U.S. patent application number 11/552853 was filed with the patent office on 2007-05-03 for devices and methods for optoelectronic manipulation of small particles.
This patent application is currently assigned to Applera Corporation. Invention is credited to Hans A. Fuernkranz, Aldrich N.K. Lau, Huan L. Phan, Steven W. Sherwood, Joon Mo Yang.
Application Number | 20070095669 11/552853 |
Document ID | / |
Family ID | 38475290 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070095669 |
Kind Code |
A1 |
Lau; Aldrich N.K. ; et
al. |
May 3, 2007 |
Devices and Methods for Optoelectronic Manipulation of Small
Particles
Abstract
A method for sorting cells in a biological sample comprising a
first type of cells and a second type of cells may comprise
introducing the biological sample into a chamber comprising a first
surface and a second surface, wherein the first surface is
associated with a transparent electrode and the second surface is
associated with a photoconductive portion of an electrode. The
method may further comprise moving incident light and the
photoconductive portion relative to one another so as to illuminate
regions of the photoconductive portion and modulate an electric
field in the chamber in proximity to the illuminated regions. The
method may further comprise separating the first type of cells from
the second type of cells in the chamber via dielectrophoretic
movement of the first type of cells and the second type of cells
caused by the modulated electric field, wherein a dielectrophoretic
characteristic of at least one of the first type of cells and the
second type of cells has been modified.
Inventors: |
Lau; Aldrich N.K.; (Palo
Alto, CA) ; Yang; Joon Mo; (Redwood City, CA)
; Phan; Huan L.; (Belmont, CA) ; Sherwood; Steven
W.; (Los Altos, CA) ; Fuernkranz; Hans A.;
(Saratoga, CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.;APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
38475290 |
Appl. No.: |
11/552853 |
Filed: |
October 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60731123 |
Oct 27, 2005 |
|
|
|
Current U.S.
Class: |
204/547 ;
204/643 |
Current CPC
Class: |
B03C 5/026 20130101;
G01N 27/305 20130101; B03C 5/005 20130101; B03C 5/028 20130101;
C07K 1/26 20130101 |
Class at
Publication: |
204/547 ;
204/643 |
International
Class: |
B03C 5/02 20060101
B03C005/02 |
Claims
1. A method for sorting cells in a biological sample comprising a
first type of cells and a second type of cells, the method
comprising: introducing the biological sample into a chamber
comprising a first surface and a second surface, wherein the first
surface is associated with a transparent electrode and the second
surface is associated with a photoconductive portion of an
electrode; moving incident light and the photoconductive portion
relative to one another so as to illuminate regions of the
photoconductive portion and modulate an electric field in the
chamber in proximity to the illuminated regions; and separating the
first type of cells from the second type of cells in the chamber
via dielectrophoretic movement of the first type of cells and the
second type of cells caused by the modulated electric field,
wherein a dielectrophoretic characteristic of at least one of the
first type of cells and the second type of cells has been
modified.
2. The method of claim 1, further comprising selecting a speed of
relative movement between the incident light and the
photoconductive portion based on dielectrophoretic movement
characteristics of at least one of the first type of cells and the
second type of cells.
3. The method of claim 2, wherein selecting the speed comprises
selecting the speed based on predetermined dielectrophoretic
movement characteristics of at least one of the first type of cells
and the second type of cells.
4. The method of claim 2, wherein selecting the speed comprises
selecting the speed based on dielectrophoretic movement
characteristics of at least one of the first type of cells and the
second type of cells observed from the separating step.
5. The method of claim 1, wherein moving the incident light and the
photoconductive portion relative to one another comprises moving
the incident light and the photoconductive portion relative to one
another in a substantially continuous loop pattern.
6. The method of claim 1, further comprising storing information
regarding the dielectrophoretic movement characteristics of at
least one of the first type of cells and the second type of
cells.
7. The method of claim 1, further comprising identifying at least
one of the first type of cells and the second type of cells from
other types of cells based on observing the dielectrophoretic
movement characteristics of at least one of the first type of cells
and the second type of cells.
8. The method of claim 1, further comprising moving the first type
of cells and the second type of cells to differing locations
outside of the chamber, wherein the moving comprises moving at
least one of the first type of cells and the second type of cells
via dielectrophoretic movement.
9. The method of claim 1, further comprising applying an electrical
potential generated via at least one of a DC power source and an AC
power source across the transparent electrode and the electrode
having a photoconductive portion so as to generate an electric
field.
10. The method of claim 1, further comprising measuring the
dielectrophoretic movement of each of the first type of cells and
the second type of cells.
11. The method of claim 10, further comprising measuring a
dielectrophoretic displacement of each of the first type of cells
and the second type of cells after separating the first type of
cells and the second type of cells.
12. The method of claim 1, further comprising moving at least one
of the first type of cells and the second type of cells via
electrophoresis.
13. The method of claim 1, further comprising altering an intensity
of the incident light so as to modulate the electric field.
14. The method of claim 13, wherein altering the intensity of the
incident light comprises altering the intensity based on a position
of the incident light relative to the photoconductive portion.
15. The method of claim 1, further comprising repeating the moving
step so as to achieve a desired separating of the first type of
cells and the second type of cells.
16. The method of claim 15, wherein repeating the moving step
comprises moving incident light of differing intensities relative
to the photoconductive portion.
17. The method of claim 1, wherein the moving step comprises
selectively applying current to an array of electroluminescent
material.
18. The method of claim 1, wherein the moving step comprises
generating an array of interdigitated virtual electrodes at
illuminated regions of the photoconductive portion.
19. The method of claim 1, wherein separating the first type of
cells from the second type of cells comprises separating tumor
cells from nontumor cells.
20. The method of claim 1, wherein introducing the biological
sample into the chamber comprises introducing the biological sample
into a chamber comprising a second patternless surface associated
with a photoconductive portion of an electrode.
21. A method for sorting cells in a biological sample comprising a
first type of cells and a second type of cells, the method
comprising: introducing the biological sample into a chamber
comprising a first surface and a second surface, wherein the first
surface is associated with a transparent electrode and the second
surface is associated with a photoconductive portion of an
electrode; receiving information that indicates dielectrophoretic
movement characteristics of the first type of cells and the second
type of cells; and selectively illuminating the second surface via
incident light based on the information so as to modulate an
electric field within the chamber and separate the first type of
cells and the second type of cells from each other.
22. The method of claim 21, wherein receiving the information
comprises receiving information corresponding to dielectrophoretic
displacement of each of the first type of cells and the second type
of cells in response to the incident light illuminating the
surface.
23. The method of claim 21, wherein selectively illuminating the
surface comprises altering a speed of relative motion between the
incident light and the surface.
24. The method of claim 21, wherein receiving the information
comprises at least one of receiving stored information and
receiving information obtained from an image of the biological
sample in the chamber.
25. A device for manipulating cells in a biological sample, the
device comprising: a chamber comprising a transparent electrode and
a photoconductive portion, wherein the chamber is configured to
receive the biological sample, wherein the transparent electrode
comprises a PEGylated transparent electrode, and wherein, upon
illumination of the photoconductive portion with a light source, an
electric field is modulated within the chamber to move the cells
via dielectrophoresis.
26. The device of claim 25, wherein the photoconductive portion
comprises a PEGylated photoconductive portion.
27. The device of claim 25, wherein the photoconductive portion
comprises a SiO.sub.2 PEGylated photoconductive portion.
28. The device of claim 25, wherein the transparent electrode
comprises a transparent gold electrode.
29. The device of claim 25, wherein the transparent electrode is
configured to transmit from about 40% to about 80% of incident
light.
30. The device of claim 25, further comprising an additional
electrode, wherein the photoconductive portion is in electrical
contact with the additional electrode.
31. The device of claim 30, wherein the additional electrode
comprises a metal electrode chosen from one of gold, indium tin
oxide, and aluminum.
32. The device of claim 25, further comprising a power source
configured to apply an electric potential to the chamber.
33. The device of claim 25, further comprising a first substrate
and a second substrate, wherein the first substrate is provided
with the transparent electrode and the second substrate is provided
with the photoconductive surface.
34. The device of claim 33, wherein the first substrate and the
second substrate are made of glass.
35. The device of claim 25, further comprising a light source
configured to scan the chamber so as to simultaneously illuminate
differing regions of the photoconductive portion, the differing
regions being illuminated at differing intensities.
36. The device of claim 35, wherein the light source comprises an
electroluminescent material.
37. The device of claim 25, further comprising a base structure,
wherein the base structure and the chamber form a disposable
cartridge assembly.
38. The device of claim 37, further comprising a fluid interface
mechanism configured to interface the disposable cartridge assembly
with fluid handling instrumentation to supply fluid to the
chamber.
39. The device of claim 25, wherein the device is configured to be
an accessory to a microscope.
40. The device of claim 39, further comprising a scanning mirror
for illuminating the photoconductive portion with light reflected
from the light source.
41. A device for separating cells in a biological sample containing
a first type of cells and a second type of cells, the device
comprising: a chamber comprising a means for generating an electric
field in the chamber, the chamber containing the biological sample;
and means for illuminating regions of the chamber by imparting
relative motion between incident light and the chamber; and means
for modulating the electric field in the chamber at locations
corresponding to the illuminated regions so as to separate the
first type of cells and second type of cells from each other by
dielectrophoretic movement of the cells.
42. The device of claim 41, wherein the means for illuminating is
configured so as to scan incident light relative to the
chamber.
43. The device of claim 41, wherein the means for modulating
comprises a photoconductive element.
44. The device of claim 43, wherein the photoconductive element
comprises a plurality of noncontiguous photoconductive
elements.
45. The device of claim 41, further comprising a means for
measuring information relating to dielectrophoretic movement of the
first type of cells and the second type of cells.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/731,123, filed Oct. 27, 2005, the entire
contents of which are incorporated by reference herein.
TECHNICAL FIELD
[0002] This invention relates to devices and methods for
manipulating small particles, such as, for example, micro-particles
and/or nano-particles. In particular, this invention relates to
devices and methods for manipulating small particles, such as
cells, including stem cells, and nucleic acids in solution.
