U.S. patent application number 15/846497 was filed with the patent office on 2018-05-03 for devices and methods for separating particles.
The applicant listed for this patent is University of Georgia Research Foundation, Inc.. Invention is credited to Mark A. Eiteman, Leidong Mao, Taotao Zhu.
Application Number | 20180117597 15/846497 |
Document ID | / |
Family ID | 49580431 |
Filed Date | 2018-05-03 |
United States Patent
Application |
20180117597 |
Kind Code |
A1 |
Mao; Leidong ; et
al. |
May 3, 2018 |
DEVICES AND METHODS FOR SEPARATING PARTICLES
Abstract
Embodiments of the present disclosure provide for devices,
methods for separating particles, and the like.
Inventors: |
Mao; Leidong; (Athens,
GA) ; Zhu; Taotao; (Athens, GA) ; Eiteman;
Mark A.; (Athens, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Georgia Research Foundation, Inc. |
Athens |
GA |
US |
|
|
Family ID: |
49580431 |
Appl. No.: |
15/846497 |
Filed: |
December 19, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15290700 |
Oct 11, 2016 |
9873126 |
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15846497 |
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13873424 |
Apr 30, 2013 |
9517474 |
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15290700 |
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61648786 |
May 18, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B03C 2201/18 20130101;
C12N 13/00 20130101; B03C 1/288 20130101; B03C 2201/26 20130101;
G01N 27/74 20130101; B03C 1/32 20130101 |
International
Class: |
B03C 1/28 20060101
B03C001/28; B03C 1/32 20060101 B03C001/32; G01N 27/74 20060101
G01N027/74; C12N 13/00 20060101 C12N013/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under
Agreement No. 5 R21 GM104528-03, awarded by the National Institutes
of Health. The Government has certain rights in the invention.
Claims
1. A device, comprising: a first fluid inlet in a flow channel for
flowing a first liquid including two or more types of particles,
wherein the first liquid includes a magnetic fluid or is mixed with
the magnetic fluid; a magnetic device configured to direct a
non-uniform magnetic force onto the magnetic fluid and the
particles; and a plurality of outlets, wherein the non-uniform
magnetic force causes the types of particles to be separated and
flow into different outlets.
2. The device of claim 1, wherein the particles are cells.
3. The device of claim 1, wherein the magnetic device includes a
magnet disposed on one side of the device to generate the
non-uniform magnetic force.
4. The device of claim 1, wherein the magnetic device is configured
to control the non-uniform magnetic force exerted on the particles
and the magnetic buoyancy force experienced by the particles.
5. The device of claim 1, wherein the plurality of outlets includes
two outlets for separating particles having two different
volumes.
6. The device of claim 1, wherein the magnetic device includes a
permanent magnet.
7. A method for separating particles, comprising: disposing at
least two types of particles in a first fluid, wherein the first
liquid includes or is mixed with the magnetic fluid; flowing the
magnetic fluid and the particles down a channel; exposing the
magnetic fluid and the particles to a non-uniform magnetic force;
and separating the types of particles.
8. The method of claim 7, wherein a magnet disposed on one side of
the channel generates the non-uniform magnetic force.
9. The method of claim 7, wherein exposing includes causing the
particles to experience a magnetic buoyancy force that causes the
particles to separate from one another based on the volume of the
particles.
10. The method of claim 7, wherein the particles are cells.
11. The method of claim 7, wherein separating includes causing the
different types of cells to flow through different outlets.
12. The method of claim 7, wherein the method is operated under a
continuous flow.
Description
CLAIM OF PRIORITY TO RELATED APPLICATION
[0001] This application is a divisional of co-pending U.S. patent
application entitled "DEVICES AND METHODS FOR SEPARATING PARTICLES"
having Ser. No. 15/290,700, filed Oct. 11, 2016 which is a
divisional of U.S. patent application entitled "DEVICES AND METHODS
FOR SEPARATING PARTICLES" having Ser. No. 13/873,424, filed on Apr.
30, 2013, now U.S. Pat. No. 9,517,474, which claims priority to
U.S. provisional application entitled "DEVICES AND METHODS FOR
SEPARATING PARTICLES" having Ser. No. 61/648,786, filed on May 18,
2012, the contents of which are incorporated herein by reference in
their entirety.
BACKGROUND
[0003] Microfluidic particle and cell sorting plays an important
role in environmental monitoring, disease diagnostics, and
therapeutics. Some techniques include labeling the particle or
cell, however, these techniques have disadvantages. Thus, there is
a need to develop alternative techniques for particle sorting.
SUMMARY
[0004] In accordance with the purpose(s) of the present disclosure,
as embodied and broadly described herein, embodiments of the
present disclosure, in one aspect, relate to a devices, methods for
separating particles, and the like.
[0005] In an embodiment, a device, among others, includes: a first
fluid inlet in a flow channel for flowing a first liquid including
two or more types of particles, wherein the first liquid includes a
magnetic fluid and/or is mixed with the magnetic fluid; a magnetic
device configured to direct a non-uniform magnetic force onto the
magnetic fluid and the particles; and a plurality of outlets,
wherein the non-uniform magnetic force causes the types of
particles to be separated and flow into different outlets.
[0006] In an embodiment, a method for separating particles, among
others, includes: disposing at least two types of particles in a
first fluid, wherein the first liquid includes and/or is mixed with
the magnetic fluid; flowing the magnetic fluid and the particles
down a channel; exposing the magnetic fluid and the particles to a
non-uniform magnetic force; and separating the types of
particles.
[0007] Other structures, compositions, methods, features, and
advantages will be, or become, apparent to one with skill in the
art upon examination of the following drawings and detailed
description. It is intended that all such additional structures,
systems, methods, features, and advantages be included within this
description, be within the scope of the present disclosure, and be
protected by the accompanying claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Many aspects of this disclosure can be better understood
with reference to the following drawings. The components in the
drawings are not necessarily to scale, emphasis instead being
placed upon clearly illustrating the principles of the present
disclosure. Moreover, in the drawings, like reference numerals
designate corresponding parts throughout the several views.
[0009] FIG. 1A illustrates a schematic representation of the
sorting device with permanent magnets and a microfluidic channel.
FIG. 1B illustrates an image of protype device. Scale bar is 10 mm.
FIG. 1C illustrates a topview of the device and relevant
dimensions. The arrows indicate direction of magnets'
magnetization. FIG. 1D illustrates a cross-section of the
device.
[0010] FIGS. 2A-2I illustrate an analytical three-dimensional
simulation of magnetic field and force distributions in
microfluidic channel, and trajectories of cells. Simulation
parameters match exact experimental conditions. FIGS. 2A-2C show
the x-y plane (z=0), FIGS. 2D-2F illustrate the y-z plane (x=0),
FIGS. 2G-2I illustrate the x-z plane (y=0) of magnetic field
strength (surface plot) (FIGS. 2A, 2D, 2G), magnetic force (surface
plot: force magnitude; arrow plot: force direction) (FIGS. 2B, 2E,
2H), and particles' trajectories (FIGS. 2C, 2F, 2I). Dots indicate
starting points, while crosses indicate ending points of cells'
trajectories. E. coli cell has volume range of 2.1-16.7 .mu.m.sup.3
and Yeast cell has volume range of 180-382 .mu.m.sup.3, resulting
in a distribution of trajectories for each type of cell. The
triangle in FIG. 2C indicates boundary between Outlets C and D.
Dots indicate starting points, while crosses indicate ending points
of cells' trajectories.
