U.S. patent application number 16/947338 was filed with the patent office on 2020-11-12 for acoustically responsive particles.
The applicant listed for this patent is Lu Gao, Leah M Johnson, Gabriel Lopez, Charles Wyatt Shields, IV. Invention is credited to Lu Gao, Leah M Johnson, Gabriel Lopez, Charles Wyatt Shields, IV.
Application Number | 20200355677 16/947338 |
Document ID | / |
Family ID | 1000004990082 |
Filed Date | 2020-11-12 |
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United States Patent
Application |
20200355677 |
Kind Code |
A1 |
Johnson; Leah M ; et
al. |
November 12, 2020 |
Acoustically Responsive Particles
Abstract
Acoustically responsive particles and methods are provided for
their use. Methods are provided for making and using tunable,
monodisperse acoustically responsive particles and negative
contrast acoustic particles, wherein the particles can contain a
functional group available for covalent modification.
Inventors: |
Johnson; Leah M; (Durham,
NC) ; Lopez; Gabriel; (Penasco, NM) ; Gao;
Lu; (Chapel Hill, NC) ; Shields, IV; Charles
Wyatt; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Johnson; Leah M
Lopez; Gabriel
Gao; Lu
Shields, IV; Charles Wyatt |
Durham
Penasco
Chapel Hill
Durham |
NC
NM
NC
NC |
US
US
US
US |
|
|
Family ID: |
1000004990082 |
Appl. No.: |
16/947338 |
Filed: |
July 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15792109 |
Oct 24, 2017 |
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16947338 |
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14388508 |
Sep 26, 2014 |
9797897 |
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PCT/US2013/032706 |
Mar 15, 2013 |
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15792109 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/538 20130101;
G01N 33/54313 20130101; G01N 33/545 20130101; C08F 30/08 20130101;
C08L 83/04 20130101; G01N 33/585 20130101; C08G 77/04 20130101 |
International
Class: |
G01N 33/545 20060101
G01N033/545; G01N 33/538 20060101 G01N033/538; G01N 33/543 20060101
G01N033/543; G01N 33/58 20060101 G01N033/58; C08L 83/04 20060101
C08L083/04; C08F 30/08 20060101 C08F030/08 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with U.S. Government support under
the National Science Foundation grant no. CBET-1050176 and
DMR-1121107. The U.S. Government has certain rights in the
invention.
Claims
1. A method for bulk synthesis of monodisperse, tunable contrast
acoustic particles, the method comprising: agitating in an acidic
aqueous solution, varying ratios of a di-functional, a
tri-functional, and a tetra-functional monomer under conditions
sufficient to allow for hydrolysis; and adding a catalyst and
continuing to agitate the solution under alkaline pH conditions
sufficient to allow for a condensation reaction and formation of
acoustic contrast particles with a narrow size distribution;
monodisperse acoustic contrast particles, wherein the tunable
acoustic contrast of the monodisperse particles formed is based on
the ratios of the di-functional, tri-functional, and
tetra-functional monomers used.
2. The method of claim 1 wherein the di-functional, a tri-
functional, and a tetra-functional monomers form two, three, or
four siloxane bonds, respectively.
3. The method of claim 1 further comprising removing a majority of
the larger non-uniform oligomers from the hydrolyzed monomers.
4. The method of claim 2, wherein the monomer comprises a
conjugative group that is available for covalent modification in
the formed monodisperse particles.
5. The method of claim 4, wherein the conjugative group comprises a
vinyl, carboxylate, hydroxyl, epoxide, sulfhydryl, amide, acrylate,
thiol, or amine.
6. The method of claim 4, wherein the conjugative group comprises a
vinyl group.
7. The method of claim 1, wherein the ratio of the tetra-functional
to the di-functional monomer is between 1:1000 and 1:2.
8. The method of claim 1, wherein the di-functional monomer is a
dimethoxydimethylsilane (DMODMS) or a vinylmethyldimethoxysilane
(VMDMOS).
9. The method of claim 1, wherein the tri-functional monomer is a
trimethoxymethylsilane (TMOMS), a vinyltrimethoxysilane (VTMOS), or
a (3-aminopropyl)trimethoxysilane (AmTMOS).
10. The method of claim 1, wherein the tetra-functional monomer is
a tetramethoxysilane (TMOS, a.k.a. tetramethyl orthosilicate) or a
tetraethoxysilane (TEOS, a.k.a. tetraethyl orthosilicate).
11. The method of claim 1, wherein the catalyst is ammonium
hydroxide or triethylamine.
12. The method of claim 1, wherein the monodisperse particles range
in size from about 0.5 .mu.m to about 5 .mu.m.
13. A monodisperse, acoustic contrast particle formed by the method
of claim 1.
14. A monodisperse, acoustic contrast particle formed by the method
of claim 1, wherein the conjugative group is covalently modified
with a moiety for binding to a target of interest.
15. The monodisperse, acoustic contrast particle of claim 14,
wherein the target of interest comprises a cell, a protein, a
virus, a receptor, an antibody, an antigen, a drug, or a
metabolite.
16. A method for acoustic-mediated bioanalysis comprising: exposing
a fluid sample suspected of containing a target of interest to a
plurality of acoustic contrast particles according to claim 14
under conditions sufficient that the moiety binds to the target;
and subjecting the fluid sample to acoustic radiation pressure from
an acoustic standing wave sufficient within an acoustic focusing
chamber to focus the particles to the acoustic antinodes such that
the target is separated from other components in the sample.
17. The method of claim 3 wherein the monomer comprises a
conjugative group that is available for covalent modification in
the formed monodisperse particles.
18. A monodisperse, acoustic contrast particle formed by the method
of claim 3, wherein the conjugative group is covalently modified
with a moiety for binding to a target of interest.
19. The monodisperse, acoustic contrast particle of claim 18,
wherein the target of interest comprises a cell, a protein, a
virus, a receptor, an antibody, an antigen, a drug, or a
metabolite.
20. A method for acoustic-mediated bioanalysis comprising: exposing
a fluid sample suspected of containing a target of interest to a
plurality of acoustic contrast particles according to claim 18
under conditions sufficient that the moiety binds to the target;
and subjecting the fluid sample to acoustic radiation pressure from
an acoustic standing wave sufficient within an acoustic focusing
chamber to focus the particles to the acoustic antinodes such that
the target is separated from other components in the sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/388,508, filed on Sep. 26, 2014 and titled
"ACOUSTICALLY RESPONSIVE PARTICLES", which is a CFR section 371
national phase application of International Patent Application No.
PCT/US2013/032706, filed on Mar. 15, 2013 and titled "ACOUSTICALLY
RESPONSIVE PARTICLES", which claims the benefit of U.S. Provisional
Patent Application No. 61/615,524, filed Mar. 26, 2012 and titled
"ELASTOMERIC PARTICLES FOR BIO-ANALYSIS AND METHODS OF USE", and
U.S. Provisional Patent Application No. 61/692,011, filed Aug. 22,
2012 and titled "FUNCTIONALIZED MONODISPERSE ACOUSTICALLY
RESPONSIVE COLLOIDS FROM NUCLEATION AND GROWTH", the disclosures of
which are incorporated herein by reference in their entireties.
TECHNICAL FIELD
[0003] The present disclosure relates to acoustically responsive
particles. Particularly, the present disclosure relates to methods
for making and using tunable, monodisperse acoustically responsive
particles and negative contrast acoustic particles, wherein the
particles can contain a functional group available for covalent
modification.
BACKGROUND
[0004] Particles respond to an applied acoustic standing wave by
transporting to specific locations along the wave (i.e., pressure
node, pressure antinode). This relocation is dictated by the
contrast factor (i.e., positive contrast, negative contrast) which
originates from differences in density and elasticity between the
particle and the surrounding media. For example, particles with
positive contrast (e.g., incompressible polystyrene beads, cells)
in aqueous media are generally transported to acoustic pressure
nodes. On the other hand, compressible, silicone elastomeric
particles (NACPs) have a negative contrast property that is
opposite to commonly used particles (e.g., polystyrene
beads).sup.1-2. Consequently, NACPs move to acoustic pressure
anti-nodes when subjected to acoustic standing waves, which is a
direction opposite from common, incompressible particles.
[0005] The capacity to relocate incompressible particles such as
cells to pressure nodes has been used in various approaches for
focusing and separation of mammalian cells..sup.3-9 For example,
the recently commercialized ATTUNE flow cytometer (LIFE
TECHNOLOGIES) substitutes traditional hydrodynamic focusing with
ultrasonic standing wave fields to focus cells into a single
flowing stream prior to laser interrogation..sup.3 To increase the
high-throughput capacity of flow cytometry, Piyasena et al.
recently developed multi-node acoustic focusing and demonstrated up
to 37 parallel flow streams..sup.4 Peterson et al. exploited the
inherent contrast factor of constituents from whole blood to
separate and sort positive contrast erythrocytes from negative
contrast lipids within an acoustofluidic device..sup.5,6
[0006] One current drawback of using negative acoustic contrast
elastomeric particles to relocate incompressible particles such as
cells to pressure nodes is that the elastomeric particles are not
amenable to covalent modification with a specifically desired
molecular recognition molecule. For example, PCT Patent Application
Publication WO 2010/132474A2 discloses `Stable Elastomeric Negative
Acoustic Contrast Particles and Their Use in Acoustic Radiation
Fields`, but does not teach preparation of stable, elastomeric
particles using starting materials with functional groups available
for covalent modification with biological moieties. For instance,
WO 2010/132474A2 only describes the use of inert silicone starting
material (i.e., polydimethyl siloxane (PDMS)) without available
groups for biofunctionalization in which to synthesize the
elastomeric particles. Recently, Cushing et al. reported using
protein adsorption as a way of modifying the surface of such
negative acoustic contrast PDMS particles for biomolecule
quantification assays..sup.10 While protein adsorption may be
convenient, such adsorption techniques often generate heterogeneous
surfaces resulting from random orientation and denaturation of
proteins on the surface..sup.11 These considerations become more
important in cell sorting applications that require high
concentrations of active, surface-presenting bioaffinity groups for
capturing rare cells and cells with a low quantity of targeted
surface antigens.
[0007] In addition to the use of negative acoustic contrast
elastomeric particles for bioseparations in acoustofluidic devices,
negative and positive acoustic contrast particles have utility in
many industrial fields such as those fields involving the
production of paints, foods, inks, coatings, films, cosmetics, and
rheological fluids. Using bulk synthetic approaches to synthesize
monodisperse colloids with useful biochemical and mechanical
properties represents a longstanding goal in synthetic chemistry,
chemical engineering, bioengineering, and mechanical engineering.
Rapid and scalable synthesis of vast quantities of monodisperse
colloids appeals to many industrial fields involving the production
of paints, foods, inks, coatings, films, cosmetics, and rheological
fluids..sup.20 Monodisperse colloids also garner significant
importance in scientific communities with examples in the
production of slurries, clays, minerals, aerosols, foams,
macromolecules, sols, semiconductor nanocrystallites, silica
colloids, and biochemical interfaces with proteins, viruses,
bacteria, and cells..sup.20
[0008] As described above, utilization of acoustic contrast
colloids in biological applications, such as diagnostic screenings
or immunological bio-marker assays, would require the presence of
ample functional groups for various binding and bio-conjugation
reactions. The ability to rapidly synthesize functional,
monodisperse colloids with controlled mechanical properties (i.e.,
specific bulk modulus and density) is desirable as it would allow
for tight responsive control in acoustic fields. Particles designed
with high bulk moduli and densities exhibit positive acoustic
contrast coefficients, indicating transport to the acoustic
pressure nodes of standing waves..sup.5 Conversely, particles
designed with low bulk moduli and densities exhibit negative
acoustic contrast coefficients, indicating transport to the
acoustic pressure antinodes of standing waves..sup.5 Predicate
models for colloid synthesis have failed to fabricate tightly
monodisperse colloids with a tunable acoustic response (i.e.,
exhibiting either positive or negative acoustic contrast by
altering the mechanism of synthesis) via bulk synthetic
methods.
[0009] Accordingly, there remains an unmet need for acoustic
contrast particles with functional groups that would allow for a
range of binding and bio-conjugation reactions. In addition, there
remains an unmet need for monodisperse acoustic contrast colloids
that can be produced via bulk synthetic methods, and also for such
monodisperse particles that can be produced with covalently
modifiable functional groups that can be produced via bulk
synthetic methods. The presently disclosed subject matter provides
such particles.
SUMMARY
[0010] Certain aspects of the presently disclosed subject matter
having been stated hereinabove, which are addressed in whole or in
part by the presently disclosed subject matter, other aspects will
become evident as the description proceeds when taken in connection
with the accompanying Examples and Figures as best described herein
below.
In accordance with an aspect provided herein, a method for
synthesizing elastomeric negative contrast acoustic particles
having a functional group available for covalent modification is
provided. The method includes emulsifying an elastomer pre-polymer
including a functional group with a catalyst in the presence of a
surfactant under conditions sufficient to produce emulsion
droplets, and curing the emulsion droplets under conditions
sufficient to form stable elastomeric negative acoustic contrast
particles that have a functional group available for covalent
modification.
[0011] In accordance with an aspect provided herein, the method
includes covalently modifying the available functional group with a
moiety for binding to a target of interest.
