U.S. patent application number 17/472168 was filed with the patent office on 2022-06-09 for hydrogel particles with tunable optical properties.
This patent application is currently assigned to SLINGSHOT BIOSCIENCES. The applicant listed for this patent is SLINGSHOT BIOSCIENCES. Invention is credited to Jeremy AGRESTI, Jeffrey KIM, Oliver LIU, Anh Tuan NGUYEN.
Application Number | 20220178810 17/472168 |
Document ID | / |
Family ID | 1000006156866 |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220178810 |
Kind Code |
A1 |
KIM; Jeffrey ; et
al. |
June 9, 2022 |
HYDROGEL PARTICLES WITH TUNABLE OPTICAL PROPERTIES
Abstract
The present disclosure relates to compositions comprising a
hydrogel particle with optical properties substantially similar to
the optical properties of a target cell, and methods for their
use.
Inventors: |
KIM; Jeffrey; (Berkeley,
CA) ; LIU; Oliver; (San Francisco, CA) ;
AGRESTI; Jeremy; (El Cerrito, CA) ; NGUYEN; Anh
Tuan; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SLINGSHOT BIOSCIENCES |
Emeryville |
CA |
US |
|
|
Assignee: |
SLINGSHOT BIOSCIENCES
Emeryville
CA
|
Family ID: |
1000006156866 |
Appl. No.: |
17/472168 |
Filed: |
September 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17163769 |
Feb 1, 2021 |
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17472168 |
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16684694 |
Nov 15, 2019 |
10942109 |
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17163769 |
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15895307 |
Feb 13, 2018 |
10481068 |
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16684694 |
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15145856 |
May 4, 2016 |
9915598 |
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15895307 |
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13858912 |
Apr 8, 2013 |
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15145856 |
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61621376 |
Apr 6, 2012 |
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61668538 |
Jul 6, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 15/1012 20130101;
G01N 21/6428 20130101; G01N 2015/1018 20130101; G01N 2015/1006
20130101; G01N 2015/149 20130101; G01N 2021/6439 20130101; G01N
15/14 20130101; G01N 2015/1415 20130101 |
International
Class: |
G01N 15/10 20060101
G01N015/10; G01N 15/14 20060101 G01N015/14; G01N 21/64 20060101
G01N021/64 |
Claims
1. (canceled)
2. A population of hydrogel particles, each hydrogel particle
comprising: (i) a polymer comprising a polymerized monomer and a
bifunctional monomer; (ii) DNA encapsulated within the hydrogel
particle; and (iii) a nanoparticle encapsulated within the
hydrogel, wherein the population hydrogel particles has an optical
property that is substantially similar to a corresponding property
of a target cell, the optical property provided by the polymerized
monomer, bifunctional monomer, or the encapsulated
nanoparticle.
3. The population of hydrogel particles of claim 2 wherein the
polymerized monomer is lactic acid, glycolic acid, acrylic acid,
1-hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol
methacrylate, acrylamide, N-vinylpyrrolidone, methyl methacrylate,
glycidyl methacrylate, glycol methacrylate, ethylene glycol,
fumaric acid, methacrylamide, N-alkylacrylamide,
N-alkylmethacrylamide, N,N-dialkylacrylamide,
N-[(dialkylamino)alkyl]acrylamide,
N-[(dialkylamino)alkyl]methacrylamide, (dialkylamino)alkyl
acrylate, or (dialkylamino)alkyl methacrylate.
4. The population of hydrogel particles of claim 2, wherein the
polymerized monomer is polymerized acrylamide.
5. The population of hydrogel particles of claim 2, where the
bifunctional monomer comprises one or more chemical side groups
that are capable of reacting with a dye molecule.
6. The population of hydrogel particles of claim 5, wherein the one
or more chemical side groups is amine, carboxyl, maleimide, epoxide
or alkyne.
7. The population of hydrogel particles of claim 2, wherein the
bifunctional monomer is allyl amine, allyl acrylate, allyl
methacrylate, allyl alcohol, allyl isothiocyanate, allyl chloride,
allyl maleimide, or bis-acrylamide.
8. The population of hydrogel particles of claim 7, wherein the
bifunctional monomer is bis-acrylamide.
9. The population of hydrogel particles of claim 8, wherein the
bisacrylamide is N,N'-methylenebisacrylamide,
N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide,
N,N'-ethylenebis-methacrylamide, N,N'propylenebisacryl amide, or
N,N'-(1,2-dihydroxyethylene)bisacrylamide.
10. The population of hydrogel particles of claim 2, wherein the
polymerized monomer is acrylamide and the bifunctional monomer is
bis-acrylamide.
11. The population of hydrogel particles of claim 2, wherein the
encapsulated nanoparticle is polymethyl methacrylate (PMMA),
polystyrene (PS), or silica.
12. The population of hydrogel particles of claim 11, wherein the
encapsulated material is PMMA.
13. The population of hydrogel particles of claim 11, wherein the
encapsulated material is PS.
14. The population of hydrogel particles of claim 11, wherein the
encapsulated material is silica.
15. The population of hydrogel particles of claim 10, wherein the
encapsulated nanoparticle is polymethyl methacrylate (PMMA),
polystyrene (PS), or silica.
16. The population of hydrogel particles of claim 15, wherein the
encapsulated material is PMMA.
17. The population of hydrogel particles of claim 15, wherein the
encapsulated material is PS.
18. The population of hydrogel particles of claim 15, wherein the
encapsulated material is silica.
19. The population of hydrogel particles of claim 2, comprising up
to 1500 .mu.g/mL of DNA.
20. The population of hydrogel particles of claim 2, comprising 40
ug/mL to 1000 ug/mL of DNA.
21. The population of hydrogel particles of claim 2, further
comprising a dye.
22. The population of hydrogel particles of claim 21, wherein the
dye is conjugated to the bifunctional monomer.
23. The population of hydrogel particles of claim 2, having a
refractive index between 1.1 and 2.9.
24. The population of hydrogel particles of claim 23, wherein the
hydrogel particles have an average refractive index of greater than
about 1.7.
25. The population of hydrogel particles of claim 2, wherein the
hydrogel particles have an average diameter in the range of from 5
.mu.m to 100 .mu.m.
