 4A shows a phosvitin complex
(1,490,000 MW) concentration (Z) in a 100 .mu.m channel (X) for 120
seconds (Y), while FIG. 4B shows a thyrogobulin (bovine) (669,000
MW) concentration (Z) in a 100 .mu.m channel (X) for 120 seconds
(Y).
[0077] Referring now to FIG. 3, T-Sensor 10 of FIG. 2 is again
used; but in this embodiment, crystallization channel 15 is filled
only partially. Exit end of channel 15 is connected to an absorbing
material 24 that absorbs, over time, a predefined quantity of
solvent mixed solution from 22, thereby increasing the
concentration of protein, and inducing it to crystallize. Again,
microseeding or temperature gradients can also be applied in this
embodiment.
[0078] A prototype for 12 PCG experiments on a single card is shown
in two different operational modes in FIGS. 5 and 6. A single
microfluidic PCG experiment embodies the following elements: a
driver fluid interface 30, two fluid reservoirs 32, 34 and
microfluidic channel/check valves 36, 38, crystallization chamber
39, harvesting chambers 40, microchannel connections 42 and
adhesive sealing means 44.
[0079] Referring now to FIG. 5, a microfluidic cartridge, generally
indicated at 50, contains a plurality of fluid reservoirs 32, 34.
Reservoirs 32 are filled with a protein sample, while reservoirs 34
are filled with a precipitant solution. Fluids in reservoirs 32, 34
are expelled by applying pressure to a fluid located within channel
30, which may be air or an inert oil. Reservoirs 32, 34 combine to
form a T-sensor structure with crystallization chamber 33. Laminar
flow ensures that the two fluids do not mix within chamber 39 other
than by mutual self-diffusion. The contents of crystallization
chamber 39 void into harvesting chamber 40. Each fluid reservoir
32, 34 is filled through a fluid inlet 52 and has microfluidic
channel/check valves 36, 38 a vent hole 54 to permit air escape
during the filling operation. Surface tension effects because of
the small diameter of the connecting to the fluid reservoirs 32, 34
prevent fluids flowing out of said reservoirs. Once loaded, fluid
reservoirs 32, 34 are carefully sealed with adhesive strip 44, as
can be seen in FIG. 6. This strip 44 can be supplied directly
bonded to cartridge 50. Harvesting chambers 40 are sealed with
another strip 44 of adhesive tape also supplied directly on
cartridge 50. In microgravity or for long-term storage prior to
fluid activation, the check valves 36, 38 minimize vapor loss from
reservoirs 32, 34. Check valves 36, 38 allow fluid flow in one
direction only such that back flow is prevented, and when
appropriately placed within a microfluidic circuit, can act as one
level of fluid containment.
[0080] External valve activation and fluid driving can be
accomplished in one of two ways: using an external driver or by air
bellows incorporated on the microfluidic cartridge. An external
fluid driver interface 60 (FIG. 6) would be an air pump to which
each card would be hooked up. Air pump 60 delivers a precise amount
of pressure to drive fluids through the circuit. Another option is
to use an air bellows 62, as shown in FIG. 5, directly manufactured
on the circuit board that can be driven by pressure to pump the
fluids into the microfluidic structures. Air bellows 62 may also
have a vent hole 64, which may be sealed by a ball bearing, and
when under pressure air bellows 62 would again act as the fluid
driver. Release of pressure due to sudden power outage would allow
air to bleed into the microfluidic circuit, allowing it to
equilibrate. Check valves 36, 38 in any event would prevent fluid
back flow and satisfy one level of containment. The advantage of
vent hole 64 on the air bellows 62 is that circuit cartridge 50,
once actuated, could be allowed to slowly return to equilibrium and
then allow facile harvesting of crystal chamber 40 contents by
applying another round of pressure on the bellows 62. It is also
possible to fill bellows 62 with inert oil to drive the fluids and
prevent vapor loss in the microfluidic cartridge 50 over the
long-term course of a PCG experiment, if this becomes
necessary.
