U.S. patent application number 13/230790 was filed with the patent office on 2012-01-05 for high precision scanning of encoded hydrogel microparticles.
This patent application is currently assigned to MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Stephen C. Chapin, Patrick Seamus Doyle, Daniel Colin Pregibon.
Application Number | 20120003755 13/230790 |
Document ID | / |
Family ID | 43381179 |
Filed Date | 2012-01-05 |
United States Patent
Application |
20120003755 |
Kind Code |
A1 |
Chapin; Stephen C. ; et
al. |
January 5, 2012 |
High Precision Scanning of Encoded Hydrogel Microparticles
Abstract
Techniques are provided for high precision scanning of hydrogel
microparticles. The high precision is achieved by one or more
modifications to the microparticle composition, or microfluidics
apparatus that align the microparticles in a detection channel, or
method of preparing a sample for introduction into the apparatus,
or some combination. An apparatus comprises a body structure having
formed therein a central channel and multiple focusing channels in
fluid communication with the central channel through multiple
junctions. A width of the central channel is smaller in a portion
downstream of each junction. A particle comprises a hydrogel matrix
and a probe molecule. The particle has an aspect ratio greater than
about three. A method includes loading into a sample fluid inlet a
mixture, wherein a number of particles lies within a range from
about 5 to about 10 particles/.mu.l.
Inventors: |
Chapin; Stephen C.; (Boston,
MA) ; Doyle; Patrick Seamus; (Boston, MA) ;
Pregibon; Daniel Colin; (Cambridge, MA) |
Assignee: |
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY
Cambridge
MA
|
Family ID: |
43381179 |
Appl. No.: |
13/230790 |
Filed: |
September 12, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12552268 |
Sep 1, 2009 |
8034629 |
|
|
13230790 |
|
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61220782 |
Jun 26, 2009 |
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Current U.S.
Class: |
436/501 |
Current CPC
Class: |
B01L 2300/123 20130101;
B01L 2400/084 20130101; B01L 3/502761 20130101; B01L 2200/0647
20130101; Y10T 428/2982 20150115; Y10T 436/2575 20150115; B01L
2300/0816 20130101; Y10T 436/117497 20150115 |
Class at
Publication: |
436/501 |
International
Class: |
G01N 21/75 20060101
G01N021/75 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0004] This invention was made with Government support under
Contract No. 21EB008814 awarded by the National Institute of
Biomedical Imaging and Bioengineering, National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. A particle comprising a hydrogel matrix and a probe molecule,
wherein: a greatest particle dimension is less than about 500
micrometers (.mu.m, 1 .mu.m=10.sup.-6 meters); an aspect ratio of
length to width of the particle is greater than about three; and
the probe molecule is selected to bind to a target molecule.
2. The particle as recited in claim 1, wherein a first longitudinal
portion of the particle is encoded with a plurality of thickness
variations.
3. The particle as recited in claim 2, wherein the first
longitudinal portion is more rigid and less porous than a different
second longitudinal portion of the particle.
4. The particle as recited in claim 3, wherein the probe molecule
is disposed only in the more porous hydrogel matrix in the
different second longitudinal portion.
5. The particle as recited in claim 2, further comprising a
fluorescent entity loaded into the first longitudinal portion.
6. The particle as recited in claim 3, wherein the hydrogel matrix
in the first longitudinal portion comprises more Poly(ethylene
glycol) (700) diacrylate and less Poly(ethylene glycol) (200) than
does the hydrogel matrix in the second longitudinal portion.
7. The particle as recited in claim 3, wherein: the hydrogel matrix
in the first longitudinal portion comprises about 20% Poly(ethylene
glycol) (700) diacrylate and about 20% Poly(ethylene glycol) (200)
called DA30; and the hydrogel matrix in the second longitudinal
portion comprises about 10% Poly(ethylene glycol) (700) diacrylate
and about 30% Poly(ethylene glycol) (200) called DA20.
8. The particle as recited in claim 2, wherein the plurality of
thickness variations vary horizontally on a scale matched to a
scanning window of an apparatus to decode the encoded
variations.
9. The particle as recited in claim 1, wherein the aspect ratio is
about 3.4.
10. The particle as recited in claim 2, wherein the first
longitudinal portion is about half the length of the hydrogel
body.
11. The particle as recited in claim 1, wherein the width is in a
range from about 10 .mu.m to about 80 .mu.m and the length is in a
range from about 80 .mu.m to about 300 .mu.m.
12. A method comprising a) providing an apparatus comprising a body
structure having formed therein a plurality of microfluidic
channels comprising a central channel in fluid communication with a
sample fluid inlet and a plurality of focusing channels in fluid
communication with the central channel through a plurality of
junctions; b) providing a plurality of particles comprising
hydrogel bodies sized to fit in the central channel, wherein each
hydrogel body comprises a first longitudinal portion encoded with a
plurality of thickness variations and a probe molecule, wherein the
probe molecule is selected to bind to a target molecule and is
associated with a particular plurality of thickness variations, and
wherein the plurality of fabricated particles includes a plurality
of different probe molecules; c) loading into the sample fluid
inlet a mixture of a test sample with the plurality of particles,
wherein a number of particles in the mixture lies within a range
from about 5 particles per microliter (particles/.mu.l, 1
.mu.l=10.sup.-6 liters) to about 10 particles/.mu.l; d) detecting
optical emissions from particles of the plurality of fabricated
particles in the central channel downstream of a last junction of
the plurality of junctions; and e) determining presence of a target
in the test sample based on the detected optical emissions.
13. The method as recited in claim 12, further comprising applying
a first pressure at the sample fluid inlet and a second pressure at
a focusing fluid inlet in fluid communication with the plurality of
focusing channels so that a sum of flow rates through the plurality
of focusing channels is about equal to a flow rate of the sample
fluid inlet.
14. The method as recited in claim 13, wherein the sample fluid
inlet is the same as the focusing fluid inlet and the first
pressure is equal to the second pressure.
15. The method as recited in claim 12, wherein the number of
particles in the mixture is about 7.5 particles per .mu.l.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit as a Divisional Continuation
of application Ser. No. 12/552,268, filed Sep. 1, 2009, the entire
contents of which are hereby incorporated by reference as if fully
set forth herein, under 35 U.S.C. .sctn.120, which claims benefit
of Provisional Appln. 61/220,782, filed Jun. 26, 2009, the entire
contents of which are hereby incorporated by reference as if fully
set forth herein, under 35 U.S.C. .sctn.119(e).
[0002] This application is related to U.S. utility application Ser.
No. 11/867,217 filed Oct. 4, 2007 and published as US Patent
Application Publication US 2008/0176216 on Jul. 24, 2008
(hereinafter Doyle I), the entire contents of which are hereby
incorporated by reference as if fully set forth herein.
[0003] This application is related to U.S. utility application Ser.
No. 11/586,197 filed Oct. 25, 2006 and published as US Patent
Application Publication US 2007/0105972 on May 10, 2007
(hereinafter Doyle II), the entire contents of which are hereby
incorporated by reference as if fully set forth herein.
BACKGROUND
[0005] The ability to accurately detect and quantify biological
molecules in a complex mixture is crucial in both basic research
and clinical settings. Advancements in the fields of genomics and
proteomics require robust technologies that can obtain high-density
information from biological samples in a rapid and cost-effective
manner. High-throughput screening for genetic analysis,
combinatorial chemistry, and clinical diagnostics benefits greatly
from multiplexed analysis, which is the simultaneous detection of
several target molecules. This approach significantly reduces the
required assay time, sample volume, and cost. However, it requires
an encoding scheme to identify which, of a large number of
immobilized probe species that bind with specific labeled target
molecules in the sample, is being detected during analysis.
EXAMPLE EMBODIMENTS
[0006] Techniques are provided for high precision scanning of
hydrogel microparticles. The high precision is achieved by one or
more modifications to the microparticle composition, or
microfluidics apparatus that align the microparticles in a
detection channel, or method of preparing a sample for introduction
into the apparatus, or some combination.
[0007] In one set of embodiments, an apparatus comprises a body
structure having formed therein multiple microfluidic channels,
each having at least one dimension in a size range from about 0.1
micron to about 500 micrometers (.mu.m, 1 .mu.m=10.sup.-6 meters).
The microfluidic channels include a central channel and multiple
focusing channels in fluid communication with the central channel
through multiple junctions. A width of the central channel is
smaller in a portion downstream of each junction than in a portion
upstream of that junction.
[0008] In another set of embodiments, a particle comprises a
hydrogel matrix and a probe molecule. The particle has a greatest
particle dimension less than about 500 micrometers (.mu.m, 1
.mu.m=10.sup.-6 meters), and an aspect ratio of length to width
greater than about three. The probe molecule is selected to bind to
a target molecule.
[0009] In another set of embodiments, a method includes providing
an apparatus comprising a body structure having formed therein
multiple microfluidic channels, including a central channel in
fluid communication with a sample fluid inlet and multiple focusing
channels in fluid communication with the central channel through
multiple junctions. The method also includes providing multiple
particles comprising hydrogel bodies sized to fit in the central
channel, wherein each hydrogel body includes a first longitudinal
portion encoded with a plurality of thickness variations and a
probe molecule. The probe molecule is selected to bind to a target
molecule and is associated with a particular plurality of thickness
variations. Among the multiple particles are multiple different
probe molecules. The method includes loading into the sample fluid
inlet a mixture of a test sample with the particles, wherein a
number of particles in the mixture lies within a range from about
15 particles per microliter (particles/.mu.l, 1 .mu.l=10.sup.-6
liters) to about 20 particles/.mu.l. The method includes detecting
optical emissions from particles in the central channel downstream
of a last one of the junctions. The method also includes
determining presence of a target in the test sample based on the
detected optical emissions. In some embodiments, determining the
presence includes determining the amount of target in the test
sample.
[0010] Still other aspects, features, and advantages are readily
apparent from the following detailed description, simply by
illustrating a number of particular embodiments and
implementations, including the best mode contemplated for carrying
out the invention. The invention is also capable of other and
different embodiments, and its several details can be modified in
various obvious respects, all without departing from the spirit and
scope of the invention. Accordingly, the drawings and description
are to be regarded as illustrative in nature, and not as
restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The present invention is illustrated by way of example, and
not by way of limitation, in the figures of the accompanying
drawings and in which like reference numerals refer to similar
elements and in which:
[0012] FIG. 1 is a block diagram that illustrates an example
hydrogel microparticle, according to an embodiment;
[0013] FIG. 2 is a block diagram that illustrates an example
elevation view of the hydrogel particle in a microfluidics channel,
according to an embodiment;
[0014] FIG. 3A is a block diagram that illustrates an example view
looking down into a microfluidics channel in a detection zone,
according to an embodiment;
[0015] FIG. 3B is a graph that illustrates an example measurement
of optical emissions at a detector in the detection zone, according
to an embodiment;
[0016] FIG. 4 is an image that illustrates a mixture of hydrogel
particles in a fluid sample, according to an embodiment;
[0017] FIG. 5A is a diagram of microfluidics channels in an example
apparatus, according to an embodiment;
[0018] FIG. 5B is an image of a portion of microfluidics channels
in an example apparatus, according to an embodiment;
[0019] FIG. 6 is a diagram of microfluidics channels in another
example apparatus, according to an embodiment;
[0020] FIG. 7 is a flow diagram of a method for using hydrogel
microparticles in an apparatus, according to an embodiment;
[0021] FIG. 8A through FIG. 8D are images of hydrogel particles in
a detection zone, according to various embodiments;
[0022] FIG. 9 is a graph that illustrates dependence of
microparticle position in a microfluidics channel on particle
composition, according to various embodiments;
[0023] FIG. 10 is a graph that illustrates dependence of
microparticle detection in a microfluidics channel on particle
composition, according to various embodiments;
[0024] FIG. 11 is a graph that illustrates distribution of
microparticle speeds in different microfluidics channels, according
to various embodiments;
[0025] FIG. 12A is a graph that illustrates restoring forces on
microparticles that are not aligned with a microfluidics channel,
according to various embodiments;
[0026] FIG. 12B is a map that illustrates fluid speeds passing a
microparticle that is not aligned with a microfluidics channel,
according to an embodiment;
[0027] FIG. 13A and FIG. 13B are graphs that illustrate stability
of microparticle alignment within a microfluidics channel based on
orientation of the microparticle, according to an embodiment;
and
[0028] FIG. 14 is a photo of a microfluidics device in operation,
according to an embodiment.