26. The population of hydrogel particles of claim 25, wherein no
more than 10% of the hydrogel particles have a diameter exceeding
the average diameter of the hydrogel particles by more than
10%.
27. The population of hydrogel particles of claim 2, wherein the
optical property comprises side scattering.
28. The population of hydrogel particles of claim 27, wherein the
side scattering is within 10% of a target cell.
29. The population of hydrogel particles of claim 2, wherein the
optical property comprises forward scattering.
30. The population of hydrogel particles of claim 29, wherein the
forward scattering is within 10% of a target cell.
31. The population of hydrogel particles of claim 2, wherein the
optical properties are measured using flow cytometry.
32. A population of hydrogel particles, each hydrogel particle
comprising: (i) a polymer comprising polymerized acrylamide and
bis-acrylamide; and (ii) DNA encapsulated within the hydrogel
particle; wherein the population hydrogel particles has an average
diameter in the range of from 5 .mu.m to 100 .mu.m, and no more
than 10% of the hydrogel particles have a diameter exceeding the
average diameter of the hydrogel particles by more than 10%.
33. The population of hydrogel particles of claim 32, having a
refractive index between 1.1 and 2.9.
34. The population of hydrogel particles of claim 33, wherein the
hydrogel particles have an average refractive index of greater than
about 1.7.+
35. The population of hydrogel particles of claim 32, wherein
having a side scattering within 10% of a target cell.
36. The population of hydrogel particles of claim 32, having a
forward scattering within 10% of a target cell.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 17/163,769, filed Feb. 1, 2021, which is a continuation of U.S.
application Ser. No. 16/684,694, filed Nov. 15, 2019, which is a
continuation of U.S. application Ser. No. 15/895,307, filed Feb.
13, 2018, which is a continuation of U.S. application Ser. No.
15/145,856, filed May 4, 2016, now U.S. Pat. No. 9,915,598, which
is a division of U.S. application Ser. No. 13/858,912, filed Apr.
8, 2013, which in turn claims priority to and benefit of
provisional App. No. 61/621,376, filed Apr. 6, 2012 and claims
priority to and benefit of provisional App. No. 61/668,538, filed
Jul. 6, 2012; the contents of each of the aforementioned
applications are incorporated herein in their entireties by
reference thereto.
2. BACKGROUND
[0002] Flow cytometry is a technique that allows for the rapid
separation, counting, and characterization of individual cells and
is routinely used in clinical and laboratory settings for a variety
of applications. The technology relies on directing a beam of light
onto a hydrodynamically-focused stream of liquid. A number of
detectors are then aimed at the point where the stream passes
through the light beam: one in line with the light beam (Forward
Scatter or FSC) and several perpendicular to it (Side Scatter or
SSC). FSC correlates with the cell volume and SSC depends on the
inner complexity of the particle (i.e., shape of the nucleus, the
amount and type of cytoplasmic granules or the membrane roughness).
As a result of these correlations, different specific cell types
exhibit different FSC and SSC, allowing cell types to be
distinguished in flow cytometry.
[0003] The ability to identify specific cell types, however, relies
on proper calibration of the instrument, a process that has relied
on the use of purified cells of the cell type of interest.
Obtaining these purified cells can require costly, laborious
procedures that are prone to batch-to-batch variation. Therefore,
there is a need in the art for synthetic compositions with tunable
optical properties that can mimic specific cell types in devices
such as flow cytometers.
3. SUMMARY
[0004] In one aspect, the present disclosure provides for
compositions comprising a hydrogel particle, wherein the hydrogel
particle has at least one optical property substantially similar to
that of a target cell, wherein the optical property is measured by
a cytometric device.
[0005] In another aspect, the present disclosure provides for
methods of producing a hydrogel particle, wherein the hydrogel
particle has optical properties substantially similar to the
optical properties of a target cell. The present disclosure also
provides for methods of producing a hydrogel particle, wherein the
hydrogel particle has pre-determined optical properties. Also
provided for is a method of calibrating a cytometric device for
analysis of a target cell, the method comprising a) inserting into
the device a hydrogel particle having optical properties
substantially similar to the optical properties of the target cell;
b) measuring the optical properties of the hydrogel particle using
the cytometric device, thereby calibrating the cytometric device
for analysis of the target cell.
4. BRIEF DESCRIPTION OF THE FIGURES
[0006] FIG. 1 illustrates the optical properties of disclosed
hydrogel particles compared to polystyrene beads;
[0007] FIG. 2 depicts the process of producing labeled hydrogel
particles of the disclosure;
[0008] FIG. 3 provides brightfield and fluorescent images of
labeled hydrogel particles of the disclosure; and
[0009] FIG. 4 illustrates the use of hydrogel particles of the
disclosure as calibrants for cell types displaying a variety of
optical scattering properties.
[0010] FIG. 5 provides dating showing correlation of inter-drop
delay for a flow cytometer with hydrogel particle diameter.
[0011] FIG. 6 provides brightfield (6A and 6C) and fluorescent (6B
and 6D) images of Chinese Hamster Ovary cells (6A and 6B) and
hydrogel particles of the disclosure (6C and 6D).
[0012] FIG. 7 provides data showing comparison of human buccal
cells to hydrogel particles encapsulating different amounts of DNA,
as measured by fluorescence-activated cell sorting (FACS),
[0013] FIG. 8 provides data for hydrogel particles encapsulating
nanoparticles at different concentrations, demonstrating tuning of
side scattering independent of forward scattering.
[0014] FIG. 9 provides data for hydrogel particles produced with
different percentages of polymer, demonstrating tuning of
refractive index measured by forward scattering.
5. DETAILED DESCRIPTION
5.1. Definitions
[0015] As used herein throughout the specification and in the
appended claims, the following terms and expressions are intended
to have the following meanings:
[0016] The indefinite articles "a" and "an" and the definite
article "the" are intended to include both the singular and the
plural, unless the context in which they are used clearly indicates
otherwise.
[0017] "At least one" and "one or more" are used interchangeably to
mean that the article may include one or more than one of the
listed elements.