[0012] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, an
antibody, an antigen, a drug, virus, nucleic acid, a polysaccharide
or a metabolite.
[0013] In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of elastomeric negative contrast acoustic
particles disclosed herein. The functional group of the particles
includes a covalently attached moiety for binding to the target of
interest, under conditions sufficient that the moiety binds to the
target. The method includes subjecting the fluid sample to acoustic
radiation pressure from an acoustic standing wave sufficient within
an acoustic focusing chamber to focus the particles to the acoustic
pressure antinodes such that the target is separated from other
components in the sample.
[0014] In accordance with an aspect provided herein, the method
includes removing any positive acoustic contrast particles from the
acoustic focusing chamber.
[0015] In accordance with an aspect provided herein, the method
includes removing the negative contrast acoustic particles from the
acoustic focusing chamber and analyzing the particles.
[0016] In accordance with an aspect provided herein, a method for
synthesizing elastomeric negative contrast acoustic particles
having a covalently functionalized surfactant for recognition of a
target of interest is provided. The method includes emulsifying an
elastomer pre-polymer with a catalyst in the presence of a
surfactant under conditions sufficient to produce emulsion
droplets, wherein the surfactant is covalently functionalized to
allow for binding to a target of interest, and curing the emulsion
droplets under conditions sufficient to form stable elastomeric
negative acoustic contrast particles having the functionalized
surfactant available for binding to the target of interest.
[0017] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, an
antibody, an antigen, a drug, polysaccharide, or a metabolite.
[0018] In accordance with an aspect provided herein, the
elastomeric negative contrast acoustic particle is functionalized
such that binds to the target of interest.
[0019] In accordance with an aspect provided herein, an elastomeric
negative contrast acoustic particle made according to any of the
methods provided herein is disclosed.
[0020] In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of elastomeric negative contrast acoustic
particles under conditions sufficient that the functionalized
surfactant binds to the target of interest, and subjecting the
fluid sample to acoustic radiation pressure from an acoustic
standing wave sufficient within an acoustic focusing chamber to
focus the particles to the acoustic pressure antinodes such that
the target is separated from other components in the sample.
[0021] In accordance with an aspect provided herein, a method for
bulk synthesis of monodisperse, tunable contrast acoustic particles
is provided. The method includes agitating varying ratios of one of
a di-functional, a tri-functional, and a tetra-functional siloxane
monomer in an aqueous solution such that hydrolysis and uniform
condensation occur upon addition of a catalyst and monodisperse
acoustic contrast particles are formed. The tunable acoustic
contrast of the monodisperse particles formed is based on the
ratios of the di-functional, tri-functional, and tetra-functional
siloxane monomers used.
In accordance with an aspect provided herein, the siloxane monomer
includes a conjugative group such that the group is available for
covalent modification in the formed monodisperse particles.
[0022] In accordance with an aspect provided herein, a
monodisperse, acoustic contrast particle made according to one or
more methods disclosed herein is provided.
[0023] In accordance with an aspect provided herein, a
monodisperse, acoustic contrast particle has a conjugative group
that is covalently modified with a moiety for binding to a target
of interest.
[0024] In accordance with an aspect provided herein, the target of
interest of the particle includes one of a cell, a protein, a
receptor, an antibody, a virus, an antigen, a drug, and a
metabolite.
[0025] In accordance with an aspect provided herein, a method for
bulk synthesis of monodisperse, tunable contrast acoustic particles
is provided. The method includes agitating in an acidic aqueous
solution varying ratios of one of a di-functional, a
tri-functional, and a tetra-functional siloxane monomer under
conditions sufficient to allow for hydrolysis and the formation of
oligomers, and adding a catalyst and continuing to agitate the
solution under alkaline pH conditions sufficient to allow for a
condensation reaction and formation of monodisperse acoustic
contrast particles. The tunable acoustic contrast of the
monodisperse particles formed is based on the ratios of the
di-functional, tri-functional, and tetra-functional siloxane
monomers used.
[0026] In accordance with an aspect provided herein, the siloxane
monomer includes a conjugative group such that the group is
available for covalent modification in the formed monodisperse
particles.
[0027] In accordance with an aspect provided herein, a
monodisperse, acoustic contrast particle made according to the one
or more methods disclosed herein is provided.
[0028] In accordance with an aspect provided herein, the
conjugative group is covalently modified with a moiety for binding
to a target of interest.
[0029] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a virus, a receptor, an
antibody, an antigen, a drug, and a metabolite.
[0030] In accordance with an aspect provided herein, a method for
synthesizing monodisperse, tunable contrast acoustic particles is
provided. The method includes agitating in an acidic aqueous
solution varying ratios of one of a di-functional, a
tri-functional, and a tetra-functional siloxane monomer under
conditions sufficient to allow for hydrolysis and the formation of
oligomers. The method may include removing a majority of the large
non-uniform oligomers from the smaller hydrolyzed oligomers and
adding a catalyst and continuing to agitate the solution under
conditions sufficient to allow for a uniform condensation reaction
and formation of monodisperse acoustic contrast particles. The
tunable acoustic contrast of the monodisperse particles formed is
based on the ratios of the di-functional, tri-functional, and
tetra-functional siloxane monomers used.
[0031] In accordance with an aspect provided herein, the siloxane
monomer includes a conjugative group such that the group is
available for covalent modification in the formed monodisperse
particles. In accordance with an aspect provided herein, the
conjugative group is covalently modified with a moiety for binding
to a target of interest.
[0032] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, a virus, an
antibody, an antigen, a drug, a polysaccharide or a metabolite.
[0033] In accordance with an aspect provided herein, a method for
synthesizing monodisperse, tunable contrast acoustic particles is
provided. The method includes agitating in an acidic aqueous
solution varying ratios of one of a tri-functional and a
tetra-functional siloxane monomer under conditions sufficient to
allow for hydrolysis and the formation of oligomers, agitating in a
separate acidic aqueous solution one or more of a di-functional
siloxane monomer and under conditions sufficient to allow for
hydrolysis and the formation of oligomers, removing from the
di-functional solution a majority of the large non-uniform
oligomers from the smaller hydrolyzed oligomers, adding a catalyst
for uniform condensation, and continuing to agitate the solution,
and removing from the tri- and tetra-functional solution a majority
of the large non-uniform oligomers from the smaller hydrolyzed
oligomers, adding the tri-and tetra-functional solution directly to
the di-functional solution, adding a catalyst and continuing to
agitate the solution under conditions sufficient to allow for a
condensation reaction and formation of monodisperse acoustic
contrast particles. The tunable acoustic contrast of the
monodisperse particles formed is based on the ratios of the
di-functional, tri-functional, and tetra-functional siloxane
monomers used.
[0034] In accordance with an aspect provided herein, the siloxane
monomer includes a conjugative group such that the group is
available for covalent modification in the formed monodisperse
particles. In accordance with an aspect provided herein, the
conjugative group is covalently modified with a moiety for binding
to a target of interest.
[0035] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, a virus, an
antibody, an antigen, a drug, a polysaccharide or a metabolite.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The foregoing summary, as well as the following detailed
description of various embodiments, is better understood when read
in conjunction with the appended figures. For the purposes of
illustration, there is shown in the Figures exemplary embodiments;
however, the presently disclosed subject matter is not limited to
the specific methods and exemplary embodiments disclosed.
[0037] FIGS. 1A-1B are schematic drawings showing a system for
acoustic cell sorting using either the negative acoustic contrast
particles (NACPs) or FMAR (functional monodisperse and acoustically
responsive) negative contrast colloids according to one or more
embodiments of the present disclosure. A) Rapid and continuous cell
sorting in acoustic field. Unlabeled "residual" cells focus in the
acoustic node within the microfluidic channel via forces from an
acoustic standing wave. Cells captured by the negative contrast
particles of the present disclosure displace to the acoustic
antinodes whereas unbound cells focus in the acoustic node. B)
Channel Cross Section. Note: components are not to scale and the
glass lid of the acoustofluidic device is not shown.
[0038] FIG. 2 is a graph showing relative colloid size of particles
made using Protocol I according to one or more embodiments of the
present disclosure. The FMAR particles were synthesized with
reproducible and tunable size ranges that extend beyond 100 to 800
nm measured by dynamic light scattering (DLS). The ratios shown on
the x axis represent the ratio of tri-functional monomers
(vinyltrimethoxysilane (VTMOS)) to di-functional monomers
(vinylmethyldimethoxysilane (VMDMOS)) used in the preparation of
the colloids represented by each bar, providing ample vinyl groups
on the surface of synthesized particles for facile conjugation
reactions.
[0039] FIG. 3 is a graph showing size distribution of FMAR colloids
made using Protocol I with different mixing conditions according to
one or more embodiments of the present disclosure. Particles were
made using the siloxane monomer trimethoxymethylsilane (TMOMS).
Particle diameter (nm) is shown on the x axis and percent
population by count is shown on the y axis. Average size measured
by a qNANO instrument was 496.2 nm and the coefficient of variance
was 12.65%.
[0040] FIGS. 4A & 4B are images of particles made using
Protocol I according to one or more embodiments of the present
disclosure. Particles were made using the siloxane monomer
vinyltrimethoxysilane (VTMOS). A) Scanning electron micrograph
(SEM) image. B) Optical microscope image.
[0041] FIGS. 5A & 5B are optical micrographs of FMAR colloids
made using the siloxane monomer trimethoxymethylsilane (TMOMS) as
described in Protocol II. A) Particles are shown with microscope
settings at a low (dark) plane of focusing. B) Particles are shown
with microscope settings at a high (bright) plane of focusing. Both
images show monodisperse colloids that do not aggregate without a
surfactant.
[0042] FIG. 6 is a graph of Zeta Potential of particles described
in FIG. 5A-5B made via Protocol II using all tri-functional species
trimethoxymethylsilane (TMOMS). The surface charge indicates that
the colloids are sufficiently stable without the use of protective
surfactants.
[0043] FIGS. 7A-7F are fluorescent images of FMAR colloids (1:100
monomer ratio of Tetramethoxysilane (TMOS): Dimethoxydimethylsilane
(DMODMS) siloxane monomers) made via Protocol II with negative
acoustic contrast in a silicon acoustofluidic chip as a
demonstration that the FMAR colloids are sufficient to displace
positive acoustic contrast particles from the acoustic node to the
acoustic antinodes according to one or more embodiments of the
present disclosure. A) Streptavidin-conjugated ALEXA FLUOR 488
incubated FMAR colloids as a positive control (PZT power=0V,
flow=15 .mu.L/min). B) The same particles as in (A) (PZT power=15V,
flow=15 .mu.L/min).
C) Pink fluorescent biotin-coated polystyrene beads (PZT power=0V,
flow=100 .mu.L/min). D) Same particles as (C) (PZT power=15V,
flow=100 .mu.L/min). E) FMAR colloids bound to polystyrene beads
used as a surrogate test (PZT power=0V, flow=100 .mu.L/min). F)
Same particles as (E) (PZT power=10V, flow=100 .mu.L/min).
[0044] FIGS. 8A-8D are SEM images of colloids synthesized using
Protocol III at various magnifications according to one or more
embodiments of the present disclosure. FMAR colloids were
synthesized via rapid bulk synthesis using the siloxane monomer
trimethoxymethylsilane (TMOMS). A) Particles are shown at a
magnification of 5000.times.. B) Particles are shown are shown at a
magnification of 15000.times.. C) Particles are shown at a
magnification of 15000.times. at a different site than shown in
(B). D) Particles are shown at a magnification of 2500.times..
[0045] FIGS. 9A-9B show positive acoustic contrast FMAR colloids
synthesized using Protocol III as described in FIG. 8 in a silicon
acoustofluidic chip as a demonstration of the acoustic tunability
of the synthesis mechanisms according to one or more embodiments of
the present disclosure. A) The acoustic field is turned off. B)
FMAR colloids responding to the acoustic field, focusing in the
center of the channel (acoustic node) indicating FMAR colloids can
be easily designed to exhibit positive acoustic contrast.
[0046] FIG. 10 is an optical micrograph of FMAR colloids
synthesized using Protocol III as described in FIG. 8.
[0047] FIGS. 11A-11B are graphs showing size distribution of FMAR
colloids synthesized using Protocol III as described in FIG. 8
characterized by a qNANO device according to one or more
embodiments of the present disclosure. Particle diameter is shown
on the x axis and percent population by count is shown on they
axis. The coefficient of variance was A) 12.39% and B) 14.01%.
[0048] FIG. 12 is an optical micrograph of colloids synthesized
using Protocol IV according to one or more embodiments of the
present disclosure. The particle cores were made using
dimethoxydimethylsilane (DMODMS) siloxane monomers and the particle
shells were made using trimethoxymethylsilane (TMOMS).
[0049] FIGS. 13A-13B are an image of a silicon acoustofluidic
channel containing FMAR colloids synthesized from Protocol IV as
described in FIG. 12 according to one or more embodiments of the
present disclosure. A) (PZT Off), Flow=100 uL/min. B) (PZT On,
V=10), Flow=turned off for 60 sec.