[0081] Activation by applying pressure on the driver fluid within
channel 30 by bellows 62 pumps the fluid reservoirs 32, 34 contents
into crystallization chamber 39 via check valves 36, 38. Check
valves 38 ensure that there is no back flow from crystallization
chamber 39 while check valves 36 ensure a further level of fluid
containment. Harvesting of a particular PCG experiment occurs by
partially peeling off the adhesive strip 44 to allow access to the
chosen harvesting chamber 40. Circuit pressurization via fluid
driver interface 60 or air bellows 62 would allow flushing with
inert oil or air of the crystallization chamber 39 contents.
Crystals are then accessible for facile transfer and/or
manipulation within harvesting chamber 40. Currently, a clear
plastic adhesive tape commercially available from Hampton Research
is used for sealing hanging drop experiments. This tape seals the
equilibration wells while at the same time holding the hanging
drop. This tape is compliant such that tape covering the crystal
harvesting chambers 40 creates a minimal backpressure once fluid is
pumped into the channel. Should compliance present a problem, it is
possible to provide a narrow vent hole on the outlet side that is
very hydrophobic, and therefore would not let any liquid escape,
only air.
[0082] The prototype crystallization chip in the PCG device would
incorporate the vented air bellows design. This greatly simplifies
testing and makes it very user-friendly. For the device, a volume
of 20 .mu.L can be used for each crystallization chamber; however,
smaller chamber volumes of 10-100 nanoliters are readily possible.
Three approaches can be used in the microfluidic circuit cartridges
to initiate protein crystallization and accompanying figures show
the conceptual design for a single PCG experiment on the prototype
board. It should be noted that all three approaches could be mixed
and matched onto a single board. The PCG techniques are:
self-diffusion of precipitants and protein across a laminar
boundary (see FIG. 7); turbulent mixing of all components--batch
mode (see FIG. 8); and vapor transport into a desiccant or
precipitant (see FIG. 9).
[0083] Referring now to FIG. 7, the interfacial diffusion approach
will consist of using 2.times.concentrations of protein and
precipitant in each fluid reservoir (volumes>10 .mu.L) and each
made up in 1.times.concentrations of same buffer, salt and
detergent. The two fluids are then injected under pressure in a 1:1
mixing ratio controlled by the diameter of microchannels 42 into
crystallization chamber 39. Chamber can be filled under laminar
flow conditions provided that it has at least one dimension of less
than roughly 500 micrometers, and chamber is filled fairly slowly
using gentle finger pressure (all other microfluidic structures
will be small enough to easily fulfill the requirements of laminar
flow). Pressure in the system is equilibrated by removing the
finger gently from vent hole 64. Air bellows 62 are then carefully
sealed with clear adhesive tape in the same way as are fluid
reservoirs 32, 34 and harvesting chamber 40. Voiding of
crystallization chamber 40 into harvesting chamber 40 involves
removal of the adhesive tape covering harvesting chamber 39 and
applying pressure on air bellows 62.
[0084] Referring now to FIG. 8, cartridge 50, which uses turbulent
mixing for all components, operates in a batch mode. A turbulent
mixing chamber 70 is inserted between fluid reservoirs 32, 34 and
crystallization chamber 39. Chamber 70 mixes the protein and
precipitant fluids into a homogeneous liquid which is transported
to chamber 39 for crystallization. This design is particularly
useful under microgravity conditions such as on a space shuttle
mission, as the viscous precipitants do not have time to mix and
induce nucleation during the duration of an extended mission.
[0085] FIG. 9 shows an example of cartridge 50 of FIG. 8 which uses
the principles of vapor diffusion to operate. In this embodiment,
crystallization chamber 39 is only partially filled after the
fluids are mixed within mixing chamber 70. A predefined desiccant
or precipitant 72 is located within harvesting chamber 40 to absorb
a fixing quantity of solution into chamber 40, increasing the
concentration of protein with chamber 39, and inducing
crystallization.