[0011] FIGS. 3A-3B illustrate cell viability test of Escherichia
coli and Saccharomyces cerevisiae. FIG. 3A, top and bottom, are
photos showing Escherichia coli and Yeast colonies formed in M9
medium and EMG 408 ferrofluids after 10.sup.6 dilution from initial
growth, respectively. FIG. 3B illustrates Colony Forming Unites
(CFU) count of Escherichia coli and Saccharomyces cerevisiae using
initial growth cell concentration.
[0012] FIGS. 4A-4I illustrate experimental composite micrographs of
sorting process. FIGS. 4A, 4D, 4G illustrate particles/cells
mixture (FIG. 4A: Escherichia coli (green) and 7.3 .mu.m particles
(red); FIG. 4D: Saccharomyces cerevisiae (red and bright-field) and
1.0 .mu.m particles (green); FIG. 4G: Escherichia coli (green) and
Saccharomyces cerevisiae (red and bright-field) before magnetic
fields were applied. FIGS. 4B, 4E, 411 illustrate micrographs of
Outlet C after magnetic fields were applied, and FIGS. 4C, 4F, 4I
were micrographs of Outlet D. Blue triangles indicate boundary
between Outlets C and D. Scale bars represent 200 .mu.m.
[0013] FIGS. 5A-5L illustrate experimental composite micrographs of
size distribution analysis, including micrographs of
particles/cells mixture collected before sorting at Inlet A and
after separation at Outlets C and D, and remaining and separation
efficiencies. FIGS. 5A-5D illustrate Escherichia coli and 7.3 .mu.m
particles mixture; FIGS. 5E-511 illustrate Saccharomyces cerevisiae
and 1.0 .mu.m particles mixture; FIGS. 5I-5L illustrate Escherichia
coli and Saccharomyces cerevisiae mixtures. The bar with normal
number on top shows remaining efficiency, while the bar with italic
number on top shows separation efficiency. Scale bars represent 200
.mu.m.
[0014] FIG. 6A illustrates a schematic representation of the
sorting device with permanent magnets and a microfluidic channel.
FIG. 6B illustrates an image of protype device. Scale bar is 10 mm.
FIG. 6C illustrates a topview of the device and relevant
dimensions. The arrows indicate direction of magnets'
magnetization. FIG. 6D illustrates a cross-section of the
device.
[0015] FIG. 7 illustrates cell viability test of HeLa cell and red
blood cell in Hank's Balanced Salt Solution (HBSS) and PEG
ferrofluids, medium 1 is HBSS, medium 2 to 4 are PEG ferrofluid
with magnetic nanoparticles volume fraction of 0.395%, 0.79%, 1%
respectively. After 0, 1, and 2 hours incubation, HeLa cell and red
blood cell viability are counted with trypan blue dye exclusion
assay.
[0016] FIGS. 8A-8J illustrate experimental composite micrographs of
focusing and sorting process. FIGS. 8A-8D illustrate HeLa cell and
red blood cells in channel before magnetic fields were applied.
FIGS. 8E-8J were micrographs after magnetic fields were applied.
FIGS. 81I-8J illustrate micrographs of Outlet 1-6. Scale bars
represent 200 .mu.m.
[0017] FIG. 9 illustrates the separation efficiency verification of
cells distribution in each outlet after separation.
[0018] FIG. 10A illustrates a schematic representation of the
sorting device with permanent magnets and a microfluidic channel.
FIG. 10B illustrates an image of protype device. Scale bar is 10
mm. FIG. 10C illustrates a topview of the device and relevant
dimensions. The arrows indicate direction of magnets'
magnetization. FIG. 10D illustrates a cross-section of the
device.
[0019] FIGS. 11A-1111 illustrates an experimental composite
micrographs of sorting process. FIGS. 11A-11D were superimposed
micrographs of 15.5 .mu.m and 5.8 .mu.m particles particles
mixture. FIGS. 11E-1111 illustrates HeLa cell and 5.8 .mu.m
particles particles mixture. FIGS. 11A-11B, 11E, and 11F
illustrates micrographs of inlets and outlets before magnetic
fields were applied. FIGS. 11C, 11D, 11G, 1111 illustrate
micrographs of inlets and outlets after magnetic fields were
applied. Scale bars represent 200 .mu.m.
DISCUSSION
[0020] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, as such may, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present disclosure
will be limited only by the appended claims.
[0021] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
(unless the context clearly dictates otherwise), between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the disclosure.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the disclosure, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the disclosure.
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0023] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present disclosure
is not entitled to antedate such publication by virtue of prior
disclosure. Further, the dates of publication provided could be
different from the actual publication dates that may need to be
independently confirmed. Terms defined in references that are
incorporated by reference do not alter definitions of terms defined
in the present disclosure or should such terms be used to define
terms in the present disclosure they should only be used in a
manner that is inconsistent with the present disclosure.
[0024] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0025] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of chemistry, material science, and
the like, which are within the skill of the art. Such techniques
are explained fully in the literature.
[0026] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
and compounds disclosed and claimed herein. Efforts have been made
to ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.), but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C., and pressure is in
atmosphere. Standard temperature and pressure are defined as
25.degree. C. and 1 atmosphere.
[0027] Before the embodiments of the present disclosure are
described in detail, it is to be understood that, unless otherwise
indicated, the present disclosure is not limited to particular
materials, reagents, reaction materials, manufacturing processes,
or the like, as such can vary. It is also to be understood that the
terminology used herein is for purposes of describing particular
embodiments only, and is not intended to be limiting. It is also
possible in the present disclosure that steps can be executed in
different sequence where this is logically possible.
[0028] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a support" includes a plurality of
supports. In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings unless a contrary intention is
apparent.
DISCUSSION
[0029] Embodiments of the present disclosure provide for devices,
methods for separating particles, and the like. An embodiment of
the present disclosure is advantageous because it has a very high
sorting efficiency (e.g., about 95% or more, about 99% or more,
about 99.9% or more) and a very high throughput (e.g., about
10.sup.7 cells/hour or more, about 10.sup.8 cells/hour or more). In
addition, the device is less expensive than other techniques (e.g.,
FACS) and is straightforward to operate.
[0030] In general, embodiments of the present disclosure include
non-uniform magnetic field-assisted processes and devices for the
separation of particles (e.g., cells) within a magnetic fluid.
Under non-uniform magnetic fields, particles such as cells can
experience the generated magnetic field direction to produce a
magnetic buoyancy force, analogous to buoyancy force, as magnitude
of the force is proportional to the volume of cell. This force can
be used to spatially separate cells of different sizes in certain
flow conditions (e.g., laminar flow). Embodiments of the present
disclosure can be label-free and/or do not require time-consuming
steps of magnetic beads conjugation. Embodiments of the present
disclosure include high-efficiency and high-throughput
continuous-flow particle separation and focusing devices using
magnetic fluid (e.g., ferrofluids) and magnets (e.g., permanent
magnets). Permanent magnet based devices are low-cost and easy to
operate and their operations does not generate heat. Magnetic
fields produced by permanent magnets are substantially larger than
the ones by current-carrying electrodes, which can increase the
sorting throughput and efficiency of embodiments of the present
disclosure.
[0031] In an exemplary embodiment, the device includes a first
fluid inlet in a flow channel for flowing a first liquid including
two or more types of particles, where the first liquid includes a
magnetic fluid. In an embodiment, additional inlets can be present
to introduce other reagents or fluids. For example, the cells can
be flowed in the first fluid inlet and the magnetic fluid can be
flowed in a second fluid inlet, where the two fluids mix in or
prior to introduction to the channel. In an embodiment, the flow
rate of the fluid(s) can be controlled and the flow rate can be
used to enhance the separation.