[0050] FIGS. 14A-14D are images of FMAR colloids made according to
one or more embodiments of the present disclosure. A) Optical
micrograph of elastomeric FMAR colloids for cell separation made
according to Protocol II using the siloxane monomers
trimethoxymethylsilane (TMOS) and the siloxane monomers
dimethoxydimethylsilane (DMODMS) at a ratio of 1:100, B)
Fluorescence micrograph of the elastomeric FMAR colloids made
according to Protocol II using functional monomers TMOS and DMODMS
at a ratio of 1:100 with adsorbed Nile red. C) SEM image of biotin
polystyrene (SPHEROTECH, INC.) bound to a KG-1 a human myeloblast
cell. D) SEM of highly monodisperse FMAR colloids made according to
Protocol III using only functional TMOMS monomers.
[0051] FIGS. 15A-15H are images of KG-1 a cell binding and
separation according to one or more embodiments of the present
disclosure. A-C) Optical micrographs of streptavidin adsorbed
elastomeric FMAR colloids (1:100 TMOS:DMODMS as described in FIG.
14A&B) binding to Calcein AM dyed KG-1a cells. D-F)
Fluorescence microscopy images of Calcein AM dyed KG-1a cells
illuminating the bound non-fluorescent FMAR colloids, demonstrating
binding (same frames as FIG. 3A-C). G) Unbound KG-1 a cells
focusing in the acoustic node of a silicon acoustofluidic channel,
H) KG-1 a cells bound to the elastomeric FMAR colloids focusing to
the acoustic antinode of the silicon acoustofluidic channel. Note:
The scale bar in A is also for B-F and the scale bar in H is also
for G.
[0052] FIGS. 16A-16D are images of the use of FMAR colloids in
silicon acoustofluidic channels to effect acoustophoretic cell
displacement according to one or more embodiments of the present
disclosure. KG-1a cells spontaneously migrate to the pressure
antinode in the presence of an acoustic standing wave when bound to
FMAR colloids as described in FIGS. 14A & 14B. A) Shows a
representative random distribution of fluorescent cells in an
acoustofluidic device without a standing wave. B-D) Show cells
responding to the primary radiation force (time step is
approximately 1 sec).
[0053] FIGS. 17A-17B are schematic diagrams showing the chemical
structure of A) a triblock co-polymer surfactant and B) the
surfactant associated with a NACP according to one or more
embodiments of the present disclosure.
[0054] FIGS. 18A-18B show brightfield (A) and fluorescent
microscope (B) images of binding between surfactant biotin
functionalized PDMS NACPs (large circles) and
streptavidin-functionalized polystyrene beads (smaller circles)
according to one or more embodiments of the present disclosure.
[0055] FIGS. 19A-19D show acoustic response of PVMS NACPs having a
biotinylated group covalently attached through the vinyl group of
the PVMS according to one or more embodiments of the present
disclosure. Brightfield image A) and corresponding fluorescence
image B) of PVMS microparticles (NACPs) functionalized with
biotin-PEG-TFPA and subsequently labelled with a fluorescent
streptavidin. The fluorescent image was acquired during a 250 ms
exposure. The scale bars represent 50 .mu.m. C, D) Fluorescence
images show a mixture of fluorescent streptavidin-functionalized
NACPs (large diffuse circles, "PVMS NACPs") and non-biotinylated
polystyrene beads (small bright circles, "polystyrene PACPs")
within a channel of an acoustofluidic C) without and D) with
activation of the PZT. Mixture contained a polystyrene: NACP ratio
of 1:7. Upon generation of an ultrasound standing wave within the
microchannel D), the incompressible polystyrene PACPs transport to
the center of channel, corresponding to the pressure node, whereas
compressible PVMS NACPs transport to the channel sidewalls,
corresponding to the pressure antinodes. Images acquired in the
absence of flow. Dashed lines are included to demarcate the channel
boundaries.
[0056] FIGS. 20A-20B are fluorescence images demonstrating the
ability to use surfactant functionalized NACPs to transport
positive acoustic contrast particles (PACPs) to the acoustic
antinode within an acoustofluidic device according to one or more
embodiments of the present disclosure. The PDMS NACPs
functionalized with biotin-PLURONIC F108 transported positive
acoustic contrast particles (PACPs) having a streptavidin label to
the acoustic antinode. A) As a negative control, non-biotinylated
PDMS NACPs encapsulated with Nile Red fluorophore (large circles)
were mixed with streptavidin polystyrene microparticles PACPs
(smaller and brighter circles). The lack of binding between the
non-biotinylated PDMS and streptavidin polystyrene particles
results in their transport to the antinode and node, respectively,
in the presence of an ultrasound standing wave. B) The high
affinity between the biotinylated PDMS NACPs and the streptavidin
functionalized polystyrene PACPs generate particle complexes that
transport collectively to the acoustic antinode within the
ultrasound standing wave. Images acquired in the absence of flow
with a 1:10 ratio of PACPs:NACPs. Dashed lines are included to
demarcate the channel boundaries. Scale bars represent 200
.mu.m.
DETAILED DESCRIPTION
[0057] The presently disclosed subject matter now will be described
more fully hereinafter with reference to the accompanying Figures,
in which some, but not all embodiments of the presently disclosed
subject matter are shown. The presently disclosed subject matter
may be embodied in many different forms and should not be construed
as limited to the embodiments set forth herein; rather, these
embodiments are provided so that this disclosure will satisfy
applicable legal requirements. Indeed, many modifications and other
embodiments of the presently disclosed subject matter set forth
herein will come to mind to one skilled in the art to which the
presently disclosed subject matter pertains having the benefit of
the teachings presented in the foregoing descriptions and the
associated Figures. Therefore, it is to be understood that the
presently disclosed subject matter is not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims.
[0058] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a subject" includes a plurality of subjects, unless the context
clearly is to the contrary (e.g., a plurality of subjects), and so
forth.
[0059] Throughout this specification and the claims, the terms
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
Likewise, the term "include" and its grammatical variants are
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that can be
substituted or added to the listed items.
[0060] For the purposes of this specification and appended claims,
the term "about" when used in connection with one or more numbers
or numerical ranges, should be understood to refer to all such
numbers, including all numbers in a range and modifies that range
by extending the boundaries above and below the numerical values
set forth. The recitation of numerical ranges by endpoints includes
all numbers, e.g., whole integers, including fractions thereof,
subsumed within that range (for example, the recitation of 1 to 5
includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g.,
1.5, 2.25, 3.75, 4.1, and the like) and any range within that
range.
[0061] The terms "particles" and "colloids" are herein used
interchangeably for the purposes of the specification and
claims.
[0062] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs.
[0063] In one aspect, the present disclosure provides negative and
positive acoustic contrast particles (NACPs) and (PACPs),
respectively, that allow for covalent modification of the particles
such that the particles can be modified with specific biomolecular
recognition molecules. In another aspect, the present disclosure
provides methods for bulk synthesis of monodisperse, tunable
contrast acoustic particles, where the tunable acoustic contrast of
the monodisperse particles formed is based on the ratios of the
di-functional, tri-functional, and tetra-functional siloxane
monomers used. The monodisperse, tunable acoustic contrast
particles are synthesized using a nucleation and growth method and
these particles can also be functionalized to allow for specific
covalent modification. Thus, the monodisperse and acoustically
responsive particles synthesized using a nucleation and growth
method may be referred to herein as monodisperse and acoustically
responsive (FMARs). The acoustic contrast particles of the present
disclosure will now be described in further detail. An important
goal is to be able to employ negative acoustic contrast particles
(NACPs) for bioanalytical techniques that require
biofunctionalization of the particle surface for binding to
specific biomolecules or cells. Common bioconjugation schemes, such
as carboiimide chemical approaches, are not feasible with NACPs
synthesized using the common silicone material (e.g., PDMS),
because the resulting PDMS NACPs lack the necessary functional
groups such as carboxylates, hydroxyls, epoxies, and amines to
introduce functionality. To address this problem, in one example,
PVMS pre-polymers were used to generate NACPs with surface vinyl
groups that would be useful for reactions such thiolene. In this
manner, a variety of chemical reactions can be employed to
functionalize the vinyl containing NACPs. In another example, NACPs
were synthesized in the presence of a covalently functionalized
surfactant to allow for binding to a target of interest.
Thus, in one aspect, the particles of the present disclosure
augment existing acoustophoretic particle sorting capabilities and
relocation of PACPs to antinodes of ultrasound standing waves. FIG.
1 illustrates the principle. Central to this bioseparation scheme
is the specific association between the engineered NACPs and
targeted PACPs to create a stable complex capable of in-tandem
transport to the acoustic antinode. This requires precise design of
biofunctional NACPs that exhibit stability and specificity for
targeted PACPs. Thus, in one aspect, the present disclosure
provides the design, preparation, and application of NACPs for
bioanalytical techniques.
[0064] The design, preparation, and utilization of biospecific
NACPs hold great potential for a platform technology. NACPs are
applicable to a large number of biomedical analytical applications
(e.g., rare cell and marker isolation, detection and analysis) as
well as therapeutic applications (continuous separation
applications for removing cellular and large molecular analytes
from complex samples such as blood). Similar to the universality of
magnetic particles, acoustic-responsive NACPs may also be employed
in a variety of applications, but with the added benefit that
nonelastic particles (i.e., components not bound to NACPs) are also
affected by the applied acoustic field, thus enabling highly
sensitive and continuous separations. In one non-limiting example,
NACPs can be employed, in conjunction with acoustic microfluidic
cells, to enrich rare cells from complex cellular admixtures.
[0065] In the present disclosure, silicone NACPs were prepared that
contained stable, covalently bound biofunctional groups on the NACP
surface. Silicone was chosen for the particle material due to its
high compressibility that enables use in acoustic-based
bioanalysis. However, silicone material is notoriously difficult to
functionalize owing to the inertness of the material. Moreover,
silicone particles often irreversibly aggregate, thereby making use
in bioassays quite difficult. The ability to create stable,
disperse NACPs that contain reactive, surface-presenting biological
moieties is the key to enabling acoustic-mediated bioseparation and
bioassays. NACPs require stability during downstream manipulation
steps (e.g., washing steps) without aggregation or loss of
bio-activity. The present disclosure enables said requirements by
teaching 1) NACP preparation from silicone starting materials that
contain functional groups (e.g., vinyl groups) to permit immediate,
covalent bioconjugation of NACP surfaces; and 2) NACP preparation
conditions that avoid particle aggregation. In one example,
polyvinylmethyl siloxane (PVMS) was employed as a starting material
that undergoes alkoxy condensation curing reactions to ultimately
generate NACPs with surface-based vinyl groups.
[0066] The preparation of stable, bio-functional NACPs is key to
permitting acoustic-based bioassays and bioseparation. The choice
of surfactant employed during NACP homogenization preparation
markedly affected NACP stability during downstream manipulation
steps involving centrifugation and biofunctionalization. For
example, surfactants such as cetyl trimethylammonium bromide (CTAB)
and Tween-20 resulted in irreversible aggregation during attempts
to re-suspend NACPs with physiological buffers (PBS, pH=7.3)
post-centrifugation. Alternatively, nonionic triblock copolymer
surfactants with hydrophile-lipophile (HLB) values >24 (e.g.,
PLURONIC F108, FIOS, PLURONIC F68) permitted preparation of stable
NACPs capable of withstanding numerous centrifugation wash cycles
without aggregation. The use of these surfactants to maintain
stable, non-aggregated NACPs in physiological buffers is a critical
finding that enables use of these NACPs in biological
applications.
[0067] The results presented herein demonstrate the ability of
negative acoustic contrast particles (NACPs) to specifically
capture and transport positive acoustic contrast particles (PACPs)
to the antinode of an ultrasound standing wave. Emulsification and
post curing of pre-polymers, either polydimethylsiloxane (PDMS) or
polyvinylmethylsiloxane (PVMS), within aqueous surfactant solution
resulted in the formation of stable NACPs that can focus onto
acoustic antinodes. Both photochemical reactions with
biotin-tetrafluorophenyl azide (biotin-TFPA) and
end-functionalization of PLURONIC F108 surfactant to
biofunctionalize NACPs was demonstrated. These biotinylated NACPs
can bind specifically to streptavidin polystyrene microparticles
(as cell surrogates) and transport them to the acoustic antinode
within an acoustofluidic chip as shown in the results and figures
provided herein.
[0068] In another aspect, the present disclosure provides methods
for bulk synthesis of monodisperse, tunable contrast acoustic
particles, where the tunable acoustic contrast of the monodisperse
particles formed is based on the ratios of the di-functional,
tri-functional, and tetra-functional siloxane monomers used.
Predicate models for colloid synthesis have failed to fabricate
tightly monodisperse colloids with a tunable acoustic response
(i.e., exhibiting either positive or negative acoustic contrast by
altering the mechanism of synthesis) via bulk synthetic methods.
The method provided in the present disclosure "nucleation and
growth" solves this problem by synthesis of particles in a two-step
process that includes 1) uniform hydrolysis of monomeric siloxane
species to form monodisperse nuclei, and 2) uniform growth of
colloids by polycondensation of monomers in solution.
[0069] Further, known methods for synthesis of monodisperse
colloids from bulk synthesis generally result in inert silicones,
which cannot be readily bio-functionalized to covalently interact
with surrounding entities. In the present disclosure, siloxane
monomers bearing at least one functional group are incorporated
during the synthesis process such that the entire microparticle
bares ample reactive sites for various conjugations. The FMAR
colloids of the present disclosure represent a unique class of
particles that exhibit three distinct and useful
characteristics.