[0086] It would also be possible to take a starting protein
solution and dialyze it against the appropriate starting fluid
composition using an H-Filter prior to the crystallization
experiment, should long term in-orbit storage in a particular
buffer be deleterious to protein stability. An H-filter setup could
be incorporated into the design to eliminate irreversible protein
aggregates. Referring now to FIG. 10, cartridge 50 contains fluid
reservoir 32 filled with a protein sample and fluid reservoir 34
containing a precipitant solution, as shown in the previous
examples. An additional fluid reservoir 80 is located on cartridge
50 which contains a protein and buffer solution. All reservoirs are
filled through inlets 52. Fluids from reservoirs 32, 80 flow
through microchannels 42 into a channel 32 which operates as an
H-Filter to separate unwanted particles into a waste reservoir 84.
The filtered solution travels through check valve 38 where it
contacts fluid from reservoir 34 to form a laminar flow stream
through crystallization chamber 39. Another option is to just
filter protein reservoir 32 contents using a 0.22 .mu. filter
directly incorporated onto cartridge 50 and placed just after the
protein fluid reservoir 32 and before check valve 38. This type of
filter is typically used to remove particulate matter for dynamic
light scattering experiments.
[0087] Referring now to FIG. 11, a high density screening
crystallization cartridge 50 is shown. Cartridge 50 contains four
crystallization chambers 39. Chambers 39 have approximately a
0.5.times.0.5 mm cross-section. Protein solutions are added at a
series of ports 86, while precipitant solutions are added at a
series of ports 88. A series of valves 90 couple air bellows 62 to
a series of filling chambers 92, which each correspond to a port
86. Each chamber 92 has a capacity of 1-10 .mu.l. A series of
harvesting chambers 40 are each coupled to one of chambers 39.
Ports 88 are each connected to a fluid reservoir 34, which in turn
are coupled to a corresponding harvesting chamber 40. Each of
harvesting chambers 40 has a corresponding vent hole 94. Each
harvesting chamber 40 has a capacity of approximately 50 .mu.l,
while each fluid reservoir has a capacity of between 0.1 and 0.5
ml.
[0088] In operation, ports 86 and 88 are filled with their
respective solutions. With valves 90 in the closed position, mixing
is achieved as the solutions contact each other within chambers 39
to establish a concentration gradient, as molecules diffuse across
the interface zone, thus diluting the protein solution. Valves 90
are then opened individually and the solutions are moved through
chambers 39 under the force provided by air bellows 62. During this
protein crystallization growth phase, vents 94 and 64, ports 86 and
88, and harvesting chambers 40 are all sealed using adhesive tape.
Harvesting occurs by opening valves 90, which forces the contents
of crystallization chambers 39 into harvesting chambers 40.
[0089] Testing the design of the microfluidic crystallization chips
requires the use of protein. Lysozyme and thaumatin PCG systems as
initial controls for evaluation of the performance of the chips and
instrumentation may be used in this embodiment.
[0090] A primary concern is the wetting of the fluid reservoirs to
efficiently expel any air bubbles formed during the filling
operation. This may be a question of having adequate pipette tips
for liquid handling and compatible fluid inlet dimensions.
Siliconization may be used to control wetting. Rounded corners,
oval or circular fluid reservoir shapes may be examined to minimize
bubble entrapment. Dimension and placement of the vent hole should
be studied as well as whether filling should be done in a position
where the cartridge is slanted to efficiently void air bubbles. All
fluids are to be degassed prior to filling.
[0091] The shape of the crystallization chamber is important to
ensure laminar flow of the two liquids during its filling. A
crystallization chamber having a T-sensor structure should be
sufficient for operation. However, for rapid inspection of PCG
results, it is advantageous to localize PCG in a smaller region.
Laminar flow in a crystallization chamber can be readily monitored
by injecting two fluids each containing a different colored
dye.
[0092] The volume of the harvesting chamber should be of sufficient
size to allow harvesting of the entire crystallization chamber
contents as well as addition of aliquots of mother liquor and
cryo-protectant buffer. The harvesting chamber shape should have
rounded corners and allow facile access for crystal harvesting.