BACKGROUND
[0003] Cellular analysis and research often requires the
manipulation of small particles, including cells, cell aggregates,
cell organelles, stem cells, nucleic acids, bacteria, protozoans,
viruses, and/or other micro- and/or nano-particles. Typically, the
small particles to be manipulated have a dimension (e.g., diameter)
ranging from approximately 0.1 micrometer to approximately several
hundred micrometers, for example from approximately 1 micrometer to
approximately 100 micrometers, or, for example, from approximately
5 micrometers to approximately 10 micrometers. By way of example
only, mammalian cells have a diameter ranging from about 5
micrometers to about 100 micrometers and a lymphocyte may be about
10 micrometers in diameter. In some cases, groups of particles
(e.g., cells, stem cells, etc.) may be separated from other
particles. The dimension of a group of particles may be as large as
about 100 micrometers.
[0004] Various devices and methods have been used to manipulate
small particles so as to identify, discriminate, sort,
characterize, quantitate, observe, move, collect, and/or otherwise
manipulate the small particles, such as, for example, live stem
cells. For example, microfluidic devices that rely on
pressure-driven flow to separate cells, for example sperm cells
from epithelial cells, have been utilized. This technique is a
passive technique and relatively cost effective for cell sorting,
however, the operation protocol is craft sensitive and must be
determined on an application by application basis In other words,
because microfluidic devices rely on pressure-induced flow to
separate cells by virtue of their size and flow rate, appropriate
operational conditions must be determined on a trial-and-error
basis, since nontarget and target cells may be of substantially the
same size. Moreover, due to the relatively narrow microfluidic
channels and cross-junctions present in such microfluidic devices,
care must be taken to avoid rupturing cells while forcing them
through the small passageways.
[0005] Flow cytometers, including fluorescence activated sorters,
for example, are relatively complex optics-based instruments that
serially analyze and isolate fluorescently-labeled cells from a
flowing stream of fluid. One such device of this class has been
used to manipulate Escherichia coli cells. This sorting device
comprises a narrow capillary T-shaped junction connected to three
reservoirs at each end for receiving aqueous sample, collection,
and waste, respectively. The device relies on electro-osmotic flow
(EOF) for cell transport and a preset fluorescence threshold to
trigger the switching of EOF direction at the T-shaped junction,
thereby resulting in cell sorting. A modified version of the
device, a microfluidic cell sorting device, has been utilized to
sort stably transfected HeLa cells. This modified version relies on
pressure-driven flow and a focused laser spot at the junction to
deflect and reroute cells by optical force gradient (optophoresis)
to a collection reservoir.
[0006] The reliance on lasers and other optics contributes to
relatively high fabrication costs of some flow cytometers. Further,
when using such devices, it may be necessary to simultaneously
optimize the optical, fluidic, electronic, and computer systems,
and efficiency may be reduced.
[0007] Aside from being relatively complex systems and having
relatively high fabrication costs, disadvantages of fluorescence
activated sorting techniques may include, among others, limited run
time due to ion depletion of the sample solution as a result of
electro-osmosis and clogging of small orifices and other
passageways. Regarding the latter, the size of the orifices at the
T-shaped junction are typically relatively small, for example about
3 to about 10 microns. Thus, depending on the types of particles
(e.g., cells) being manipulated, some particles may be too large to
pass through the junction. Moreover, passive adsorption of proteins
and/or other material may occur on the surfaces at the junction,
causing a build up of such materials on the surface and potentially
result in clogging of the device. Further, if polydimethylsiloxane
(PMDS) is used to fabricate a flow cytometer, performing a surface
modification on PMDS in order to reduce nonspecific adsorption of
biomolecules poses challenges.
[0008] Another potential drawback may include the use of dyes to
label for recognition various cells of interest. In the case of
stem cells, for example, using dyes and other labeling techniques
potentially could harm and/or otherwise stress the cells.
Similarly, in cases where relatively high intensity lasers are
used, such lasers could harm and/or cause stress to the cells.
Proliferating potentially stressed cells and possibly implanting
the proliferated cells back into a patient could possibly pose
potential health and/or other risks. Moreover, the sorting
throughput of flow cytometers may be limited in that such devices
typically operate on a cell-by-cell manipulation basis. Although,
the manipulation of each cell occurs relatively rapidly, due to the
cell-by-cell manipulation scheme, the amount of time it may take to
manipulate all of the cells in a sample may be relatively
large.
[0009] Other techniques for manipulating small particles include
the use of a dielectrophoretic force. Dielectrophoresis (DEP)
refers to the motion imparted on uncharged objects as a result of
polarization induced by a spatially nonuniform electric field. An
analytical expression of the dielectrophoretic force, {right arrow
over (F)}.sub.DEP, acting on a particle (T. B. Jones,
Electromechanics of Particles, Cambridge University Press, 1995) is
set forth below: F -> DEP = 2 .times. .pi. .times. .times. r 3
.times. m .times. .alpha. r .times. .gradient. -> .times. ( E
-> RMS 2 ) , .times. .alpha. r .ident. Re .function. ( p * - m *
p * + 2 .times. m * ) ##EQU1##
[0010] In the above equation, r is the radius of the particle, the
factor in parentheses in the first line of the equation is the RMS
value of the electric field, and .alpha..sub.r is the real part of
the Clausius-Mosotti factor which relates the complex permittivity
of the object .epsilon..sub.p and the complex permittivity of the
medium .epsilon..sub.m. The star (*) denotes that the complex
permittivity is a complex quantity. The Clausius-Mosotti factor may
have any value between 1 and -1/2, depending on the applied AC
frequency and the complex permittivity of the object and medium. If
it is less than zero, the dielectric force is negative and the
particle moves toward a lower electric field. If the
Clausius-Mosotti factor is greater than zero, the dielectric force
is positive and the particle moves toward a stronger electric
field. In other words, if the object (e.g., particle, cell, etc.)
is more polarizable than its surroundings, it may be pulled toward
relatively strong field regions ("positive DEP") and if it is less
polarizable, it may be pulled toward relatively weak field regions
("negative DEP").
[0011] If the particles are charged, then under DC current or low
frequency AC current, electrophoresis (EP) occurs, instead of DEP.
EP refers to the lateral motion imparted on charged objects in a
nonuniform or uniform electric field.
[0012] DEP has been used to manipulate particles, such as cells,
for example, via a traveling wave generated by a series of
patterned electrodes lining up and charged with phase-shifted AC
signals. The electrodes can be patterned in an independently
controlled array to provide the traveling wave. For examples of
such a technique, reference is made to Pethig et al., "Development
of biofactory-on-a-chip technology using excimer laser
micromachining," Journal of Micromechanics and Microengineering,
vol. 8, pp. 57-63, 1998, and Green et al., "Separation of
submicrometer particles using a combination of dielectrophoretic
and electrohydrodynamic forces," Journal of Physics D: Applied
Physics, vol. 31, L25-L30, 1998. In one technique, disclosed by Das
et al., "Dielectrophoretic Segregation of Different Cell Types on
Microscope Slides," Anal. Chem. May 1, 2005, vol. 77, pp.
2708-2719, incorporated by reference herein, a glass slide is
patterned with an electrode array in which the electric field
frequency decreases in one direction along the length of the slide,
which in turn results in a variation of generated DEP forces along
the length of the slide. For other examples of the use of DEP
particle manipulation via a traveling wave, reference is made to
Hagendorn, et. al., "Traveling-wave dielectrophoresis of
microparticles," Electrophoresis, vol. 12, pp. 49-54, 1992 and
Talary et al., "Electromanipulation and separation of cells using
traveling electric fields," J. Phys. D: Appl. Phys., vol. 29, pp.
2198-2203 (1996), the entire contents of both of which are
incorporated by reference herein.
[0013] The use of DEP for separating differing cell types in a
device wherein electrode arrays are used to create the nonuniform
electric field also has been disclosed, for example, in U.S. Pat.
No. 6,790,330 B2, which issued on Sep. 14, 2004, U.S. Pat. No.
6,641,708 B1, which issued on Nov. 4, 2003, and U.S. Pat. No.
6,287,832 B1, which issued on Sep. 11, 2001, the entire disclosure
of each of which is incorporated by reference herein. These patents
disclose various devices and methods relying on DEP induced by
electrodes for cell separation.
[0014] Another technique for manipulating cells includes the use of
optophoresis to manipulate cells in a surrounding medium, such as,
for example, an aqueous suspension. Devices and methods utilizing
optophoresis rely on a radiation pressure force generated by
laser-induced optical gradient fields to capture and manipulate
micrometer-scale particles in the aqueous suspension. Devices and
methods relying on optophoresis and high intensity lasers to
directly trap a single particle have been dubbed "optical
tweezers." For exemplary applications utilizing the principles of
optical tweezers, reference is made to Ashkin et al., "Optical
trapping and manipulation of single cells using infrared laser
beams," Nature, vol. 330, December 1987, pages 769-771; and Arai et
al, "Tying a molecular knot with optical tweezers," Nature, vol.
399, June 1999, pages 446-448, each of which is incorporated by
reference herein.
[0015] Aside from the optical tweezers optophoretic technique,
another technique employing optophroetic principles uses a
fast-scan optophoresis device for recognizing, identifying, and
quantifying one type of cells from among others. Such a technique
is discussed, for example, in U.S. Application Publication No.
2002/0160470 A1, published Oct. 31, 2002; U.S. Application
Publication No. 2005/0164372 A1, published Jul. 28, 2005; Hoo et
al., "A Novel Method for Detection of Virus-infected Cells Through
Moving Optical Gradient Fields Using Adenovirus as a Model System,"
Cytometry Part A, 58A, February 2004, pages 140-146; and Forster et
al., "Use of moving optical gradient fields for analysis of
apoptotic cellular responses in a chronic myeloid leukemia cell
model," Analytical Biochemistry, 327, 2004, pages 14-22, the entire
disclosure of each of which is incorporated by reference
herein.
[0016] In an embodiment, the fast-scan optophoresis device includes
a CCD (charge couple device) camera and a coherent Nd-YAG 1064 nm
laser beam, operating at 18.3 kW/cm.sup.2 at a focused point,
scanning across the surface of a thin-layer cell in which a
suspension of various types of particles (e.g., cells) are
contained. Under a given set of conditions, all of the particles in
the suspension are swept across the thin-layer cell by the laser
beam until the laser beam's scanning speed reaches a threshold.