[0018] Unless otherwise indicated, it is to be understood that all
numbers expressing quantities, ratios, and numerical properties of
ingredients, reaction conditions, and so forth, used in the
specification and claims are contemplated to be able to be modified
in all instances by the term "about".
5.2. General Overview
[0019] Several critical calibration measurements for flow
cytometers require precise time resolution, such as setting the
offset time between lasers, and calculating the delay time between
detection and sorting of an object. Due to the fluidic conditions
within the instrument, precise setting of these timing parameters
requires the use of calibration particles that are the same size as
the cells to be analyzed. Timing calibrations are typically
performed using polystyrene beads with variable fluorescent
intensities to calibrate the response of an excitation source and
to set the inter-laser timing delay and sorting delay. Flow
cytometers can also be calibrated using forward and side scatter
signals which are general measures of size and granularity or
complexity of the target sample. These calibrations are crucial for
the accurate performance of the cytometer and for any downstream
analysis or sorting of cell populations. The disclosed hydrogel
particles exhibit tuned scatter properties and are suitable for use
as calibration reagents for a range of mammalian or bacterial cell
types. Scattering is a standard metric for distinguishing cell
types in heterogenous mixtures for clinical, food safety, and
research purposes.
[0020] Although polystyrene particles can be used to set
inter-laser and sorting delays for some applications, many
eukaryotic cell types fall outside of the size range of
commercially available polystyrene particles (1-20 .mu.m) making it
nearly impossible to accurately calibrate a flow cytometer for
these targets. Also, as shown in FIG. 1, polystyrene particles are
fundamentally limited in the optical properties that can possess
such as side scattering, which is a general measure of cellular
complexity. Polystyrene particles are therefore limited in the two
most important passive optical measurements used in flow cytometry:
FSC (forward scattering), and SSC (side scattering) which measure
the size and complexity of the target respectively. Due to these
limitations of polystyrene, users must rely on purified cell lines
to calibrate fluorescent intensity, inter-laser delay, sort delays,
size and cellular complexity for experiments. This is a lengthy and
labor-intensive process that increases the cost of flow cytometry
validation and research pipelines significantly. More importantly,
these calibration cell lines introduce biological variation,
causing disparities in the interpretation of data.
[0021] Accordingly, the present disclosure provides for
compositions comprising a hydrogel particle, wherein the hydrogel
particle has optical properties substantially similar to the
optical properties of a target cell. The inventors have
unexpectedly discovered that optical properties of a hydrogel
particle can be independently modulated by altering the composition
of the hydrogel particle. For example, side scattering (SSC) can be
modulated without substantially affecting forward scattering (FSC),
and vice versa. Furthermore, the optical properties (e.g.
refractive index) of hydrogel particles can be tuned without having
a substantial effect on density of the particle. This is a
surprising and useful feature, as hydrogel particles that serve as
surrogates for cells in cytometric methods such as flow cytometry
or (fluorescence-activated cell sorting) FACS require a minimal
density in order to function in those assays.
[0022] The present disclosure also provides for methods of
producing a hydrogel particle, wherein the hydrogel particle has
optical properties substantially similar to the optical properties
of a target cell. The present disclosure also provides for methods
of producing a hydrogel particle, wherein the hydrogel particle has
pre-determined optical properties. Also provided for is a method of
calibrating a cytometric device for analysis of a target cell, the
method comprising a) inserting into the device a hydrogel particle
having optical properties substantially similar to the optical
properties of the target cell; b) measuring the optical properties
of the hydrogel particle using the cytometric device, thereby
calibrating the cytometric device for analysis of the target cell.
Cytometric devices are known in the art, and include commercially
available devices for performing flow cytometry and FACS.
5.3. Hydrogels
[0023] Hydrogel particles of the disclosure comprise a hydrogel. A
hydrogel is a material comprising a macromolecular
three-dimensional network that allows it to swell when in the
presence of water, to shrink in the absence of (or by reduction of
the amount of) water but not dissolve in water. The swelling, i.e.,
the absorption of water, is a consequence of the presence of
hydrophilic functional groups attached to or dispersed within the
macromolecular network. Crosslinks between adjacent macromolecules
result in the aqueous insolubility of these hydrogels. The
cross-links may be due to chemical (i.e., covalent) or physical
(i.e., Van Der Waal forces, hydrogen-bonding, ionic forces, etc.)
bonds. While some in the polymer industry may refer to the
macromolecular material useful in this invention as a "xerogel" in
the dry state and a "hydrogel" in the hydrated state, for purposes
of this patent application the term "hydrogel" will refer to the
macromolecular material whether dehydrated or hydrated. A
characteristic of a hydrogel that is of particular value is that
the material retains the general shape, whether dehydrated or
hydrated. Thus, if the hydrogel has an approximately spherical
shape in the dehydrated condition, it will be spherical in the
hydrated condition.
[0024] Disclosed hydrogels can comprise greater than about 30%,
greater than about 40%, greater than about 50%, greater than about
55%, greater than about 60%, greater than about 65%, greater than
about 70%, greater than about 75%, greater than about 80%, greater
than about 85%, greater than about 90%, or greater than about 95%
water. Synthetically prepared hydrogels can be prepared by
polymerizing a monomeric material to form a backbone and
cross-linking the backbone with a crosslinking agent. Common
hydrogel monomers include the following: lactic acid, glycolic
acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl
methacrylate, propylene glycol methacrylate, acrylamide,
N-vinylpyrrolidone, methyl methacrylate, glycidyl methacrylate,
glycol methacrylate, ethylene glycol, fumaric acid, and the like.
Common cross linking agents include tetraethylene glycol
dimethacrylate and N,N'-15 methylenebisacrylamide. In some
embodiments, a hydrogel particle of the disclosure is produced by
the polymerization of acrylamide.
[0025] In some embodiments, a hydrogel comprises a mixture of at
least one monofunctional monomer and at least one bifunctional
monomer.