[0032] In an embodiment the channel can have a constant diameter
along its length. In another embodiment, the channel can have a
tapered diameter. In an embodiment, the flow chamber can be
designed to optimize the separation of the particles.
[0033] In an embodiment, the channel can have a length of about
5000 .mu.m to 20000 .mu.m prior to splitting into two or more
outlets (e.g., 2 to 100), and a diameter of about 1000 to 2000
.mu.m. In an embodiment, the outlets (e.g., outlet channels) can
have the same or different diameters and can independently have a
diameter of about 500 to 2000 .mu.m. In an embodiment, the outlets
can be designed (e.g., diameter, three-dimensional orientation
relative to the channel (e.g., offset from the axis of the
channel), and the like) to enhance the separation of the
particles.
[0034] In an exemplary embodiment of the device, a magnetic device
configured to direct a non-uniform magnetic force onto particles is
positioned at a point of the channel. In an embodiment, the
magnetic device can be positioned relative to the split from the
channel to the outlets. In an embodiment, the magnetic device is
configured to direct the non-uniform magnetic force onto particles
from one side of the channel. As noted above, the design of the
device (e.g., the position of the magnetic device and/or the
outlets) can take into consideration the various components, the
particles to be separated, and/or the magnetic fluid, to achieve
the desired separation efficiency and/or throughput.
[0035] In an embodiment, the magnetic energy can be produced using
a magnetic device that includes one or more permanent magnets
positioned to produce a non-uniform magnetic field in at least a
portion of the channel. In an embodiment, one permanent magnet is
disposed on one side of the channel to generate the non-uniform
magnetic force. The strength of the magnetic field can be selected
based upon the configuration of the device, the particles to be
separated (e.g., the volume of the particles), and the like. In
another embodiment, the magnetic device includes three or more
magnets (e.g., 3, 4, 5, 6, 7, and so on) that can be used to form a
non-uniform magnetic field within an area of the channel. The
design, number of magnets used, the non-uniform magnetic field
generated, and the like, can be designed to separate particles.
[0036] As noted above, the device includes a plurality of outlets.
Once the non-uniform magnetic force acts upon the particles, the
particles flow in the first fluid is altered so that certain types
of particles flow into one outlet and another type of particle
flows into a different outlet.
[0037] In an embodiment where many different types of particles are
to be separated, then the outlets can be spaced apart along the
length of the channel and/or more than one magnet can be used along
the length of the channel in conjunction with the spacing of the
outlet. Many different types of configurations are envisioned that
are consistent with the teachings of the present disclosure and are
intended to be covered by claims of this and future
application.
[0038] In an embodiment, the particles can experience non-uniform
magnetic force and are biologically compatible with the magnetic
fluid. In particular, the particles can be separated by the
magnetic buoyancy force exerted upon them. In an embodiment, the
particles can include cells, polystyrene microparticles, and a
combination thereof. In an embodiment, the cells can include
bacterial cells, yeast cells, blood cells, cancer cells, neural
cells, sperm cells, eggs, as well as types of cells that have size
difference can be distinguished by this technique. In an
embodiment, the cells can include Escherichia coli, Saccharomyces
cerevisiae, Lactobacillus casei, red blood cells, Jurkat cells, and
HeLa cells. In an embodiment, the volume of the cells can be about
5 to 3000 .mu.m.sup.3.
[0039] In an embodiment, the particles are mixed with a magnetic
fluid (e.g., prior to introduction to the device and/or within the
device). In an embodiment, the magnetic fluid is a colloidal
mixture of nano-size magnetic particles (e.g., about 5 to 10 nm in
diameter), covered by a surfactant, suspended in a compatible
carrier medium. In an embodiment, the magnetic particles can be
iron oxide particles, cobalt particles, cobalt ferrite particles,
iron particles, and FePt particles, or a combination thereof, where
the amount of the magnetic particles in the magnetic fluid can be
about 1% (v/v) to 10% (v/v). In an embodiment, the surfactant can
include electric double layer surfactant, polymer surfactant,
inorganic surfactant, or a combination thereof. In an embodiment,
the carrier medium can include water, hydrocarbon oil, kerosene, or
a combination there. In an embodiment, the magnetic fluid can be a
ferrofluid, paramagnetic solution, or a combination thereof.
[0040] As mentioned above, embodiments of the present disclosure
include a method for separating particles, where the device
described herein can be used to perform steps of the method. In an
embodiment, the method is a continuous flow method. In an
embodiment, the at least two types of particles are disposed in
fluid including a magnetic fluid (e.g., a first fluid). In an
embodiment, the fluid including the particles and the magnetic
fluid are flowed down the channel. At a position in the channel
(e.g., a first area), the fluid is exposed to a non-uniform
magnetic force, where a magnetic device can be used to generate the
non-uniform magnetic force. In an embodiment, the particles
experience a magnetic buoyancy force that causes the particles to
separate from one another based on the volume of the particles. In
an embodiment, the particles can be separated from one another into
two or more outlets.
[0041] In an embodiment, this process can be repeated for the
particles that are separated to increase efficiency and/or separate
particles having similar characteristics (e.g., volume). For
example, the separated flow can be recirculated through the same
channel or can be flowed through a different channel is a device
include two or more channels and magnets.
EXAMPLES
[0042] Now having described the embodiments of the disclosure, in
general, the example describes some additional embodiments. While
embodiments of the present disclosure are described in connection
with the example and the corresponding text and figures, there is
no intent to limit embodiments of the disclosure to these
descriptions. On the contrary, the intent is to cover all
alternatives, modifications, and equivalents included within the
spirit and scope of embodiments of the present disclosure.
Example 1
Brief Introduction
[0043] A new sorting scheme based on ferrofluid hydrodynamics
(ferrohydrodynamics) was used to separate mixtures of particles and
live cells simultaneously. Two species of cells, including
Escherichia coli and Saccharomyces cerevisiae, as well as
fluorescent polystyrene microparticles were studied for their
sorting throughput and efficiency using a commercial ionic
ferrofluid. To separate mammalian cells including blood cells,
cervical cancerous cells and epithelial cells, a lab-customized
ferrofluid was used. Ferrofluids are stable magnetic nanoparticles
suspensions. Under external magnetic fields, magnetic buoyancy
forces exerted on particles and cells lead to size-dependent
deflections from their laminar flow paths and result in spatial
separation. We report the design, modeling, fabrication and
characterization of the sorting devices. This scheme is simple,
low-cost and label-free compared to other existing techniques.
INTRODUCTION
[0044] Microfluidic particle and cell sorting plays an important
role in environmental monitoring (Liu et al. 2004; Beyor et al.
2008; Dharmasiri et al. 2010), disease diagnostics (Nagrath et al.