(1) FMAR colloids provide ample reactive groups on the colloidal
surface (or within the colloid) for various reactions with
biological moieties (e.g., bio-conjugations) or with synthetic
compounds.
[0070] (2) The FMAR colloids of the present disclosure can meet
specific size requirements as possessing average diameters within a
predefined threshold of variance. Precise definitions of
monodispersity range, though the National Institute of Standards
and Technology (NIST) defines monodispersity as a population of
particles possessing a radial coefficient of variance of 5% or
less. The bulk synthetic approaches of the present disclosure
provide a tight limit to the overall size dispersity, and the
colloids synthesized according to these methods have a
monodispersity defined as having a radial coefficient of variance
of 15% or less.
(3) The FMAR colloids of the present disclosure possess specific
bulk moduli (i.e., compressibility) and densities such as to be
considered acoustically responsive. Acoustically responsive is
defined herein as spatially displacing either to the node or the
antinode of an acoustic pressure standing wave. The FMAR colloids
presented herein can be designed to exhibit either positive or
negative acoustic contrast, allowing the particles to spontaneously
align in the acoustic node and antinode of a standing wave,
respectively. The type of acoustic response in the colloid can be
controlled by varying the degree of crosslinking density. For
example, incorporating large ratios of tri- and tetra-functional
monomeric species (monomers containing three or four siloxane
bonds) into the synthesis of FMAR colloids leads to a high
crosslinking density and particles exhibit more rigid
characteristics, yielding a positive acoustic contrast factor.
Conversely, incorporating large ratios of di- and tri-functional
monomeric species into the synthesis of FMAR can lead to a ow
crosslinking density and particles exhibit compressible
characteristics, yielding a negative acoustic contrast factor.
[0071] The FMAR colloids of the present disclosure exhibit several
key qualities that enable their use in a variety of applications.
The functional groups on the microparticles allow for the stable
covalent conjugation of synthetic materials and biomolecules on or
within the FMAR colloids. Notwithstanding, various functional and
non-functional surfactants can be used to coat the particles to
increase stability and prevent aggregation for a variety of
additional uses. The FMAR colloids of the present disclosure
harness the potential for application in a myriad of industrial
pursuits due to the ability to use a bulk synthesis production
method for production of the monodisperse microparticles. These
industries include, but are not limited to paints, foods, inks,
coatings, films, cosmetics, rheological fluids, slurries, clays,
minerals, aerosols, foams, macromolecules, sols, semiconductor
nanocrystallites, silica colloids, and biochemical interfaces.
[0072] The functionalized monodisperse acoustically responsive
(FMAR) colloids of the present disclosure describe a range of
microparticle characteristics in one uniform colloidal suspension.
The FMAR colloids can be easily synthesized to possess ample
functional groups throughout the polymer construct for facile
bio-conjugations and synthetic reactions in biosensing, screening,
separation, marking, coating, and signaling applications, FMAR
colloids can be synthesized in bulk to exhibit size monodispersity
within a predefined threshold, allowing for direct incorporation to
industrial products including, but not limited to, paints, foods,
cosmetics, aerosols, coatings, and films. The synthesis procedure
for FMAR colloids can allow for the ability to control the
mechanical properties of colloids such that either positive
acoustic contrast or negative acoustic contrast characteristics are
attained, depending on the application of interest.
[0073] The functionalized monodisperse acoustically responsive
(FMAR) colloids of the present disclosure can to bind to specific
analytes (i.e., rigid moieties such as molecules, rigid polystyrene
beads, or cells) and relocate those captured analytes from the
acoustic pressure node to the antinode for collection as shown in
the results and figures provided herein.
[0074] In accordance with an aspect provided herein, a method for
synthesizing elastomeric negative contrast acoustic particles
having a functional group available for covalent modification is
provided. The method includes emulsifying an elastomer pre-polymer
including a functional group with a catalyst in the presence of a
surfactant under conditions sufficient to produce emulsion
droplets, and curing the emulsion droplets under conditions
sufficient to form stable elastomeric negative acoustic contrast
particles that have a functional group available for covalent
modification.
[0075] In accordance with an aspect provided herein, the elastomer
pre-polymer includes a silicone material.
[0076] In accordance with an aspect provided herein, the functional
group includes one of vinyl, carboxylate, hydroxyl, epoxide,
sulfhydryl, amide, acrylate, thiol, azide, maleimide, isocyanate,
aziridine, carbonate, N-hydroxysuccinimide ester, imidoester,
carbodiimide, anhydride, succinimidyl carbonate, and amine, and
combinations thereof.
[0077] In accordance with an aspect provided herein, the elastomer
pre-polymer includes polyvinylmethylsiloxane (PVMS).
In accordance with an aspect provided herein, the surfactant is a
nonionic surfactant including one of PLURONIC F108, PLURONIC F68,
PLURONIC P103, PLURONIC F98, PLURONIC P84, PLURONIC F127, PLURONIC
F88, PLURONIC F77, PLURONIC P84, and FIOS, or an ionic surfactant
including one of CHAPS, betaines, lecithin, phosphates, cetyl
trimethylammonium bromide (CTAB), hexadecyl trimethyl ammonium
bromide, cetyl trimethylammonium chloride (CTAC), cetylpyridinium
chloride (CPC), benzalkonium chloride (BAC), Benzethonium chloride
(BZT), ammonium lauryl sulfate, and sodium lauryl sulfate, and
combinations thereof.
[0078] In accordance with an aspect provided herein, the nonionic
surfactant includes a PLURONIC surfactant having greater than 50
end groups of polyethylene oxide (PEO).
[0079] In accordance with an aspect provided herein, the nonionic
surfactant includes a tribiock copolymer surfactant having a
hydrophile-lipophile (HLB) value greater than 24.
[0080] In accordance with an aspect provided herein, the method
includes covalently modifying the available functional group via a
thiolene reaction, a thermal-initiated reaction, or a
photo-initiated reaction.
[0081] In accordance with an aspect provided herein, the method
includes covalently modifying the available functional group with a
moiety for binding to a target of interest.
[0082] In accordance with an aspect provided herein, the moiety
includes a biotin or a streptavidin for binding to the target of
interest that includes a binding partner for the biotin or the
streptavidin.
[0083] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, an
antibody, an antigen, a drug, virus, nucleic acid, a polysaccharide
or a metabolite.
[0084] In accordance with an aspect provided herein, an elastomeric
negative contrast acoustic particle made according to the one or
more methods disclosed herein is provided. The functional group is
covalently modified with a moiety for binding to a target of
interest.
[0085] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, an
antibody, an antigen, a drug, virus, nucleic acid, a polysaccharide
or a metabolite.
[0086] In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of elastomeric negative contrast acoustic
particles disclosed herein. The functional group of the particles
includes a covalently attached moiety for binding to the target of
interest, under conditions sufficient that the moiety binds to the
target. The method includes subjecting the fluid sample to acoustic
radiation pressure from an acoustic standing wave sufficient within
an acoustic focusing chamber to focus the particles to the acoustic
pressure antinodes such that the target is separated from other
components in the sample.
In accordance with an aspect provided herein, the method includes
removing any positive acoustic contrast particles from the acoustic
focusing chamber.
[0087] In accordance with an aspect provided herein, the method
includes removing the negative contrast acoustic particles from the
acoustic focusing chamber and analyzing the particles.
[0088] In accordance with an aspect provided herein, a method for
synthesizing elastomeric negative contrast acoustic particles
having a covalently functionalized surfactant for recognition of a
target of interest is provided. The method includes emulsifying an
elastomer pre-polymer with a catalyst in the presence of a
surfactant under conditions sufficient to produce emulsion
droplets, wherein the surfactant is covalently functionalized to
allow for binding to a target of interest, and curing the emulsion
droplets under conditions sufficient to form stable elastomeric
negative acoustic contrast particles having the functionalized
surfactant available for binding to the target of interest.
[0089] In accordance with an aspect provided herein, the elastomer
pre-polymer includes a silicone material.
[0090] In accordance with an aspect provided herein, the surfactant
is functionalized with one of a vinyl, carboxylate, hydroxyl,
epoxide, sulfhydryl, amide, acrylate, thiol, azide, maleimide,
isocyanate, aziridine, carbonate, N-hydroxysuccinimide ester,
imidoester, carbodiimide, anhydride, succinimidyl carbonate, and
amine, and combinations thereof.
[0091] In accordance with an aspect provided herein, the elastomer
pre-polymer includes polydimethylsiloxane (PDMS) or
polyvinylmethylsiloxane (PVMS).
[0092] In accordance with an aspect provided herein, the surfactant
is a nonionic surfactant including one of PLURONIC F108, PLURONIC
F68, PLURONIC P103, PLURONIC F98, PLURONIC P84, PLURONIC F127,
PLURONIC F88, PLURONIC F77, PLURONIC P84, and FIOS, and
combinations thereof, or an ionic surfactant including one of
CHAPS, betaines, lecithin, phosphates, cetyl trimethylammonium
bromide (CTAB), hexadecyl trimethyl ammonium bromide, cetyl
trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC),
benzalkonium chloride (BAC), Benzethonium chloride (BZT), ammonium
lauryl sulfate, and sodium lauryl sulfate, and combinations
thereof.
[0093] In accordance with an aspect provided herein, the nonionic
surfactant includes a PLURONIC surfactant having greater than 50
end groups of polyethylene oxide (PEO).
[0094] In accordance with an aspect provided herein, the nonionic
surfactant includes a tribiock copolymer surfactant having a
hydrophile-lipophile (HLB) value greater than 24.
[0095] In accordance with an aspect provided herein, the
functionalized surfactant includes a linking target that binds to
the target of interest.
[0096] In accordance with an aspect provided herein, the linking
target is one of a biotin, a biotinylated tetrafluorophenyl azide,
a streptavidin, a fluorescent streptavidin, nucleic acid, an
antibody, or an antigen.
[0097] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, an
antibody, an antigen, a drug, polysaccharide, or a metabolite.
[0098] In accordance with an aspect provided herein, the
elastomeric negative contrast acoustic particle is functionalized
such that binds to the target of interest.
In accordance with an aspect provided herein, an elastomeric
negative contrast acoustic particle made according to any of the
methods provided herein is disclosed.
[0099] In accordance with an aspect provided herein, the target of
interest of the elastomeric negative contrast acoustic particle
includes one of a cell, a protein, a receptor, an antibody, an
antigen, a drug, a virus, a nucleic acid, a polysaccharide, or a
metabolite.
In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of elastomeric negative contrast acoustic
particles under conditions sufficient that the functionalized
surfactant binds to the target of interest, and subjecting the
fluid sample to acoustic radiation pressure from an acoustic
standing wave sufficient within an acoustic focusing chamber to
focus the particles to the acoustic pressure antinodes such that
the target is separated from other components in the sample.
[0100] In accordance with an aspect provided herein, the method
includes removing any positive acoustic contrast particles from the
acoustic focusing chamber.
[0101] In accordance with an aspect provided herein, the method
includes removing the negative contrast acoustic particles from the
acoustic focusing chamber and analyzing the particles.
[0102] In accordance with an aspect provided herein, a method for
bulk synthesis of monodisperse, tunable contrast acoustic particles
is provided. The method includes agitating varying ratios of one of
a di-functional, a tri-functional, and a tetra-functional siloxane
monomer in an aqueous solution such that hydrolysis and uniform
condensation occur upon addition of a catalyst and monodisperse
acoustic contrast particles are formed. The tunable acoustic
contrast of the monodisperse particles formed is based on the
ratios of the di-functional, tri-functional, and tetra-functional
siloxane monomers used.
[0103] In accordance with an aspect provided herein, the siloxane
monomer includes a conjugative group such that the group is
available for covalent modification in the formed monodisperse
particles. In accordance with an aspect provided herein, the
conjugative group includes a vinyl, carboxylate, hydroxyl, epoxide,
sulfhydryl, amide, acrylate, thiol, or amine.
[0104] In accordance with an aspect provided herein, the
conjugative group includes a vinyl group.
[0105] In accordance with an aspect provided herein, the method
includes a co-solvent for miscibility.
[0106] In accordance with an aspect provided herein, the co-solvent
is ethanol.
[0107] In accordance with an aspect provided herein, the ratio of
the co-solvent to water ranges between about 1:100 and 50:50.
[0108] In accordance with an aspect provided herein, the method
includes washing the particles.
[0109] In accordance with an aspect provided herein, the method
includes heating the solution to about 70.degree. C. such that
produced alcohol groups are boiled out of solution.
[0110] In accordance with an aspect provided herein, the ratio of
the di-functional to the tri-functional siloxane monomer is one of
0:100, about 25:75, about 50:50, or about 75:25.
[0111] In accordance with an aspect provided herein, the
di-functional monomer is a dimethoxydimethylsilane (DMODMS) or a
vinylmethyldimethoxysilane (VMDMOS).
In accordance with an aspect provided herein, the tri-functional
monomer is a trimethoxymethylsilane (TMOMS) or a
vinyltrimethoxysilane (VTMOS).
[0112] In accordance with an aspect provided herein, the catalyst
is ammonium hydroxide.
[0113] In accordance with an aspect provided herein, the
monodisperse particles range in size from about 100 nm to about 800
nm.