[0026] A monofunctional monomer can be a monofunctional acrylic
monomer. Non-limiting examples of monofunctional acrylic monomers
are acrylamide; methacrylamide; N-alkylacrylamides such as
N-ethylacrylamide, N-isopropylacrylamide or N-tert-butylacrylamide;
N-alkylmethacrylamides such as N-ethylmethacrylamide or
N-isopropylmethacrylamide; N,N-dialkylacrylamides such as
N,N-dimethylacrylamide and N,N-diethyl-acrylamide;
N-[(dialkylamino)alkyl]acrylamides such as
N-[3dimethylamino)propyl]acrylamide or
N-[3-(diethylamino)propyl]acrylamide;
N-[(dialkylamino)alkyl]methacrylamides such as
N-[3-dimethylamino)propyl]methacrylamide or
N-[3-(diethylamino)propyl]methacrylamide; (dialkylamino)alkyl
acrylates such as 2-(dimethylamino)ethyl acrylate,
2-(dimethylamino)propyl acrylate, or 2-(diethylamino)ethyl
acrylates; and (dialkylamino)alkyl methacrylates such as
2-(dimethylamino)ethyl methacrylate.
[0027] A bifunctional monomer is any monomer that can polymerize
with a monofunctional monomer of the disclosure to form a hydrogel
as described herein that further contains a second functional group
that can participate in a second reaction, e.g., conjugation of a
fluorophore.
[0028] In some embodiments, a bifunctional monomer is selected from
the group consisting of: allyl amine, allyl alcohol, allyl
isothiocyanate, allyl chloride, and allyl maleimide.
[0029] A bifunctional monomer can be a bifunctional acrylic
monomer. Non-limiting examples of bifunctional acrylic monomers are
N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide,
N,N'-ethylenebisacrylamide, N,N'-ethylenebis-methacrylamide,
N,N'propylenebisacrylamide and
N,N'-(1,2-dihydroxyethylene)bisacrylamide.
[0030] Higher-order branched chain and linear co-monomers can be
substituted in the polymer mix to adjust the refractive index while
maintaining polymer density, as described in U.S. Pat. No.
6,657,030, incorporated herein by reference in its entirety.
[0031] In some embodiments, a hydrogel comprises a molecule that
modulates the optical properties of the hydrogel. Molecules capable
of altering optical properties of a hydrogel are discussed further
below.
[0032] Naturally occurring hydrogels useful in this invention
include various polysaccharides available from natural sources such
as plants, algae, fungi, yeasts, marine invertebrates and
arthropods. Non-limiting examples include agarose, dextrans,
chitin, cellulose-based compounds, starch, derivatized starch, and
the like. These generally will have repeating glucose units as a
major portion of the polysaccharide backbone.
[0033] Polymerization of a hydrogel can be initiated by a
persulfate. The persulfate can be any water-soluble persulfate.
Non-limiting examples of water soluble persulfates are ammonium
persulfate and alkali metal persulfates. Alkali metals include
lithium, sodium and potassium. In some preferred embodiments, the
persulfate is ammonium persulfate or potassium persulfate, more
preferably, it is ammonium persulfate.
[0034] Polymerization of a hydrogel can be accelerated by an
accelerant. The accelerant can be a tertiary amine. The tertiary
amine can be any water-soluble tertiary amine. Preferably, the
tertiary amine is N,N,N',N'tetramethylethylenediamine or
3-dimethylamino)propionitrile, more preferably it is
N,N,N',N'tetramethylethylenediamine (TEMED).
5.4. Hydrogel Particles
[0035] In one aspect, a hydrogel particle of the disclosure
comprises a hydrogel and is produced by polymerizing a droplet (see
FIG. 2). Microfluidic methods of producing a plurality of droplets,
including fluidic and rigidified droplets, are known, and described
in US Patent Publication No. 2011/0218123 and U.S. Pat. No.
7,294,503, incorporated herein by reference in their entireties.
Such methods provide for a plurality of droplets containing a first
fluid and being substantially surrounded by a second fluid, where
the first fluid and the second fluid are substantially immiscible
(e.g., droplets containing an aqueous-based liquid being
substantially surrounded by an oil based liquid).
[0036] A plurality of fluidic droplets (e.g., prepared using a
microfluidic device) may be polydisperse (e.g., having a range of
different sizes), or in some cases, the fluidic droplets may be
monodisperse or substantially monodisperse, e.g., having a
homogenous distribution of diameters, for instance, such that no
more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or
about 0.01% of the droplets have an average diameter greater than
about 10%, about 5%, about 3%, about 1%, about 0.03%, or about
0.01% of the average diameter. The average diameter of a population
of droplets, as used herein, refers to the arithmetic average of
the diameters of the droplets.
[0037] Accordingly, the disclosure provides population of hydrogel
particles comprising a plurality of hydrogel particles, wherein the
population of hydrogel particles is substantially monodisperse.
[0038] The term microfluidic refers to a device, apparatus or
system including at least one fluid channel having a
cross-sectional dimension of less than 1 mm, and a ratio of length
to largest cross-sectional dimension perpendicular to the channel
of at least about 3:1. A microfluidic device comprising a
microfluidic channel is especially well suited to preparing a
plurality of monodisperse droplets.
[0039] Non-limiting examples of microfluidic systems that may be
used with the present invention are disclosed in U.S. patent
application Ser. No. 11/246,911, filed Oct. 7, 2005, entitled
"Forming and Control of Fluidic Species," published as U.S. Patent
Application Publication No. 2006/0163385 on Jul. 27, 2006; U.S.
patent application Ser. No. 11/024,228, filed Dec. 28, 2004,
entitled "Method and Apparatus for Fluid Dispersion," published as
U.S. Patent Application Publication No. 2005/0172476 on Aug. 11,
2005; U.S. patent application Ser. No. 11/360,845, filed Feb. 23,
2006, entitled "Electronic Control of Fluidic Species," published
as U.S. Patent Application Publication No. 2007/000342 on Jan. 4,
2007; International Patent Application No. PCT/US2006/007772, filed
Mar. 3, 2006, entitled "Method and Apparatus for Forming Multiple
Emulsions," published as WO 2006/096571 on Sep. 14, 2006; U.S.
patent application Ser. No. 11/368,263, filed Mar. 3, 2006,
entitled "Systems and Methods of Forming Particles," published as
U.S. Patent Application Publication No. 2007/0054119 on Mar. 8,
2007; U.S. Provisional Patent Application Ser. No. 60/920,574,
filed Mar. 28, 2007, entitled "Multiple Emulsions and Techniques
for Forming"; and International Patent Application No.