2007; Adams et al. 2008; Hoshino et al. 2011), and therapeutics
(Toner and Irimia 2005; Yung et al. 2009). Compared to
high-specificity and label-based cell sorting techniques such as
fluorescence-activated cell sorter (FACS) (Bonner et al. 1972) and
magnetic-activated cell sorter (MACS) (Miltenyi et al. 1990),
microfluidic sorting is mostly label-free, relying on cells'
intrinsic properties such as size, shape, density, deformability,
electric and magnetic properties for manipulation specificity
(Pamme 2007; Tsutsui and Ho 2009; Gossett et al. 2010; Lenshof and
Laurell 2010). When applicable, microfluidic sorting is favored
over label-based ones, because they are inexpensive and require
minimal user training for operation (Gossett et al. 2010). Among
them, those based on channel design including pinched flow
fractionation (Yamada et al. 2004) and deterministic lateral
displacement (Huang et al. 2004; Davis et al. 2006) combine laminar
flows with mechanical structures to direct particles of different
sizes into separate streamlines. Continuous inertial separation
uses balance between inertial lift force and Dean drag force in
curved channels for size-dependent sorting of particles and cells
(Di Carlo 2009). On the other hand, external energy inputs such as
acoustic, electric and magnetic forces have also been used to
manipulate cells in microfluidic systems. Depending on the
application, their simpler channel geometry and faster manipulation
speed may outweigh the complications of integrating electrodes in
their designs. For example, acoustophoresis can separate particles
and cells according to their size, density, as well as
compressibility (Laurell et al. 2007; Shi et al. 2009; Wang and Zhe
2011). Dielectrophoresis (DEP), arising from interactions of cells'
dipoles and their surrounding electric fields, can realize low-cost
and integrated devices for cell manipulation (Voldman 2006).
Magnetophoresis (MAP) takes advantages of paramagnetic nature of
red blood cells and magnetotactic bacteria and applies non-uniform
magnetic fields to separate them from non-magnetic objects
(Zborowski et al. 2003; Lee et al. 2004). However, most
applications of magnetophoresis use functionalized magnetic beads
for labeling (Pamme 2006; Liu et al. 2009; Gijs et al. 2010). The
label-based methods are manually intensive and time-consuming. The
magnetic moments of these beads, even from the same batch, can vary
dramatically due to their manufacturing procedure, making scaling
of the method scaling difficult (Hafeli et al. 1997; Miller et al.
2001; Rife et al. 2003; Mihajlovic et al. 2007; Shevkoplyas et al.
2007).
[0045] To address problems with label-based magnetophoresis, a
label-free technique that uses reverse magnetophoresis to
manipulate and sort cells has been developed recently based on
ferrofluid hydrodynamics (ferrohydrodynamics) (Yellen et al. 2005;
Kose et al. 2009; Zhu et al. 2010; Zhu et al. 2011a; Kose and Koser
2012). Ferrofluids are colloidal suspensions of magnetic
nanoparticles, typically magnetite (Fe.sub.3O.sub.4) with
approximately 10 nm diameters (Rosensweig 1985). They are covered
by either electrostatic or steric surfactants to keep them from
agglomeration due to van der Waals force and in suspension within a
water or oil medium. Ferrohydrodynamics studies mechanics of
ferrofluid motion under external magnetic fields (Rosensweig 1985;
Odenbach and Editor 2002). Its applications in microfluidics,
recently reviewed by Nguyen (Nguyen 2012), include miniaturized
polymerase chain reaction (PCR) (Sun et al. 2007; Sun et al. 2008),
traveling-wave magnetic field pumping (Mao and Koser 2006; Mao et
al. 2011), micro-scale mixing (Mao and Koser 2007), micropump
(Hatch et al. 2001; Love et al. 2004), and droplet manipulation
(Nguyen et al. 2006; Zhang et al. 2011b, a).
[0046] In applications of cell manipulation, the purpose of using
ferrofluids is to induce effective magnetic dipole moments within
cells. Under non-uniform magnetic fields, cells will experience in
the weaker field direction a magnetic buoyancy force, analogous to
buoyancy force, as magnitude of the force is proportional to the
volume of cell (Rosensweig 1985). Many groups have been working on
adapting this principle to particles and cells sorting. For
example, Whitesides' group separated synthetic particles according
to their densities' difference using paramagnetic salt solutions
(Winkleman et al. 2007; Mirica et al. 2009). Pamme's group
demonstrated continuous particle and cell manipulation using
paramagnetic salt solution in microfluidic devices (Peyman et al.
2009; Rodriguez-Villarreal et al. 2011). Xuan's group studied the
transport of particles in both paramagnetic solutions and
ferrofluids through a rectangular microchannel embedded with
permanent magnets (Liang et al. 2011; Zhu et al. 2012). Park's
group recently sorted human histolytic lymphoma monocytes cells
from red blood cells using gadolinium diethylenetriamine
pentaacetic acid (Gd-DTPA) solution (Shen et al. 2012). However,
magnetic susceptibility of paramagnetic salt solutions is
inherently small, about 5 orders of magnitude weaker than that of a
ferrofluid (Krebs Melissa et al. 2009), rendering slower
manipulation speed and low throughput. As a result of the higher
susceptibility of ferrofluids, Koser's group was able to use an
integrated microfluidic platform for sorting of microparticles and
live cells within a citrate stabilized cobalt-ferrite ferrofluid in
static flow conditions (Kose et al. 2009). The same device was also
applied to continuous-flow frequency-adjustable particles
separation (Kose and Koser 2012). Our group developed
high-efficiency and high-throughput continuous-flow particle
separation and focusing devices using commercial ferrofluids and
hand-held permanent magnets (Zhu et al. 2010; Zhu et al. 2011b; Zhu
et al. 2011a). Permanent magnet based devices are low-cost and easy
to operate; their operations do not generate heat. Magnetic fields
produced by permanent magnets are substantially larger than the
ones by current-carrying electrodes.
[0047] High throughput, label-free and selective cell sorting
realized in a single automated device can have profound impacts on
environmental monitoring, diagnostics and therapeutics. Although
continuous-flow ferrohydrodynamic sorting has been demonstrated
with microparticles, it has not previously been reported with live
cells (Zhu et al. 2010). The potential for live cell applications
of continuous-flow ferrohydrodynamic sorting motivates the study
presented here. We developed a microfluidic device that could
continuously sort cells of different sizes based on
ferrohydrodynamics, which involved manipulation of cells within
ferrofluids via external non-uniform magnetic fields. When cell
mixtures and ferrofluids were injected into the channel by a
pressure-driven flow, deflections of cells from their laminar flow
paths would occur because of the magnetic field gradient and
resulting magnetic buoyance force. This deflection will lead to
spatial separation of cells of different sizes at the end of
channel.
[0048] In the following sections, we first summarize materials and
methods used in this study, followed by results from a
three-dimensional theoretical study of cells' transport in the
microfluidic device. In the first embodiment, cell viabilities of
Escherichia coli and Saccharomyces cerevisiae in a commercial
ferrofluid are discussed. Afterwards, calibration of the sorting
device with fluorescent polystyrene microparticles is performed.
Escherichia coli and Saccharomyces cerevisiae are sorted in the
device, and cells distribution is analyzed on samples collected
from channel outlets. In the second embodiment, cell viabilities of
red blood cells, HeLa cells, in the customized ferrofluid are
tested. A stronger magnetic field gradient is applied to enable
particles focusing. Calibration of the sorting device with
polystyrene microparticles of comparable size is performed before
sorting the cells. Cell distribution is analyzed on samples
collected from six channel outlets. In the third embodiment, a new
one-step separation micro-device without the need of washing cells
after enrichment was developed. This device takes in cells sample
in its natural reagent or ferrofluids, separate them based on their
sizes, and return purified cells to an outlet. It eliminates the
preparation and washing steps typically associated with our device.
In the end we will discuss outlook of ferrohydrodynamic
sorting.