DETAILED DESCRIPTION
[0029] A method and apparatus are described for high precision
scanning of encoded hydrogel microparticles. In the following
description, for the purposes of explanation, numerous specific
details are set forth in order to provide a thorough understanding.
It will be apparent, however, to one skilled in the art that some
embodiments may be practiced without these specific details. In
other instances, well-known structures and devices are shown in
block diagram form in order to avoid unnecessarily obscuring the
illustrated embodiments.
[0030] Some embodiments are described below in the context of
detecting, with a single wavelength detector, both particle codes
and fluorescently-labeled long molecule targets associated with
processes in biological organisms. However, the invention is not
limited to this context. In other embodiments, the apparatus,
particles, or method, or some combination, can be used to decode
particles or quantify targets based on fluorescence,
chemiluminescence, magnetic properties, radioactivity, radio
frequency, electrical resistance, opacity, or colorimetry, among
other means. For optical approaches, single or multiple wavelengths
may be used in a given assay. In addition, multiple detectors or
excitation sources may be used. Targets may include biological
entities such as proteins, nucleic acids, cytokines, lipids, whole
cells, enzymes, antibodies, bioterror threats, or any range of
chemicals from polymers to small molecules. Some specific
applications include drug discovery, biomarker discovery,
expression profiling, combinatorial chemistry, clinical
diagnostics, or monitoring environmental samples for microorganisms
or chemical agents.
[0031] Particle-based assay platforms exhibit several advantages
over planar arrays in applications that involve the detection of
low to medium target densities (1 to about 1000 different targets),
or demand rapid modification of the probes to be fixed in the
particles, or necessitate high-throughput processing of samples.
Compared to planar arrays, the use of micrometer-sized particles
(called microparticles herein, 1 micrometer, .mu.m, =1
micron=10.sup.-6 meters) leads to faster probe-target binding
kinetics due to mixing during incubation, more efficient separation
and washing steps, and higher degrees of reproducibility. (See for
example, M. Evans, C. Sewter and E. Hill, "An Encoded Particle
Array Tool for Multiplex Bioassays," Assay and Drug Development
Technologies, 2003, vol. 1, pp 199-207; and Z. Zhi, Y. Morita, Q.
Hasan and E. Tamiya, "Micromachining Microcarrier-based
Biomolecular Encoding for Miniaturized and Multiplexed
Immunoassay," Analytical Chemistry, 2003, vol. 75, pp 4125-4131.)
The vast majority of particles used in suspension arrays are
optically encoded latex microspheres with diameters between 0.3 and
10 microns that can be interrogated and decoded with laser-based
flow cytometry (measurement of cell sized particles). Optical
encoding is accomplished by swelling the spheres with fluorescent
organic dyes with different emission spectra. Although used
extensively, this scheme requires multiple excitations and is
limited to the multiplexed sensing of only about 100 targets (also
called analytes, herein) due to spectral overlap of particle
encoding fluorescence and target-detection fluorescence. (See for
example, R. J. Fulton, R. L. McDade, P. L. Smith, L. J. Kienker and
J. R. K. Jr., "Advanced Multiplex Analysis with the Flowmetrix
System," Clin. Chem., 1997, vol. 43, pp 1749-1756; and K. L. Kellar
and M. A. Iannone, "Multiplexed Microsphere-based Flow Cytometric
Assays," Exp. Hematol., 2002, vol. 30, pp 1227-1237.)
Sub-micrometer rods with multiple metal stripes that serve as a
graphical code for multiplexing have also been developed. (See for
example, S. R. Nicewarner-Pena, R. G. Freeman, B. D. Reiss, L. He,
D. J. Pena, I. D. Walton, R. Cromer, C. D. Keating and M. J. Natan,
"Submicrometer Metallic Barcodes," Science, 2001, vol. 294, pp
137-141; and M. Sha, I. Walton, S. Norton, M. Taylor, M. Yamanaka,
M. Natan, C. Xu, S. Drmanac, S. Huang, A. Borcherding, R. Drmanac
and S. Penn, "Multiplexed SNP Genotyping Using Nanobarcode Particle
Technology," Anal Bioanal Chem, 2006, vol. 384, pp 658-666.)
However, the high density of such rods leads to rapid settling in
solution and thus requires the rods to be vigorously mixed during
assays, a procedure which can damage fragile biological molecules
like antibodies. Moreover, a feasible high-throughput
quantification and decoding strategy for the rods has not been
introduced as of this writing, thereby significantly limiting the
applicability of such rods in clinical or research settings.
[0032] According to various embodiments, particle based assays use
coded hydrogel particles in an apparatus that effectively focuses
and aligns the particles for efficient optical detection and high
throughput. A hydrogel (also called aquagel) is a network of
polymer chains that are water-insoluble. A polymer is a large
molecule (macromolecule) composed of repeating structural units
typically connected by covalent chemical bonds. Hydrogels are
highly absorbent (they can contain over 99% water) and possess a
degree of flexibility due to their significant water content.
Because of this flexibility, hydrogel particles present special
challenges for focusing and alignment in microfluidics channels, as
are described in more detail below.
Particle
[0033] FIG. 1 is a block diagram that illustrates an example
hydrogel microparticle 100, according to an embodiment. The
particle has a longest dimension given by length L 102, a shortest
dimension perpendicular to the length given by thickness T 106, and
a dimension perpendicular to both given by width W 104. The
particle is a microparticle, with all dimensions in a range from
about 0.1 micrometers (.mu.m, also called microns, 1
.mu.m=10.sup.-6 meters) to about 500 microns. The particle is
shaped to align in microfluidics channel by having a length L 102
greater than a width W 104 greater than a thickness T 106. The
particle 100 is made of a hydrogel matrix that includes one or more
probe molecules loaded into a longitudinal portion along the length
102 of the particle 100 called a probe portion 120. The probe
molecules are selected to bind to a particular type or types of
target molecules.
[0034] In the same or different longitudinal portion called an
encoded portion 110, a code is emplaced in the particle. Any method
may be used to impart a code to the encoded portion. In an
illustrated embodiment, the encoded portion is different from the
probe portion and the thickness of the particle in the encoded
portion decreases varying amounts to impart a spatial pattern that
can be detected. The spatial pattern in the encoded portion 110 is
associated with the probe molecules loaded into the probe portion
120.
[0035] Any spatial pattern may be used, such as ridges, gulleys and
peaks. For example, in the illustrated embodiment the spatial
pattern is a set of zero or more holes through the thickness of the
particle. The light emitted from the encoded portion (e.g., via
reflectance, transmittance or fluorescence) depends on the
thickness of the particle in an optical scanning window. In an
example embodiment, the hydrogel matrix in the encoded portion is
loaded with a fluorescent entity. In some embodiments, different
materials are deposited into one or more thin portions to enhance
detectability, either with optical or other detectors, such as
magnetic detectors, radio frequency detectors, and temperature
detectors, among others. In some embodiments, different materials
or structures are loaded into the hydrogel matrix to impart a code
to be detected on each particle.
[0036] The horizontal scale 112 of thickness variations, i.e., in a
horizontal plane perpendicular to the thickness, is related to the
aperture of the optics or other detector used to detect the
particle 100. In the illustrated embodiment, the hole size and
spacing between holes are on the order of an optical window used to
detect the particles, which in some embodiments is anticipated to
be in a range from about 2 microns to about 8 microns.
[0037] The number of possible hole positions in the encoded portion
determines the number of different codes that can be represented.
In example embodiments, three to five hole positions are allowed in
a column across the width of the particle and five to ten column
positions are allowed at different positions along the length of
the particle in the encoded portion 110. In the illustrated
embodiment, up to four hole positions are depicted spaced in each
column and six columns are depicted spaced longitudinally in the
encoded portion 110 (allowing. Thus the illustrated embodiment has
an encoded portion 110 that is about 18 to 80 microns wide and
about 26 to 100 microns long, accounting for the size of spaces
between holes as well as the holes. For an encoded portion with
three hole positions per column and 7 columns, over 3000 unique
codes can be provided.
[0038] In some embodiments, the probe portion 120 is distinct from
the encoded portion. An advantage of this arrangement is that the
same detector can be used to detect both the code and the binding
of target molecules to probe molecules. For a particle on which the
encoded portion 110 is one half to one quarter the length of the
particle, the particle lengths are about 50 to 400 microns.
[0039] The thickness T 106 is chosen to be sufficient to provide
structural integrity to the particle, while still being thin enough
to enable the formation of thickness variations, such as holes,
which are of sufficient magnitude to be detected. In example
embodiments, the thickness is in a range from about 10 microns to
about 40 microns to enable the formation of holes during the
cross-linking process used to fabricate the hydrogel matrix.
Thickness variations may be formed in any manner. In some
embodiments thickness variations are generate, at least in part,
using lock release lithography (LRL, see Ki Wan Bong, Daniel C.
Pregibon and Patrick S. Doyle, "Lock release lithography for 3D and
composite microparticles," Lab on a Chip, The Royal Society of
Chemistry, London, v 9, pp 863-86, 2009).
[0040] According to several embodiments, an aspect ratio of length
L 102 to width W 104 of particle 100 is greater than three (3). For
example, in some embodiments, described in more detail below, the
aspect ratio is about 3.4. An advantage of aspect ratio greater
than 3 is that such particles tend to align with the direction of
flow even in wider regions of microfluidics channels in an
apparatus used to observe the particles. The alignment of the
particles with the flow makes it easier to get the particles
positioned for the detector, as described in more detail below.
[0041] According to some embodiments, the encoded portion 110 is
more rigid and less porous than the rest of the particle, including
the probe portion 120. This is an advantage because the encoded
portion better retains its structure to reduce errors in reading
the code, while the increased porosity of the remaining portion
allows more probe molecules to be loaded into the hydrogel matrix
for a better signal strength related to binding. The hydrogel is
largely transparent; and the deeper the penetration of probe
molecules, the more binding opportunities when the particle is
exposed to a sample with target molecules, and the more emitted
light indicative of binding can reach the detector.