PCT/US2006/001938, filed Jan. 20, 2006, entitled "Systems and
Methods for Forming Fluidic Droplets Encapsulated in Particles Such
as Colloidal Particles," published as WO 2006/078841 on Jul. 27,
2006, each incorporated herein by reference.
[0040] Droplet size is related to microfluidic channel size. The
microfluidic channel may be of any size, for example, having a
largest dimension perpendicular to fluid flow of less than about 5
mm or 2 mm, or less than about 1 mm, or less than about 500 .mu.m,
less than about 200 .mu.m, less than about 100 .mu.m, less than
about 60 .mu.m, less than about 50 .mu.m, less than about 40 .mu.m,
less than about 30 .mu.m, less than about 25 .mu.m, less than about
10 .mu.m, less than about 3 .mu.m, less than about 1 .mu.m, less
than about 300 nm, less than about 100 nm, less than about 30 nm,
or less than about 10 nm.
[0041] Droplet size can be tuned by adjusting the relative flow
rates. In some embodiments, drop diameters are equivalent to the
width of the channel, or within about 10%, 15%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 100% the width of the channel.
[0042] The dimensions of a hydrogel particle of the disclosure are
substantially similar to the droplet from which it was formed.
Therefore, in some embodiments, a hydrogel particle has a diameter
of less than about 1 .mu.m, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450,
500, 600, 800, or less than 1000 .mu.m in diameter. In some
embodiments, a hydrogel particle has a diameter of more than about
1 .mu.m, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or
greater than 1000 .mu.m in diameter. In typical embodiments, a
hydrogel particle has a diameter in the range of 5 .mu.m to 100
.mu.m.
[0043] In some embodiments, a hydrogel particle of the disclosure
is spherical in shape.
[0044] In some embodiments, a hydrogel particle of the disclosure
has material modulus properties (e.g., elasticity) more closely
resembling that of a target cell as compared to a polystyrene bead
of the same diameter.
[0045] In some embodiments, a hydrogel particle of the disclosure
does not comprise agarose.
5.5. Optical Properties
[0046] 5.5.1 Passive Optical Properties
[0047] The three primary modes of deconvolution for flow cytometry
are the two passive optical properties of a particle (forward
scattering, FSC, corresponding to the refractive index, or RI; and
side scattering, SSC) and biomarkers present on the surface of a
given cell type. Therefore, compositions that allow hydrogel
particles of the disclosure to mimic specific cell types with
respect to these three modes are useful for providing synthetic,
robust calibrants for flow cytometry.
[0048] In some embodiments, the refractive index (RI) of a
disclosed hydrogel particle is greater than about 1.10, greater
than about 1.15, greater than about 1.20, greater than about 1.25,
greater than about 1.30, greater than about 1.35, greater than
about 1.40, greater than about 1.45, greater than about 1.50,
greater than about 1.55, greater than about 1.60, greater than
about 1.65, greater than about 1.70, greater than about 1.75,
greater than about 1.80, greater than about 1.85, greater than
about 1.90, greater than about 1.95, greater than about 2.00,
greater than about 2.10, greater than about 2.20, greater than
about 2.30, greater than about 2.40, greater than about 2.50,
greater than about 2.60, greater than about 2.70, greater than
about 2.80, or greater than about 2.90.
[0049] In some embodiments, the refractive index (RI) of a
disclosed hydrogel particle is less than about 1.10, less than
about 1.15, less than about 1.20, less than about 1.25, less than
about 1.30, less than about 1.35, less than about 1.40, less than
about 1.45, less than about 1.50, less than about 1.55, less than
about 1.60, less than about 1.65, less than about 1.70, less than
about 1.75, less than about 1.80, less than about 1.85, less than
about 1.90, less than about 1.95, less than about 2.00, less than
about 2.10, less than about 2.20, less than about 2.30, less than
about 2.40, less than about 2.50, less than about 2.60, less than
about 2.70, less than about 2.80, or less than about 2.90.
[0050] The SSC of a disclosed hydrogel particle is most
meaningfully measured in comparison to that of target cell. In some
embodiments, a disclosed hydrogel particle has an SSC within 30%,
within 25%, within 20%, within 15%, within 10%, within 5%, or
within 1% that of a target cell, as measured by a cytometric
device.
[0051] The FSC of a disclosed hydrogel particle is most
meaningfully measured in comparison to that of target cell. In some
embodiments, a disclosed hydrogel particle has an FSC within 30%,
within 25%, within 20%, within 15%, within 10%, within 5%, or
within 1% that of a target cell, as measured by a cytometric
device.
[0052] SSC can be tuned for a hydrogel by incorporating a
high-refractive index molecule in the hydrogel. Preferred
high-refractive index molecules include colloidal silica, alkyl
acrylate and alkyl methacrylate. Thus in some embodiments, a
hydrogel particle of the disclosure comprises alkyl acrylate and/or
alkyl methacrylate.
[0053] Alkyl acrylates or Alkyl methacrylates can contain 1 to 18,
1 to 8, or 2 to 8, carbon atoms in the alkyl group, such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl,
2-ethylhexyl, heptyl or octyl groups. The alkyl group may be
branched or linear.
[0054] High-refractive index molecules can also include vinylarenes
such as styrene and methylstyrene, optionally substituted on the
aromatic ring with an alkyl group, such as methyl, ethyl or
tert-butyl, or with a halogen, such as chlorostyrene.
[0055] In some embodiments, FSC is modulated by adjusting the
percentage of monomer present in the composition thereby altering
the water content present during hydrogel formation.
[0056] FSC is related to particle volume, and thus can be modulated
by altering particle diameter, as described herein.
[0057] SSC can be engineered by encapsulating nanoparticles within
hydrogels to mimic organelles in a target cell. In some
embodiments, a hydrogel particle of the disclosure comprises one or
more types of nanoparticles selected from the group consisting of:
polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS)
nanoparticles, and silica nanoparticles.