Materials and Methods:
[0049] The prototype polydimethylsiloxane (PDMS) microfluidic
device was fabricated through a standard soft-lithography approach
and attached to a flat surface of another piece of PDMS, as shown
in FIGS. 1A-1B. A mask of the device pattern was created using
AutoCAD 2008 (Autodesk Inc., San Rafael, Calif.) and printed by a
commercial photo-plotting company (CAD/Art Services Inc, Bandon,
Oreg.). In the first embodiment, dimensions of the microfluidic
channel are listed in FIGS. 1C-1D. Thickness of the device was
measured to be 38 .mu.m by a profilometer (Dektak 150, Veeco
Instruments Inc., Chadds Ford, Pa.). Before attachment, PDMS
surfaces were treated with plasma (PDC-32G plasma cleaner, Harrick
Plasma, Ithaca, N.Y.) at 11.2 Pa 02 partial pressure with 18 W
power for 1 minute. A stack of four NdFeB permanent magnets was
embedded into PDMS channel with their magnetization direction
vertical to the channel during curing stage. Each magnet is 5 mm in
width, 5 mm in length and 2 mm in thickness. The magnet stack was
placed 2 mm away from the channel. Flux density at the center of
magnets stack's surface was measured to be 470 mT by a Gauss meter
(Model 5080, Sypris, Orlando, Fla.) and an axial probe with 0.381
mm diameter of circular active area. In the second embodiment,
dimensions of the microfluidic channel are listed in FIGS. 6C-6D.
Thickness of the device was measured to be 50 .mu.m by a
profilometer (Dektak 150, Veeco Instruments Inc., Chadds Ford,
Pa.). A NdFeB permanent magnets was embedded into PDMS channel with
their magnetization direction vertical to the channel during curing
stage. The magnet is 2.54 cm in width, 6.35 mm in length and
thickness. The magnet stack was placed 1 mm away from the channel.
Flux density at the center of magnets stack's surface was measured
to be 470 mT. In the third embodiment, dimensions of the
microfluidic channel are listed in FIGS. 10C-10D. Thickness of the
device was measured to be 50 .mu.m by a profilometer (Dektak 150,
Veeco Instruments Inc., Chadds Ford, Pa.). A NdFeB permanent
magnets was embedded into PDMS channel with their magnetization
direction vertical to the channel during curing stage. The magnet
is 2.54 cm in width, 3.175 mm in length and thickness. The magnet
stack was placed 2 mm away from the channel. Flux density at the
center of magnets stack's surface was measured to be 470 mT. Before
liquid injection, the device was treated with plasma for 10 minutes
to render PDMS surfaces hydrophilic. This step ensured both cells
and microparticles would not attach onto PDMS surfaces during
sorting.
[0050] In the first embodiment, we used a commercial water-based,
pH .about.7 magnetite ferrofluid coated with anionic surfactants
(EMG 408, Ferrotec Co., NH). Volume fraction of magnetite particles
in this ferrofluid is 1.1%. Mean diameter of nanoparticles has been
determined from Transmission Electron Microscopy (TEM) images to be
.about.10 nm. Initial magnetic susceptibility was measured to be
0.26; saturation magnetization was 60 Gauss; viscosity was
1.2.times.10.sup.-3 kg/ms. Escherichia coli (strain MG1655) and
Saccharomyces cerevisiae (Baker's yeast), and two fluorescent
microparticles (green 1.0 .mu.m diameter, Thermo Fisher Scientific
Inc., Waltham, Mass., and red 7.3 .mu.m diameter, Bangs
Laboratories Inc., Fishers, Ind.) were used in sorting. In the
second and third embodiment, we used a water-based, pH .about.7
maghemite ferrofluid coated with polyethylene glycol copoplymer
(ATLOX4913, Croda. Inc). Volume fraction of magnetite particles in
this ferrofluid is 1%. Mean diameter of nanoparticles has been
determined from Transmission Electron Microscopy (TEM) images to be
.about.5.13 nm. Initial magnetic susceptibility was measured to be
0.098; saturation magnetization was 2.93 kA/m.
[0051] Ferrofluid and particles/cells mixture injected into
microchannel were maintained at tunable flow rates using a syringe
pump (Nexus 3000, Chemyx Inc., Stafford, Tex.). Sorting was
conducted on the stage of an inverted microscope (Zeiss Axio
Observer, Carl Zeiss Inc., Germany). Micrographs of cells and
particles were recorded through either a green fluorescent filter
set (41001 FITC, Chroma Technology Corp., Rockingham, Vt.), or a
red filter set (43HE, Carl Zeiss Inc., Germany), and a CCD camera
(SPOT RT3, Diagnostic Instruments, Inc., Sterling Heights, Mich.).
Cell samples collected from channel outlets were pipetted onto
microscope slides and analyzed using a high-resolution CCD camera
(AxioCam HR, Carl Zeiss Inc., Germany) for size distributions to
quantitatively evaluate efficiency of this approach. ImageJ.RTM.
software was used to count the number of cells.
[0052] Saccharomyces cerevisiae (Baker's yeast) cells were first
grown in a 10 ml test tube containing 2 ml of YPG medium (10 g/l
yeast extract, 20 g/l glucose, 20 g/l glucose) overnight. They were
then transferred into a 100 ml shake flask containing 20 ml of YPG
medium. After 4 h growth at 30.degree. C. and 250 rpm, cells in the
flask were stained with fluorophores. Escherichia coli (strain
MG1655) cells were first grown in a 10 ml test tube containing 2 ml
of Luria-Bertani (LB) medium overnight. They were then transferred
into a 100 ml shake flask containing 20 ml of LB medium (25 g/l
LB). After 4 h growth at 37.degree. C. and 250 rpm, cells were
stained with fluorophores. Nucleic acid stains SYTO9 (green) and
SYTO17 (red) (Molecular Probes Inc., Eugene, Oreg.) were used in
cell staining.
[0053] Hela cells were cultured in culture flasks (BD Falcon)
containing 12 mL of DMEM medium with 10% (v/v) fetal bovine serum,
100 U/mL penicillin, and 100 .mu.g/mL streptomycin. All cell lines
were incubated (5% CO2, 90% humidified) at 37.degree. C. in an
incubator (Innova-Co 170; New Brunswick Scientific, U.K.) prior to
use. Cells were subcultured at a ratio of 1:5 every 3 days to
maintain cells in the exponential growth phase. Cells were detached
from the flask with the treatment of 0.25% (w/v) trypsin EDTA
solution (Gibco) for 3 min for harvest. Cells were then suspended
in the culture media at a concentration of 2.times.10.sup.6
cells/mL before use.
[0054] To study of viability of Escherichia coli and Saccharomyces
cerevisiae cells exposed to EMG 408 ferrofluids, nominally
2.times.10.sup.9 cells Escherichia coli and 2.times.10.sup.7 cells
Saccharomyces cerevisiae grown as described above were centrifuged
twice at 4.degree. C. and washed in defined M9 medium (6.78 g/l
Na.sub.2HPO.sub.4, 3.0 g/l KH.sub.2PO.sub.4, 0.5 g/l NaCl, 1.0 g/l
NH.sub.4Cl) without carbon source. For either cell type in
duplicate, the washed cell pellet from centrifugation was combined
with either 2 ml of EMG 408 ferrofluid or 2 ml M9 medium as a
control. After 2 hours of incubation at room temperature in these
fluids, cell density was determined in triplicate using standard
microbial serial dilutions (10.sup.6 dilution for Escherichia coli,
and 10.sup.4 dilution for Saccharomyces cerevisiae), with the
transferring of known volumes to Petri plates and counting of
Colony Forming Units (CFU) after 24 hours.