In accordance with an aspect provided herein, a monodisperse,
acoustic contrast particle made according to one or more methods
disclosed herein is provided.
[0114] In accordance with an aspect provided herein, a
monodisperse, acoustic contrast particle has a conjugative group
that is covalently modified with a moiety for binding to a target
of interest.
[0115] In accordance with an aspect provided herein, the target of
interest of the particle includes one of a cell, a protein, a
receptor, an antibody, a virus, an antigen, a drug, and a
metabolite.
[0116] In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of acoustic contrast particles according to
claim 48 under conditions sufficient that the moiety binds to the
target, and subjecting the fluid sample to acoustic radiation
pressure from an acoustic standing wave sufficient within an
acoustic focusing chamber to focus the particles to the acoustic
antinodes such that the target is separated from other components
in the sample.
[0117] In accordance with an aspect provided herein, a method for
bulk synthesis of monodisperse, tunable contrast acoustic particles
is provided. The method includes agitating in an acidic aqueous
solution varying ratios of one of a di-functional, a
tri-functional, and a tetra-functional siloxane monomer under
conditions sufficient to allow for hydrolysis and the formation of
oligomers, and adding a catalyst and continuing to agitate the
solution under alkaline pH conditions sufficient to allow for a
condensation reaction and formation of monodisperse acoustic
contrast particles. The tunable acoustic contrast of the
monodisperse particles formed is based on the ratios of the
di-functional, tri-functional, and tetra-functional siloxane
monomers used.
In accordance with an aspect provided herein, the siloxane monomer
includes a conjugative group such that the group is available for
covalent modification in the formed monodisperse particles.
[0118] In accordance with an aspect provided herein, the
conjugative group includes a vinyl, carboxylate, hydroxyl, epoxide,
sulfhydryl, amide, acrylate, thiol, or amine.
[0119] In accordance with an aspect provided herein, the
conjugative group includes a vinyl group.
[0120] In accordance with an aspect provided herein, the method
includes washing the particles.
[0121] In accordance with an aspect provided herein, the method
includes heating the solution to about 150.degree. C. for a length
of time sufficient to harden the particles.
[0122] In accordance with an aspect provided herein, the ratio of
the tetra-functional to the di-functional siloxane monomer is one
of 1:100, about 1:10, about 1:20, or about 1:4.
In accordance with an aspect provided herein, the di-functional
siloxane monomer is a dimethoxydimethylsilane (DMODMS) or a
vinylmethyldimethoxysilane (VMDMOS).
[0123] In accordance with an aspect provided herein, the
tri-functional siloxane monomer is a trimethoxymethylsilane (TMOMS)
or a vinyltrimethoxysilane (VTMOS).
[0124] In accordance with an aspect provided herein, the
tetra-functional siloxane monomer is a trimethoxysilane (TMOS) or a
(3-Aminopropyl)trimethoxysilane (AmTMOS).
[0125] In accordance with an aspect provided herein, the catalyst
is ammonium hydroxide.
[0126] In accordance with an aspect provided herein, the
monodisperse particles range in size from about 0.5 .mu.m to about
5 .mu.m.
[0127] In accordance with an aspect provided herein, a
monodisperse, acoustic contrast particle made according to the one
or more methods disclosed herein is provided.
[0128] In accordance with an aspect provided herein, the
conjugative group is covalently modified with a moiety for binding
to a target of interest.
[0129] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a virus, a receptor, an
antibody, an antigen, a drug, and a metabolite.
In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of acoustic contrast particles according to
claim 65 under conditions sufficient that the moiety binds to the
target and subjecting the fluid sample to acoustic radiation
pressure from an acoustic standing wave sufficient within an
acoustic focusing chamber to focus the particles to the acoustic
antinodes such that the target is separated from other components
in the sample.
[0130] In accordance with an aspect provided herein, a method for
synthesizing monodisperse, tunable contrast acoustic particles is
provided. The method includes agitating in an acidic aqueous
solution varying ratios of one of a di-functional, a
tri-functional, and a tetra-functional siloxane monomer under
conditions sufficient to allow for hydrolysis and the formation of
oligomers. The method may include removing a majority of the large
non-uniform oligomers from the smaller hydrolyzed oligomers and
adding a catalyst and continuing to agitate the solution under
conditions sufficient to allow for a uniform condensation reaction
and formation of monodisperse acoustic contrast particles. The
tunable acoustic contrast of the monodisperse particles formed is
based on the ratios of the di-functional, tri-functional, and
tetra-functional siloxane monomers used.
In accordance with an aspect provided herein, the siloxane monomer
includes a conjugative group such that the group is available for
covalent modification in the formed monodisperse particles.
[0131] In accordance with an aspect provided herein, the
conjugative group includes a vinyl, carboxylate, hydroxyl, epoxide,
sulfhydryl, amide, acrylate, thiol, or amine.
[0132] In accordance with an aspect provided herein, the
conjugative group includes a vinyl group.
[0133] In accordance with an aspect provided herein, the method
includes washing the particles.
[0134] In accordance with an aspect provided herein, the method
includes sonicating the particles.
[0135] In accordance with an aspect provided herein, the particles
are sonicated in ethanol.
[0136] In accordance with an aspect provided herein, the acidic
aqueous solution is less than pH 3.
[0137] In accordance with an aspect provided herein, the method is
performed with only the tetra-functional siloxane monomer.
[0138] In accordance with an aspect provided herein, the
di-functional siloxane monomer is a dimethoxydimethylsilane
(DMODMS) or a vinylmethyldimethoxysilane (VMDMOS).
[0139] In accordance with an aspect provided herein, the
tri-functional siloxane monomer is a trimethoxymethylsilane (TMOMS)
or a vinyltrimethoxysilane (VTMOS).
In accordance with an aspect provided herein, the tetra-functional
siloxane monomer is a trimethoxysilane (TMOS).
[0140] In accordance with an aspect provided herein, the catalyst
is triethylamine.
[0141] In accordance with an aspect provided herein, the
monodisperse particles range in size from about 0.5 .mu.m to about
50 .mu.m.
In accordance with an aspect provided herein, the conjugative group
is covalently modified with a moiety for binding to a target of
interest.
[0142] In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, a virus, an
antibody, an antigen, a drug, a polysaccharide or a metabolite.
[0143] In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of acoustic contrast particles according to
claim 83 under conditions sufficient that the moiety binds to the
target and subjecting the fluid sample to acoustic radiation
pressure sufficient from an acoustic standing wave within an
acoustic focusing chamber to focus the particles to the acoustic
antinodes such that the target is separated from other components
in the sample.
[0144] In accordance with an aspect provided herein, a method for
synthesizing monodisperse, tunable contrast acoustic particles is
provided. The method includes agitating in an acidic aqueous
solution varying ratios of one of a tri-functional and a
tetra-functional siloxane monomer under conditions sufficient to
allow for hydrolysis and the formation of oligomers, agitating in a
separate acidic aqueous solution one or more of a di-functional
siloxane monomer and under conditions sufficient to allow for
hydrolysis and the formation of oligomers, removing from the
di-functional solution a majority of the large non-uniform
oligomers from the smaller hydrolyzed oligomers, adding a catalyst
for uniform condensation, and continuing to agitate the solution,
and removing from the tri- and tetra-functional solution a majority
of the large non-uniform oligomers from the smaller hydrolyzed
oligomers, adding the tri-and tetra-functional solution directly to
the di-functional solution, adding a catalyst and continuing to
agitate the solution under conditions sufficient to allow for a
condensation reaction and formation of monodisperse acoustic
contrast particles. The tunable acoustic contrast of the
monodisperse particles formed is based on the ratios of the
di-functional, tri-functional, and tetra-functional siloxane
monomers used.
[0145] In accordance with an aspect provided herein, the siloxane
monomer includes a conjugative group such that the group is
available for covalent modification in the formed monodisperse
particles.
[0146] In accordance with an aspect provided herein, the
conjugative group includes a vinyl, carboxylate, hydroxyl, epoxide,
sulfhydryl, amide, acrylate, thiol, or amine.
[0147] In accordance with an aspect provided herein, the
conjugative group includes a vinyl group.
[0148] In accordance with an aspect provided herein, the
di-functional siloxane monomer is a dimethoxydimethylsilane
(DMODMS) or a vinylmethyldimethoxysilane (VMDMOS).
[0149] In accordance with an aspect provided herein, the
tri-functional siloxane monomer is a trimethoxymethylsilane (TMOMS)
or a vinyltrimethoxysilane (VTMOS).
[0150] In accordance with an aspect provided herein, the
tetra-functional siloxane monomer is a trimethoxysilane (TMOS).
[0151] In accordance with an aspect provided herein, the catalyst
is triethylamine.
[0152] In accordance with an aspect provided herein, the
monodisperse particles range in size from about 0.5 .mu.m to about
10 .mu.m.
[0153] In accordance with an aspect provided herein, the
conjugative group is covalently modified with a moiety for binding
to a target of interest.
In accordance with an aspect provided herein, the target of
interest includes one of a cell, a protein, a receptor, a virus, an
antibody, an antigen, a drug, a polysaccharide or a metabolite.
[0154] In accordance with an aspect provided herein, a method for
acoustic-mediated bioanalysis is provided. The method includes
exposing a fluid sample suspected of containing a target of
interest to a plurality of acoustic contrast particles according to
claim 97 under conditions sufficient that the moiety binds to the
target and subjecting the fluid sample to acoustic radiation
pressure from an acoustic standing wave sufficient within an
acoustic focusing chamber to focus the particles to their acoustic
antinodes such that the target is separated from other components
in the sample.
EXAMPLES
[0155] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject
matter.
Example 1
Methods for Synthesis of FMAR Colloids Using Nucleation and
Growth
[0156] Described below are four distinct approaches (Protocols I,
II, III, and IV) for synthesizing FMAR colloids from nucleation and
growth. In general, colloid particles were prepared using a
vinyl-siloxane such as, for example, to list a few
vinyltrimethoxysilane, triethoxyvinylsilane,
vinylmethyldiethoxysilane, and vinylmethyldimethoxysilane. The
vinyl-siloxane was added to acidic aqueous solution and agitated
(e.g., stirred, vortexed, sonicated) to permit hydrolysis. During
this step other siloxanes may be added such as, for example, to
list a few tetramethylorthosilicate, methyltrimethoxysilane, and
dimethoxydimethylsilane. At this point, a catalyst was added to
begin the condensation reaction and formation of monodisperse
particles. In another example, vinyl-siloxanes such as, for
example, to list a few vinyltrimethoxysilane, triethoxyvinylsilane,
vinylmethyldiethoxysilane, and vinylmethyldimethoxysilane and other
siloxanes such as, for example, to list a few
tetramethylorthosilicate, methyltrimethoxysilane, and
dimethoxydimethylsilane were added to a mixture of water and
co-solvent. At this point, a catalyst was added and the mixture was
agitated. A variety of modifications may be made to this nucleation
and growth protocol such as altering the temperature, resuspending
in a non-reactive medium, or adding surfactant to increase particle
stability.
[0157] Fabrication of acoustofluidic device. An acoustofluidic
device or otherwise referred to as an acoustic focusing chamber was
prepared to characterize the FMARs. The acoustofluidic device was
prepared using standard photolithography, deep reactive-ion etching
(DRIE), anodic bonding and plasma bonding. The device contained a
downstream collection module and an acoustic actuation component
(i.e., lead zirconate titanate piezoelectric element or PZT). The
channel width was designed to operate at a half wavelength resonant
mode (e.g., 252 .mu.m and driving frequency of 2.94 MHz or 272
.mu.m and 2.72 MHz) resulting in an antinode at both channel walls
and a single node in the channel center line. For the experiments,
an electric signal with peak-to-peak voltage of 31V is applied to
the PZT.
Each Protocol for synthesizing the FMARs is now described in
further detail below containing a list of materials, methods, and
representative results for each of the types of colloids
produced.
Generalized Materials List
[0158] Di-functional species: [0159] Dimethoxydimethylsilane
(DMODMS) [0160] Vinylmethyldimethoxysilane (VMDMOS) [0161]
Vinylmethyldiethoxysilane (VMDEOS) [0162]
3-Aminopropyl(diethoxy)methylsilane [0163] (AmDEOMS) [0164] Any
other di-functional siloxane monomer containing at least one
functional group [0165] Tri-functional species: [0166]
Trimethoxymethylsilane (TMOMS) [0167] Vinyltrimethoxysilane (VTMOS)
[0168] Triethoxyvinylsilane (VTEOS) [0169]
(3-Aminopropyl)trimethoxysilane (AmTMOS) [0170] 3
Trimethoxysilyl)propylacrylate (AcTMOS) [0171]
[3-(Diethylamino)propyl]trimethoxysilane (DAmTMOS) [0172] Any other
tri-functional siloxane monomer containing at least one functional
group [0173] Tetra-functional species: [0174]
Tetraethylorthosilicate (TEOS) [0175] Tetramethoxysilane (TMOS)
[0176] Tetrapropylorthosilicate (TPOS) [0177] Catalyst: [0178]
Ammonium (NH.sub.4OH) [0179] Tin octoate [0180] Triethylamine (TEA)
[0181] Co-solvent: [0182] Ethanol (EtOH) [0183] Methanol [0184]
Polysorbate [0185] Polyethylene glycol [0186] Sodium dodecyl
sulfate (SDS) [0187] Acid: Hydrochloric acid (HCl) [0188] Any other
acid
Protocol I (Stober-Based Method)
[0189] This approach employs fundamental features from the Stober
method (Stober, W. & Fink, A. (1969). However, two distinct
features that make the method disclosed herein different are 1) the
ratios of the di-, tri-, and tetra-functional monomers
incorporated, which results in colloids having various
cross-linking densities and bulk moduli useful for various acoustic
behaviors, and 2) the colloids that are synthesized contain many
functional groups.