[0058] 5.5.2 Functionalization of Hydrogel Particles
[0059] Hydrogel particles can be functionalized, allowing them to
mimic optical properties of labeled cells. In some embodiments, a
hydrogel particle comprises a bifunctional monomer, and
functionalization of the hydrogel particle occurs via the
bifunctional monomer. In typical embodiments, a functionalized
hydrogel particle comprises a free amine group.
[0060] A hydrogel particle can be functionalized with any
fluorescent dye known in the art, including fluorescent dyes listed
in The MolecularProbes.RTM. Handbook--A Guide to Fluorescent Probes
and Labeling Technologies, incorporated herein by reference in its
entirety. Functionalization can be mediated by a compound
comprising a free amine group, e.g. allylamine, which can be
incorporated into a hydrogel particle during the formation
process.
[0061] Non-limiting examples of known fluorescent dyes include:
6-carboxy-4', 5'-dichloro-2',7'-dimethoxyfluorescein
succinimidylester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein;
6-carboxyfluoresccin; 5-(and-6)-carboxyfluoresccin;
5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether,-alanine-c-
arboxamide, or succinimidyl ester; 5-carboxyfluoresceinsuccinimidyl
ester; 6-carboxyfluorescein succinimidyl ester;
5-(and-6)-carboxyfluorescein succinimidyl ester;
5-(4,6-dichlorotriazinyl) aminofluorescein;
2',7'-difluorofluorescein; eosin-5-isothiocyanate;
erythrosin5-isothiocyanate; 6-(fluorescein-5-carboxamido)hexanoic
acid or succinimidyl ester;
6-(fluorescein-5-(and-6)-carboxamido)hexanoic acid or
succinimidylester; fluorescein-5-EX succinimidyl ester;
fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate;
OregonGreen.RTM. 488 carboxylic acid, or succinimidyl ester; Oregon
Green.RTM. 488isothiocyanate; Oregon Green.RTM. 488-X succinimidyl
ester; Oregon Green.RTM.500 carboxylic acid; Oregon Green.RTM. 500
carboxylic acid, succinimidylester or triethylammonium salt; Oregon
Green.RTM. 514 carboxylic acid; Oregon Green.RTM. 514 carboxylic
acid or succinimidyl ester; RhodamineGreen.TM. carboxylic acid,
succinimidyl ester or hydrochloride; Rhodamine Green.TM. carboxylic
acid, trifluoroacetamide or succinimidylester; Rhodamine
Green.TM.-X succinimidyl ester or hydrochloride; RhodolGreen.TM.
carboxylic acid, N,0-bis-(trifluoroacetyl) or succinimidylester;
bis-(4-carboxypiperidinyl) sulfonerhodamine or
di(succinimidylester); 5-(and-6)carboxynaphtho fluorescein,
5-(and-6)carboxynaphthofluorescein succinimidyl ester;
5-carboxyrhodamine 6G hydrochloride;
6-carboxyrhodamine6Ghydrochloride, 5-carboxyrhodamine 6G
succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester;
5-(and-6)-carboxyrhodamine6G succinimidyl ester;
5-carboxy-2',4',5',7'-tetrabronosulfonefluorescein succinimidyl
ester or bis-(diisopirpylethylammonium) salt;
5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine;
5-(and-6)-carboxytetramethylrhodamine;
5-carboxytetramethylrhodamine succinimidyl ester;
6-carboxytetramethylrhodamine succinimidyl ester;
5-(and-6)-carboxytetramethylrhodamine succinimidyl ester;
6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester;
6-carboxy-Xrhodamine succinimidyl ester;
5-(and-6)-carboxy-Xrhodaminesuccinimidyl ester;
5-carboxy-X-rhodamine triethylammonium salt; Lissamine.TM.
rhodamine B sulfonyl chloride; malachite green; isothiocyanate;
NANOGOLD.RTM. mono(sulfosuccinimidyl ester); QSY.RTM. 21 carboxylic
acid or succinimidyl ester; QSY.RTM. 7 carboxylic acid or
succinimidyl ester; Rhodamine Red.TM.-X succinimidyl ester,
6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid;
succinimidyl ester; tetramethylrhodamine-5-isothiocyanate;
tetramethylrhodamine-6-isothiocyanate;
tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red.RTM.
sulfonyl; Texas Red.RTM. sulfonyl chloride; Texas Red.RTM.-X STP
ester or sodium salt; Texas Red.RTM.-X succinimidyl ester; Texas
Red.RTM.-X succinimidyl ester; and
X-rhodamine-5-(and-6)-isothiocyanate.
[0062] Other examples of fluorescent dyes include BODIPY.RTM. dyes
commercially available from Invitrogen, including, but not limited
to BODIPY.RTM. FL; BODIPY.RTM. TMR STP ester; BODIPY.RTM. TR-X STP
ester; BODIPY.RTM. 630/650-X STPester, BODIPY.RTM. 650/665-X STP
ester;
6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propi-
onic acid succinimidyl ester;
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
acid;
4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-pentanoic
acid succinimidyl ester;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionic
acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionic
acid; sulfosuccinimidyl ester or sodium salt;
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionyl)am-
ino)hexanoic acid;
6-((4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)am-
ino)hexanoic acid or succinimidyl ester;
N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cys-
teic acid, succinimidyl ester or triethylammonium salt;
6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora3a,
4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid;
4,4-difluoro-5,7-diphenyl-4-bora3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid; succinimidyl ester;
6-((4,4-difluoro-5-phenyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)amino)-
hexanoic acid or succinimidyl ester;
4,4-difluoro-5-(4-phenyl-1,3butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-p-
ropionic acid succinimidyl ester;
4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styry-
loxy)acetyl)aminohexanoic acid or succinimidyl ester;
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid; 4,4-difluoro-5-styryl-4-bora-3a,
4a-diaza-s-indacene-3-propionic acid; succinimidyl ester;
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propioni-
c acid;
4,4-difluoro-1,3,5,7-tetramethyl-4bora-3a,4a-diaza-s-indacene-8-pr-
opionic acid succinimidyl ester;
4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diazas-indacene-3-yl)phen-
oxy)acetyl)amino)hexanoic acid or succinimidyl ester; and
6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryl-
oxy)acetyl)aminohexanoic acid or succinimidyl ester.