[0055] To study the viability of HeLa, red blood cells exposed to
PEG-ferrofluids, nominally 2.times.10.sup.6 cells grown as
described above were centrifuged twice at 4.degree. C. and washed
in Hank's buffer solution (HBSS). For either cell type in
duplicate, the washed cell pellet from centrifugation was combined
with either 1 ml of PEG ferrofluid or 1 ml HBSS as a control. After
2 hours of incubation at room temperature in these fluids, cell
viability was determined with trypan blue staining and counted with
a haemocytometer.
Theory and Simulation:
[0056] Previously, we reported both two-dimensional (2D) and
three-dimensional (3D) analytical models for microfluidic
transports of microparticles in ferrofluids (Zhu et al. 2011a; Zhu
et al. 2011b). In this work, we applied the 3D analytical model to
predict cells' sorting in permanent magnet based device. Briefly,
we obtained cells' trajectories by first calculating magnetic
buoyancy force on cells using a 3D analytical model of magnetic
fields (Furlani and Sahoo 2006) and a nonlinear magnetization model
of ferrofluids (Rosensweig 1985), and then solving governing
equations of motion for cells in laminar flow condition (Brody et
al. 1996). All relevant parameters used in our simulation are
listed in FIGS. 1A-1D and Materials and Methods section. In
addition, we calculated volume of a single rod-shape Escherichia
coli cell with short axis of 0.5-1 .mu.m and long axis of 2-4 .mu.m
to be 2.1-16.7 .mu.m.sup.3 (Kaya and Koser 2009), and volume of a
single sphere-shape Saccharomyces cerevisiae cell with diameter of
7-9 .mu.m to be 180-382 .mu.m.sup.3 (Jorgensen et al. 2002).
[0057] FIGS. 2A-2I summarizes simulated distribution of magnetic
fields and magnetic buoyance forces in the sorting channel, as well
as 3D trajectories of Escherichia coli and Saccharomyces cerevisiae
cells. The surface plot in FIG. 2A shows magnitude of magnetic
fields of x-y plane at z=0. Magnetic fields decayed rather quickly
from the surface of the magnet and formed a gradient that resulted
in magnetic buoyance force on cells in both x and y directions, as
indicated in FIG. 2B. Consequently, cells experiencing such force
when entering the sorting channel would decelerate in x direction
and accelerate in y direction. Force computed on a spherical
microparticle of 7.3 .mu.m diameter, with its total volume
(.about.200 .mu.m.sup.3) close to that of a single Saccharomyces
cerevisiae cell, is on the order of 10 pN. Cell mixtures were
quickly sorted by magnetic buoyancy force towards the end of
channel, as shown in FIG. 2C with simulated cells' trajectories
considering their natural size variations. All Escherichia coli
cells, having much smaller size and volume compared to
Saccharomyces cerevisiae cells, exited the channel through Outlet
D, while all Saccharomyces cerevisiae cells migrated towards Outlet
C. FIGS. 2D-2F illustrate distribution of magnetic fields and
forces, as well as trajectories of cells of y-z plane at x=0; FIGS.
2G-2I depict the cases of x-z plane at y=0. We are interested in 3D
trajectories of cells, in part due to the opaqueness of ferrofluids
and difficulty in recording cells' weak fluorescence in the
channel, especially the red fluorescent from Saccharomyces
cerevisiae cells, as shown later in the results. In a concentrated
ferrofluid (.about.10% v/v), particles and cells are visible only
when they are very close (.about.1 .mu.m) to the surface of channel
(Zhu et al. 2011b). Visibility was a less of a problem when diluted
ferrofluids (.about.1% v/v) and thin microchannel were used in our
device. Simulation results from FIG. 2F and FIG. 2I indicated in
our current setup all cells were pushed towards the channel bottom
surface, which would enhance visibility of stained cells.
Results and Discussions:
Cell Viability
[0058] FIG. 3A shows the CFU in both M9 medium and EMG 408
ferrofluids after incubation. Counts of CFU for each case were
averaged over 3 plates and plotted in FIG. 3B. We observed a slight
increase in cell density after 2 hours of incubation in the
ferrofluid compared to the M9 medium control for both cell types,
suggesting a possibility that either the EMG 408 ferrofluid acted
as a cell protectant or the cells continued to grow in this
ferrofluid during incubation. Nonetheless, this ferrofluid was not
detrimental to the viability of both cell types after 2 hours of
exposure, which allowed enough time to carry out the sorting
procedure.
[0059] FIG. 7 shows the viability of HeLa cells and mouse red blood
cells in both HBSS and PEG ferrofluids after incubation. We counted
the cell numbers with trypan blue viability staining after 2 hours
of incubation in the ferrofluid compared with the HBSS control for
both cell types,
Cells Sorting:
[0060] In the first embodiment, we first calibrated the sorting
device using a mixture of Escherichia coli cells and red
fluorescent 7.3 .mu.m particles, which have similar total volume of
Saccharomyces cerevisiae cells. Washed Escherichia coli cell pellet
from centrifugation as described above was stained with 1 .mu.l of
green nucleic acid stain SYTO9. Both particles and cells have
concentrations of .about.10.sup.7 counts/ml. We introduced
microparticles/cells mixture into microfluidic channel Inlet A at a
constant flow rate of 1.5 .mu.l/min. The mixture was
hydrodynamically focused into a narrow stream by sheath flow from
Inlet B at a flow rate of 6 .mu.l/min. The observation window was
located right before the channel outlets, as indicated in FIG. 1C.
When magnetic fields were off, particles and cells were observed in
fluorescent mode flowing together near sidewall of the channel and
exiting through Outlet D, as shown in composite micrograph of FIG.
4A. When magnetic fields were on, magnetic buoyancy forces
deflected particles from their laminar flow paths towards Outlet C,
as shown in FIG. 2B. On the other hand, forces on smaller
Escherichia coli cells were inadequate to deflect them to Outlet C;
therefore they exited the channel through Outlet D still, as shown
in FIG. 2C. This resulted in spatial separation of particles/cells
mixture at the end of channel. We were able to separate
.about.10.sup.6 particles from .about.10.sup.6 cells per hour with
1.5 .mu.l/min flow rate. Simply increasing the flow rate can
further increase sorting throughput. Current microfluidic sorting
schemes use flow rates ranging between .about.10 .mu.l/min and
.about.1 ml/min (Gossett et al. 2010). With such flow rates and
10.sup.7-10.sup.8 cells/ml concentration, maximum sorting
throughput of our device in theory can go up to 10.sup.9 cells per
hour.
[0061] Secondly, we calibrated the device using a mixture of
Saccharomyces cerevisiae cells and green fluorescent 1.0 .mu.m
particles, which have similar volume as Escherichia coli cells.
Saccharomyces cerevisiae were stained with red nucleic acid stain
SYTO17. Both particles and cells again have concentrations of
.about.10.sup.7 counts/ml. Due to weak red fluorescence from SYTO17
in our setup, we chose to use a combination of bright-field and
fluorescent modes microscopy to record the sorting process. FIG. 4D
shows merged composite micrograph of green fluorescent 1.0 .mu.m
particles and bright-field particles/Saccharomyces cerevisiae
mixture, both of which exited channel through Outlet D when
magnetic fields were off. Sorting of this mixture was achieved as
soon as magnetic fields were on, as depicted in FIGS. 4E-4F. Cells
distribution analysis presented in the following section confirmed
a close to 100% sorting efficiency. Sorting throughput was
.about.10.sup.6 cells per hour. Here we demonstrated that
combination of bright-field and fluorescent microscopy can
successfully circumvent recording issues originating from
opaqueness of ferrofluids and weak fluorescence from stained live
cells.