[0093] Plastic clear tape should be used for sealing the fluid
reservoirs and harvesting chamber and tested for long-term
stability and compatibility with the microfluidic circuit
cartridge. Attention should be paid in PCG trials to ease of
peeling off the tape from the circuit boards. It may also be
advantageous for efficient handling to provide a backing to the
plastic clear adhesive tape that peels off exposing the adherent
surface for subsequent sealing. Visual cues can be provided on the
circuit cartridges of where to place the sealing tape.
[0094] The microfluidic cards are made of plastic laminates bonded
together with adhesive. The plastic laminate composition is mylar,
which is a very resistant material. The fluid compatibility and
long-term fluid integrity, however, needs to be assessed and is
addressed in the work packages. Problems with PCG fluid
compatibility are not anticipated with either the laminate adhesive
and mylar. Alternatively, glass or silicon can be used if the fluid
incompatibility is severe. Under these circumstances, it should be
possible to perform all fluidic development using the laminate
method; however, when it comes to mass production, it may be
desirable to make the structures out of glass or silicon.
[0095] The microfluidic integrated circuit cartridges, when sealed
with the covering adhesive film, comprise one level of fluid
containment. The fluid driver interface connection on the circuit
cartridges is airtight, while the air bellows design does not
compromise the containment level. Fifty (50) microfluidic
integrated circuit cards containing up to 20 individual PCG
experiments each or 1000 PCG experiments in all could fit with
external controllers into a sealed container within the volume of a
mid-deck locker that provides the second level of containment and,
if required, temperature control.
[0096] Usually, microfluidic systems require some kind of fluidic
driver to operate, e.g., piezoelectric pumps, micro-syringe pumps,
electroosmotic pumps, etc. In two previous patent applications,
U.S. patent application Ser. No. 09/415,404 and U.S. patent
application Ser. No. 60/189,163, which applications are hereby
incorporated by reference, there are shown microfluidic systems
that are entirely driven by an inherently available force such as
gravity, capillary action, absorption in porous materials,
chemically induced pressures or vacuums (e.g., by a reaction of
water with a drying agent), or by vacuum or pressure generated by
simple manual action. Such devices are extremely simple and cheap,
do not require electricity, can be manufactured, for example,
entirely out of a single material such as plastic, with a method
such as injection molding, and are simple to operate.
[0097] One embodiment of a device according to the present
invention would comprise a hydrostatic pressure-driven cartridge,
in which the hydrostatic pressure heads are manufactured as part of
the cartridge itself. The cartridge would then be placed on its
side so that the gravity pulls the liquids through the
channels.
[0098] Another embodiment comprises a cartridge on which air spaces
under a flexible membrane are in fluid connection with the
microfluidic fluid circuit. These compressible airspaces can then
be used to aspirate liquids into the channels, or to apply pressure
to push liquids to various points on the cartridge, for example, to
prime a microfluidic circuit or to siphon fluids until it starts
working by gravitational force.
[0099] Another embodiment contains chambers in which certain
chemical liquids (e.g., ethanol, butane, carbon dioxide, organic
solvents, etc. or any substance which has a partial pressure at
operating temperature that generates enough force to push liquids
through a microfluidic system at desired flow rates) are present in
equilibrium with their gaseous phases. These spaces are in fluid
connection with parts of the microfluidic circuit and the other
reagents, and the pressure in these chambers push the reagents and
samples through the channels of the microfluidic circuit.
[0100] In addition to filling by gravity or syringe, bellows-driven
microfluidic structures have been manufactured in which the bellows
are integrated into the laminate as either aspiration or
pressurization bubbles. Vents can be placed at various places on
the cartridges to allow directional flow of the fluids.
[0101] It is also possible to prefill cartridges during
manufacturing. A predefined volume of fluid can be placed on a
reservoir on an open laminate, which is then sealed with tape, or a
cover layer. This action can also be used to drive the fluid to
where it should be inside the microfluidic circuit.
[0102] While the present invention has been shown and described in
terms of a preferred embodiment thereof, it will be understood that
this invention is not limited to this particular embodiment and
that many changes and modifications may be made without departing
from the true spirit and scope of the invention as defined in the
appended claims.
* * * * *