Above that threshold, one type of particles escapes and is left
behind the sweeping laser beam due to various forces, including
drag forces, acting on that particle type. Software is used to
measure the optophoretic distance that each particle travels and
accumulated statistics may be used for identification and
quantitation. This device has been used to analyze chronic myeloid
leukemia cells and HeLa human ovarian carcinoma cells. Due to the
use of high power lasers and focusing optics, manufacturing costs
associated with such optophoretic scanning devices may be
relatively high. Moreover, the use of high intensity lasers may
potentially harm and/or otherwise stress the cells. In the case of
stem cells, for example, that may be proliferated after being
sorted and collected and then implanted into a patient, there may
be a risk associated with such cell stressing.
[0017] Another more recently developed particle manipulation
technique includes so-called "optoelectronic tweezers," which have
been used to attract or expel a plurality of small particles by
application of an optically activated DEP force. In contrast to
optical tweezers, optoelectronic tweezers can use a low power
incoherent light source, for example, on the order of 1
.mu.W/cm.sup.2, instead of the high intensity laser used by optical
tweezers. By way of example, optoelectronic tweezers may utilize a
light source that has a power approximately ten orders of magnitude
less than that of the high intensity lasers typically employed in
optical tweezers. In the optoelectronic tweezers technique, by
projecting the low power incoherent light source onto a
photoconductive surface, a liquid suspension containing various
particles, e.g., cells, sandwiched between a patternless
photoconductive surface and another patternless surface may be
subject to a nonuniform electric field resulting from the
illumination of the photoconductive layer. In turn, a
dielectrophoretic force is created and acts on the particles.
Particles may then be attracted by or repelled from the illuminated
area depending upon, among other things, the particles' dielectric
properties.
[0018] One device that employs the above-described principles
includes a manipulation chamber comprising a top indium tin oxide
transparent glass electrode, a bottom substrate coated with
photoconductive material to complete the circuitry, and an aqueous
layer containing particles of interest sandwiched between the
surfaces. A focused incoherent light spot creates a nonuniform
electric field by which the particles (e.g., live cells) in the
aqueous sandwiched layer are manipulated based on their respective
dielectric constants and sizes.
[0019] For further explanation of the operation principles of
optoelectronic tweezers, including various devices and techniques
employing those principles, reference is made to Pei Yu Chiou et
al., "Massively parallel manipulation of single cells and
microparticles using optical images," Nature, vol. 436:21, July
2005, pages 370-372; PCT publication number WO 2005/100541,
entitled "Optoelectronic Tweezers for Microparticle and Cell
Manipulation," which claims priority to U.S. Provisional
Application No. 60/561,587, filed on Apr. 12, 2004; U.S.
application Ser. No. 10/979,645, entitled "Surface Modification For
Non-Specific Adsorption Of Biological Material," filed Nov. 1,
2004, in the name of Aldrich Lau; and U.S. Provisional Application
No. 60/692,528, entitled "Optoelectronic separation of
biomolecules: Separation of dye-labeled DNA, RNA, proteins, lipids,
terpenes, and polysaccharides," filed Jun. 30, 2005, in the name of
Aldrich Lau, the entire contents of each of which are incorporated
by reference herein.
[0020] Conventional optoelectronic tweezers are typically employed
by providing a manipulation chamber on a microscope stage and
targeting predetermined cells of interest. Once the cells of
interest are in view, the light source can be mapped onto the
manipulation chamber and the predetermined cells can be captured.
Thus, the existing devices use previsualization in order to capture
known targets of interest.
[0021] Based on current techniques for manipulating small
particles, including sorting, identifying, characterizing,
quantifying, moving and/or otherwise manipulating small particles,
it may be desirable to provide a technique for manipulating small
particles that is relatively inexpensive to make and/or use and/or
is disposable. It may be desirable to provide a manipulation device
that is relatively easy to fabricate. For example, it may be
desirable to provide a technique that may not require patterned
electrodes, microchannels, capillary junctions, capillary orifices,
relatively expensive lasers or optics, high power lasers, and/or
other elements that are relatively expensive and/or intricate to
fabricate. It also may be desirable to provide a device that relies
on DEP to manipulate particles and achieves greater flexibility and
control over modulation of the electric field than conventional
device that utilize patterned electrodes. Moreover, it may be
desirable to provide a particle manipulation technique that reduces
potential clogging that can occur in device having relatively small
junctions and/or orifices through which particles must pass.
[0022] Further, it may be desirable to provide a technique that
achieves high sorting throughput, purity, and/or the recovery of
undamaged (e.g., uncontaminated and/or unstressed) cells. It may
further be desirable to provide a technique that achieves the
recovery of live, unstressed mammalian cells. For example, it may
be desirable to provide a technique that sorts stem cells from
other cells, such as mouse feeder cells, and recovers the stem
cells uncontaminated and/or unstressed. It also may be desirable to
provide a particle manipulation technique that does not require the
cells to be chemically labeled and/or exposed to high intensity
laser radiation. Although it may be desirable to provide a
manipulation technique that does not require chemical (e.g.,
including dyes and other fluorescence labeling), it also may be
desirable to provide a manipulation technique that can work in
conjunction with conventional detection methods, including the use
of fluorescence signal detection, for example.
[0023] It may be desirable to provide a technique that permits
visualization of the manipulation (e.g., sorting) of cells via a
microscope, a camera, or other visualization tool.
[0024] It also may be desirable to provide a technique which
selectively sorts cells based on various cell properties, such as,
for example dielectric constant and size, and which may be
automated. Moreover, it may be desirable to provide a technique
that permits surface modification of the device, for example, to
alter nonspecific and/or specific adsorption, and/or surface
modification of the particles being manipulated. Regarding the
former, surface modification of the device may be beneficial to
reduce or enhance nonspecific adsorption of, for example, proteins,
lipids, cells, and/or other biomolecules. Regarding the latter, it
may be desirable to provide a technique that permits reversible
surface modification of the particles so as to alter the particles'
size, dielectric constant, polarity, and/or other properties.
[0025] It may also be desirable to improve upon existing devices
that utilize optoelectronic tweezers principles in order to
manipulate cells. For example, it may be desirable to provide a
device that improves adhesion of the photoconductive and/or
electrode layer to the glass substrate, improves robustness, and/or
enables operation at a relatively low AC frequency or via direct
current. It also may be desirable to reduce nonspecific adsorption
of biomolecules. It also may be desirable to provide a device that
enables surface modification of the substrates so as to, among
other things, reduce nonspecific adsorption and permit the use of
surface active agents (e.g., ligands, etc.) to differentiate
particles.
[0026] Further, it may be desirable to utilize the principles
associated with optoelectronic tweezers and/or other optoelectronic
manipulation techniques and chambers in conjunction with existing
manipulation techniques. In other words, it may be desirable to
provide an optoelectronic manipulation chamber as an accessory to a
microscope or portable medical device. It may also be desirable to
provide a device that utilizes the principles of optoelectronic
tweezers in combination with conventional manipulation techniques,
such as for example, laser pressure catapulting, laser
microdissection, laser microinjection, eletroporation,
microcapillaries, microdissector, microinjection,
micromanipulators, piezoelectric microdissection, drug
interaction/cell response, ion channel conductivity measurement
(patch clamp), and/or other types of manipulation techniques. To
achieve such a combination, it may be desirable to provide an
optoelectronic manipulation chamber that permits insertion of an
instrument or other external element into the liquid sample cavity
containing the particles to be manipulated.
[0027] A further desirable aspect may include particle manipulation
techniques that may be automated.
SUMMARY
[0028] Devices and methods according to exemplary aspects of the
present invention may satisfy one or more of the above-mentioned
desirable features. Other features and advantages will become
apparent from the detailed description which follows.
[0029] According to an exemplary aspect, the invention may include
the use of low-power optoelectronic tweezer principles in lieu of
the high power laser of a fast-scan optophoresis technique. In
other words, a scanning low power incoherent light source may be
used to optically create a DEP force which acts to entrain some
small particles in the scanning light source path or allow other
particles to escape from the scanning light source path so as to
sort particles.
[0030] According to an exemplary aspect of the invention, as
embodied and broadly described herein, the invention may include a
method for sorting cells in a biological sample comprising a first
type of cells and a second type of cells comprising introducing the
biological sample into a chamber comprising a first surface and a
second surface, wherein the first surface is associated with a
transparent electrode and the second surface is associated with a
photoconductive portion of an electrode. The method may further
comprise moving incident light and the photoconductive portion
relative to one another so as to illuminate regions of the
photoconductive portion and modulate an electric field in the
chamber in proximity to the illuminated regions. The method may
further comprise separating the first type of cells from the second
type of cells in the chamber via dielectrophoretic movement of the
first type of cells and the second type of cells caused by the
modulated electric field, wherein a dielectrophoretic
characteristic of at least one of the first type of cells and the
second type of cells has been modified.
[0031] According to yet another exemplary aspect, the invention may
include a method for sorting cells in a biological sample
comprising a first type of cells and a second type of cells,
comprising introducing the biological sample into a chamber having
a surface with a photoconductive portion and receiving information
that indicates dielectrophoretic movement characteristics of the
first type of cells and the second type of cells. The method may
further comprise selectively illuminating the surface via incident
light based on the information so as to modulate an electric field
within the chamber and separate the first type of cells and the
second type of cells from each other.
[0032] In yet another exemplary aspect, the invention may include a
device for manipulating cells in a biological sample, the device
comprising a chamber comprising a transparent electrode and a
photoconductive portion, wherein the chamber is configured to
receive the biological sample, and a light source configured to
illuminate the photoconductive portion so as to modulate an
electric field within the chamber, the electric field being
configured to move the cells via dielectrophoresis. The transparent
electrode may comprise a PEGylated transparent electrode.
[0033] Yet another exemplary aspect of the invention includes a
device for separating cells in a biological sample containing a
first type of cells and a second type of cells comprising a chamber
comprising a means for generating an electric field in the chamber,
the chamber containing the biological sample. The device may
further comprise means for illuminating regions of the chamber by
imparting relative motion between incident light and the chamber
and means for modulating the electric field in the chamber at
locations corresponding to the illuminated regions so as to
separate the first type of cells and second type of cells from each
other by dielectrophoretic movement of the cells.