[0062] Finally, sorting of Escherichia coli and Saccharomyces
cerevisiae cells were carried out in the same device at the same
time. Escherichia coli cells were stained with green fluorescence
while Saccharomyces cerevisiae were stained with red fluorescence.
Both types of cells were adjusted to .about.10.sup.7 cells/ml
concentration in initial mixture. It is clearly shown in FIG. 4G
that all cells exited from the channel through Outlet D when there
was no magnetic field. Both bright-field and fluorescent mode
micrographs of cells were recorded and merged to form FIG. 4G.
Saccharomyces cerevisiae cells were successfully sorted from the
initial cell mixture with the application of magnetic fields, as
demonstrated in FIGS. 4H-4I.
[0063] In the second embodiment, mouse red blood cell pellet were
collected from centrifugation of whole blood. Both red blood cells
and Hela cells have concentrations of 2.times.10.sup.6 counts/ml.
We introduced cells mixture into microfluidic channel Inlet A at a
constant flow rate of 8 .mu.l/min. The mixture was hydrodynamically
focused into a narrow stream by sheath flow from Inlet B at a flow
rate of 14 .mu.l/min. The observation window was located at four
different sections, as indicated in FIGS. 8A-8D. When magnetic
fields were off, particles and cells were flowing together near
sidewall of the channel and exiting through Outlet 1, as shown in
composite micrograph of FIG. 8D. When magnetic fields were on,
magnetic buoyancy forces deflected particles towards the inlet wall
as shown in FIG. 8E, then hydrodynamically focused into a narrow
stream as shown in FIG. 8F. HeLa Cells were separated due to the
larger size as shown in FIG. 8G and deflected towards outlet 5-6,
as shown in FIG. 8J. On the other hand, forces on smaller red blood
cells were inadequate; therefore they exited the channel through
Outlet 1-4, as shown in Figures FIGS. 8H-8I. This resulted in
spatial separation of cells mixture at the end of channel.
[0064] FIGS. 11A-11D were superimposed micrographs of 15.5 .mu.m
and 5.8 .mu.m particles particles mixture. FIGS. 11E-1111
illustrates HeLa cell and 5.8 .mu.m particles particles mixture.
FIG. 11A, FIG. 11B, FIG. 11E, and FIG. 11F illustrates micrographs
of inlets and outlets before magnetic fields were applied. FIG.
11C, FIG. 11D, FIG. 11G, and FIG. 1111 illustrate micrographs of
inlets and outlets after magnetic fields were applied.
[0065] In the third embodiment, we demonstrated sorting process
using a mixture of 5.8 .mu.m and 15.5 .mu.m polystyrene particles.
Both particles and cells have concentrations of
.about.2.times.10.sup.6 counts/ml. We introduced
microparticles/cells mixture in HBSS or ferrofluids into
microfluidic channel Inlet A at a constant flow rate of 1.5
.mu.l/min. The mixture was hydrodynamically focused into a narrow
stream by sheath flow from Inlet B at a flow rate of 4 .mu.l/min
and a third HBSS flow at a flow rate of 6 .mu.l/min. The
observation window was located right before the channel outlets, as
indicated in FIG. 10C. When magnetic fields were off, particles and
cells were flowing together near sidewall of the channel and
exiting through Outlet 1, as shown in composite micrograph of FIG.
11B. When magnetic fields were on, magnetic buoyancy forces
deflected particles from their laminar flow paths towards Outlet 3,
as shown in FIG. 11D. On the other hand, forces on smaller
Escherichia coli cells were inadequate to deflect them to Outlet 3;
therefore they exited the channel through Outlet 1 and 2, as shown
in FIG. 11D. This resulted in spatial separation of particles/cells
mixture at the end of channel. Simply increasing the flow rate can
further increase sorting throughput. Current microfluidic sorting
schemes use flow rates ranging between .about.10 .mu.l/min and
.about.1 ml/min (Gossett et al. 2010). With such flow rates and
10.sup.7-10.sup.8 cells/ml concentration, maximum sorting
throughput of our device in theory can go up to 10.sup.9 cells per
hour.
Cell Sorting Efficiency:
[0066] In order to precisely evaluate sorting efficiency, in the
first embodiment, we collected samples from both Outlets C and D
and analyzed them for size distributions off chip. We stained cells
in distinctive fluorescence and counted them using ImageJ.RTM.
software. Specifically, in first calibration, Escherichia coli
cells were green and 7.3 .mu.m particles were red; in second
calibration, Saccharomyces cerevisiae cells were red and 1.0 .mu.m
particles were green; in cells sorting, Saccharomyces cerevisiae
cells were red and Escherichia coli cells were green. Fluorescent
mode was chosen for distribution analysis to avoid miscounting of
cell types in bright-field micrographs. A magnetic field was
applied to push all particles and cells onto a surface of glass
slide to increase visibility. We define remaining efficiency as
ratio of number of particles or cells exiting from Outlet D after
magnetic field application to their initial number before magnetic
field application. Similarly, sorting efficiency is defined as the
ratio of number of particles or cells exiting from Outlet C after
magnetic field application to their initial number before magnetic
field application. FIG. 5A shows a representative composite
micrograph of Escherichia coli cells and 7.3 .mu.m particles
collected from Inlet A before sorting. 100% of 7.3 .mu.m particles
migrated to Outlet C and 98.8% Escherichia coli cells remained in
Outlet D, as depicted in FIGS. 5B-5C. Remaining and separation
efficiencies for both particles are plotted in FIG. 5D. FIGS.
5E-511 and FIGS. 5I-5L show micrographs and efficiencies for
Saccharomyces cerevisiae cells/1.0 .mu.m particles mixture sorting
and Saccharomyces cerevisiae cells/Escherichia coli cells mixture
sorting, respectively. Both cases have 100% efficiencies. It should
be noted that samples collected from Outlets C and D were greatly
diluted by ferrofluid sheath flow from Inlet B, rendering much
lower particles and cells concentration for distribution analysis.
A possible solution to this problem is integration of cell focusing
(Zhu et al. 2011a) and sorting steps on one chip.
[0067] In the second embodiment, we collected samples from multiple
outlets and analyzed them for size distributions with a
haemocytometer. Phase contrast microscopy was used to visualize
cells in bright-field micrographs. FIG. 9 shows the cells
distribution in each outlet. 100% of HeLa cells migrated to Outlet
5 and 6 while 100% mouse red blood cells migrated to Outlet
1-4.
[0068] In the third embodiment, 100% of 15.5 .mu.m particles
migrated to Outlet 3 and 100% 5.8 .mu.m particles migrated to
Outlet 1 and 2, as counted with a haemocytometer.
Outlooks of Ferrohydrodynamic Sorting:
[0069] Ferrohydrodynamic cell sorting offers the potential for high
throughput (.about.10.sup.7 cells/hour in this study and
.about.10.sup.9 cells/hour in theory) and high separation
efficiency (.about.100%) that are comparable to existing
microfluidic sorting techniques but without the use of labels. The
associated device is inexpensive and simple, only requiring a
channel and hand-held permanent magnets. Sorting specificity of
this approach is not limited to size difference only; it is also
sensitive to cells' shape and deformability (Kose et al. 2009). In
adapting it to miniaturized flow cytometry, ferrohydrodynamic
manipulation can first focus cells into single cell streams before
sorting, eliminating needs for excessive sheath flow and preventing
sample dilution (Zhu et al. 2011a). Compared to paramagnetic
solution based sorting, ferrofluid offers much higher magnetic
susceptibility, eliminating needs for either microfabricated
ferromagnetic structures to enhance field gradient or hypertonic
concentrations of paramagnetic salts that are not biocompatible for
live cell manipulation.