Materials
[0190] Di-functional specie(s) [0191] Tri-functional specie(s)
[0192] Tetra-functional specie(s) [0193] Catalyst [0194]
Co-solvent
Methods
[0194] [0195] 1. Mix 4 mL co-solvent and 4.5 mL DI H.sub.2O (or
smaller ratios of co-solvent: DI H.sub.2O for slower reactions)
[0196] 2. Add varying ratios of di-, tri-, and tetra-functional
species that equal to 100 .mu.L [0197] 3. Pipette 1 mL catalyst
[0198] 4. Vigorously agitate via shaking or mixing [0199] 5. Place
on a hot plate at 70.degree. C. (optional)
Results
[0200] Colloids of varying compressibilities were produce with this
Protocol having diameters on the order of 100-800 nm within minutes
after the addition of a catalyst--Note: colloid size and
compressibility depends on monomer ratio, monomer concentration,
hydrolysis time, and catalyst strength. For example, FIG. 2 is a
graph showing relative colloid size of particles made using
Protocol I. The particles were synthesized with reproducible and
tunable size ranges that extend beyond 100 to 800 nm measured by
dynamic light scattering (DLS). The ratios shown on the x axis
represent the ratio of tri-functional monomers
(vinyltrimethoxysilane (VTMOS)) to di-functional monomers
(vinylmethyldimethoxysilane (VMDMOS)) used in the preparation of
the colloids represented by each bar. The particles in FIGS. 2-4
were produced specifically according to Protocol I as follows:
Materials
[0201] Di-functional species "silica initiator" [0202]
Dimethoxydimethylsilane (DMODMS) [0203] Vinylmethyldimethoxysilane
(VMDMOS) [0204] Tri-functional species "silica cross-linker" [0205]
Trimethoxymethylsilane (TMOMS) [0206] Vinyltrimethoxysilane (VTMOS)
[0207] Catalyst: [0208] i.e., Ammonia 880 (NH.sub.4OH) [0209]
Co-solvent: [0210] i.e., 200-proof ethanol (EtOH)
Methods
[0211] Mix 4 mL EtOH and 4.65 mL DI H.sub.2O in 20 mL glass vial
(or 100 .mu.L EtOH and 8.55 mL DI H.sub.2O for slower
reactions)
[0212] 1. Add 50 .mu.L di-functional species and 50 .mu.L
tri-functional species [0213] OR 25 .mu.L di-functional species and
75 .mu.L tri-functional species [0214] OR 75 .mu.L di-functional
species and 25 .mu.L tri-functional species [0215] OR 100 .mu.L
tri-functional species
[0216] 2. Pipette 1 mL NH.sub.4OH
[0217] 3. Shake for 30 min with a vortex speed of 4
[0218] 4. Place on a hot plate at 70.degree. C. to boil out
produced methanol and keep ethanol (optional) [0219] This protocol
produces semi-incompressible colloids with diameters on the order
of 100-800 nm within minutes after the addition of a catalyst (or
hours if following parenthetical option in methods step 1)
[0220] FIG. 3 is a graph showing size distribution of colloids made
using Protocol I with different mixing conditions according to one
or more embodiments of the present disclosure. Particles were made
using the siloxane monomer trimethoxymethylsilane (TMOMS). Particle
diameter (nm) is shown on the x axis and percent population by
count is shown on the y axis. Average size measured by a qNANO
instrument was 496.2 nm and the coefficient of variance was
12.65%.
[0221] FIGS. 4A & 4B are images of particles made using
Protocol I according to one or more embodiments of the present
disclosure. Particles were made using the siloxane monomer
vinyltrimethoxysilane (VTMOS). A) Scanning electron micrograph
(SEM) image. B) Optical microscope image.
Protocol II (Acidic Hydrolysis Method)
[0222] This protocol is distinct from the previous method due to
the variation in the synthesis procedure, which affects the nature
of the microparticles produced. Here, functional siloxane monomers
are hydrolyzed in a low pH medium and polycondensated in a high pH
medium instead of hydrolyzing and polycondensating in the same high
pH medium. These particles are also stable, monodisperse,
functional, and acoustically active.
Materials
[0223] Di-functional specie(s) [0224] Tri-functional specie(s)
[0225] Tetra-functional specie(s) [0226] Catalyst [0227] Acid
Methods
[0227] [0228] 1. Dilute concentrated stock HCl or other strong acid
(dilutions will range) [0229] 2. Dilute concentrated stock
NH.sub.4OH or other catalyst (dilutions will range) [0230] 3. Add
varying ratios of and tetra-functional species that equal 1.25 mL
to 7.5 mL DI H.sub.2O [0231] 4. Keep at 4.degree. C. (optional)
[0232] 5. Add 5 .mu.L diluted HCl or other strong acid [0233] 6.
Vigorously stir or mix solution for 5 hrs [0234] 7. Add 500 .mu.L
diluted catalyst and continue stirring for 10 min [0235] 8. Wash
particles and resuspend in a stable solution [0236] 9. Heat to
150.degree. C. for 12 hrs (optional)
Results
[0237] Colloids produced using this method have varying
compressibilities with diameters on the order of 0.5-5 .mu.m
several minutes after the addition of a catalyst. Colloid size and
compressibility depends on monomer ratio, monomer concentration,
hydrolysis time, and catalyst strength. Colloids produced using
this method are shown in FIGS. 5-7. The colloids were produced
specifically according to the following method.
TABLE-US-00001 Reactants Molar Ratio Siloxane monomers (must add up
to 1) 1 Tri-functional: Trimethoxymethylsilane (TMOMS)
Vinyltrimethoxysilane (VTMOS) Di-functional:
Dimethoxydimethylsilane (DMODMS) Vinylmethyldiethoxysilane (DMODMS)
Tetra-functional: Trimethoxysilane (TMOS) Ammonia 880 (NH.sub.4OH)
7 .times. 10.sup.-3 Hydrochloric acid (37% in water, concentrated
stock) 5 .times. 10.sup.-5 DI water 50
Methods
[0238] 1. Dilute 50 .mu.L 37% stock HCl solution into 5.254 mL DI
H.sub.2O to obtain 0.35% HCl [0239] 2. Dilute 50 .mu.L 25% stock
NH.sub.4OH solution into 3341 mL DI H.sub.2O to obtain 0.37%
NH.sub.3 [0240] 3. Add 1.366 mL TMOMS to 7.343 mL DI H.sub.2O
[0241] or 65.4 uL TMOS and 1.151 mL DMODMS (for a 1:100 ratio of
tetra- to di-silicone) [0242] or 130.8 uL TMOS and 1.090 mL DMODMS
(for a 1:10 ratio of tetra- to di-silicone) [0243] or 13.08 uL TMOS
and 1.313 mL VMDMOS (for a 1:100 ratio of tetra- to di-vinyl)
[0244] or 65.4 uL TMOS and 1.260 mL VMDMOS (for a 1:20 ratio of
tetra- to di-vinyl) [0245] or 261.7 uL TMOS and 1.061 VMDMOS (for a
1:4 ratio of tetra- to di-vinyl) [0246] or 13.08 uL TMOS and 1.520
mL AmTMOS (for a 1:100 ratio of tetra- to di-amine) [0247] 4. Keep
at 4.degree. C. (optional) [0248] 5. Add 3.9 .mu.L of 0.35% HCl
[0249] 6. Vigorously stir for 5 hrs with a stir bar at 600 rpm
[0250] 7. Add 646.7 .mu.L of 0.37% NH.sub.4OH and continue stirring
for 4 hrs [0251] 8. Separate the particles from the suspension by
centrifuging and resuspending in 1.times.PBS [0252] 9. Can harden
particles by heating to 150.degree. C. for 12 hours (optional)
[0253] FIGS. 5A & 5B are optical micrographs of FMAR colloids
made using the siloxane monomer trimethoxymethylsilane (TMOMS) as
described in Protocol II. A) Particles are shown with microscope
settings at a low (dark) plane of focusing. B) Particles are shown
with microscope settings at a high (bright) plane of focusing. Both
images show monodisperse colloids that do not aggregate without a
surfactant.
[0254] FIG. 6 is a graph of Zeta Potential of particles described
in FIG. 5A-5B made via Protocol II using all tri-functional species
trimethoxymethylsilane (TMOMS). The surface charge indicates that
the colloids are sufficiently stable without the use of protective
surfactants.
[0255] FIGS. 7A-7F are fluorescent images of FMAR colloids (1:100
monomer ratio of Tetramethoxysilane (TMOS): Dimethoxydimethylsilane
(DMODMS) siloxane monomers) made via Protocol II with negative
acoustic contrast in a silicon acoustofluidic chip as a
demonstration that the FMAR colloids are sufficient to displace
positive acoustic contrast particles from the acoustic node to the
acoustic antinodes according to one or more embodiments of the
present disclosure. A) Streptavidin-conjugated ALEXA FLUOR 488
incubated FMAR colloids as a positive control (PZT power=0V,
flow=15 .mu.L/min). B) The same particles as in (A) (PZT power=15V,
flow=15 .mu.L/min). C) Pink fluorescent biotin-coated polystyrene
beads (PZT power=0V, flow=100 .mu.L/min). D) Same particles as (C)
(PZT power=15V, flow=100 .mu.L/min). E) FMAR colloids bound to
polystyrene beads used as a surrogate test (PZT power=0V, flow=100
.mu.L/min). F) Same particles as (E) (PZT power=10V, flow=100
.mu.L/min).
Protocol III (Standard Nucleation & Growth Method)
[0256] The synthesis of colloids according to this Protocol III is
distinct from the previous two Protocols because the functional
siloxane monomers are hydrolyzed in a low pH medium for a short
period of time (compared to Protocol II) and undergo size
separation (typically through centrifugation) to remove large
non-uniform oligomers from the hydrolyzed nuclei prior to the
catalyst-induced polycondensation. The colloids that are produced
using this method exhibit tight size uniformity and stability in
suspensions like all methods described.
Materials
[0257] Di-functional specie(s) [0258] Tri-functional specie(s)
[0259] Tetra-functional specie(s) [0260] Catalyst [0261] Acid
Methods
[0261] [0262] 1. Modestly dilute concentrated stock HCl or other
strong acid (dilutions will range) [0263] 2. Rigorously dilute a
separate batch of concentrated stock HCl or other strong acid
(dilutions will range) [0264] 3. Add varying ratios of di-,tri-,
and tetra-functional species that equal 1 mL to 10 mL DI H.sub.2O
[0265] 4. Add modestly dilute HCl or other strong acid to DI
H.sub.2O with siloxane monomers [0266] 5. Stir or mix for 18 hrs
[0267] 6. Centrifuge at 2000.times.g for 5 min
[0268] 7. Extract 7.5 mL of supernatant and add 7.5 mL of
rigorously diluted HCl or other strong acid
[0269] 8. Add 15 .mu.L concentrated catalyst to supernatant-acidic
water solution
[0270] 9. Continue stirring or mixing for 30 min then centrifuge at
2000.times.g for 5 min
[0271] 10. Wash particles and resuspend in a stable solution
Results
[0272] This protocol produces colloids of varying compressibilities
with diameters on the order of 0.5-50 .mu.m several minutes after
the addition of a catalyst. Colloid size and compressibility
depends on monomer ratio, monomer concentration, hydrolysis time,
and catalyst strength. The colloids shown in FIGS. 8-11 were
produced according to this Protocol III and specifically according
to the following procedure.
Materials
[0273] Siloxane monomers [0274] Tri-functional: [0275]
Trimethoxymethylsilane (TMOMS) [0276] Vinyltrimethoxysilane (VTMOS)
[0277] Di-functional: [0278] Dimethoxydimethylsilane (DMODMS)
[0279] Vinylmethyldiethoxysilane (DMODMS) [0280] Triethylamine
(TEA) [0281] 200-proof ethanol (EtOH) [0282] Hydrochloric acid (37%
in water, concentrated stock)
Methods
[0282] [0283] 1. Prepare 10 mL of 0.1 M HCl (pH=1) by adding 83.3
.mu.L 37% HCl to 9.917 mL DI H.sub.2O [0284] 2. Prepare 7.5 mL of
3.16.times.10.sup.-4 M (pH=3.5) by adding 23.7 .mu.L 0.1 M HCl to
7.476 mL DI H.sub.2O [0285] 3. Add 1 mL of TMOMS to 10 mL of
H.sub.2O or various volume ratios of di-, tri-, and
tetra-functional species [0286] 4. Add 219.5 .mu.L of 0.1 M HCl to
DI H.sub.2O with monomers to obtain net pH of 2.7 [0287] 5. Stir at
500 rpm for 18 hrs [0288] 6. Centrifuge at 2000.times.g for 5 min
[0289] 7. Extract 7.5 mL of supernatant and add 7.5 mL of
3.1.times.10.sup.-4 M HCl [0290] 8. Add 15 .mu.L TEA to
supernatant-acidic water solution [0291] 9. Continue stirring for
30 min at 500 rpm [0292] 10. Centrifuge at 2000.times.g for 5 min
[0293] 11. Resuspend pellet in 10 mL EtOH and sonicate
[0294] FIGS. 8A-8D are SEM images of colloids synthesized using
Protocol III at various magnifications according to one or more
embodiments of the present disclosure. FMAR colloids were
synthesized via rapid bulk synthesis using the siloxane monomer
trimethoxymethylsilane (TMOMS). A) Particles are shown at a
magnification of 5000.times., B) Particles are shown are shown at a
magnification of 15000.times.. C) Particles are shown at a
magnification of 15000.times. at a different site than shown in
(B). D) Particles are shown at a magnification of 2500.times..