[0042] Example hydrogel compositions that provide acceptable
rigidity and porosity difference between the encoded portion 110
and different longitudinal portions of the particle are described
in more detail below and in Table 1. The more rigid hydrogel
composition includes about 30% Poly(ethylene glycol) (700)
diacrylate and about 30% Poly(ethylene glycol) (200), called DA30
hereinafter, where 700 and 200 refer to the molecular weights of
the corresponding polymers. The more porous hydrogel composition
includes about 20% Poly(ethylene glycol) (700) diacrylate and about
40% Poly(ethylene glycol) (200), called DA20 hereinafter. Thus the
hydrogel matrix in the first longitudinal portion comprises more
Poly(ethylene glycol) (700) diacrylate and less Poly(ethylene
glycol) (200) than does the hydrogel matrix in the second
longitudinal portion.
[0043] The hydrogel particles of such composition may be formed in
any manner known in the art. In some embodiments, the hydrogel
particles with loaded fluorescent entities or probes or both are
formed using techniques described below or in Doyle I or Doyle II,
cited and incorporated by reference above.
Apparatus.
[0044] FIG. 2 is a block diagram that illustrates an example
elevation view 200 of the hydrogel particle in a microfluidics
channel, according to an embodiment. The microfluidics channel 212
is formed in a lower substrate 210, e.g., by photolithography,
etching or other means, well known in the art, and covered with an
upper structure 220 of the same or different material.
[0045] Any method may be used to construct the apparatus. For
example the channels and chambers are constructed on a planar
substrate by etching, injection molding, embossing, or stamping.
Lithographic and chemical etching processes developed by the
microelectronics industry are used routinely to fabricate
microfluidic apparatus on silicon and glass substrates. Similar
etching processes also can be used to construct microfluidic
apparatus on various polymeric substrates as well. After
construction of the network of microfluidic channels and reservoirs
on the substrate, the substrate typically is attached to one or
more planar sheets that seal channel and chamber tops and/or
bottoms while providing access holes for fluid injection and
extraction ports Any material appropriate for a particular use may
be employed for substrate 210 and upper structure 220. For example,
in various embodiments the materials are selected from a group
including elastomer, glass, a silicon-based material, quartz, fused
silica, sapphire, polymeric material, and mixtures thereof. The
polymeric material may be a polymer or copolymer including, but not
limited to, polymethylmethacrylate (PMMA), polycarbonate,
polytetrafluoroethylene (e.g., TEFLON.TM.), polyvinylchloride
(PVC), polydimethylsiloxane (PDMS), polysulfone, and mixtures
thereof. Such polymeric substrate materials are desirable for their
ease of manufacture, low cost, and disposability, and because they
tend to be inert.
[0046] The channel has width 214 in the y direction 202 that is
horizontal and perpendicular to the horizontal direction of flow,
and depth 216 in the vertical z direction 204. Although particular
orientations are referred to as horizontal and vertical for
purposes of illustration, there is no requirement that the channel
have a particular orientation with respect to the gravitational
field of the Earth.
[0047] A fluid 290 bearing one or more particles 100 flows through
channel 212. Any fluid may be used. It is generally desirable that
the fluid not degrade the particle 100 or the probe molecule or
target molecule, if any, bound to the probe. It is also desirable
for the fluid to have viscosity and density that favors laminar
flow in the channels of the substrate, such as channel 212. Many
fluids used as buffers for biological molecules are suitable,
including fluids described in more detail below.
[0048] The particle 100 has width 104 less than channel width 214
and thickness 106 less than channel depth 216.
[0049] At least one of the cross sectional dimensions of channel
212, i.e., width 214 or depth 216, is in the range from 0.1 microns
to 500 microns. In the illustrated embodiments, both
cross-sectional dimensions are in the range from 0.1 microns to 500
microns. The distance from a side wall of the channel to the
particle is called the side gap 230; and the height from the bottom
of the channel to the bottom of the particle is called the height
gap 232. It is desirable that an apparatus be configured so that
the side gap 230 and height gap 232 are well controlled and well
known when the encoded portion and probe portion of the particle
are to be detected.
[0050] FIG. 3A is a block diagram that illustrates an example view
300 looking down into a microfluidics channel in a detection zone,
according to an embodiment. The channel 212 in the substrate 210
has width 214 in the y direction 202 that is horizontal and
perpendicular to the horizontal x direction 302 of flow indicated
by the open arrow. At a particular x position along the channel 212
is an optical scanning window 310 that is depicted extending across
the entire channel width and reaching down to the bottom of the
channel 212, but limited in the x direction of flow. The length in
the x direction is the scale of the scanning window for purposes of
determining the scale 112 of thickness variations in the particle.
The particle 100 moving with the flow is about to intersect the
optical scanning window 310.
[0051] For purposes of illustration, it is assumed that the
particle 100 is not perfectly aligned, i.e., parallel, to the side
walls of the channel 212, but deviates by an angle .alpha. 306.
This results in a side gap, represented by the symbol h that varies
with distance in the x direction. The side gap at the trailing edge
of the particle is given by h0 308a, at the leading edge of the
particle by hi 308b, and by an intermediate position x by h(x)
308c. As a result of the tip by angle .alpha. 306, the distance the
particle cover when passing the optical scanning window, or any
other point x, is L cosine .alpha., as shown by line segment 307.
These parameters .alpha. and h(x) are referenced later when
describing the restoring forces acting to align the particle
100.
[0052] FIG. 3B is a graph 320 that illustrates an example
measurement of optical emissions at a detector in the detection
zone, according to an embodiment. The horizontal axis 324 indicates
time and the vertical axis 324 indicates signal strength increasing
upwards. The strength of the signal resulting from measuring
optical emissions, such as fluorescence, in the optical scanning
window 310 is presented as trace 330. To more readily depict the
relation of peak signal strength on trace 330 to longitudinal
position on particle 100 moving left to right, time on axis 322
decreases to the right. The leading portion detected first is shown
on the right as the peak 322 corresponding to target bound to the
probe portion of particle 100. This is followed by the fluorescence
signal 334 detected in the encoded portion, which varies depending
on the number of holes in a column. The fewer holes, the more
fluorescence and the greater the signal strength. No holes in a
column corresponds to highest signal strength level 340c, all four
holes corresponds to lowest signal strength 340a, and one or two
holes corresponds to intermediate signal strength 340b.
[0053] Particle grouping or misalignment in the optical scanning
window, e.g., due to varying side gaps and height gaps, or
deformation of these flexible particles, can lead to signal
variations that are misinterpreted as target signatures or code
changes. In some embodiments, the scanning window is limited in the
cross channel y direction as well as the long channel x direction;
and, is thus capable of detect individual rows of holes. Several
such scanners can be used to detect all rows of the encoded
portion. Such scanning is especially susceptible to small errors in
the cross channel position y of the particle as it passes the
scanner. Thus an apparatus is needed to align the particles 100 in
a relatively narrow channel 212 in a detection zone with a scanning
window 310. The task is challenging because the particles are
oriented and spaced at random in the sample where the probe
molecules are exposed to any targets that might be in the
sample.
[0054] FIG. 4 is an image 400 that illustrates a mixture of
hydrogel particles in a fluid sample, according to an embodiment.
Particles are shown to be oriented at random not only in the plane
of the image, as indicated by particles 410a and 410b, but also in
a dimension perpendicular to that plane, as indicated by the out of
focus blurs of different lengths, such as blur 410c.
[0055] FIG. 5A is a diagram of microfluidics channels in an example
apparatus 500, according to an embodiment. The microfluidics
channels include a sample mixture inlet chamber 510 and outlet
chamber 518 in fluid communication with a central channel
comprising a first portion 512a, a second portion 512b, a third
portion 512c, a fourth portion 512d and a fifth portion 512e,
collectively called portions of a central channel 512 hereinafter.
The flow of particles is in a flow direction from the sample
mixture inlet chamber 510 to the outlet chamber 518, induced by an
applied pressure difference between inlet chamber 510 and outlet
chamber 518. Although called a central channel to emphasize the
desired position of the particles in the center of the channel, the
name does not imply a requirement that the central channel be
located in the center of the apparatus or midway between any or all
portions of the other channels or chambers in the apparatus.
[0056] The apparatus 500 also includes multiple focus fluid
channels that are in fluid communication with the central channel
512 at junctions 514a, 514b, 514c and 514d, collectively referenced
hereinafter as junctions 514. A pair of focus fluid channels
connects to the central channel at each junction. As depicted,
focus fluid channels 522a and 523a connect to the central channel
at junction 514a. Similarly, focus fluid channels 522b and 523b
connect to the central channel at junction 514b; focus fluid
channels 522c and 523c connect to the central channel at junction
514c; and, focus fluid channels 522d and 523d connect to the
central channel at junction 514d. The focus fluid channels 522a
through 522d and 523a through 523d, collectively referenced
hereinafter as focus fluid channels 522, are also in fluid
communication with a focus fluid inlet 520.
[0057] The central channel portion (e.g. portion 512e) downstream
of the last junction (e.g., junction 514d) is called the detection
zone 506. In the detection zone 506, the particles are sufficiently
aligned for high precision detection of target binding to the probe
portion and high precision detection of the code in the encoded
portion. The width of the central channel in the detection zone is
called the detection width Wd 513. According to some embodiments,
the detection width Wd 513 is about twice the width of the
particles. An advantage of this relationship, as described in more
detail below, is that the flexible hydrogel particles are less
likely to be deformed as they enter this portion of the central
channel.
[0058] Between the sample mixture inlet chamber 510 and the
detection zone 506 is the alignment zone 504 where the focus fluid
channels join the central channel at multiple junctions 514.
[0059] According to various embodiments, the width of the central
channel 512 decreases downstream of each junction 514 to the width
Wd 513 in the detection zone. An advantage of this feature, as
described in more detail below, is that particles are less likely
to cluster at the mouth of the detection zone of the central
channel.
[0060] In some embodiments, as described in more detail below, the
change in channel width from one portion of the central channel to
another is limited to be no greater than about twice the length of
the particle, e.g., about 400 microns in an illustrated embodiment.
An advantage of this constraint, as described in more detail below,
is that the flexible hydrogel particles are even less likely to
cluster. In the illustrated embodiment, the decrease in width is
abrupt at the junction and the width is constant in each portion of
the central channel between successive junctions.
[0061] According to some embodiments, the distance between
junctions, called the junction spacing Lf 528, is greater than two
particle lengths, e.g. Lf 528 is about one millimeter (1000
microns) or more. As described in more detail below, an advantage
of longer focus channel spacing is that the particles appear to
have more time to separate and line up parallel to the central
channel, especially in the wider portions of the central
channel.
[0062] This apparatus 500 has been shown effective in aligning
hydrogel particles in a detection zone for high precision
detection, as described in more detail below.