[0034] In the following description, certain aspects and
embodiments will become evident. It should be understood that the
invention, in its broadest sense, could be practiced without having
one or more features of these aspects and embodiments. It should be
understood that these aspects and embodiments are merely exemplary
and explanatory and are not restrictive of the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0035] The drawings of this application illustrate exemplary
embodiments of the invention and, together with the description,
serve to explain certain principles.
In the drawings:
[0036] FIG. 1 is a side view of an exemplary embodiment of an
optoelectronic manipulation chamber;
[0037] FIG. 2A is a top perspective schematic view of the
optoelectronic manipulation chamber of FIG. 1;
[0038] FIG. 2B is a side perspective view of the optoelectronic
manipulation chamber of FIG. 1;
[0039] FIG. 3A is an exemplary schematic representation of a light
beam scanning across a particle;
[0040] FIG. 3B is a schematic representation of various forces
acting on the particle of FIG. 3A in accordance with various
exemplary embodiments;
[0041] FIGS. 4A-4C are schematic illustrations showing exemplary
embodiments of optoelectronic manipulation of particles;
[0042] FIGS. 5A-5C are schematic illustrations showing another
exemplary embodiment of optoelectronic manipulation of
particles;
[0043] FIGS. 6A-6C are schematic illustrations showing yet another
exemplary embodiment of optoelectronic manipulation of
particles;
[0044] FIGS. 7A-7D are schematic illustrations showing yet another
exemplary embodiment of optoelectronic manipulation of
particles;
[0045] FIG. 8 is a schematic top view of an exemplary embodiment of
a particle sorting device;
[0046] FIGS. 9A-9H are schematic illustrations of an exemplary
embodiment of the use of the device of FIG. 8;
[0047] FIGS. 10A-10B are schematic illustrations of yet another
exemplary embodiment of a particle sorting device;
[0048] FIGS. 11A-11C are schematic illustrations of another
exemplary embodiment of use of the device of FIG. 8;
[0049] FIGS. 12A-12D are cross-sectional, side views of an
exemplary embodiment of an optoelectronic manipulation chamber and
use of the manipulation chamber;
[0050] FIG. 13 is a cross-sectional, side view of yet another
exemplary embodiment of an optoelectronic manipulation chamber;
[0051] FIG. 14A is a cross-sectional, side view of yet another
exemplary embodiment of an optoelectronic manipulation chamber;
[0052] FIG. 14B is a top view of the manipulation chamber of FIG.
14A from the perspective of line B-B shown in FIG. 14A;
[0053] FIG. 15 is a side view of another exemplary embodiment of an
optoelectronic manipulation chamber;
[0054] FIG. 16 is a schematic representation of exemplary steps for
fabricating a substrate with a PEGylated transparent gold electrode
layer;
[0055] FIGS. 17A-17C show perspective views of a substrate surface
after being subjected to the various surface treatment steps of
FIG. 16
[0056] FIG. 18 is a partial, perspective view of a PEGylated
transparent gold electrode substrate surface after having been
exposed to 10.times.BSA (bovine serum albumin);
[0057] FIG. 19 is a chart comparing surface roughness measurements
of various surfaces;
[0058] FIG. 20 is a chart comparing wettability of various
surfaces;
[0059] FIG. 21 is a chart comparing transparency measurements of
various surfaces;
[0060] FIGS. 22A-22D show snapshots in time of particle
manipulation using an optoelectronic manipulation chamber according
to an exemplary embodiment;
[0061] FIG. 23 is a graph showing manipulation speed as a function
of applied AC frequency for various exemplary embodiments of
optoelectronic manipulation chambers;
[0062] FIGS. 24A and 24B show schematic representations of
exemplary steps for fabricating a substrate with a PEGylated
SiO.sub.2 photoconductive layer;
[0063] FIG. 25 is a graph showing manipulation speed as a function
of applied AC frequency for yet further exemplary optoelectronic
manipulation chamber embodiments;
[0064] FIGS. 26A and 26B are schematic illustrations showing yet
additional exemplary embodiments of optoelectronic manipulation of
particles;
[0065] FIGS. 27A and 27B are schematic illustrations showing yet
further exemplary embodiments of optoelectronic manipulation of
particles;
[0066] FIG. 28A is a side view of yet another exemplary embodiment
of an optoelectronic manipulation chamber;
[0067] FIG. 28B is a perspective view taken along line 28B-28B of
FIG. 28A;
[0068] FIGS. 29A-29D are top schematic views of an exemplary
embodiment of a technique for sorting particles utilizing
optoelectronic scanning;
[0069] FIG. 30 is a schematic view of an exemplary embodiment of a
particle sorting device that uses an optoelectronic scanning
chamber;
[0070] FIG. 30A is a cross-sectional view of the device of FIG. 30
taken through line 30A-30A;
[0071] FIG. 31 is an exemplary embodiment of sorting and collecting
particles using the device of FIG. 30;
[0072] FIG. 32 is a force diagram of a particle in FIG. 31;
[0073] FIG. 33 is a perspective view of an exemplary embodiment of
an optoelectronic manipulation chamber integrated with a cartridge
assembly;
[0074] FIGS. 33A and 33B are isometric top and bottom views,
respectively, of the exemplary embodiment of FIG. 33;
[0075] FIGS. 34A and 34B are isometric top and bottom views,
respectively, of another exemplary embodiment of an optoelectronic
manipulation chamber integrated with a cartridge assembly;
[0076] FIGS. 35A and 35B are perspective top and bottom views,
respectively, of an exemplary embodiment of an interface mechanism
and a cartridge assembly of FIGS. 33-34; and
[0077] FIG. 36 is an exemplary embodiment of an optoelectronic
scanning chamber that utilizes a scanning mirror for illuminating
the chamber.
DETAILED DESCRIPTION
[0078] FIG. 1 schematically illustrates an exemplary embodiment of
a manipulation chamber which relies on optically activated DEP
particle manipulation for use in optoelectronic scanning and other
manipulation techniques, in accordance with various exemplary
aspects of the present invention. The optically activated DEP
manipulation chamber also is referred to herein as an
optoelectronic manipulation chamber. The chamber 100 may include
two substrates, 20 and 30, disposed in a spaced relationship so as
to be configured to contain therebetween a sample for analysis. By
way of example the substrates 20 and 30 may be spaced from each
other by a distance ranging from about 10 microns to about 200
microns. The edges of the substrates may be provided with a seal
40, such as, for example, a gasket (e.g., a rubber gasket such as a
silicon rubber gasket, or a fluorinated elastomer (Viton.RTM.)
gasket), an adhesive (e.g., a pressure sensitive adhesive (PSA)),
and/or other sealing mechanisms, so as to contain the sample liquid
in the chamber.
[0079] The chamber also may be provided with various ports and/or
valves (not shown in FIG. 1), for example, input and output ports
and/or valves, to allow for introduction of sample or other
materials, including manipulation tools, for example, to the
chamber, flushing of sample and/or particles from the chamber,
and/or collection of particles from the chamber. By way of example,
the input and output ports of the chamber may form an interface
with instrumentation separate from the chamber via O-rings in a
clamping fixture or via resealable elastomeric material, such as,
for example, a septum. The septum may permit a needle to pass
therethrough for sample addition and/or removal. Instrumentation
which may interface with the chamber may include, but is not
limited to, valves, such as, for example, pinch or solenoid valves,
and/or pumps, such as, for example, a peristaltic pump or a syringe
pump for microfluidic control (e.g., sample introduction and
collection).
[0080] The sample layer 50 may comprise, for example, a liquid
suspension containing a plurality of small particles of differing
types (for example, differing cell types) labeled A and B in the
exemplary embodiment of FIG. 1. It should be understood that the
liquid suspension may contain any number of differing types of
particles and the use of two particle types A and B herein is for
ease of reference and explanation. According to exemplary aspects,
the small particles may be suspended in an aqueous medium, such as,
for example, a phosphate buffer, a phosphate-buffered saline (PBS),
which may contain about 1% bovine serum albumin (BSA), for example,
a saline solution having a pH ranging from about 6.5 to about 8.5
and a conductivity ranging from less than about 10 mS/m to several
hundred mS/m, a potassium chloride solution, or other suitable
mediums, such as mediums that are biologically compatible with
cells and iso-osmotic. By way of example, the medium may also
comprise a HEPES (N-2-Hydroyxyethylpiperazine-N'-2-ethanesulfonic
acid) buffer, sugars, such as sucrose or dextrose, for osmotic
stability, and/or solutes for modifying the medium permittivity,
such as, for example, .epsilon.-amioncaproic acid.
[0081] According to exemplary aspects, the first and second
substrates 20 and 30 may be made of a transparent, insulating
material, such as, for example, glass, silica, plastic, ceramic, or
other suitable transparent and insulating material. Further, in an
exemplary aspect, the surfaces of the substrates 20 and 30 facing
the chamber interior may be modifiable and/or provided with an
adhesive promoter so as to enhance adhesion of the electrode layers
thereon. Depending on where the light source is positioned for
illuminating the photoconductive material 34, one or both of the
substrates 20 or 30 need not be transparent. For example, in the
embodiment shown in FIG. 1, wherein the light source is positioned
so as to transmit light through the first substrate 20, the second
substrate 30 need not be made of a transparent material, and vice
versa if the light source is positioned so as to transmit light
through the second substrate 30. Those having ordinary skill in the
art would recognize a variety of configurations and materials for
the first and second substrates, and other elements of the
manipulation chamber in accordance with aspects of the
invention.
[0082] The first substrate 20 may comprise a transparent electrode
22 facing the cavity 50. The second substrate 30 also may comprise
an electrode 32, such as, for example a metal electrode. In various
alternative embodiments, the substrate 30 adjacent the electrode 32
may be nontransparent and constructed of any material that can
withstand the processing conditions for deposition of a
photoconductive material. The electrode 22 and the electrode 32 may
be electrically coupled to a power supply 60, which may be AC or
DC.
[0083] In various exemplary embodiments, the transparent electrode
22 may be, for example, gold, indium tin oxide (ITO), or other
suitable transparent electrode material. The term "transparent" in
this context means that at least some light can pass through the
layer. For example, due to a nonuniform deposition of the electrode
layer on the substrate, at least some regions may have no electrode
deposited thereon or a very thin layer, and light may pass through
those regions. In an exemplary aspect, the transparent electrode
layer may be such that from approximately 20% to approximately 95%
of the incident light may pass through the electrode layer. In an
exemplary embodiment, which is described below in more detail, the
electrode 22 may be a transparent gold electrode with a PEGylated
surface. The term PEGylated refers to a surface that has been
processed so as to covalently attach PEG (poly(ethylene glycol))
and/or its derivatives thereto.