[0063] Fluorescent dyes can also include for example, Alexa fluor
dyes commercially available from Invitrogen, including but not
limited to Alexa Fluor.RTM. 350 carboxylic acid; Alexa Fluor.RTM.
430 carboxylic acid; Alexa Fluor.RTM. 488 carboxylic acid; Alexa
Fluor.RTM. 532 carboxylic acid; Alexa Fluor.RTM. 546 carboxylic
acid; Alexa Fluor.RTM. 555 carboxylic acid; Alexa Fluor.RTM. 568
carboxylic acid; Alexa Fluor.RTM. 594 carboxylic acid; Alexa
Fluor.RTM. 633 carboxylic acid; Alexa Fluor.RTM. 647 carboxylic
acid; Alexa Fluor.RTM. 660 carboxylic acid; and Alexa Fluor.RTM.
680 carboxylic acid. Fluorescent dyes the present invention can
also be, for example, cyanine dyes commercially available from
Amersham-Pharmacia Biotech, including, but not limited to Cy3 NHS
ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.
5.6. Target Cells
[0064] Hydrogel particles of the disclosure behave similarly to
target cells in procedures such as staining and analysis by flow
cytometry or FACS.
[0065] In some embodiments, a target cell is an immune cell.
Non-limiting examples of immune cells include B lymphocytes, also
called B cells, T lymphocytes, also called T cells, natural killer
(NK) cells, lymphokine-activated killer (LAK) cells, monocytes,
macrophages, neutrophils, granulocytes, mast cells, platelets,
Langerhans cells, stem cells, dendritic cells, peripheral blood
mononuclear cells, tumor infiltrating (TIL) cells, gene modified
immune cells including hybridomas, drug modified immune cells, and
derivatives, precursors or progenitors of any of the cell types
listed herein.
[0066] In some embodiments, a target cell encompasses all cells of
a particular class of cell with shared properties. For example, a
target cell can be a lymphocyte, including NK cells, T cells, and B
cells. A target cell can be an activated lymphocyte.
[0067] In some embodiments, a target cell is a primary cell,
cultured cell, established cell, normal cell, transformed cell,
infected cell, stably transfected cell, transiently transfected
cell, proliferating cell, or terminally differentiated cells.
[0068] In one embodiment, a target cell is a primary neuronal cell.
A variety of neurons can be target cells. As non-limiting examples,
a target cell can be a primary neuron; established neuron;
transformed neuron; stably transfected neuron; or motor or sensory
neuron.
[0069] In other embodiments, a target cell is selected from the
group consisting of: primary lymphocytes, monocytes, and
granulocytes.
[0070] A target cell can be virtually any type of cell, including
prokaryotic and eukaryotic cells.
[0071] Suitable prokaryotic target cells include, but are not
limited to, bacteria such as E. coli, various Bacillus species, and
the extremophile bacteria such as thermophiles.
[0072] Suitable eukaryotic target cells include, but are not
limited to, fungi such as yeast and filamentous fungi, including
species of Saccharomyces, Aspergillus, Trichoderma, and Neurospora;
plant cells including those of corn, sorghum, tobacco, canola,
soybean, cotton, tomato, potato, alfalfa, sunflower, etc.; and
animal cells, including fish, birds and mammals. Suitable fish
cells include, but are not limited to, those from species of
salmon, tout, tilapia, tuna, carp, flounder, halibut, swordfish,
cod and zebrafish. Suitable bird cells include, but are not limited
to, those of chickens, ducks, quail, pheasants and turkeys, and
other jungle foul or game birds. Suitable mammalian cells include,
but are not limited to, cells from horses, cows, buffalo, deer,
sheep, rabbits, rodents such as mice, rats, hamsters and guinea
pigs, goats, pigs, primates, marine mammals including dolphins and
whales, as well as cell lines, such as human cell lines of any
tissue or stem cell type, and stem cells, including pluripotent and
non-pluripotent, and non-human zygotes.
[0073] Suitable cells also include those cell types implicated in a
wide variety of disease conditions, even while in a non-diseased
state. Accordingly, suitable eukaryotic cell types include, but are
not limited to, tumor cells of all types (e.g., melanoma, myeloid
leukemia, carcinomas of the lung, breast, ovaries, colon, kidney,
prostate, pancreas and testes), cardiomyocytes, dendritic cells,
endothelial cells, epithelial cells, lymphocytes (T-cell and B
cell), mast cells, eosinophils, vascular intimal cells,
macrophages, natural killer cells, erythrocytes, hepatocytes,
leukocytes including mononuclear leukocytes, stem cells such as
haemopoetic, neural, skin, lung, kidney, liver and myocyte stem
cells (for use in screening for differentiation and
de-differentiation factors), osteoclasts, chondrocytes and other
connective tissue cells, keratinocytes, melanocytes, liver cells,
kidney cells, and adipocytes. In certain embodiments, the cells are
primary disease state cells, such as primary tumor cells. Suitable
cells also include known research cells, including, but not limited
to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCC cell
line catalog, hereby expressly incorporated by reference.
[0074] In some embodiments, a target cell is a tumor microvesicle
or tumor macrovesicle. Tumor microvesicles, also known as
tumor-secreted microvesicles or tumor-secreted exosomes, can be
found in circulating blood and may have immune-suppressive
activities. Tumor microvesicles typically range in size from 30-200
nm in diameter. Larger tumor microvesicles may be referred to as
tumor macrovesicles, and can range in size from 3-10 .mu.m in
diameter.
6. Examples
Example 1: Generation of Hydrogel Particles
[0075] Photomasks for UV lithography were sourced from CADart
Services Inc. and were designed using AutoCad (AutoDesk, Inc.).
SU-8 photo resist (Microchem, Inc.) was photo crosslinked on 4''
silicon wafers using a collimated UV light source (OAI, Inc.) to
create masters for microfluidic device fabrication. PDMS
(polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed
using standard published methods for soft lithography and
microfluidic device fabrication (See, McDonald J C, et al., 2000,
Electrophoresis 21:27-40).