[0063] FIG. 5B is an image of a portion of microfluidics channels
in an example apparatus, according to an embodiment. The image
depicts portion 512c and portion 512d of the central channel, the
junction 514c between those portions and the focus fluid channels
522c and 523c that join the central channel in junction 514c. Also
depicted are portions of focus fluid channels 522b and 523b that
join the central channel at the junction immediately upstream of
junction 514c, and portions of focus fluid channels 522d and 523d
that join the central channel at the junction immediately
downstream of junction 514c. FIG. 5B clearly depicts the abrupt
change in width of the central channel at junction 514c, from width
560a upstream of the junction to smaller width 560b downstream of
the junction, and the constant width in each of central channel
portions 512c and 512d. FIG. 5B also indicates that the upstream
width 560a is 350 microns and the downstream width 560b is 250
microns in the illustrated embodiment. The difference is 100
microns, which satisfies the constraint that the width change at
the junction not be greater than about 400 microns.
[0064] FIG. 6 is a diagram of microfluidics channels in another
example apparatus 600, according to an embodiment. In this
embodiment there is not a separate inlet for focusing fluid.
Instead, fluid from the sample mixture is used as the focus fluid.
Similar to apparatus 500, apparatus 600 includes a sample mixture
inlet chamber 610 and outlet chamber 618 in fluid communication
with a central channel. The central channel includes a different
number of portions of constant width, including portion 612a,
portion 612b, portion 612c, portion 612d, portion 612e, portion
612f, and portion 612g, collectively called portions of a central
channel 612 hereinafter. The flow of particles is in a flow
direction from the sample mixture inlet chamber 610 to the outlet
chamber 618, induced by an applied pressure difference between
inlet chamber 610 and outlet chamber 618.
[0065] Like apparatus 500, apparatus 600 also includes multiple
focus fluid channels that are in fluid communication with the
central channel 612 at junctions 614a, 614b, and 614c, collectively
referenced hereinafter as junctions 614. A pair of focus fluid
channels connects to the central channel at each junction. As
depicted, focus fluid channels 622a and 623a connect to the central
channel at junction 614a. Similarly, focus fluid channels 622b and
623b connect to the central channel at junction 614b; and focus
fluid channels 622c and 623c connect to the central channel at
junction 614c. The focus fluid channels 522a through 522d and 523a
through 523d are collectively referenced hereinafter as focus fluid
channels 622.
[0066] Unlike apparatus 500, however, focus fluid channels 622 are
not in communication with a separate focus fluid inlet. Instead,
the focus fluid channels 622 are in fluid communication with the
sample mixture inlet chamber 610 through the central channel 612
and multiple inlet junctions, including inlet junction 621a, inlet
junction 621b and inlet junction 621c, collectively referenced
hereinafter as inlet junctions 621. In the illustrated embodiment
the focus fluid channels 622 are narrower than the particle widths
to prevent particles from entering the focus fluid channels 622 at
the inlet junctions 621.
[0067] An advantage of the arrangement of apparatus 600 is that it
is simpler to make and to operate than apparatus 500 that includes
a separate focus fluid inlet chamber 510. A particular ratio of
focus fluid flow rate to sample mixture flow rate can be achieved
by fixing the widths of the focus fluid channels 622 compared to
the central channel portion widths. An advantage of apparatus 500,
is that the focus fluid can be driven at a separate pressure to
obtain more or time-variable ratios of flow rates of the focus
fluid relative to the sample mixture.
Method
[0068] FIG. 7 is a flow diagram of a method 700 for using hydrogel
microparticles in an apparatus, according to an embodiment.
Although shown in a particular order for purposes of illustration,
in other embodiments one or more steps, or portions, thereof, may
be performed in a different order or overlapping in time, whether
in series or parallel, or one or more steps may be omitted or one
or more other steps added, or the method may be changed in some
combination of ways.
[0069] The method 700 includes, in step 703, providing an
apparatus, e.g., apparatus 500 or apparatus 600 or other apparatus
known in the art, to focus the hydrogel particles in a fluid flow
past a detector, such as an optical detector.
[0070] In step 705, multiple particles of one or more types are
provided. For example, 1000 hydrogel microparticles, each with a
first set of one or more probes in a longitudinal portion separate
from an encoded portion, are provided. In some embodiments, step
705 includes providing multiple particles of each of two or more
types. Each different type includes a different code and a
different set of one or more probes associated with that code. For
a given type, all particles have the same code and same set of one
or more probes.
[0071] In step 707, the particles provided are mixed with a sample
at effective particle concentrations. In an illustrated embodiment,
mixtures are formed that result in particle concentrations within a
range from about 16 particles per microliter (particles/.mu.l, 1
.mu.l=10.sup.-6 liters) to about 23 particles/.mu.l. It has been
determined that for particles several tens of microns in width and
length, higher particle concentrations lead to excessive particle
volume compared to fluid volume, which interferes with either
binding between probe and target or flow of particles through an
apparatus, such as apparatus 500 or apparatus 600, or some
combination. It has also been determined that for hundreds of
different targets, and consequently different particle types, or
more, in a typical 50 .mu.l sample volume, lower concentrations
lead to so few particles for each type as to reduce redundancy and,
thus, increase measurement errors to undesirable levels.
[0072] In step 709, the mixture of sample and particles is loaded
into the sample inlet chamber of an apparatus at a first pressure
difference from the outlet chamber. Any method may be used to
obtain the pressure difference, including increasing the pressure
applied to the inlet chamber, or forming a vacuum at the outlet
chamber, or both. In some embodiments, the mixture is formed by
exposing the particles to a sample for a certain incubation time,
and then rinsing the sample off the particles and providing the
exposed particles in a mixture fluid that does not include target
molecules that are not bound to probe molecules in a particle.
[0073] In step 711, a focusing fluid is loaded into the focus fluid
inlet of an apparatus at a second pressure difference from the
outlet chamber. Again any method may be used to cause the pressure
difference. In some embodiments, the apparatus does not include a
separate focusing fluid or focus fluid inlet; and, step 711 is
omitted.
[0074] In some embodiments, the pressure applied during step 709,
or during steps 709 and 711, is chosen so that a fluid flow rate of
the mixture fluid down the central channel is about equal to a sum
of fluid flow rates through all the focus fluid channels. Thus, in
these embodiments, steps 709 and step 711 include applying a first
pressure at the sample fluid inlet and a second pressure at a
focusing fluid inlet in fluid communication with the plurality of
focusing channels so that a sum of flow rates through the plurality
of focusing channels is about equal to a flow rate from the sample
mixture fluid inlet.
[0075] In step 713 optical emission are detected in a detection
zone of the apparatus. In other embodiments, other measurements are
made in the detection zone during step 713, such as magnetic
measurements, or thermal measurements. In step 715, the presence of
the target molecule in the sample is determined based on the
detections, e.g., the optical emissions. For example, in some
embodiments only particles of one type are used and the apparatus
is so effective at focusing the particles that the particles travel
past the detector at a repeatable and known rate. The signal
expected form the encoded portion, if any, can be removed from the
measured signal to deduce the percentage or range of signal
strengths of particles that exhibit binding to a target, for
qualitative or quantitative analysis of the sample, respectively.
In some embodiments, multiple particle types are used, and the code
on each particle is also detected and used to determine the
presence or absence of target molecules in the sample.
Example Embodiments Fabrication
[0076] In the embodiments described in this and following sections,
one or more of the following methods were used to fabricate the
particle fabrication devices, the particles or the
particle-focusing devices.
[0077] The microfluidic devices used to synthesize particles and to
generate ordered particle flows were fabricated in
polydimethylsiloxane (PDMS) (Sylgard 184.TM. from DOW CORNING.TM.
of Midland, Mich.) using soft lithography methods. PDMS is widely
used for fabrication of microfluidic devices; it is inexpensive and
it can be fashioned to have complex channel structures. Master
molds for the devices were created by spin-coating a clean silicon
wafer with negative photoresist (SU-8 25.TM. from MICROCHEM.TM. of
Newton, Mass.). High-resolution photomasks (10,000 dpi, CAD ART
SERVICES.TM. of Bandon, Oreg.) were then used to selectively expose
the coated wafers to UV light, thus creating the desired patterns.
Following treatment with SU-8 developer (MICROCHEM), the wafers
were flood exposed to UV light and baked. A profilometer
(DEKTAK.TM. from VEECO INSTRUMENTS INC..TM. of Plainview, N.Y.) was
used to determine the heights of features located on the left,
right, and center portions of the wafer. The wafers were then
treated with a fluorosilane
((tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane, UNITED
CHEMICAL TECHNOLOGIES, INC..TM. of Bristol, Pa.) under vacuum for
60 min. PDMS pre-polymers (10 parts base, 1 part curing agent) were
poured over the molds to a depth of 5 mm and allowed to cure in an
oven at 65.degree. C. for 12 hours. Individual channel designs were
cut from the mold with a scalpel. Inlet and outlet holes were
punched with blunt 15-gauge luer stub adapters (CLAY ADAMS.TM. from
BD.TM. of Franklin Lakes, N.J.). The devices were then rinsed with
water and ethanol, dried with Ar gas, placed channel-side down on
PDMS-coated slides, and baked in an oven at 65.degree. C. for 5
hours. Channels used for particle synthesis had heights between
37.4 and 39.9 .mu.m, while those used for particle flowing were
between 38.2 and 38.6 .mu.m in height.
[0078] Hydrogel microparticles were photo-polymerized at rates up
to 18,000 particles per hour with 75 millisecond (ms, 1
ms=10.sup.-3 seconds) ultraviolet (UV) exposures using the
stop-flow lithography (SFL) method. A power meter (Model 1815-C
from NEWPORT CORPORATION.TM. of Irvine, Calif.) and appropriate
adjustment of the UV lamp strength were used to ensure a consistent
UV intensity (0.8 .mu.W mm.sup.-2) during the course of the
experiments. This step was taken to avoid unexpected variations in
polymerization extent due to the intensity changes over the
lifetime of the mercury bulb. Microfluidic devices with one to four
inlets were connected to a compressed air source by Tygon tubing
with modified 10 and 200 .mu.l pipette tips (BioSciences from
LIFESPAN.TM. of Seattle, Wash.) attached to one end.
[0079] Two different pre-polymer solutions, "DA20" and "DA30", were
used in the course of the experiments; the composition of each is
given in Table 1. Pre-polymer solutions were mixed 9:1 with
1.times.TE to mimic the monomer
TABLE-US-00001 TABLE 1 Composition by volume of pre-polymers used
in particle synthesis. Constituent DA 20 DA 30 Poly(ethylene
glycol) (700) diacrylate (PEG-DA) 20% 30% Poly(ethylene glycol)
(200) (PEG) 40% 30% 3X TE buffer 35% 35% Darocur 1173
photoinitiator 5% 5%
formulation used in previous nucleic acid detection experiments.
When synthesizing particles with multiple chemistries, food
coloring was added to the DA30 at 2% of the final monomer solution
volume to create a contrast difference that could be exploited for
stream visualization in the synthesis process using a charge couple
device (CCD) camera (KP-M1A from HITACHI.TM. of Tokyo, Japan).
Atomic force microscopy (AFM) was used to determine the elastic
moduli of particles synthesized with DA20 and DA30.