[0084] The electrode 32 may be a transparent or nontransparent
electrode. By way of example, the electrode 32 may be made of
indium tin oxide, gold, aluminum, copper, nickel, chromium, a metal
alloy, or any other suitable conductive material.
[0085] The power supply 60 may be AC or DC. According to various
exemplary aspects, an AC current may having a relatively high
frequency ranging from approximately 1 kHz to 10 MHz may be used.
Alternatively, the AC current may have a relatively low frequency
ranging from less than approximately 10 Hz to less than
approximately 1 kHz.
[0086] A photoconductive material 34 may be provided in a layer
over the electrode 32 so as to close the circuit. In an exemplary
aspect, the photoconductive material 34 may be separated from the
sample layer 50 by a transparent material layer 36, such as, for
example, a polymer dielectric, insulating Spin-on-Glass (SOG), a
semiconductive SOG, a semiconductive transparent film, a silicon
nitride film, a silicon dioxide with a PEGylated surface or other
surface-PEGylated layer, a silicon dioxide with surface-grafted
poly(acrylamides), any material that exhibits reduced nonspecific
adsorption of biomolecules, for example, Teflon-AF.RTM. (DuPont),
Cytop (Asahi Glass), or fluorinated/perfluorinated polymers, and/or
any material capable of being surface-modified so as to reduce
nonspecific adsorption of biomolecules. In an exemplary embodiment,
which is explained in more detail below, the second substrate 30
may be provided with a PEGylated silicon dioxide photoconductive
layer over the metal electrode layer.
[0087] A variety of materials may be used for the various elements
of the manipulation chamber, and the various layers may be treated
(e.g. via surface modification) so as to alter performance of the
chamber. By way of example, in various exemplary embodiments, one
or more surfaces of the chamber may be subject to a surface
modification to either enhance non-specific adsorption of cells
(e.g., using poly-1-lysine may be used to modify the surface) or
enhance selective adsorption of particular particle (e.g., cell)
types (e.g., using antibodies, lectins, ligands, smart polymers).
Further, differing areas on a surface may be subject to differing
modifications such that different cell types can bind to the
different areas. The materials discussed above are exemplary only
and other feasible embodiments of the manipulation chamber can be
found in U.S. patent application Ser. No. 10/979,645, incorporated
by reference herein. Moreover, in the Example which follows below,
an exemplary embodiment of an optoelectronic manipulation chamber,
including how to make such a manipulation chamber and various data
of interest relating to the chamber, is described in further
detail.
[0088] Moreover, although in various embodiments described herein,
the manipulation chamber is disclosed as comprising approximately
planar substrates sandwiching a spacer (e.g., a seal such as PSA),
sample liquid, and material layers, it should be understood that
various other configurations may be envisioned and are considered
within the scope of the invention. In general, any device
configuration may be utilized such that a light source illuminating
a photoconductive surface generates a nonuniform electric field and
a corresponding DEP force on the particle solution within the
manipulation device.
[0089] In accordance with various exemplary aspects, a scanning
light beam 700 may be used to illuminate a portion of the
photoconductive material 34 and thereby close the circuit between
the transparent electrode 22 and the electrode 32. Transmitting the
light onto the photoconductive surface 34 converts the illuminated
region of that surface to a virtual electrode, thus generating
(e.g., modulating) a nonuniform electric field and corresponding
DEP force that acts upon the particles A and B in the sample layer
50. A nonuniform electric field is modulated as a result of the
difference in areas of the electrode 22 and the virtual electrode
created by the illuminated region of the photoconductive surface
34. Due to the differing dielectric properties and size of each
particle type A and B, the differing particle types A and B
experience differing forces, including DEP forces, so as to allow
manipulation of the particles as is explained further below.
[0090] FIGS. 2A and 2B schematically illustrate a perspective view
and a top view of the optoelectronic scanning assembly according to
various aspects of the invention. Referring to FIG. 2A, the
manipulation chamber 100, such as, for example, any of the
manipulation chamber embodiments in accordance with aspects of the
invention, including the manipulation chamber embodiments described
with reference to FIG. 1, may be illuminated by a two-dimensional
light beam 700. The light beam 700 may extend in a transverse
direction to the direction of scanning (shown by the arrows in
FIGS. 2A and 2B) of the light beam across the manipulation chamber
100. As discussed above, the light beam may be from an incoherent
light source. As the light beam scans across the manipulation
chamber 100, a DEP force is created due to illumination of the
photoconductive surface and closing of the electrode circuit. That
DEP force either attracts or repels the particles (e.g., cells)
that are suspended in the liquid layer (e.g., aqueous medium) of
the manipulation chamber 100.
[0091] Thus, optoelectronic manipulation chambers may use
patternless surfaces to generate an electric field gradient. In
lieu of patterned electrodes, the patternless surfaces may utilize
deposited electrode layers and a photoconductor that completes the
circuit via illumination from a light source, thereby creating
"virtual", photoactivated electrodes.
[0092] A variety of light sources may be used to illuminate
manipulation chambers according to aspects of the invention,
including but not limited to, lasers, incoherent light sources,
light emitting diodes (LEDs). According to various exemplary
embodiments, the light source may be an incoherent light source. In
various exemplary embodiments, the incident light may range from
visible to UV range and may enable visualization through inherent
fluorescence characteristics of some particles (e.g., cells). The
operating at a power of the incident light may range from about
0.01 .mu.W/cm.sup.2 to about several hundred W/cm.sup.2, for
example.
[0093] By way of example, other suitable light sources that may be
used to illuminate the chamber for various embodiments disclosed
herein and in accordance with exemplary aspects of the invention
include, but are not limited to, LEDs, phosphor coated LEDs,
organic LEDs (OLED), phosphorescent OLEDs (PHOLED),
inorganic-organic LEDs, LEDs using quantum dot technology, and LED
arrays. Alternatively, suitable light sources may include, but are
not limited to, white light sources, halogen lamps (e.g., xenon or
mercury arc lamps), lasers, solid state lasers, laser diodes,
micro-wire lasers, diode solid state lasers (DSSL), vertical-cavity
surface-emitting lasers (VCSEL), thin-film electroluminescent
devices (TFELD), filament lamps, arc lamps, gas lamps, and
fluorescent tubes. Also by way of example, suitable mechanisms for
causing the light source to scan include, but are not limited to,
galvanometers and digital light projectors (DLP).
[0094] Moreover, by way of example, the footprint of the incident
light, such as light beam 700, illuminating the photoconductive
surface of an optoelectronic manipulation chamber may be achieved
by overlapping several beams of light, for example, rectangular
beams or other shaped beams, generated from multiple sources. As an
alternative, a single, projecting light source may provide a light
projection having, for example, the configuration illustrated in
FIGS. 2A and 2B. Those skilled in the art would understand a
variety of ways in which to generate incident light footprints of a
variety of configurations (patterns), as well as various ways
incident light could scan the chamber. Regarding the latter, by way
of example only, relative movement may be imparted between the
incident light and the chamber, by moving either the chamber, the
incident light, or both. As a further example, various independent
light sources could be turned on and off to create a scanning of
the incident light relative to the chamber.
[0095] As mentioned above, in various exemplary embodiments,
electroluminescence may be used to generate the light for
illuminating the photoconductive surface instead of a light source
external to the chamber. FIGS. 28A and 28B depict an exemplary
embodiment of an optoelectronic chamber wherein an array of
electroluminescent material 75 is used as a light source. FIG. 28B
shows a plan view of the electroluminescent material from the
perspective 28B-28B in FIG. 28A. Those with ordinary skill in the
art would understand a variety of arrangements of the
electroluminescent light source relative to the chamber in order to
illuminate the photoconductive surface and the arrangement shown in
FIGS. 28A and 28B should be understood as exemplary and not
limiting. The array may be provided in a configuration that permits
generation of spatially discrete illumination patterns on the
photoconductive surface, thereby permitting the modulation of
spatially discrete electric fields within the chamber. According to
various exemplary aspects, the electroluminescent layer may be in
the form of an array of small LEDs, quantum dots, and/or other
arrangements suitable for generating electroluminescent light. By
way of example, suitable electroluminescent materials that may be
used include, but are not limited to, FLATLITE.RTM. and GLOWIRE.
The electroluminescent material may be applied via adhesive or
other suitable securement mechanisms.
[0096] Electronic circuitry 77 may be provided so as to provide an
electric current to activate the electroluminescent material in a
way that modulates spatially discrete electric fields for DEP
movement of particles. As illustrated in FIGS. 28A and 28B, the
electronic circuitry may comprise electrical contacts 77a, 77b
configured to supply an electric current from a current source to
the array 75 of electroluminescent material. Examples of various
illumination patterns 79a, 79b, 79c are illustrated in FIG. 28B,
however it should be understood that virtually any illumination
pattern may be achieved by selectively applying current to the
arrayed electroluminescent material. Those having skill in the art
would understand how to configure electronic circuitry to apply
current to the electroluminescent material so as to achieve the
desired illumination patterns on the photoconductive surface. In
exemplary aspects, either the same power source used for biasing
the electrodes of the manipulation chamber could be used as the
power source for the electroluminescent material or a different
power source could be used. Modulation of electric fields may be
controlled both spatially and temporally as desired by controlling
the timing and locations of illumination via the electroluminescent
material. By way of example, a scanning of light relative to the
photoconductive surface may occur by consecutively activating
adjacent rows of the electroluminescent materials.
[0097] Using electroluminescence as the mechanism by which to
illuminate the photoconductive surface permits the light source to
be an integral part of the manipulation chamber (e.g., the
electroluminescent array may be applied in a layer adhered to a
surface of the chamber) and can achieve scanning and movement of
the light relative to the chamber without the need for moving parts
(e.g., parts to move either the chamber and/or the light source).