[0076] Droplets were formed using flow-focusing geometry where two
oil channels focus a central stream of aqueous monomer solution to
break off droplets in a water-in-oil emulsion. A fluorocarbon-oil
(Novec 7500 3M, Inc.) was used as the outer, continuous phase
liquid for droplet formation. To stabilize droplets before
polymerization, a surfactant was added at 0.5% w/w to the oil phase
(ammonium carboxylate salt of Krytox 157 FSH, Dupont). To make the
basic polyacrylamide gel particle, a central phase of an aqueous
monomer solution containing N-acrylamide (1-20% w/v), a
cross-linker (N,N'-bisacrylamide, 0.05-1% w/v), an accelerator, and
ammonium persulfate (1% w/v) was used. An accelerator,
(N,N,N',N'-tetramethylethylenediamine 2% vol %) was added to the
oil-phase in order to trigger hydrogel particle polymerization
after droplet formation.
[0077] Several co-monomers were added to the basic gel formulation
to add functionality. Allyl-amine provided primary amine groups for
secondary labeling after gel formation. We modulated forward
scatter by adjusting the refractive index of the gel by adding
co-monomers allyl acrylate and allyl methacrylate. Side scattering
of the droplets was tuned by adding a colloidal suspension of
silica nanoparticles and/or PMMA (poly(methyl methacrylate))
particles (.about.100 nm) to the central aqueous phase prior to
polymerization.
[0078] Stoichiometric multiplexing of the hydrogel particles was
achieved by utilizing co-monomers containing chemically orthogonal
side groups (amine, carboxyl, maleimide, epoxide, alkyne, etc.) for
secondary labeling.
[0079] Droplets were formed at an average rate of 5 kHz and were
collected in the fluorocarbon oil phase. Polymerization was
completed at 50.degree. C. for 30 minutes, and the resulting
hydrogel particles were washed from the oil into an aqueous
solution.
Example 2: Generation and Visualization of 12 .mu.m Hydrogel
Particles
[0080] Water containing 5% acylamide, 0.25% bisacrylamide, 0.05%
allyl amine, and 0.1% ammonium persulfate was flowed through a
center channel and focused by oil containing 0.1% TEMED through a
10 micron nozzle to produce 10 .mu.m hydrogel particles, shown in
FIG. 3A. Following polymerization, the particles were washed in
water, shown in FIG. 3B, and conjugated to dyes of interest. The
fluorescent hydrogel particles were visualized with fluorescence
microscopy, shown in FIG. 3C.
Example 3: Multidimensional Tuning of Hydrogel Particle Optical
Properties
[0081] As depicted in FIG. 4, hydrogel particles are tuned in
multiple dimensions to match specific cell types unlike polystyrene
beads. Cells are deconvolved using combinations of optical
parameters such as FSC and SSC (FIG. 4A) or secondary markers.
Hydrogel particles are tuned to exactly match the SSC and FSC of
specific cell types unlike polystyrene beads (brown) which are
limited in size (FSC) and side scattering (FIG. 4B). Hydrogel
particles are further functionalized with stoichiometrically tuned
ratios of specific chemical side-groups and secondary labels
allowing any cell type to be precisely matched without suffering
from biological noise as fixed cell lines do (FIG. 4C).
Example 4: Flow Cytometer Delay Time as a Function of Hydrogel
Particle Diameter
[0082] As shown in FIG. 5, the inter-drop delay for a flow
cytometer can be precisely correlated to hydrogel particle
diameter. Data are shown for hydrogel particles of 3, 6, 10, 32,
and 50 .mu.m diameters using flow cytometer nozzle sizes of 70 and
100 .mu.m.
Example 5: Comparison of Hydrogel Particles with Encapsulated DNA
to Cells
[0083] To form hydrogel particles with encapsulated DNA, 40
.mu.g/mL-1000 .mu.g/mL of reconstituted calf thymus DNA was added
to a polymer mix containing 20% 19:1 (acrylamide:bis-acrylamide)
and 0,1% allyl amine in water. 0.4% ammonium persulfate was added
to the mix prior to droplet formation. Hydrogel particles were
formed as described in Example 1. Hydrogel particles with 200
.mu.g/mL of encapsulated calf thymus DNA displayed cell-like
staining using propidium iodide as visualized using a commercial
imaging cytometer and compared to Chinese Hamster Ovary cells
stained using the same procedure. Images were obtained using a
Nexcelom Cellometer.TM. (FIG. 6).
[0084] Cells obtained from a buccal swab were washed in PBS and
stained with propidium iodide. In parallel, populations of hydrogel
particles containing a range of DNA concentrations were also
stained in the same manner. Both the cell and particle suspensions
were analyzed on a flow cytometer (488/590 nm excitation/emission).
Flow cytometry analysis of cheek cells and the same range of
encapsulated DNA particles showed that the particles display a
range of cell-like fluorescent properties (FIG. 7, left panel). The
intensity of staining shows a linear correlation with the median
intensity as measured by flow cytometry (FIG. 7, right panel).
Example 6: Tuning of Hydrogel Particle Side Scattering
[0085] Colloidal silica was added at 12.5%, 6.25%, 3.125% and 0% to
the aqueous fraction of the polymer mix and hydrogel particles were
formed as described in Example 1 Forward and side scattering data
were obtained using a flow cytometer. The results showed that side
scatter signal (FIG. 8, left panel) increased with higher
percentages of encapsulated nanoparticles while forward scatter
(FIG. 8, right panel) remained generally unchanged, demonstrating
the independent tuning of side scatter and forward scatter.
Example 7: Tuning of Hydrogel Particle Forward Scattering
[0086] In this experiment, the percentage of
acrylamide:bis-acrylamide in the hydrogel composition was varied
from between 10 and 40% to tune the refractive index of the
hydrogel particles as measured by forward scattering in a flow
cytometer. As shown in FIG. 9, the forward scattering increased
with increasing percentages of acrylamide:bis-acrylamide as a
fraction of water.
[0087] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
[0088] While various specific embodiments have been illustrated and
described, it will be appreciated that various changes can be made
without departing from the spirit and scope of the invention.
* * * * *