[0080] Particles were flushed down the synthesis channel and
collected in a 0.6-.mu.l Eppendorf tube filled with 300 .mu.l of
TET (1.times.TE with 0.05 vol % Tween-20 surfactant from SIGMA
ALDRICH.TM. St. Louis, Mo.). TE is a common buffer solution that
consists of Tris (pH buffer) and EDTA (chelating agent) and is
often used to prevent the degradation of nucleic acids by limiting
the efficacy of nucleases. 100.times.TE is obtained from EMD
CHEMICALS INC..TM. of San Diego, Calif. and then dilute to 1.times.
with DI water. TET was then used to rinse the particles of
un-reacted monomer as well as PEG and food coloring in a series of
five washing steps that involved manual aspiration facilitated by
centrifugal separation of the dense particles. Particles were
stored in TET at final concentrations of about 10 particles per
micro liter (.mu.l, 1 ml=10.sup.-6 liters) in a refrigerator (at
4.degree. C.). Unless otherwise noted, particle dimensions cited
herein refer to the size expected from the transparency mask used.
The length of each dimension could be up to 4% greater for
particles made from DA20 due to swelling.
[0081] The tablet-shaped hydrogel particles in these example
embodiments are substantially larger (about 250 microns long by
about 80 microns wide by about 35 microns thick) and more flexible
than latex microspheres commonly used in other suspension arrays.
Pressure-driven flow is used to carry the particles through
rectangular cross-section PDMS channels, while sets of side streams
called focus fluid channels and abrupt contractions in width at
junctions serve to orient the particles prior to their entrance
into a narrow "detection zone," as described above. After exposure
to a sample with one type of target molecules, the particles are
rinsed and suspended in PTET carrier fluid (5.times.TET with 25% by
volume PEG (400)), e.g., during step 709 above, for introduction
into the particle focusing device.
Particle Focusing Device Operation.
[0082] Particle focusing devices (also called flow devices herein)
with two inlets and one outlet were used for all experimental
embodiments. Different candidate particles were never mixed
together; each flow trial consisted of particles of identical shape
and composition. FIG. 14 is a photo of a microfluidics device 1400
in operation, according to an embodiment. A focus fluid reservoir
container 1452 is connected to focus fluid inlet chamber 520 using
metal tubing 1420. Similarly a fluid collection container 1458 is
connected to fluid outlet chamber 518 using metal tubing 1418. A
sample mixture reservoir container 1462 is connected to sample
mixture inlet chamber 510. Tube 1454 is connected to one pressure
source to drive the focus fluid through the device 1400; and tube
1464 is connected to an independent pressure source to drive the
sample mixture through the device 1400.
[0083] Prior to being loaded into the flow device, particles were
removed from the refrigerator, rinsed 4 times in PTET, and allowed
to sit at room temperature for 90 min. PTET was used to obtain
better density-matching between the particles and the liquid medium
in order to minimize the effects of sedimentation in the loading
process. The PTET was subjected to sonic agitation (i.e.,
"sonicated") for 1 minute before use to eliminate polymer
agglomerations that could disrupt particle flow. Once the particles
were diluted to the appropriate concentration (10 to 20 particles
per .mu.l) with PTET, 20 to 30 .mu.l of the mixture was loaded into
a pipette-capped 1462 length of Tygon tubing 1464. The pipette was
then inserted into sample mixture inlet 510. A modified Eppendorf
tube 1452 containing a 2% solution of food coloring in PTET was
connected to focus fluid inlet 520 via metal tubing 1420. Tygon
tubing 1454 was inserted through a hole in the Eppendorf cap to
provide driving pressure. The tubing 1454 and 1464 from the inlets
was then attached to two separate pressure regulators (Omega) to
provide independent control over the two streams.
[0084] Particle focusing devices (e.g., device 1400) were able to
be reused up to 50 times without any decrease in performance. The
compressed air source used to drive the flows was a plastic
canister with a hand pump. Upon sufficient pumping, a two-way valve
connecting the canister with the regulators was opened, thereby
inducing flow. Pressures of 9 pounds per square inch (psi) were
able to be maintained for more than a minute using this simple
setup.
Theoretical Considerations
[0085] In a rectangular channel with a high cross-sectional aspect
ratio, it is expected that a parabolic velocity distribution will
develop along the small dimension. This flow profile can inhibit
the performance of flow measurement devices (called cytometers) for
biological cell-sized particles by generating non-uniform particle
velocities. Although the short dimension can be further reduced to
physically confine the particles, this lowers flow velocity and
throughput. Higher driving pressures can be used to counteract this
decrease in some embodiments, but such an approach may lead to
deformation of the channel and/or the particles. While the velocity
distribution in the longer cross-sectional dimension (called
channel width) is nearly uniform in the center of the channel,
large gradients develop in a boundary layer near the side walls of
the channel. Particles passing through this layer will be slowed
significantly. It has previously been shown that rapid decreases of
channel cross-section can enhance the focusing of deformable blood
cells by introducing regions of high shear adjacent to the walls
that produce strong hydrodynamic lift forces. However, it is not
apparent that the tablet shaped particles will respond similarly as
such disc-shaped cells to such contractions.
[0086] As described above and demonstrated below, focusing streams
from focus fluid channels and multiple contractions in channel
width are used in the illustrated embodiments to disturb the
developed flow along the walls and eject particles into the center
flow region for better performance in cytometers. The following
theoretical considerations are presented to more thoroughly explain
some of the embodiments. However, the embodiments are not limited
by the completeness or accuracy of these theoretical
considerations.
[0087] For flexible bodies like blood cells that deform at constant
volume and surface area, it was found that the pressure in the thin
"height gaps" above and below the body (e.g., see FIG. 2) are
uniform. Small deformations in the body height were shown to
produce a gap distance that varied with spanwise (y) position only;
the gap distance was constant along lines parallel to the flow.
Blood cell velocity was predicted to be much less than that of the
bulk fluid for channels with small height gaps and with channel
widths (also called span herein) much larger than cell width. This
effect can be attributed to the ability of the fluid to easily
bypass the cell by moving through the relatively large "side gaps."
In contrast, for tightly fitting bodies in cylindrical tubes, the
driving pressure is concentrated across the particle, leading to
bulk fluid velocities that are lower than body velocities and even
leading to "leakback" of fluid in the opposite direction.
[0088] For the narrow detection portions of the channel (e.g.,
central channel portion 512e or 612g), a lubrication approximation
is utilized to determine the lift forces on the particle that arise
from the bypass flow in the side gaps just described. This analysis
is used to rationally support the discovered design for both the
particle and the channel that gave rise to forces and torques that
most effectively position and align the particle for proper
scanning.
[0089] In a reference frame moving with the particle, the fluid
velocity and pressure drop in the side gap are given by Equation 1
and Equation 2.
v x ( x , y ) = y 2 .mu. P x ( y - h ( x ) ) + U w ( y h ( x ) - 1
) ( 1 ) P x = - 12 .mu. q h ( x ) 3 - 6 .mu. U w h ( x ) 2 ( 2 a )
##EQU00001##
[0090] where .mu. is the dynamic viscosity, P is the dynamic
pressure, U.sub.w is the wall velocity, and q is the volumetric
flow rate per unit width. As shown in FIG. 3A, y represents the
spanwise distance across the width of the channel, x the distance
along the channel, .alpha. the deflection of the particle from a
line parallel to the channel, and h(x) the side gap as a function
of x. The geometric and dynamic criteria required for application
of the lubrication analysis are expressed by the inequalities 2a
and 2b.
tan .alpha.<<1 (2b)
and
(q/v)tan .alpha.<<1. (2c)
Neglecting deformation and any three-dimensional effects from flow
in the height gap, these conditions are met by the nearly
unidirectional bypass flow established in the side gaps when an
oblong particle is passing through the relatively narrow regions of
the channel at small .alpha.. In analyzing the stress exerted by
the fluid on a rigid particle surface, the normal viscous stresses
are zero, and the components of the stress vector s are thus given
by Equation 3 and Equation 4.
s x = - h x P - .mu. .differential. v x .differential. y ( 3 ) s y
= P . ( 4 ) ##EQU00002##
The torque tensor G (per unit width) about a position at the center
of a trailing edge of the particle, given by the vector r.sub.0,
can be calculated approximately as given by Equation 5.
G = .intg. x 1 x 2 ( r - r 0 ) .times. s ( n ) x . ( 5 )
##EQU00003##
Where r is a position vector for a point on the surface of the
particle, n is a vector normal to the particle surface, and s is
the stress vector. The torque was calculated about r.sub.0 because
high-speed videos of poorly aligned particles in the upstream
portion of the detection region were observed to most often rotate
about this point in their movement into a properly aligned
orientation. For all calculations with this lubrication
approximation, the effect of the upper gap was modeled in an
analogous fashion to that just described for the lower gap and the
contributions summed for the determination of the total force and
torque. The primary difference between the two situations is that
the lower gap involved a contraction while the upper gap involved a
symmetric expansion.
[0091] Upon entrance into the detection region of the central
channel in the example embodiments, the observed angle of
deflection, .theta., for a typical particle is between 0.degree.
and 5.degree., thereby giving a maximum value of 0.087 for tan
.theta. and satisfying the geometric requirement for lubrication
approximation. Meanwhile, based on a typical volumetric flow rate
per width, q, of 2.times.10.sup.-5 meters squared per second
(m.sup.2s.sup.-1) and a kinematic viscosity, .nu., of
1.times.10.sup.-5 m.sup.2s.sup.-1 for the PTET carrier fluid, the
q/.nu. factor in Equation 2a has a value of 2; and thus the maximum
value of the center expression in Equation 2a is 0.174. These
values suggest that the use of the lubrication approximation is
indeed valid within this side gap region of the detection zone of
the central channel in the example embodiments.
[0092] For the wider portions of the channel (e.g., central channel
portions upstream of channel portion 512c or upstream of channel
portion 612g), the rotational and translational tendencies of
particles are better understood by the principle of gradient
minimization, which dictates that the oblong particles will tend to
rotate until the velocity gradient across their rear surface can no
longer be reduced. For rectangular particles with high aspect
ratios, this condition is met once the short and long dimensions
have been oriented perpendicular and parallel, respectively, to the
flow direction. The large particles used in the example embodiments
are expected to impact the time scale of this orientation process
by altering local velocity profiles and generating significant wake
flows in areas of high particle concentration.
Alignment Testing Procedures.
[0093] The capability of a particle focusing apparatus to align
hydrogel particles for scanning was tested by imaging various
embodiments of the particles in the detection zone of various
embodiments of the particle focusing device. Once the properties of
a particular flow device are determined, it is anticipated that the
device would be deployed with a narrow window scanner (as depicted
in FIG. 3A) and not with a more complex imaging system as described
next for testing the device.
[0094] To accomplish testing, flow devices were mounted on an
inverted microscope (TE2000U, Nikon) for visualization with
10.times. and 4.times. objectives. A high-speed Phantom camera
(Vision Research) captured images at rates ranging from 4,000 to
15,000 frames per second (fps). Still images from the movies were
analyzed using Phantom Cine Viewer software (2.24 microns per pixel
for 10.times., and 12.44 microns per pixel for 4.times.). Particle
velocities were calculated in the detection zone by measuring the
time required for a fixed point on the particle to move a known
distance in the channel. Detection zone measurements of velocity,
position, and alignment were always made 750 microns upstream of
the outlet chamber (e.g., outlet chamber 518) to ensure
consistency.