Moreover, because light is generated by electric current through
the electroluminescent material, flexibility in spatial
distribution and/or movement of the photoactivated electric field
may be achieved through relatively simple, inexpensive electronic
circuitry and signal generation. Electroluminescence also requires
relatively low power to generate light, thereby reducing the power
consumption of the chamber. Additionally, the wavelength of light
may be modulated based on the electroluminescent materials used. It
is envisioned that more than one electroluminescent may be used in
a manipulation chamber in order to provide greater control over the
electric field modulation, for example, at differing locations
within the chamber.
[0098] In another exemplary embodiment for performing
optoelectronic scanning, light patterns may be generated using a
programmable scanning mirror, such as, for example, a piezoelectric
or galvanometric scanning mirror. Such an approach may be
relatively simple in operation and provide opto-mechanical
interfacing with conventional upright microscopes. Moreover, as
explained further below, the approach may permit isolation of the
scanning beam from the epi-fluorescence imaging pathway of a
microscope such that scanning and simultaneous collection of
multi-color fluorescence cell images may occur. Although
optoelectronic scanning may permit markerless particle
identification, sorting, and/or collection, the ability to combine
optoelectronic scanning with multicolor fluorescence,
DEP-signature, and/or cell morphology techniques utilizing
integrated instrumentation also may be desirable.
[0099] With reference to FIG. 36, a schematic representation of an
exemplary embodiment of using a scanning mirror to generate light
patterns for optoelectronic scanning and an epi-fluorescence
optical pathway for imaging the optoelectronic scanning chamber is
illustrated. In FIG. 36, a scanning mirror 3610 is positioned so as
to reflect light from a light source 3615 to illuminate an
optoelectronic scanning chamber 3600 with patterned light. The
optoelectronic scanning chamber 3600 may have a configuration
consistent with the teachings herein such that light illuminating a
photoconductive portion of the chamber, for example, located on a
bottom of the chamber 3600 in FIG. 36, modulates an electric field
and creates a DEP force that acts on particles within the chamber.
The mirror 3610 may be configured to scan such that the light
reflected to illuminate the chamber 3600 moves relative to the
chamber 3600 so as to achieve optoelectronic scanning in accordance
with the teachings herein. According to various exemplary
embodiments, the mirror 3610 may be a piezoelectrically-driven
mirror, a galvanometric mirror, and/or other mirrors capable of
performing scanning of the light across the chamber 3600, as known
to those having skill in the art. In exemplary embodiments, the
scanning mirror 3610 may be configured to generate relatively
complex light patterns to illuminate the chamber 3600, including
movement of light in three dimensions within the chamber. The light
source 3615 that transmits light to the mirror 3610 may be from an
upright or inverted microscope and may emit light at a wavelength
that ranges from about 600 nanometers (nm) to about 700 nm, for
example, at about 633 nm.
[0100] A series of filters 3620, 3625, and 3630 may be positioned
relative to the chamber 3600 so as to provide optical isolation of
the scanning light beam fluorescence from an imaging pathway. The
filters may include, for example, spectrally-sensitive filters, for
example a short pass filter 3620 and a band pass filter 3625, and
dichroic filters, for example, a dichroic filter 3630, such that
the scanning light is spectrally-isolated from the
epifluorescence-imaging pathway. By way of example, filter 3620 may
be a 633 nm short pass filter and filter 3625 may be a 500-600 nm
band pass filter. In another exemplary embodiment, a notch filter
may be used in lieu of the short pass filter 3620. By way of
nonlimitative example, the various filters 3620, 3625, and 3635 may
be configured to permit the passage of light emitted from particles
(e.g., cells) labeled with dyes that are useful in biological
and/or other analysis, such as, for example FITC having a peak
emission of 525 nm, PE having a peak emission of 568 nm, YoYo1
having a peak emission of 514 nm, tetramethylrhodamine having a
peak emission of 546 nm, and DilC18 having a peak emission of 546
nm.
[0101] A fluorescence source 3635 may be disposed so as to transmit
light through the filters 3620, 3625, and 3630 to the chamber 3600
so as to perform fluorescence detection and analysis of particles
in the chamber, as is known to those skilled in the art. By way of
example, the fluorescence source may have a wavelength of, for
example, between 400-500 nm, for example, 407 nm or 488 nm.
Examples of suitable fluorescence sources include, but are not
limited to, lasers or arc lamps. Selection of the fluorescence
source, and corresponding wavelength, may depend on, among other
things, the required peak excitation of the flourophores (common
dyes noted above). The fluorescence detection and/or imaging of the
particles may occur via detection through a CCD camera 3640
configured and positioned so as to detect the fluorescence emitted
by the particles of interest in the chamber 3600. One or more
detectors and/or controllers 3645 may be positioned so as to detect
beam alignment and measure the output of the scanning beam 3615 and
to control the scanning mirror 3610.
[0102] It is envisioned that a conventional microscope may be
modified by attaching the various components of the exemplary
embodiment of FIG. 36 thereto, thereby providing a relatively
simple device for use in providing simultaneous light scanning and
detection/imaging of an optoelectronic scanning chamber. In another
exemplary arrangement, light could be projected from above the
optics of an inverted microscope. For example, a projector could be
used as the light source to produce a pattern of light, for
example, in the 633-635 nm range, and a lens may be used to
demagnify the projected light and transmit the demagnified light
image via a mirror to the chamber sitting on a microscope stage.
This arrangement utilizes the projector, lens, and mirror to turn
light projected from a side of the microscope onto the chamber, for
example, at a ninety degree angle and permits viewing of the
chamber via the microscope viewing mechanism. In some cases, it is
possible to modify a microscope by removing the transmitted light
condenser and place the projector and lens on top of the chamber.
In such an arrangement, the use of a mirror can be dispensed with.
In various exemplary embodiments, the projector may be programmable
so as to project a desired light pattern relative to the
optoelectronic scanning chamber.
[0103] FIG. 3A schematically illustrates a side partial view of an
exemplary particle P being scanned by an incident light beam 700 in
an optoelectronic manipulation device according to exemplary
aspects of the invention. FIG. 3B illustrates a force analysis on
the particle P of FIG. 3A as it is subject to and dragged by the
DEP force created by the scanning light beam and the manipulation
chamber. If the particle P has a positive Clausius-Mosotti value,
the particle P is attracted to the relatively strong electric field
that occurs proximate the scanning light and if it has a negative
Clausius-Mosotti value, it will be repelled by the relatively
strong electric field proximate the light beam. In either case,
however, the force analysis is the same. As shown in FIG. 3B, as
the light beam 700 scans across the manipulation chamber and
illuminates the photoconductive surface, a DEP force is created and
acts on the particle P, as shown by the arrow labeled F.sub.DEP in
FIG. 3B, substantially in the direction of the scanning light. The
other forces acting on the particle P simultaneously with the DEP
force are gravity F.sub.g, buoyancy F.sub.b, surface force F.sub.s,
and viscous drag, F.sub.d. Each of these various forces are labeled
in FIG. 3B with the direction each force acts being indicated by
corresponding arrows.
[0104] The relative magnitudes of the various forces described with
reference to FIG. 3B depend on factors such as, for example, the
characteristics, such as, for example, dielectrophoretic
characteristics, of the particle P, the characteristics of the
surrounding medium in which the particle is suspended, the
intensity of the light beam, and the applied voltage. Thus, the
various forces may be controlled, at least to an extent, by
selecting suitable mediums, strength of DEP force, scanning speed,
and other factors. In addition, by providing various agents and/or
other techniques to modify the surface of the particle, the medium
in which the particle is suspended, and/or the surfaces of the
manipulation chamber, the forces acting on the particles also may
be controlled. By way of example only, it may be possible to
reversibly modify particles to control whether the particle has a
negative DEP force (is repelled by the scanning light source) or a
positive DEP force (is attracted by the light source) and/or to
modify the strength of the DEP force, for example, by modifying the
charge and/or other dielectrophoretic characteristics of a
particle. Further, it may be possible to modify the size of the
particle by coating or other similar surface modification
techniques.
[0105] For example, the liquid medium in which a particle of
interest is suspended may be selected so as to balance the buoyant
force with gravity. When manipulating cells, the medium preferably
is iso-osmotic. For low-ionic strength media, a variety of agents,
such as, for example, sucrose, mannitol, polysaccharides, and other
similar agents, may be used to modify the medium in order so as to
provide a relatively electrically neutral medium. Moreover, the
surface force may be altered, for example, minimized, by providing
a surface coating on one or both of the surfaces in contact with
the sample layer in the manipulation chamber. In an exemplary
aspect, the wettability of one or both surfaces in contact with the
sample layer may be altered so as to make the surface either more
or less hydrophilic or hydrophobic.
[0106] According to various exemplary aspects, a viscosity enhancer
may be added to the liquid medium carrying the particles in order
to control (e.g., increase) the viscous drag force acting on the
particle P. In various embodiments, the viscous drag forces may be
altered by the addition of a viscosity enhancer chosen from
neutral, anionic, or cationic enhancers. By way of example,
suitable viscosity enhancers may be selected from polymeric
materials, including but not limited to, celluloses, such as, for
example, hydroxymethycellulose and 2-hydroxyethyl cellulose;
polysaccharides, such as, for example, chitosan; agar and agarose;
ethyleneglycol and its derivatives; homopolymers, such as, for
example, polyacrylamide, poly(N,N-dimethylacrylamide), poly(vinyl
alcohol), polyoxazoline, poly(N-vinyl pyrrolidone),
poly(N-vinylimidazole), poly(4-vinyl pyridine), poly(2-hydroxyethyl
(meth)acrylate), poly(vinyl methyl ether), salts of polyaspartic
acid; copolymers of the preceding monomeric units; and combinations
thereof.
[0107] Further, in various exemplary embodiments, the viscosity
enhancers may be chosen from inorganic materials, including, but
not limited to, fume silica for example. According to yet further
exemplary embodiments, the viscosity enhancers may be chosen from
organic solvents, including, but not limited to, glycerol, dimethyl
sulfoxide (DMSO), and N-methyl-2-pyrrolidone (NMP), and mixtures
thereof, for example. In yet further exemplary embodiments, the
viscosity enhancers may be chosen from proteins, such as, for
example, bone morphogenetic protein (BMP). Those skilled in the art
would understand that various viscosity enhancers may be selected
and that factors such as, for example, chemical structure,
molecular weight, and concentration of an enhancer may alter the
viscosity of a particular formulation.