[0070] On the other hand, using water-based ferrofluids for cell
manipulation is a work in progress. Diagnostic and research
applications directed towards simply purifying or isolating cells
of interest from complex mixtures such as blood and exfoliated
cytology specimens are exciting. For instance, blood cells obscure
the detection of the larger but rare abnormal cervical cells from
Pap test specimens and metastatic epithelial tumor cells
circulating in blood (Moriarty et al. 2009; Yu et al. 2011).
Misinterpreted cervical cytology ranks third among causes of
medical negligence claims against pathologist (Frable 2007). A
simple, low-cost tumor cell enrichment platform would benefit
cancer screening. However, two issues, cell visibility and
biocompatibility of mammalian cells in ferrofluids, limit
applications of ferrohydrodynamic manipulation. Ferrofluids are
opaque due to light diffraction from their high concentration of
magnetic nanoparticles. Fluorescent cells need to be close to
channel surface for microscopic recording. In order to address this
issue, ferrofluids with low solid content, as well as shallow
microfluidic channel, are favored for cell manipulation. In
addition, magnetic fields can be used to push cells onto channel
surface, increasing visibility of cells in fluorescent mode. In
this study, we used a combination of both bright-field and
fluorescent modes microscopy to circumvent the opaqueness issue.
Cells were readily visible in a shallow channel in bright-field
micrographs. Another potential issue is biocompatibility of
ferrofluids. Our next step is to extend this methodology to
mammalian cells, particularly human specimens such as blood and
other bodily fluids, exfoliated musical cells, and tumor aspirates.
The requirements of mammalian cells may differ from Escherichia
coli and Saccharomyces cerevisiae. For cell manipulation,
materials, pH value, and surfactants of ferrofluids need to be
rendered biocompatible, at the same time the overall colloidal
system of ferrofluids must be maintained. Typically, nanoparticles
within ferrofluids for cell applications are made of magnetite
(Pankhurst et al. 2003). pH value of ferrofluids needs to be
compatible with cell culture and maintained at 7.4. Salt
concentration, tonicity, and surfactant must be carefully chosen
close to physiological conditions to reduce cell death. Although
these are stringent requirements, progress has been made towards
synthesizing biocompatible ferrofluids. For example, Koser's group
used citrate to stabilize cobalt-ferrite nanoparticles for live red
blood cell and Escherichia coli cell sorting (Kose et al. 2009).
Yellen's group used Bovine Serum Albumin (BSA) to stabilize
magnetite nanoparticles for human umbilical vein endothelial cells
manipulation (Krebs Melissa et al. 2009). Viability tests from both
studies have shown cells were able to retain their viability for up
to several hours in ferrofluids. In our study, a commercially
available pH .about.7 magnetite ferrofluid was able to sustain
viability of both Escherichia coli and Saccharomyces cerevisiae
cells for at least 2 hours.
CONCLUSION
[0071] In conclusion, we have developed a label-free and
continuous-flow ferrohydrodynamic cell sorting device and applied
it in separating Escherichia coli and Saccharomyces cerevisiae
cells. A commercial magnetite ferrofluid was used to separate
particle and cell mixtures. A lab customized ferrofluid was used to
separate mammalian cells. Construction of our device is simple and
low-cost; we choose to use permanent magnets instead of integrated
electrodes to eliminate complex microfabrication process and
auxiliary power supply. Current sorting throughput is 10.sup.7
cells/hour, and sorting efficiency is close to 100%. We envision
this device can achieve up to two orders higher throughput while
still maintaining current sorting efficiency.
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Example 2
[0139] Currently, we are developing a type of water based
ferrofluids that can facilitate cervical cancer cells sorting. To
maintain the nonmagnetic properties of cancer cells, cellular
uptake of magnetic nanoparticles should be minimized. Interaction
between cells and magnetic nanoparticles were known to be caused by
endocytosis and physical attraction (Verma and Stellacci 2010),
which are dictated by the surface properties. Polyethyene glycol
and phosphorylcholine based copolymer was chosen as the surfactant
to stabilize magnetite (Fe.sub.3O.sub.4) nanoparticles for their
excellent biocompatibility (Yuan, Armes et al. 2006; Jozefczak,
Hornowski et al. 2009). Biomemetic phospholipid polar group were
also proven to inhibit non-selective cellular uptake of
nanoparticles (Ishihara and Takai 2009). Copolymer structure
provides more flexibility with anchoring group and functional
group, however, multiple groups can also easily interact with
several nanoparticles leading to flocculation (Boyer, Whittaker et
al. 2010). Once the colloidal stable ferrofluids are developed,
cancer cells viability and cellular uptake of nanoparticles will be
measured (Samanta, Yan et al. 2008). Positive results will enable
the application of ferrofluids combing microfluidic platform as the
cancer cells sorter. [0140] Boyer, C., M. R. Whittaker, et al.
(2010). "The design and utility of polymer-stabilized iron-oxide
nanoparticles for nanomedicine applications." NPG Asia Mater 2:
23-30. [0141] Ishihara, K. and M. Takai (2009). "Bioinspired
interface for nanobiodevices based on phospholipid polymer
chemistry." Journal of The Royal Society Interface 6(Suppl 3):
S279-S291. [0142] Jozefczak, A., T. Hornowski, et al. (2009).
"Effect of poly (ethylene glycol) coating on the magnetic and
thermal properties of biocompatible magnetic liquids." Journal of
Magnetism and Magnetic Materials 321(10): 1505-1508. [0143]
Samanta, B., H. Yan, et al. (2008). "Protein-passivated
Fe.sub.3O.sub.4 nanoparticles: low toxicity and rapid heating for
thermal therapy." Journal of Materials Chemistry 18(11): 1204-1208.
Verma, A. and F. Stellacci (2010). "Effect of Surface Properties on
Nanoparticle--Cell Interactions." Small 6(1): 12-21. [0144] Yuan,
J. J., S. P. Armes, et al. (2006). "Synthesis of Biocompatible
Poly[2-(methacryloyloxy)ethyl phosphorylcholine]-Coated Magnetite
Nanoparticles." Langmuir 22(26): 10989-10993.
[0145] It should be noted that ratios, concentrations, amounts, and
other numerical data may be expressed herein in a range format. It
is to be understood that such a range format is used for
convenience and brevity, and thus, should be interpreted in a
flexible manner to include not only the numerical values explicitly
recited as the limits of the range, but also to include all the
individual numerical values or sub-ranges encompassed within that
range as if each numerical value and sub-range is explicitly
recited. To illustrate, a concentration range of "about 0.1% to
about 5%" should be interpreted to include not only the explicitly
recited concentration of about 0.1 wt % to about 5 wt %, but also
include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and
the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the
indicated range. In an embodiment, the term "about" can include
traditional rounding according to the measurement technique and the
type of numerical value. In addition, the phrase "about `x` to `y`"
includes "about `x` to about `y`".
[0146] Many variations and modifications may be made to the
above-described embodiments. All such modifications and variations
are intended to be included herein within the scope of this
disclosure and protected by the following claims.
* * * * *