[0095] A "successful" scan was defined to be one in which the
horizontal distance between the centers of any two holes in a given
column of the encoded portion was less than 5 microns. In addition,
particles with any measurable lateral (y-direction) drift from the
center of the channel in the detection zone were deemed
"unsuccessful" passages. This conservative definition of success is
based on properties of a scanner expected to be used during normal
operations, e.g., a photo-multiplier tube (PMT) with a sampling
rate of one MegaHertz (MHz, 1 MHz=10.sup.6 Hertz, Hz, 1 Hz=1 sample
per second) and an excitation beam width of about 1 micron to about
10 microns. Quoted throughput values include both successful and
unsuccessful particles, and unless otherwise noted, each flow trial
involved the imaging of 100 particles.
Experimental Embodiments
[0096] To separate the contributions of particle composition,
channel design, and device operation, several experimental
embodiments were constructed and tested using the procedures or
theories described above. In a first set of embodiments, a
canonical hydrogel particle was used under a variety of operating
conditions and channel designs to determine how to minimize
clogging and disruptive particle-particle interactions in the
detection zone. In another set of experimental embodiments,
hydrogel particle size, shape and composition were varied to
demonstrate the impact of those particle properties on alignment
tendency and mechanical stability in a particular particle focusing
apparatus. In yet another set of experimental embodiments, methods
of operating a particle focusing device with a revised hydrogel
particle design suitable for bioassays were determined to balance
high throughput and success rate with maintaining a high degree of
reproducibility.
Example Particle Focusing Devices
[0097] Embodiments with different detection-zone width (Wd), number
(N) of junctions, junction spacing (Lf), and forcing pressures were
implemented for a fixed canonical particle design. The canonical
particles were photo-polymerized from DA20 pre-polymer, had
dimensions of roughly 270 by 90 by 33 microns, and featured 10 by
10 micron holes spread evenly throughout the particle. The
composition was selected based on earlier work; while the hole
design was chosen for its symmetry and for its use in alignment
measurements. Sample mixture loading concentration was fixed at 10
particles per .mu.l.
[0098] Initial trials were performed with a simple channel design
with one pair of focus fluid channels impinging at one junction
("1-junction device," N=1) as presented in previous related work
(Doyle I). The focus fluid that flows along each wall of the
central channel downstream of the junction is called a "side flow,"
"side stream," "sheath flow," or "sheath stream." The central
channel decreased in width from 500 to 100 microns at the junction,
producing a large velocity gradient in the flow direction. The
detection zone was 2.3 mm in length, with Wd=100 microns. The
forcing pressures of the particle and sheath streams were matched,
and they varied between 4 and 9 psi. Average particle velocities
for this pressure range were between 10 and 30 centimeters per
second (cm/s, 1 cm=10.sup.-2 meters). Image analysis of the
detection region revealed a tendency for particles to appear in
clusters with poor alignment and slight deformations of the leading
and trailing particle edges. Moreover, flow in the channel would
temporarily decrease at times, producing wild variations in
particle velocities over short periods of time.
[0099] These observations implied that the particles were jamming
at the contraction point of the channel. Subsequent investigation
of this region revealed sporadic instances in which groups of 2 to
5 particles traveled closely together and lodged tightly in the
contraction zone, thus impeding flow and leading to an accumulation
and compression of particles. After 100 to 1000 milliseconds (ms),
the flexible hydrogel particles would eventually squeeze past one
another and eliminate the blockage--producing clumps observed
further downstream. It should be noted that the deformations were
elastic and the clogging never led to permanent (plastic)
deformation. Particles collected in an exit reservoir did not
exhibit any substantial structural abnormalities.
[0100] Various subsequent embodiments included devices with 2
junctions and 3 junctions, with Wd=100 microns and Lf=400 microns.
The frequency and duration of the blockage events were reduced, but
only by small amounts. Video imaging of the flow patterns in the
500 micron wide portion of the central channel of the device with
one junction revealed a tendency for some particles to travel
slowly along channel walls. Such behavior is consistent with the
flow profile of a channel with a high aspect ratio, as discussed
earlier. When the same observations were made in the 500 micron
wide portions of the central channel for the 2- and 3-junction
devices, the "wall-huggers" in these channels were reliably coerced
off the wall prior to the detection zone by the local velocity
increase created by the impinging side streams.
[0101] It was postulated that increasing Lf from 400 to 1000
microns would better enable particles to adopt a lengthwise flow
orientation prior to the junctions. This was based on the belief
that rotation into the lengthwise orientation arose from a tendency
for the particle to minimize the velocity gradient of the flow
impinging upon it. The disordered flow patterns in the wakes of
particles severely limited this effect, making congested areas less
likely to produce well-aligned particles. Embodiments with larger
Lf seemed to confirm this hypothesis. Based on observations of both
upstream and detection zones, the longer residence time of the
particles in the wide portions led to a nearly complete elimination
of blockage events and elimination of drastic velocity variations
in the embodiment with 3-junctions and Lf=1000 microns. Thus for
particle lengths of 270 microns, junction spacing greater than 400
microns, such as 1000 microns, is advantageous. Note that 400
microns is less than 540 microns (twice the particle length) and
1000 microns is greater. Thus these embodiments show it is
advantageous to have junction spacing of greater than about twice
the particle length.
[0102] The above embodiment included detection zone width (Wd) of
100 microns and particles widths (W) of 90 microns. Even though
clustering and velocity fluctuations were eliminated by increasing
Lf, particle deformations were observed, especially at higher
forcing pressures. Particles approaching the final width
contraction from 200 microns to 100 microns in the 3-junction
device were seen to distort violently if the particle approached
the contraction from a position far from a centerline of the
channel. Furthermore, the small side gap between the particle and
channel walls (5 microns on each side) led to a large pressure drop
across the particle length and introduced substantial lift forces
that compressed the particle in the direction perpendicular to flow
and elongated it in the direction parallel to flow (about 10%
increase in length).
[0103] Increasing detection zone widths Wd to 150 microns did not
eliminate the deformations. FIG. 8A through FIG. 8D are images of
hydrogel particles in a detection zone, according to various
embodiments of the particle. Scale bar 802 in all images is 50
microns long. Each embodiment involved a particle made of DA 20
with the whole length covered in coding holes. Each image shows an
example of a failure of the particle to be aligned properly for
accurate detection in a narrow scanning window. FIG. 8A illustrates
a failure mode of a 190 micron long by 90 micron wide particle 810
(with aspect ratio, AR, =2.11) with poor alignment. FIG. 8B, FIG.
8C and FIG. 8D illustrate failure modes of three 270 micron long by
90 micron wide particles 820, 830, 840, respectively (AR=3.00) with
drastic deformations that would preclude reading of the
barcode.
[0104] It was determined that embodiments with detection zone
channel widths Wd roughly twice as wide as the particle width would
suffer fewer particle deformations. It is believed that this is due
to increasing bypass flow and lowering the pressure drop.
[0105] These embodiments of the particle focusing device indicate
that it is advantageous to use multiple sets of side streams (i.e.,
multiple junctions) to constrict the central channel to the
detection zone width, to separate the junctions by a sufficient
distance (e.g., more than about two particle lengths), as well as
to have a detection zone width that permits substantial bypass flow
(e.g., about twice the particle width) for shape preservation,
alone or in some combination.
Particle Properties.
[0106] An embodiment of a particle focusing device called the
canonical flow device was used for different embodiments of the
hydrogel particle. The canonical flow device included 3-junctions
with Lf=1000 microns and Wd=150 microns. All embodiments for using
the device described in this section were performed with a pressure
of 9 psi for both inlets, leading to particle velocities of 25 to
35 cm/s.
[0107] The alignment of the tablet-shaped (e.g., rounded rectangle)
particles observed in initial experiments was far more reliable
than that of additional morphologies that were also investigated
(oblong particles with pointed ends, as well as tear-drop and
bullet shapes). Thus different embodiments of the tablet-shaped
particles are described in this section.
[0108] The effect of particle aspect ratio (AR) was explored by
flowing 90 micron wide particles (DA20 composition) with four
different lengths: 190, 230, 270, and 310 microns. Flow trials
involved measuring the lateral position and success rate of 100
particles of each aspect ratio and the results listed in Table 2a
and Table 2b for DA20 particles and DA30 particles,
respectively.
TABLE-US-00002 TABLE 2a Effect of AR on DA20 particles in device
with N = 3, Wd = 150 microns Aspect Success Mean dist from
centerline ratio rate (microns) Main failure mode 2.11 91% 4.5
Lateral movement 2.56 95% 2.6 Front deformation 3 82% 3.2
Front/back deformation 3.44 99% 1.4 None
TABLE-US-00003 TABLE 2b Effect of AR on DA30 particles in device
with N = 3, Wd = 150 microns Aspect Success Mean dist from
centerline ratio rate (microns) Main failure mode 2.11 82% 9.2
Lateral movement 2.56 94% 6.9 Lateral movement 3 88% 6.5 Poor
alignment 3.44 100% 3.6 None
[0109] The position was calculated as the distance from the center
of the particle to the centerline of the channel. It was observed
that several of the shortest particles, AR=2.11, exhibited
significant lateral movement and had slanted front and rear edges
upon reaching the detection zone (FIG. 8A). Particles with AR=2.56
flowed more closely along the centerline of the channel and with
virtually no lateral movement, but several had leading edges that
were slightly blurred or compressed at driving pressures of 9 psi.
Particles with AR=3.00 exhibited drastic deformations (FIG. 8B to
FIG. 8D), leading to the lowest success rate of the four particle
types. In many instances, the front edge of the particle was bent
towards one of the walls, thereby disrupting the alignment of the
holes in the code region. Several particles (10%) with this AR also
exhibited curved side walls. The particles with AR=3.44 suffered
from none of the problems that plagued the other designs. The sole
failure in this trial was one particle that was twisted into a
U-shape. It is believed that this is due to a detection zone width
Wd=150 microns less than twice (180 microns) the particle width (90
microns).
[0110] The results of the trials with these embodiments of the
particles suggested two potential sources of failure. One source,
at low AR, is a reduced tendency to orient into and maintain a flow
alignment that was conducive to scanning. As discussed earlier, the
generation of lateral forces in the small side gap between the
channel wall and the particle edge will tend to rotate the particle
into an orientation such that the major axis of the particle is
aligned with the centerline of the channel. This effect becomes
more pronounced as the particle length-to-channel width ratio
(represented by the symbol b) becomes larger. Longer particles
experience a larger net torque, and smaller side gaps generate
greater lift. Another source, at more moderate AR, is a higher
susceptibility to deformations of a leading edge. A visual
inspection of the images of the particles entering the final
contraction in the 3-junction device revealed that many of the
particles of mid-range AR were poorly oriented and thus forced to
bend significantly to enter the more narrow detection width. In
contrast, particles with the lowest AR appeared to resist such
deformation and were able to enter with imperfect alignment due to
their smaller size. Particles with the highest AR were already
aligned sufficiently so that they did not experience distortion
upon their entrance into the narrow channel of the detection
zone.