[0108] Regardless of the viscosity enhancer that may or may not be
used, the viscous drag force acting on a particle being moved
through the liquid medium by the DEP force may be expressed by the
Stokes Equation in the case of low Reynolds number flow and
assuming the particle has a spherical shape, as follows:
F.sub.d=3.pi..mu.dV
[0109] Where .mu. is the viscosity of the liquid medium, d is the
diameter of the particle, and V is the velocity of the particle.
Under a prescribed set of conditions, such as, for example,
constant AC voltage, constant frequency, constant intensity of
light, etc., the DEP force is a constant force for a given particle
type in a given buffer medium. Under the same set of conditions,
therefore, the above equation implies that the viscous drag is
directly proportional to the velocity of the particle. Thus, at a
sufficiently large scanning velocity, the viscous drag may exceed
the DEP force, and consequently, the particle will not move at the
same speed as the scanning light beam. In other words, the particle
is left behind (escapes) the scanning light source.
[0110] For a given AC frequency, a given applied voltage, and a
given liquid medium, F.sub.DEP remains constant, independent of the
scanning speed and hence the driven particle velocity. The drag
force, F.sub.d, however, increases as scanning speed and particle
speed increases. Above a threshold velocity, F.sub.d exceeds
F.sub.DEP and a given particle type is left behind (escapes) the
scanning incident light. The threshold velocity differs for
differing particle types and depends on the dielectrophoretic
property of the particle (e.g, including particle size and
dielectric constant (permittivity), as can be seen by the DEP force
equation above). For a given particle (e.g., cell) type, the
dielectrophoretic property of the particle is determined by the
membrane of the cell, including it capacitance, permittivity,
conductivity, for example, and size. For a given particle type,
therefore, the dielectrophoretic property of the cell is unique and
constant.
[0111] FIGS. 4A-4C illustrate a schematic perspective top view of
an exemplary embodiment of optoelectronic scanning for two particle
types A and B having positive Clausius-Mosotti factors, which make
the particles A and B attracted to the light beam (e.g., positive
DEP). FIGS. 4A-4C respectively show the effect on the particles A
and B for three different scanning speeds of a light beam 700
across the manipulation chamber in the direction of the large
arrows shown in each of the figures.
[0112] Referring first to FIG. 4A, a relatively low scanning speed
of the light beam 700 is illustrated, with the direction of the
scanning light being indicated by the arrow in the figure. As
illustrated, at a relatively low scanning speed, both particle
types A and B are swept along the length of the manipulation
chamber 100 with the scanning light beam. In other words, in FIG.
4A, the DEP force acting on both particle types A and B is greater
or equal to the viscous drag force experienced by each of the
particle types A and B. Therefore, the particles move across the
manipulation chamber in response to the DEP force at a velocity
that is approximately equal to the scanning speed of the light beam
700, and thus stay "trapped" in the light beam 700 as it scans
across the chamber 100.
[0113] The illustration in FIG. 4B shows the effect on the particle
types A and B for a medium scanning speed of the light beam 700
across the manipulation chamber. The scanning speed of FIG. 4B is
higher than both the scanning speed of FIG. 4A and the threshold
speed corresponding to the particle type A, but is at or below the
threshold speed corresponding to the particle type B. In this
situation, therefore, the particle type A escapes the scanning
light beam 700 while the particle type B continues to move along
with the scanning light beam, as shown in FIG. 4B. As shown in FIG.
4B, the DEP force created by the scanning light beam 700 continues
to move particle type A along the manipulation chamber 100, as
illustrated by the displacement between positions Ia and IIa shown
for the particle type A.
[0114] At the scanning speed illustrated in the exemplary
embodiment of FIG. 4C, which is higher than the scanning speeds of
FIGS. 4A and 4B, the scanning speed exceeds the respective
threshold speeds of particle types A and B. In this case, both
particle types A and B are left behind (escape) the scanning light
beam 700. However, as shown by the positions Ib and IIb in FIG. 4C,
at a given time, the displacement of particle type B is larger than
the displacement of particle type A due to the difference in DEP
force acting on each particle type A and B, which is a result of
the differing dielectric properties and/or size of each particle
type. Thus, differing particle types exhibit differing
dielectrophoretic movement characteristics based on the particle's
dielectric properties and/or size, for example. In other words, the
motion imparted as a result of a DEP force differs for differing
particle types and therefore different types of particles exhibit
unique dielectrophoretic movement characteristics. For example,
such dielectrophoretic movement characteristics include the
displacement (dielectrophoretic displacement) of a particle type
and the speed of manipulation (dielectrophoretic speed) of the
particle type as a result of an applied DEP force resulting from a
moving incident light relative to the particle type.
[0115] It should be noted that in various exemplary embodiments,
the scanning velocity may be constant with time, may increase
linearly with time, may be a triangular function of time, or may be
a square function of time. Those skilled in the art will understand
that various waveforms may be used to control the scanning speed as
a function of time, and that the particular function used will
depend on, among other things, the scanning application.
[0116] According to various exemplary embodiments, optoelectronic
scanning principles, such as in the manner described with reference
to FIGS. 4B and 4C, may be used to perform cell identification and
in turn create a database regarding the behavior of differing
particle (e.g., cell) types when subject to optoelectronic scanning
under predetermined conditions. That is, optoelectronic-driven DEP
forces may be studied for various cell types of interest and a
functional relationship between the displacement of a cell type and
the light beam scanning speed may be determined. Further, the
scanning speed at which a cell type can be trapped (e.g., swept
along with) and/or the threshold speed at which a cell type escapes
the scanning light beam also may be determined. This information
may be collected and stored for the purpose of creating a database
for use in providing an automated technique to identify, sort,
collect, and/or otherwise manipulate various particle types. For
example, once information regarding displacement characteristics of
a particular cell type has been determined and stored in a
database, the identity of that particular cell type in a large
population of other cell types can be determined by selecting an
appropriate scanning speed and measuring the displacement of the
various cell types over a predetermined time period. By matching
the measured displacement with the stored displacement
characteristics, the particular cell type can be identified.
[0117] According to various exemplary aspects, the
dielectrophoretic movement characteristics, such as, for example,
displacement, of the various particles in the sample layer of a
manipulation chamber may be determined by capturing images of the
sample in the chamber before, after and/or during scanning using a
CCD camera and image processing software. Multiple light scans may
be performed on the sample to improve resolution. Speed of the
various particles may be another dielectrophoretic movement
characteristic that may be measured. As used herein,
"dielectrophoretic movement characteristic" may refer to any
parameter that may serve to characterize the movement of a particle
as a result of dielectrophoresis (e.g., DEP force acting on the
particle). Examples of such dielectrophoretic movement
characteristics may include speed, displacement, and acceleration,
for example.
[0118] According to an exemplary embodiment, in order to perform
image processing, the manipulation chamber may be placed on a
translation stage and a CCD camera (either monochrome or color) may
be used to capture images through an optical imaging system, which
may be a microscope. Such a system may be configured to magnify the
chamber and generate and image. The image could be an image chosen
from phase contrast, differential interference contrast,
reflectance, light scatter, fluorescence, or other types of images.
In an exemplary aspect, the CCD camera may be interfaced with a
computer so as to achieve image processing capabilities and to
store various data. Software for image processing could include,
for example, Universal Imaging Metamorph, Image Pro, or other image
processing software. Images also may be captured via a spatially
sensitive photodiode.
[0119] Using a CCD camera with an objective lens, a field of view,
for example, approximately 0.5 mm.times.0.5 mm can be generated,
and either brightfield or darkfield illumination may be used. By
moving the translation stage while the CCD camera remains
stationary, or vice versa, images of multiple locations of the
manipulation chamber can be captured and processed.
[0120] It is contemplated that in addition to capturing still
images of the manipulation chamber at various snapshots in time
during the scanning process, real-time moving images also may be
taken.
[0121] Aside from utilizing optoelectronic scanning to identify the
presence of particular particle types from among other particle
types in a sample, optoelectronic scanning may be used to sort and
collect particle types. In an exemplary aspect, the sorting and
collecting steps may follow an identification step. FIGS. 5A-5C,
FIGS. 6A-6C, and 7A-7C depict various exemplary embodiments of
utilizing optoelectronic manipulation principles (e.g., including
optoelectronic scanning) to sort and collect particle types.
[0122] FIGS. 5A-5C schematically illustrate an exemplary embodiment
for sorting two differing particle types A and B contained in a
suspension 50, with both particle types A and B having positive
Clausius-Mosotti values such that both are attracted to the
scanning light beam 700. In the exemplary embodiment of FIGS.
5A-5C, due to the dielectrophoretic properties of each particle
type, at a given velocity, particle type A has a relatively small
DEP force F.sub.DEP,A and a relatively large drag force F.sub.d,A
acting on it, whereas particle type B has a relatively large DEP
force F.sub.DEP,B and a relatively small drag force F.sub.d,B
acting on it. As indicated in the equations above, for a given
particle type, the DEP force remains constant, while the drag force
may change based on the speed of the particle (e.g., the speed of
the particle as it is moved by the DEP force).
[0123] FIG. 5A illustrates a portion of the scanning mode (e.g.,
time=0) when, due to the speed of scanning which has not yet
exceeded the threshold velocity of either particle type, both
particle types A and B are attracted to the stationary light beam
and are thus trapped within the light beam. As the light beam
reaches above a threshold scanning speed corresponding to particle
type A, which speed may be predetermined, for example, based on
previously gathered and stored displacement/scanning speed
relationships, particle type A escapes the scanning light beam 700
and is left behind while particle type B remains trapped and swept
along with the scanning light beam 700. FIG. 5B illustrates a
snapshot of the scanning assembly at a time after the light beam
has reached the threshold scanning speed at which particle type A
escapes the light beam 700.
[0124] Following the trapping of one or more particles of particle
type A, a collection scheme, for example, as illustrated in the
exemplary embodiment of FIG. 5C may be utilized to sort the
particles and collect them for further processing, manipulation,
and/or disposal. According to various exemplary embodiments, a
collection scheme may include illuminating the sorted particles,
e.g., types A and B, with separate beams of light (e.g., footprints
of focused light which may have a solid circular or other
configuration) focused so as to respectively trap one or more
particles of particle type A and one or more particles of particle
type B, for example. Although FIGS. 5A-5C show two differing
particle types, it should be understood that several differing
particle types may be present and sorted. The segregated particle
types trapped respectively by the focused light beams 701 and 702
can now be moved out of the scanning assembly to collection
reservoirs or other desirable locations by moving each light beam
701 and 702 at a speed such that the particle types A and B remain
trapped by the light beams 701 and 702, respectively.