[0295] FIG. 9 shows positive acoustic contrast FMAR colloids
synthesized using Protocol III as described in FIG. 8 in a silicon
acoustofluidic chip as a demonstration of the acoustic tunability
of the synthesis mechanisms according to one or more embodiments of
the present disclosure. A) The acoustic field is turned off. B)
FMAR colloids responding to the acoustic field, focusing in the
center of the channel (acoustic node) indicating FMAR colloids can
be easily designed to exhibit positive acoustic contrast.
[0296] FIG. 10 is an optical micrograph of FMAR colloids
synthesized using Protocol III as described in FIG. 8.
FIGS. 11A-11B are graphs showing size distribution of FMAR colloids
synthesized using Protocol III as described in FIG. 8 characterized
by a qNANO device according to one or more embodiments of the
present disclosure. Particle diameter is shown on the x axis and
percent population by count is shown on they axis. The coefficient
of variance was A) 12.39% and B) 14.01%.
Protocol IV (Core-Shell Method)
Materials
[0297] Di-functional specie(s) [0298] Tri-functional specie(s)
[0299] Tetra-functional specie(s) [0300] Catalyst [0301] Acid
Methods
[0301] [0302] i. Acid preparation [0303] 1. Modestly dilute
concentrated stock HCl or other strong acid (dilutions will range)
[0304] 2. Rigorously dilute a separate batch of concentrated stock
HCl or other strong acid (dilutions will range) [0305] ii.
Semi-rigid shell hydrolysis [0306] 1. Add 1 mL of varying ratios of
tri- and tetra-functional species to 10 mL of DI H.sub.2O [0307] 2.
Add 200 .mu.L of modestly dilute HCl or other strong acid to DI
H.sub.2O solution with tri-/tetra-functional monomers [0308] 3.
Vigorously stir or mix for 18 hrs [0309] iii. Emulsion core
hydrolysis [0310] 1. Add 1 mL of di-functional species to 10 mL DI
H.sub.2O [0311] 2. Add 200 .mu.L of modestly dilute HCl or other
strong acid to DI H.sub.2O solution with di-functional monomers
[0312] 3. Vigorously stir or mix for 6 hrs [0313] iv. Emulsion core
synthesis [0314] 1. Centrifuge di-functional monomer-containing
solution at 2000.times.g for 5 min [0315] 2. Extract 7.5 mL of
supernatant and add 7.5 mL of rigorously dilute HCl or other strong
acid [0316] 3. Add 30 .mu.L concentrated catalyst to
supernatant-acidic water solution [0317] 4. Continue stirring or
mixing for 30 min [0318] v. Semi-rigid shell polycondensation
[0319] 1. Centrifuge at 2000.times.g for 5 min [0320] 2. Extract
7.5 mL of supernatant and add 7.5 mL of 3.1.times.10.sup.-4 M HCl
and mix [0321] 3. Directly add the 10 mL acidic supernatant to the
emulsion core solution, continue stirring [0322] 4. Immediately add
10 .mu.L concentrated catalyst [0323] 5. Continue stirring or
mixing for 30 min
Results
[0324] This protocol produces colloids of varying compressibilities
with diameters on the order of 0.5-10 .mu.m several minutes after
the addition of a catalyst. Colloid size and compressibility
depends on monomer ratio, monomer concentration, hydrolysis time,
and catalyst strength. The colloids shown in FIGS. 12 and 13 were
produced using this Protocol. Specifically, the colloids shown in
FIGS. 12 and 13 were produced according to the following
procedure.
Materials
[0325] Siloxane Monomers [0326] Tri-functional: [0327]
Trimethoxymethylsilane (TMOMS) [0328] Vinyltrimethoxysilane (VTMOS)
[0329] Di-functional: [0330] Dimethoxydimethylsilane (DMODMS)
[0331] Vinylmethyldiethoxysilane (DMODMS) [0332] Triethylamine
(TEA) [0333] 200-proof ethanol (EtOH) [0334] Hydrochloric acid (37%
in water, concentrated stock)
Methods
[0334] [0335] A. Acid Preparation [0336] 10. Prepare 10 mL of 0.1 M
HCl (pH=1) by adding 83.3 .mu.L 37% HCl to 9.917 mL H.sub.2O [0337]
11. Prepare 7.5 mL of 3.16.times.10.sup.-4 M (pH=3.5) by adding
23.7 .mu.L of 0.1 M HCl to 7.476 mL H.sub.2O [0338] B. Semi-Rigid
Shell Hydrolysis (start at 10 pm Day 1, finish 10:15 pm Day 1)
[0339] 4. Add 1 mL of TMOMS to 10 mL of H.sub.2O or various volume
ratios of tri- and tetra-functional species [0340] 5. Add 219.5
.mu.L of 0.1 M HCl to H.sub.2O with TMOMS to obtain net pH of 2.7
[0341] 6. Stir at 500 rpm for 18 hrs [0342] C. Emulsion Core
Hydrolysis (start at 9:30 am Day 2) [0343] 1. Add 1 mL of DMODMS to
10 mL of H.sub.2O [0344] 2. Add 219.5 .mu.L of 0.1 M HCl to
H.sub.2O with DMODMS to obtain net pH of 2.7 [0345] 3. Stir at 500
rpm for 6 hours [0346] D. Emulsion Core Synthesis (start at 3:30 pm
Day 2, finish 4:15 pm Day 2) [0347] 5. Centrifuge DMODMS H.sub.2O
at 2000.times.g for 5 min [0348] 6. Extract 7.5 mL of supernatant
and add 7.5 mL of 3.1.times.10.sup.-4 M HCl [0349] 7. Add 30 .mu.L
TEA to supernatant-acidic water solution [0350] 8. Continue
stirring for 30 min at 500 rpm [0351] 9. Dispose 5 mL of the
solution [0352] E. Semi-Rigid Shell Polycondensation (start at
.about.4:00 pm Day 2, finish 4:15 pm Day 2) [0353] 1. Centrifuge at
2000.times.g for 5 min [0354] 2. Extract 7.5 mL of supernatant and
add 7.5 mL of 3.1.times.10.sup.-4 M HCl and mix [0355] 3. Directly
add the 10 mL acidic supernatant to the emulsion core solution,
continue stirring [0356] 4. Immediately add 10 .mu.L TEA [0357] 5.
Continue stirring for 30 min at 500 rpm
[0358] FIG. 12 is an optical micrograph of colloids synthesized
using Protocol IV according to one or more embodiments of the
present disclosure. The particle cores were made using
dimethoxydimethylsilane (DMODMS) siloxane monomers and the particle
shells were made using trimethoxymethylsilane (TMOMS).
[0359] FIG. 13 is an image of a silicon acoustofluidic channel
containing FMAR colloids synthesized from Protocol IV as described
in FIG. 12 according to one or more embodiments of the present
disclosure. A) (PZT Off), Flow=100 uL/min. B) (PZT On, V=10),
Flow=turned off for 60 sec.
Example 2
Use of FMAR Colloids in Silicon Acoustofluidic Channels to Effect
Acoustophoretic Cell Displacement
[0360] The following examples show the utility of using the FMAR
colloids synthesized according to the Protocols described above to
separate cells in a silicon acoustofluidic chip. FIG. 14 shows
images of FMAR colloids made according to one or more embodiments
of the present disclosure. In FIG. 14A, an optical micrograph is
shown of elastomeric FMAR colloids for cell separation made
according to Protocol II using tetra-functional functional monomers
trimethoxymethylsilane (TMOS) and di-functional
dimethoxydimethylsilane (DMODMS) at a ratio of 1:100. FIG. 14B
shows a fluorescence micrograph of the elastomeric FMAR colloids
with adsorbed Nile red in a silicon acoustofluidic chip. When the
acoustic field is turned on, the FMAR colloids respond to the
acoustic field by focusing in at the acoustic antinodes indicating
FMAR colloids can be easily designed to exhibit negative acoustic
contrast. FIG. 14C shows an SEM image of biotin polystyrene
(SPHEROTECH, INC.) bound to a KG-1a human myeloblast.
FIGS. 15A-15H are images of KG-1 a cell binding and separation
according to one or more embodiments of the present disclosure.
A-C) Optical micrographs of streptavidin adsorbed elastomeric FMAR
colloids (1:100 TMOS:DMODMS as described in FIG. 14A&B) binding
to Calcein AM dyed KG-1 a cells, D-F) Fluorescence microscopy
images of Calcein AM dyed KG-1a cells illuminating the bound
non-fluorescent FMAR colloids, demonstrating binding (same frames
as FIG. 3A-C). G) Unbound KG-1 a cells focusing in the acoustic
node of a silicon acoustofluidic channel. H) KG-1 a cells bound to
the elastomeric FMAR colloids focusing to the acoustic antinode of
the silicon acoustofluidic channel. Note: The scale bar in A is
also for B-F and the scale bar in H is also for G.
[0361] FIGS. 16A-D are images of the use of FMAR colloids in
silicon acoustofluidic channels to effect acoustophoretic cell
displacement according to one or more embodiments of the present
disclosure. KG-1a cells spontaneously migrate to the pressure
antinode in the presence of an acoustic standing wave when bound to
FMAR colloids as described in FIGS. 14A & 14B. A) Shows a
representative random distribution of fluorescent cells in an
acoustofluidic device without a standing wave, B-D) Show cells
responding to the primary radiation force (time step is
approximately 1 sec).
Example 3
Preparation & Characterization of Negative Acoustic Contrast
Particles (NACPs) Having a Functional Group Available for Covalent
Modification
[0362] Preparation of stable, biofunctionalized elastomeric
particles (NACPs): An important goal is to be able to employ NACPs
for bioanalytical techniques that require biofunctionalization of
the particle surface for binding to specific biomolecules or cells.
Common bioconjugation schemes, such as carboiimide chemical
approaches, are not feasible with NACPs synthesized using the
common silicone material (e.g., polydimethylsiloxane "PDMS"),
because the resulting PDMS NACPs lack the necessary functional
groups such as carboxylates, hydroxyls, epoxies, and amines to
introduce functionality. To address this problem,
polyvinylmethylsiloxane (PVMS) pre-polymers were used to generate
NACPs with surface vinyl groups that would be useful for reactions
such thiolene. In this manner, a variety of chemical reactions
could be employed to functionalize the vinyl containing NACPs. For
example, thiolene click reactions using biotinylated thiols and a
water soluble azothermal initiator (VA-50, WAKO) or photoactivated
biotin-benzophenone could be employed to covalently
biofunctionalize the vinyl-containing NACPs.
[0363] Below is an example of a protocol that was used to prepare
such biofunctional NACPs with PVMS. A mixture of 1.02 g of
hydroxyl-terminated PVMS, 0.07 g vinylmethoxysiloxane homopolymer
(GELEST), were thoroughly stirred and combined with a solution of
0.1 gram PLURONIC F108 (ALDRICH) in 15 mL of MILLIQ water, briefly
vortexed, and homogenized using a POLYTRON PT 1200E homogenizer for
5 min at 6500 rpm. After stirring at 50.degree. C. for 4 hrs, the
polydisperse emulsion was permitted to cure at ambient conditions
for at least 7 days before being passed through a 12 .mu.m
polycarbonate filter (WHATMAN) and stored at ambient conditions
until use. Biotinylation of PVMS microparticles occurred by first
washing 7.2.times.10.sup.7 microparticles with 1.times.PBS by
centrifuging at 8000.times.g, 2min and resuspending the pellet with
a final volume of 2 mL of 1.times.PBS. The microparticles were
transferred to a glass vial with a stir bar and 3.3 mg
Biotin-PEG-tetrofluorophenyl azide (Biotin-PEG-TEPA, QUANTA
BIODESIGN) in 100 .mu.L of dimethylacetamide (DMAC) was added. The
Biotin-PEG-TFPA is a photoaffinity molecule that reacts with a
variety of croups (e.g., C--H bonds) via nitrene formation. A light
source (OMINICURE) with light guide was placed 500 mm above the
stirring solution for 30 minutes at a light intensity of 100
mW/cm.sup.2 at 365 nm as measured by a power meter. The resultant
slightly yellow solution was stored at 4.degree. C. until use. The
biotinylation of the EP surfaces in this manner permits linking of
commercially available biotinylated antibodies and other molecules
via streptavidin protein. The NACPs synthesized as described above
using biotinylated tetrafluoraphenyl azide (Biotin-PEG-T FPA)
proved successful for biofunctionalization. For example, the NACPs
modified with Biotin-PEG-TFPA remained stable, dispersed, and able
to bind Streptavidin (data not shown).