[0111] It is well known that the mechanical strength of a hydrogel
depends to a great extent on the number and nature of the
cross-links present. By using a pre-polymer with a higher
proportion of a cross-linking active monomer agent (PEG-DA), it was
found to be possible to generate hydrogel particles with higher
cross-linking densities that are more resistant to deformation. The
AR study was thus repeated with embodiments of particles
polymerized from DA30 pre-polymer to investigate effects on flow
characteristics (Table 2b).
[0112] From atomic force microscopy (AFM) measurements on particle
regions without coding holes, the elastic modulus of the DA20
hydrogel was found to be 10.1.+-.0.4 kilo Pascals (kPa, 1
kPa=10.sup.3 Pascals, 1 Pascal=1 Newton per square meter), while
that of the DA30 hydrogel was found to be 19.6.+-.1.2 kPa. The
impact of the added rigidity of the DA30 particles was noticeable,
with significantly less deformation at all values of AR. DA30
particles did not exhibit the bent leading edges, curved side
walls, or compressions that plagued the more flexible DA20
particles. Images of the final contraction revealed particles with
no perceptible shape changes, in sharp contrast to the DA20
embodiments.
[0113] Atomic force microscopy (AFM, AGILENT TECHNOLOGY.TM. of
Santa Clara Calif.) was incorporated within an optical microscope
(IX 81 from OLYMPUS CORPORATION.TM. of Tokyo, Japan) to enable
positioning of AFM cantilevered probes above particle samples.
Calibration of AFM cantilevers of nominal spring constant k=0.01
nanoNewtons per nanometer (nN per nm, 1 nN=10.sup.-9 Newtons, 1
nm=10.sup.-9 meters) and probe radius R=25 nm (Veeco) was
conducted. Briefly, inverse optical lever sensitivity in nm per
Volt (InvOLS) was measured from deflection-displacement curves
recorded on rigid glass substrates. For each measurement of elastiC
moduli, at least 25 replicate indentations were acquired to maximum
depths of 20 nm. Acquired probe deflection-displacement responses
were converted to force-depth responses using measured spring
constants and InvOLS (Scanning Probe Imaging Processor from IMAGE
METROLOGY.TM. of Horsholm, Denmark). Elastic moduli, E, were
calculated by applying a modified Hertzian model of spherical
contact to the loading segment of the force-depth response with the
scientific computing software Igor Pro (WAVEMETRICS.TM. of Lake
Oswego, Oreg.).
[0114] While the higher cross-linking density solved one flow
problem, it seemed to exacerbate the other. The stiffening of DA30
particles with ARs of 2.11 and 2.56 led to an increase in poorly
aligned particles that were also more susceptible to moving
laterally in the detection zone. FIG. 9 is a graph 900 that
illustrates dependence of microparticle position in a microfluidics
channel on particle composition, according to various embodiments.
The horizontal axis 902 is particle length L in microns. The
vertical axis 904 is lateral position of particle measured from the
center of the channel span in microns. The particle width is fixed
at 90 microns. Each plotted point represents an average of 100
particles, and all measurements were made from channel centerline
to the central most point of the particle. The diamonds 910
represent DA20 values and the squares 920 the DA30 values. At all
lengths, the softer DA20 particles 920 exhibit superior positioning
and a reduced tendency to move laterally. Longer particles are
observed to settle into stable flow trajectories closer to the
centerline than shorter particles.
[0115] The use of DA30 raised the success rates of the two higher
ARs, but actually led to a decrease in the success rates of the two
lower ARs. In the case of DA30 particles with ARs of 2.11 and 2.56,
the additional rigidity arising from higher cross-link density,
when combined with the compact morphology, leads to a regime of
rigid-body motion within the channel. This effectively eliminates
the temporary hydrogel deformations induced by the surrounding
focusing flow that play a significant role in coercing the particle
into the preferred position and alignment. Any attempt to tune the
flow behavior with this design parameter must balance the desire
for structural integrity with the need for efficient focusing and
orientation by hydrodynamic forces. Thus, the particle embodiments
with a high aspect ratio and a stiff gel network have advantages
over embodiments with lower aspect ratio.
Bifunctional Embodiments
[0116] The channel and particle embodiments with the better
observed performance characteristics were refined and combined to
create embodiments of a flow-through system that could be operated
reliably at high-throughput with hydrogel designs that were capable
of extracting and displaying information from bioassays.
[0117] Three different particle focusing device embodiments (A, B,
C) were used, each with L.sub.f=1000 .mu.m, as listed in Table
3.
TABLE-US-00004 TABLE 3 Three particle focusing device embodiments
Wd junctions, Name (.mu.m) N Upstream widths (.mu.m) A 100 4 450,
350, 250, 150, 100 B 125 4 450, 350, 250, 150, 125 C 150 3 450,
350, 250, 150
[0118] Various embodiments of the hydrogel particle with different
shapes and compositions were tested to find a combination that more
often resulted in proper alignment, durability in high-velocity
flows, and compatibility with bioassays. Using new masks and stop
flow lithography (SFL), 9 micron by 9 micron holes were limited to
one half of the particle, thus creating a "probe" portion and an
"encoded" portion (e.g., FIG. 1), also called regions. Columns and
rows of holes were separated by 9 microns. This design allows the
capture and quantification of target(s) on one end and the display
of code associated with probe identity on the other (see, Doyle I).
To create "bifunctional" particles, the code region was polymerized
from DA30 to ensure mechanical stability, while the probe region
was polymerized from DA20 to produce a pore size consistent with
that featured on particles employed in high-sensitivity assays in
previous work (e.g., Doyle I). Furthermore, the use of DA20
imparted a flexibility that would aid in orientation within the
channel, as seen in the earlier particle study. The new particles
were 235 microns long by 65 microns wide by 35 microns thick, and
featured a redesigned hole setup capable of 3,072 distinct codes.
The AR of this hydrogel particle embodiment (3.62) was higher than
any used in previous embodiments. The narrower width also permitted
the detection zone widths of device embodiments B and C (Wd=125,
150, respectively) to satisfy the desirable condition that the
detection zone channel width be about twice the particle width.
[0119] Bifunctional particles were flowed through the three device
embodiments at loading concentrations of 10 particles per .mu.l;
and performance was compared to that of particles with probe and
code regions both synthesized from DA30. Higher success rates were
recorded for the bifunctional particles in all three channels. FIG.
10 is a graph 1000 that illustrates dependence of microparticle
detection in a microfluidics channel on particle composition,
according to various embodiments. The horizontal axis 1002
indicates detection zone width in microns. The vertical axis 1004
indicates success rate in percent. Data is collected at three
increasing values of Wd, corresponding to the three device
embodiments A, B and C, respectively. The trace connecting diamonds
1010 indicates the results for pure DA30 particles, while the trace
connecting squares 1020 indicates the results for the bifunctional
particles.
[0120] Bifunctional particles also flowed more closely along the
centerline than their pure DA30 counterparts in all channel types,
with the best positioning achieved in design C. With the encoded
portion only occupying about half of the particle length, a
preference for "probe-first" flow was observed, with 91% of
bifunctional and 79% of pure DA30 particles doing so. In addition,
higher velocities (from about 35 cm/s to about 60 cm/s) were
recorded for these slender particles of 65 micron width. Compared
to the previously used 90 micron wide embodiments, the 65 micron
wide embodiments, closer to half the detection zone width Wd,
provided larger side gaps that effectively reduced the pressure
drop across the particle, thereby diminishing the tendency to
deform in the detection region. For the 600 total hydrogel
particles analyzed, probe-first particles traveled about 10% faster
than code-first particles, with a larger difference being measured
for the pure DA30 design.
[0121] The effect of hole spacing was investigated, again using the
same three particle focusing device embodiments. Seeking to
increase throughput, the loading concentration was increased from
10 to 15 particles per .mu.l for these embodiments. Bifunctional
particle embodiments with column spacing of 9, 7, and 5 microns
were studied (all row spacing remained at 9 microns). Shorter
column spacing reduces the particle area needed for coding and thus
increases the area available for probe immobilization. However, the
desire to minimize the code area is advantageously balanced by the
desire to maintain structural integrity. Nine trials with
bifunctional particle embodiments having three different hole
spacing in the three channels revealed a noticeable reduction in
success rate for holes with 5-.mu.m spacing. For the 900 hydrogel
particles studied, the average success rates of the 9 micron
spading, 7 micron spacing, and 5 micron spacing were 97%, 99%, and
88%, respectively. Most failures of the smallest spacing were due
to large compressions of holes in particles traveling code-first.
Mean throughput at the higher loading concentration was 29
particles/s; while mean velocity was 51 cm/s.
[0122] The high success rates achieved in device embodiments B and
C (100% and 99%, respectively) with 7 micron hole spacing were
explored further by conducting additional trials. Five new batches
of bifunctional particles, with the 7 micron hole spacing were
synthesized, and five devices of each embodiment were constructed.
To investigate the reproducibility of the earlier results, each
batch was sent through one of the five sets of devices (i.e., batch
1 through B-1 and C-1, batch 2 through B-2 and C-2, etc.) at a
loading concentration of 15 particles per .mu.l. The results of
these trials (first rows of Table 4) indicated a high degree of
repeatability, including inter-trial coefficients of variance (COV)
less than 4% for the mean velocity, as well as mean success rates
over 99%.
TABLE-US-00005 TABLE 4 Particle alignment results for various
device embodiments and loading concentrations. Mean Mean
Inter-trial Loading Mean through- velocity velocity Probe-
(particles/.mu.l) Device Trials success rate put (s.sup.-1) (cm/s)
COV first 15 B 5 99.80% 30.2 46.3 3.29% 84.40% C 5 99.20% 24.9 35
3.91 84.60% 17.5 B 5 98.40% 40.2 48.1 2.02% 75.60% C 5 98.80% 35.4
35.4 1.44 79.80% 20 B 1 92% 47.4 50.8 NA 86% C 1 91% 40.3 38.5 NA
71%
[0123] FIG. 11 is a graph 1100 that illustrates distribution of
microparticle speeds in different microfluidics channels, according
to various embodiments. Graph 1100 is a histogram of detection-zone
velocities of 1,000 bifunctional particles in devices B and C. The
horizontal axis 1102 indicates velocity bins in cm/s with 1 cm/s
resolution; and the vertical axis 1104 indicates the number of
particles observed traveling within a particular velocity bin. The
speeds observed in device embodiment B are substantially faster
than those observed in device embodiment C. The small spread of
velocities for each device indicates a high degree of order and
repeatability. Results are compared from trials run in five B and
five C devices with five different particle batches. The tails on
the left side of each spike can be attributed to the small
percentage (about 15% to about 20%) of particles that flowed
"code-first." For the bifunctional design, these particles were
typically 10% slower than their probe-first counterparts. This
uniformity of particle speed is advantageous for high-fidelity
signal analysis; and signifies the establishment of well-ordered
flows with minimal particle-particle interactions. As in earlier
trials, particles preferentially adopted a probe-first orientation
by the time they had entered the detection zone, with 76% flowing
in this manner in device embodiment B compared to 80% in device
embodiment C.
[0124] In order to maximize throughput, an attempt was made to
determine higher loading concentration that could still produce
well-ordered, single-file flows. A reproducibility study similar to
the one just described was conducted at a loading concentration of
17.5 particles/.mu.l. Once again, a high degree of repeatability
was achieved, as shown in Table 4, with a minor success-rate
decrease that was outweighed by a noticeable increase in mean
throughput. As in the previous study, device embodiment B performed
slightly better, with a higher mean throughput. Trials were then
performed at a loading concentration of 20 particles/.mu.l. At this
concentration, a more significant drop-off in the success rate
occurred, as shown in Table 4, with crowding in the channel
significantly disrupting the upstream focusing and even leading to
the partial overlap of particles in the detection region. It was
concluded that the increase in throughput at 20 particles/.mu.l was
not worth the decrease in success rate, and a preferred loading
concentration was determined to be 17.5 particles/.mu.l.
[0125] The hydrodynamic forces acting on the encoded particles in
the detection region of device B were explored by applying
Equations 1 through 5 to the nearly unidirectional side-gap flow to
determine the torque involved in lengthwise alignment. COMSOL
Multiphysics' Incompressible Navier Stokes module was used to model
the two dimensional (2-D) fluid flow in the microfluidic device
embodiments for particle focusing. For all simulations, a
stationary nonlinear solver was used with the Direct (UMFPACK)
linear system solver. Relative tolerance for the solver was at
least 1.0.times.10.sup.-4 for all converged flow profiles, and high
mesh densities were used to increase resolution in areas of
particular interest. Based on a "full-device" simulation without
particles present, it was determined that devices with Wd=125
microns and N=4 had a mean fluid velocity of 6.79 m/s at the point
of particle measurement in the detection region. This full-device
simulation specified no-slip boundary conditions at all walls of
the channel except the inlet port (normal pressure condition set at
9 psi) and the outlet port (neutral condition set). With a channel
Reynolds number defined as Re.sub.c=U Dh/.nu. (U is mean flow
velocity, Dh is hydraulic diameter of channel, .nu. is kinematic
viscosity of fluid), these simulations demonstrate that
Re.sub.c.apprxeq.40 for typical detection-zone geometries.
[0126] The same module was also utilized for the study of
hydrodynamic forces on particles in the detection region of the
channel. Using pressure estimates from the full-device simulation,
flow profiles were solved for a 1.5 psi drop across a 700 micron
long detection zone with Wd=125 microns, which contained a single
particle with various values of .theta.=-.alpha.. No-slip boundary
conditions were set at the two side walls, a normal pressure
condition was set at the inlet, and a neutral condition was set at
the outlet. In addition, a normal flow velocity condition was set
at the boundary of the particle to match the typical velocity of
the particles in the detection region (50 cm/s). To determine the
forces acting on the particle surface, the post-processing feature
of software from COMSOL.TM. of Burlington, Mass. was used to export
data on drag and lift forces at each point of the line segments
used to model the particle. This data was then combined to
numerically calculate the torque with a script using MATLAB.TM.
from The MathWorks, Inc. of Natick, Mass.
[0127] FIG. 12A is a graph that illustrates restoring forces on
microparticles that are not aligned with a microfluidics channel,
according to various embodiments. The horizontal axis 1202
indicates the deflection angle .theta. in degrees. The vertical
axis indicates the torque at the middle of the trailing edge of the
particle in picoNewtons (pN) per meter (1 pN=10.sup.-12 Newtons).
The numerical result is given by the diamonds 1210. The ratio of
particle length-to-channel width is given by the parameter b. The
lubrication approximation for values of b equal to 1.88, 1.41 and
0.94 are given as traces 1220, 1230 and 1240, respectively.
Calculations are based on a 1.5 psi drop across a 700-.mu.m long
detection zone containing a single particle. Re of the flow
upstream of the particle is .apprxeq.15.
[0128] As expected, the torque about a central point on the
particle's trailing edge increased as the deviation from lengthwise
orientation grew. This torque always acted to restore the
lengthwise orientation, and its magnitude was greater for particles
with higher values of b. This trend reinforces earlier observations
regarding alignment tendencies and particle dimensions. For the
case b=1.88, results of the lubrication approximation agree well
with the torques calculated for various values of .theta. using 2-D
COMSOL simulations. FIG. 12B is a map that illustrates fluid speeds
passing a microparticle that is not aligned with a microfluidics
channel, according to an embodiment. Here the deflection angle
.theta. is 5 degrees, and speeds peak at 4.9 m/s in jets induced
where the particle comes closest to the channel wall.
[0129] To further understand the flow in device embodiment B, the
orientation of particles was recorded by measuring the acute angle
(.theta.) between the major axis of the particle and the centerline
of the channel as particles moved through the upstream contraction
points (.theta.=-.alpha., see FIG. 1). An indication of alignment
tendency can be obtained by measuring the standard deviation in
this angle at the end of each constant width portion of the central
channel for a collection of 85 particles. FIG. 13A and FIG. 13B are
graphs that illustrate stability of microparticle alignment within
a microfluidics channel based on orientation of the microparticle,
according to an embodiment. FIG. 13A is a plot 1300 of deviation of
.theta. with channel width in the upstream region for particles
that flow code-first or probe-first. The horizontal axis 1302
indicates channel width Wd in microns. The vertical axis 1304
indicates the standard deviation of .theta. in degrees. The code
first flows are indicated by the diamonds 1310; and the probe-first
flows are indicated by squares 320. The larger deviation exhibited
by the code-first particles suggests that such an orientation is
less stable than the probe-first orientation. Measurements were
taken at the end of the corresponding constant width region prior
to contraction.
[0130] The results indicate a nearly linear decrease in deviation
as the channel width decreases for both of the orientations. The
larger deviations in .theta. for code-first particles suggest this
orientation is not driven to alignment in the flow as strongly as
probe-first particles. Indeed, of the 85 particles studied, four
switched from code-first to probe-first during flow, while none
made the transition from probe-first to code-first. A closer
examination of one particle of each orientation reinforces this
induction. For each frame of video, the full angle between the long
axis and the centerline (.phi.) was measured to preserve
orientation information, with code-first corresponding to
.phi.=0.degree. and .phi. increasing in the counter-clockwise
direction. FIG. 13B is a plot 1350 of .phi. over the course of the
travels of two particles through the upstream portions of the
central channel. The horizontal axis 1352 indicates time in
microseconds (.mu.s, 1 .mu.s=10.sup.-6 seconds), and the vertical
axis 1354 indicates the orientation .phi. of the particle (0 for
code first, and 180 for probe first). Trace 1360 indicates the
orientation of a first particle that starts nearly in a code first
orientation; and trace 1370 indicates the orientation of a
different particle that starts nearly in a probe forward
orientation.
[0131] The probe-first orientation is seen to be achieved in a
smoother and more predictable fashion than the code-first
orientation, again suggesting that the probe-first orientation is
more stable. While the probe-first particle experiences limited
disruptions in the regions of constant width (shaded boxes), the
code-first particle experiences two sharp alterations in alignment.
From left to right, the constant-width regions measure 350, 250,
and 150 microns in width, respectively.
[0132] Despite starting only 20.degree. from a lengthwise
orientation, the code-first particle nearly moves into a widthwise
orientation before rotating back into a code-first alignment that
oscillates quite dramatically as the channel width approaches 150
.mu.m. Meanwhile, the probe-first particle behaves in a much more
controlled and predictable manner, with a rapid transition from
widthwise to lengthwise orientation before a relatively mild
oscillation about .phi.=180.degree..
[0133] These analyses provide insight into the preferential
orientation that was observed in every trial with the
half-probe/half-code particle embodiments, including those in which
both regions were polymerized from DA30. Of the 3,200 bifunctional
particles analyzed in A, B, and C device embodiments, 83% flowed
probe-first, compared to 79% of the 300 pure-DA30 particles. This
indicates that the flexibility difference in the bifunctional
design plays, at most, a minor role in the preference. The
measurements of rotational tendency in the upstream region imply
that the resistance to fluid flow in the thin height gaps (each
about 1 micron to about 2 microns) above and below the code region
is different from the resistance in the height gaps above and below
the probe. This disparity can be observed directly by comparing the
lateral wobbles of a code-first particle to the smooth, settled
flow of a probe-first particle in the upstream regions of a channel
shown in FIG. 13B. While it is tempting to attribute this
resistance difference to the relative stiffness of the code region,
the data from the pure DA30 trials refute this suggestion.
[0134] The holes that constitute the graphical code are the only
other source of asymmetry in the particle and thus appear to give
rise to the resistance difference. It seems that the flow pattern
and the resulting pressure gradient along the length of the
particle in the height gap depend on whether the holes are on the
leading or the trailing edge of the particle. This hypothesis is
supported by the observed velocity disparity between code-first and
probe-first particles noted earlier for bifunctional particles as
well as pure DA30 particles.
[0135] It has been shown that a plurality of sufficiently spaced
side-focusing streams, a detection zone of ample width, and a
moderate particle loading concentration are advantageous for
high-throughput flow alignment of graphically encoded hydrogel
microparticles. In addition, the reliable alignment of soft
particles in high-speed flows (without deformation or clogging) is
greatly enhanced by balancing the mechanical properties and
morphology of the particles to ensure efficient focusing by
hydrodynamic forces while still maintaining overall structural
integrity in areas of high shear. The high throughputs achieved in
the example embodiments (40 particles/s) compare favorably with
those of currently available technologies for analyzing hard-sphere
suspension arrays. The use of multiple probe strips on each
particle in some embodiments has the potential to greatly augment
this processing capacity.
Other Embodiments
[0136] Additional flow trials were conducted in two channel
embodiments that did not use sheath streams (N=0). These
embodiments featured only a central channel (of the same length as
the central channel in A, B, and C) that gradually tapered to a
final width of either 100 microns (D) or 150 microns (E). The final
width persisted for 2.6 mm in D and 3.4 mm in E. Bifunctional
particles with 7 micron hole column spacing were used for these
trials. At throughputs of only about 20 particles/s, success rates
were lower (83% for D, 97% for E) than those in channels with side
streams and abrupt contractions. A throughput of about 40
particles/s in E led to only a 92% rate of success. Analysis of the
upstream behavior in these simple taper channels revealed a
disordered flow of tumbling particles, as well as particles that
slowly traveled along the walls of the channel (a behavior that was
seen earlier in single-focus devices).
[0137] While many of these particles were able to eventually adopt
a proper orientation further downstream, the disordered upstream
tendencies led to a flow pattern and velocity distribution in the
detection zone that exhibited more variability than those of
designs with sheath flow. Indeed, for comparable throughputs, the
standard deviation of particle velocity in D was 40% greater than
in A and the deviation in E was 47% greater than in C. In many
instances, consecutive particles in the detection zone in D and E
were touching one another, with some even wedging a small portion
of their probe region under the code region traveling ahead of it.
These observations indicate poor conditioning of the particles in
the upstream region and underscore the importance of side streams
for the reliable establishment of well-ordered, single-file
particle flows.
[0138] In the foregoing specification, the invention has been
described with reference to specific embodiments thereof. It will,
however, be evident that various modifications and changes may be
made thereto without departing from the broader spirit and scope of
the invention. The specification and drawings are, accordingly, to
be regarded in an illustrative rather than a restrictive sense.
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