[0364] In another experiment, surfactant PLURONIC F108 (ALDRICH)
was functionalized with the Biotin-PEG-TFPA described above, and
then the biotinylated surfactant was used during the synthesis of
the NACPs as described above using PVMS NACPs. FIGS. 17A and B are
schematic diagrams showing the chemical structure of the F108
triblock co-polymer surfactant and the surfactant associated with
an NACP, respectively. The resulting compressible, surfactant
biofunctionalized PVMS NACPs were used in an experiment in an
acoustic channel with incompressible cells without a label that had
been stained with calein AM for imaging purposes. The purpose of
this experiment was to show that PVMS NACPs and cells (when not
bound) respond differently to an ultrasonic standing wave. The
results showed that the surfactant biofunctionalized NACPs
segregated to the periphery of the acoustic channel under an
applied frequency of 2.93 MHz while at the same time, the
incompressible cells stained with calein AM located to the central
axis of the microfluidic channel (data not shown).
[0365] In a related experiment, PDMS NACPs were prepared with
biotinylated-F108. The surfactant biotin functionalized NACPs were
incubated with microparticles functionalized with Streptavidin
(Polysciences, Green microparticles, 6 .mu.m diameter) for 30 min
and washed and imaged. The streptavidin with its four biotin
binding sites serves as a linker between the biotinylated NACPs and
the streptavidin-functionalized particles FIG. 18 shows brightfield
(left panel) and accompanying fluorescent microscope (right panel)
images of binding between NACPs functionalized with
biotin-surfactant (large circles) and the
streptavidin-functionalized polystyrene beads (smaller
circles).
[0366] A CD34+ cell line (Kg-1 a) was employed as a model system to
investigate acoustic-mediated cell separation using bio
functionalized NACPs. Separation, collection, and analysis of CD34+
cells is significant for reasons including repopulation of marrow
for autologous stem cell transplantation and characterization of
malignancies such as acute lymphoblastic leukemia. CD34+ cells were
pre-functionalized with biotinylated monoclonal antibodies (CD34,
mouse anti-human) and fluorescent ALEXAFLUOR-488 streptavidin
before incubation with the biotinylated PVMS NACPs described above.
The streptavidin with its four biotin binding sites serves as a
linker between the biotinylated NACPs and the streptavidin-labeled
cells. Some binding between cells and particles was observed (data
not shown). Next, the EP-cell complexes were introduced into the
aforementioned acoustic chip with an applied frequency of 2.93 MHz.
Observation using fluorescent microscopy through the glass lid of
the acoustic chip showed migration of the NACP-bound CD34+ cells
from the acoustic node to the periphery (antinodes) of the acoustic
channel (data not shown).
In another example, biofunctionalized PVMS NACPs were prepared
according to the following procedure. A mixture of 1.0 g of
hydroxyl-terminated PVMS,.sup.14 0.07 g vinylmethoxysiloxane
homopolymer (GELEST), and 0.02 g tin octoate catalyst (GELEST) was
thoroughly stirred and combined with a solution of 0.5 or 0.7 wt %
PLURONIC F108 (ALDRICH) in ultrapure water (Mill-Q, 18M.OMEGA.
resistivity). The mixture was briefly vortexed, homogenized using a
PT 1200E homogenizer (POLYTRON) with a 3 mm rotor for 5 min at
18,750 rpm, and stirred for at least 2 hrs at 50.degree. C. The
polydisperse emulsion was permitted to cure via alkoxy condensation
of silanol-terminated PVMS with vinylmethoxysiloxane. Particles
were filtered through a 12 .mu.m polycarbonate membrane (WHATMAN,
CYCLOPORE) and stored at ambient conditions until use.
[0367] Preparing PDMS particles: A mixture comprising a 1:10 weight
ratio of curing agent: base of SYLGARD 184 (DOW CHEMICAL) was
thoroughly mixed and subsequently combined with 1 wt % of F108. The
mixture was homogenized as previously described. The emulsion was
incubated at 45.degree. C., stirring for at least 1.5 hrs and
subsequently left at ambient conditions for at least 12 hrs to
permit curing.
[0368] Functionalization: For reactions biotin-PEG-TFPA,
.about.5.times.10.sup.7 PVMS microparticles were washed with
1.times.PBS by centrifuging and resupending the pellet in a final
volume of 2 mL of 1.times.PBS. The microparticles were transferred
to a cylindrical glass vial (2.5 cm diameter) and 3 mg
biotin-PEG-TFPA in 100 .mu.L of dimethylacetamide was added. Light
irradiation occurred using an OMNICURE S1000 equipped with a high
pressure mercury lamp and an internal 320-500 nm filter. The
associated light guide was placed .about.5 mm above the stirring
solution for 30 min at a light intensity of .about.100 mW/cm.sup.2
at a wavelength of 365 nm, (as measured by POWERMAX USB SENSOR,
COHERENT). The resultant yellow solution was stored at 4.degree. C.
until use. Biotinylation of F108 surfactant followed a similarly
reported protocol..sup.18 Briefly, hydroxyl end groups on F108 were
modified to succinimidyl carbonate using N,N'-disuccinimidyl
carbonate (ALDRICH) and 4-(dimethylamino)pyridine (Aldrich) and
subsequently reacted with biotin-hydrazide (ALDRICH). Once
biotinylated, F108 was used to prepare silicone emulsions as
previously described. Subsequent addition of streptavidin
(ALEXAFLUOR 488 OR ALEXAFLUOR 546) to NACPs occurred by washing
particles at least three times by centrifuging and resupending the
pellet in 1.times.PBS, and incubating with either 1 .mu.M or 1.7
.mu.M of streptavidin for 30 min at room temperature.
[0369] Characterization of negative acoustic contrast materials and
microparticles. Attenuated total reflection-Fourier transform
infrared (ATR-FTIR) spectra were acquired using a THERMO ELECTRON
NICOLET 8700 spectrometer (Ge crystal, 32 scans, 4 cm.sup.2
resolution). Scanning electron microscopy (SEM) images were
obtained using model FEI XL 30 SEM under ultra-high resolution mode
after sputter coating the samples with approximately 6 nm of gold.
Optical microscopy images were obtained using an upright ZEISS AXIO
IMAGER A2 microscope with appropriate filter set (ex 470/40, em
525/50 or ex 545/25, em 605/70 or ex 395, em 445/150).
[0370] Bioseparation studies. Binding between streptavidin
polystyrene microparticles (POLYSCIENCES, YG microspheres, 6 .mu.m)
and PDMS NACPs (encapsulated with rhodamine B, functionalized with
biotin-F108) occurred by combining .about.10.sup.6 polystyrene
particles and .about.10.sup.7 PDMS particles and incubating with
end-over-end rotation for 30 min at room temperature. PDMS NACPs
were washed three times with 1.times.PBS before combining with the
polystyrene microparticles. Polystyrene particles were added
directly from the manufacture's stock without washing.
Bioseparation events within a channel were monitored through the
glass lid of the acoustofluidic device prepared as described in
Example 1 using fluorescent microscopy.
[0371] Results: Silicone elastomers offer properties suitable for
NACPs such as compressibility at mild temperature (e.g., Young's
modulus .about.1 MPa for typical PDMS formulations)..sup.12 Here,
all NACPs were prepared by emulsifying silicone pre-polymers in
aqueous surfactant solutions and subsequently curing to produce
solid microparticles. Because homogenization produces polydisperse
particles, filtration or centrifugation was employed to narrow the
breadth of particle size distributions. In one example, filtration
of NACPs with a 12 .mu.m polycarbonate filter resulted in an
average particle diameter of 6.+-.3 .mu.m (data not shown).
Although a variety of surfactants enabled formation of
silicone-in-water emulsions, the importance of surfactant type
became evident when attempting to re-suspend cured NACPs in
surfactant-free buffer, which often resulted in irreversible
particle aggregation. Here, it was found that the block copolymer
surfactant, F108, stabilizes silicone microparticles likely due to
the strong association of the hydrophobic polypropylene oxide block
with silicone..sup.13 This stable association was further exploited
by end-functionalizing F108 with biotin (see FIGS. 17 and 18).
Biotin-functionalized F108 enables use of the streptavidin protein
as a linker between NACPs and any biotinylated analyte (e.g., cells
labelled with biotinylated antibodies).
[0372] The feasibility of direct modification of NACPs was also
evaluated. Typically, surface modification of PDMS is accomplished
by employing modification methods such as ultraviolet (UV)/ozone
irradiation,.sup.14 UV graft polymerization,.sup.11 oxygen plasma
treatment,.sup.15 and adsorption..sup.16 These modification
approaches are usually performed on macroscopic silicone surfaces
not held to the unique stringencies required to functionalize
NACPS. For the NACPs described herein, conditions were avoided that
resulted in significant change in modulus or irreversible
microparticle aggregation. For instance, modification of PDMS
surfaces via oxygen plasma results in the formation of brittle
silica layers.sup.17 which could affect the negative acoustic
contrast property. Here, to evaluate direct, covalent modification
of particles, we used PVMS which contains vinyl groups and can be
functionalized chemically without forming a silica-like
crust..sup.14
[0373] To first evaluate and compare chemical groups in both PDMS
and PVMS, bulk samples were prepared and characterized using
ATR-FTIR (data not shown). PVMS material displays characteristic
vinyl peaks at .about.960 cm.sup.-1 (C.dbd.C twist, .dbd.CH.sub.2
wagging), .about.1,410 cm.sup.-1 (.dbd.CH.sub.2 scissors), and
.about.1,590 cm.sup.-1 (C.dbd.C stretch). While vinyl groups are
versatile for various chemical reactions (e.g., thiolene or
methathesis coupling), studies described herein revealed that
relatively simple photochemical reaction with biotin-PEG-TFPA
results in biofunctionalization of PVMS particles. Photoreacting
biotin-PEG-TFPA with PVMS microparticles and subsequently adding
fluorescent streptavidin resulted in significant differences in
fluorescent signal between positive and negative samples (data not
shown). For example, signal to background values (S/B) of
fluorescent images of PVMS microparticles functionalized with
biotin-PEG-TFPA and fluorescent streptavidin was 22.+-.2, whereas
the negative control reaction without light irradiation was
9.0.+-.0.3, suggesting a biotinylation reaction of NACPs occurred.
These studies demonstrate the utility of using biotin-PEG-TFPA for
bio-functionalization of silicone microparticles.
[0374] The acoustic responsiveness of these PVMS silicone
microparticles was evaluated and the images are shown in FIG.
19A-19D. The results described herein show that microparticles
prepared from PVMS function as NACPs within aqueous media (see
FIGS. 19A-D). For example, a mixture of biotinylated PVMS NACPs and
non-biotinylated polystyrene microparticles randomly distribute
within an acoustofluidic channel in the absence of a standing wave
field (FIG. 19C). Upon application of an operating frequency of
2.98 MHz to generate an ultrasound standing wave within the
microchannel (wavelength=2.times.channel width), polystyrene and
PVMS microparticles rapidly separate (FIG. 19D). Incompressible
positive acoustic contrast polystyrene particles transport to the
center of channel, corresponding to the pressure node, whereas
compressible PVMS NACPs transport to the channel sidewalls,
corresponding to the pressure antinodes. The capacity for PVMS to
function as NACPs (see FIGS. 19A-D) illustrates the versatility of
using silicone elastomers with different chemical compositions.
[0375] The utility of the NACPs having a group that can be modified
covalently with a specific biomolecular recognition group was
investigated in cell separations. To this end, polystyrene
microparticles were employed as surrogates for mammalian cells
(i.e. as NACPs) and the separation characteristics using NACPs
prepared from PDMS and a functionalized surfactant were
investigated in an acoustofluidic device. As described previously
and shown in the brightfield (left panel) and fluorescent (right
panel) images of FIG. 18, streptavidin coated polystyrene and PDMS
microparticles functionalized with biotin-PLURONIC F108 do
associate when they are added together in solution. FIG. 20 shows
that the NACP-polystyrene microparticle complexes (PDMS:polystyrene
complexes), when placed within the acoustofluidic device in the
presence of an acoustic wave, transport in unison to the acoustic
antinode (FIG. 20B). This shows that NACPs can serve as vehicles
for specific transport of positive acoustic contrast particles.
Conversely, non-biotinylated PDMS microparticles did not bind the
streptavidin polystyrene particles (see FIG. 20A where the
non-biotinylated PDMS particles (large diffuse circles) transported
to the acoustic antinode and the polystyrene microparticles (small
bright circles) aligned at the acoustic node. Thus, in the absence
of fluid flow NACPs accumulate at the acoustofluidic channel walls
(acoustic antinode) during PZT activation (see FIGS. 19 and 20). By
permitting laminar flow within the channel, NACPs will maintain
their position at the acoustic antinode while simultaneously moving
in a streamline flow to the downstream trifurcation, as recently
demonstrated..sup.10 This capacity to couple relocation with
downstream sample collection facilitates continuous sorting
applications.
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applications. Advanced Materials, 12(10): 693-713.
[0402] Although the foregoing subject matter has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be understood by those skilled in
the art that certain changes and modifications can be practiced
within the scope of the appended claims.
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