[0125] Another exemplary embodiment of a sorting and collection
scheme that may be useful in manipulating particles having negative
Clausius-Mosotti values, leading to a negative DEP force which
causes the particles to be repelled by a light source and the
electric filed generated by it, is illustrated schematically in
FIGS. 6A-6C. In this exemplary embodiment, particles of particle
type A and particle type B have negative Clausius-Mosotti values
and thus during a portion of the scanning mode, such as the start
of the scanning mode (e.g., time=0) when the scanning speed of the
light source has not reached the threshold speed corresponding to
either particle type A or B, as illustrated in FIG. 6A, both
particle types are repelled by the scanning light beam 700 and the
DEP force acts to move the particles A and B in a direction away
from the scanning light beam 700.
[0126] Due to differences in their respective dielectrophoretic
properties (e.g., size and dielectric constant), particle type A is
influenced by a relatively large drag force F.sub.d,A and a
relatively small DEP force F.sub.DEP,A, whereas particle type B is
under the action of a relatively small DEP force F.sub.DEP,B and a
relatively large drag force F.sub.d,B. The scanning speed of the
light beam 700 can be adjusted such that it is above the threshold
velocity of particle type A, thereby causing the drag force acting
on the particle type A to become larger than the DEP force acting
on that particle type so that at a later time (e.g., time=t) during
scanning particle type A is left behind the scanning light beam 700
(e.g., particles of particle type A escape the scanning light
source). On the other hand, the scanning speed is selected such
that it is below the threshold speed corresponding to particle type
B such that the DEP force acting on particle type B continues to be
larger than the drag force acting on that particle type. In this
case, particle type B continues to be expelled from the light
source and thus particles of particle type A and particle type B
are separated from one another (e.g., divided by the light beam
700), as shown in FIG. 6B.
[0127] As discussed above with reference to FIGS. 5A-5C, the
scanning speed of the light beam 700 in the exemplary embodiments
of FIGS. 6A-6C may be predetermined based on information known
about the varying particle types that are being scanned. By way of
example, the scanning speed sufficient to separate particle type A
from particle type B, as shown in FIG. 6B for example, may be
predetermined based on information collected during an
optoelectronic scanning identification process such as that
described with reference to FIGS. 4A-4C, for example.
[0128] Once the differing particle types A and B have been
separated, as illustrated in FIG. 5B, each particle type may be
collected or otherwise manipulated separately from the other
particle type. By way of example, FIG. 6C illustrates an exemplary
embodiment of a collection scheme that may be used to collect one
or more particles of particle type A for further processing and/or
manipulation and to collect one or more particles of particle type
B for further processing and/or manipulation. It should be
understood that the collection of the particles may include
removing the particles from the manipulation chamber 100.
[0129] As discussed above, both particle types A and B in the
exemplary embodiment of FIGS. 6A-6C have negative Clausius-Mosotti
values. By encircling each of the segregated particle types A and B
with separate light beams 703 and 704 focused into ring-like
configurations, the particles of each particle type A and B become
trapped within the respective light rings 703 and 704 due to the
negative DEP, repelling force acting on them. Once trapped within
each ring of light 703 and 704, the differing particle types A and
B may be moved to different collection reservoirs or other
locations by moving the light rings 703 and 704 at a speed such
that the respective particle types A and B trapped therein are not
able to escape.
[0130] In various exemplary embodiments, optoelectronic scanning
also may be employed to separate and/or otherwise manipulate
particle types of both positive and negative Clausius-Mosotti
values contained in the same sample layer. By way of example, FIGS.
7A-7D schematically illustrate an exemplary embodiment of using
optoelectronic scanning to identify and sort particles in a sample
containing at least a first particle type having a positive
Clausius-Mosotti value and at least a second particle type having a
negative Clausius-Mosotti value. In the exemplary embodiment of
FIGS. 7A-7D, particle type A has a positive Clausius-Mosotti value
and particle type B has a negative Clausius-Mosotti value.
[0131] As scanning begins and the light beam 700 scans the
manipulation chamber 100 so as to be moved toward the particles A
and B shown in FIG. 7A, particles of particle type A will be
attracted to the scanning light beam 700 due to their positive
Clausius-Mosotti values. In contrast, particles of particle type B
will be repelled by the scanning light beam 700. FIG. 7A
illustrates an exemplary snapshot of the manipulation chamber 100
at the beginning of scanning, wherein the DEP forces F.sub.DEP,A
and F.sub.DEP,B are shown acting on each particle type A and B. As
shown in FIG. 7A, F.sub.DEP,A acts in a direction to move particle
type A toward the light beam 700 and F.sub.DEP,B acts in a
direction to move particle type B away from the light beam 700.
[0132] Due to the attraction of particle type A toward the light
beam 700, at a later time during the scanning process and with the
scanning speed of the light being controlled, particles of particle
type A become captured by (e.g., trapped in) the light beam 700 and
move along with that incident light 700 as it scans across the
manipulation chamber 100 in the direction shown by the arrow in
FIG. 7B. Assuming the scanning speed does not exceed particle type
A's threshold velocity, more and more particles of particle type A
will become trapped by the light beam 700 as it scans across such
particles during its travel across the manipulation chamber 100. At
the same time, particles of particle type B continue to be repelled
by the light beam 700 and therefore move along the manipulation
chamber 100 ahead of the scanning light beam 700, also in the
direction of the arrow shown in FIG. 7B.
[0133] Once the scanning light beam 700 is swept across the
manipulation chamber 100, the direction of scanning of the light
beam 700 relative to the chamber may be reversed such that the
light beam 700 scans in the direction shown in FIG. 7C. By scanning
in the reverse direction, separation of particle type A and
particle type B may be improved by further separating (e.g.,
increasing the distance between) particle type A particles from
particle type B particles. That is, particle type B, which is
repelled by the light, may remain at its position prior to the
reverse scanning, which may be at the right hand side of the
chamber if the light beam 700 scans across the entire chamber in
the direction shown in FIG. 7B. Particle type A, on the other hand,
will continue to be attracted by the light beam 700 during the
reverse scan shown in FIG. 7C, and thus further separation between
particle type A and particle type B may be achieved.
[0134] Once the desired separation between particle types A and B
is achieved and/or at some time after reverse scanning of the light
beam 700, the scanning mode may be stopped and focused light beams
used to independently move particles of particle type A and
particles of particle type B, for example, to collection reservoirs
or the like. As illustrated in FIG. 7D, a focused light beam having
a solid circular configuration may be used to collect particles of
particle type A having a positive Clausius-Mosotti value. A focused
light beam having a ring-like configuration can be used to encircle
and collect particles of particle type B having a negative
Clausius-Mosotti value.
[0135] It should be understood that the number of scanning
iterations, including reverse scanning, of FIGS. 5-7 can occur
numerous times as needed to achieve desired separation of
particles. Further, the reverse scanning step of the exemplary
embodiment of FIG. 7 could occur numerous times during the scanning
process. In other words, the reverse scanning can take place at any
point during the scanning of the manipulation chamber and need not
occur only once the light has scanned the entire chamber in one
direction. Moreover, the speed of forward and reverse scanning may
vary independently with time during the course of scanning.
[0136] It should be understood that in the exemplary embodiments
described herein, any number of particles of each particle type may
be in the sample layer introduced into the manipulation chamber and
may be separated from the other particle types and collected, and
that the number of each particle types illustrated in the figures
is exemplary only. Further, it also should be understood that the
exemplary embodiments could be used to separate and/or collect more
than two differing particle types and that the use of two particle
types A and B is for ease of reference and explanation. Moreover, a
variety of focused light configurations, other than rings or solid
circles, for example, may be used to move the sorted particles to
respective collection reservoirs. And it is envisioned that groups
(e.g., clusters) of trapped particles may be moved together via a
focused light beam.
[0137] Also, in the description of FIGS. 5-7 and otherwise herein,
when incident light is referred to as moving (e.g., scanning or
otherwise) relative to the chamber, it should be understood that
such moving is intended to imply relative movement between the
incident light and the chamber. It is envisioned that such relative
movement may be achieved by moving the light while the chamber
remains stationary, moving the chamber while the light remains
stationary, moving both the chamber and the light source, or
independently illuminating a plurality of stationary light sources
configured in an array so as to achieve movement of incident light
relative to the chamber. It also should be understood that the
velocity of the relative movement of incident light relative to the
chamber (e.g., the scanning speed) may be varied as a function of
time.
[0138] FIG. 8 schematically illustrates an exemplary embodiment of
a device that may be used to identify particles of interest (e.g.,
target particles) in a sample containing a plurality of differing
particle types and to separate and collect those particles of
interest utilizing the principles of optoelectronic manipulation
(e.g., optoelectronic scanning) described herein. As shown in FIG.
8, an exemplary optoelectronic identification and sorting device
200 may comprise an optoelectronic manipulation chamber 110
comprising an inlet 120 and at least two outlets 130, 140. A first
outlet 130 may lead, for example, to a waste collection region in
flow communication with the manipulation chamber 110 and a second
outlet 140 may lead to a channel 144 configured to collect the
target particles from the manipulation chamber 110. The channel 144
may be in flow communication with a collection reservoir 146
configured for collecting one or more target particles after those
particles are moved from the manipulation chamber 110 and through
the collection channel 144. A third outlet 148 may be provided in
flow communication with the collection reservoir 146 to pass the
collected target particles from the optoelectronic sorting device
200 to other instrumentation and/or locations for further
processing and/or other manipulation of the collected target
particles.
[0139] One or more of the various inlets and outlets may be
associated with valves, for example, microfluidic valves 122, 132,
and 142, so as to control flow through those inlets and outlets.
Although the valves 122, 132, and 142 of FIG. 8 are shown as being
placed within the sorting device 200, it should be understood that
one or more of the valves could be placed outside of the device 200
in conjunction with other instrumentation, such as, for example,
reservoirs, pumps, including microfluidic pumps, or other
instrumentation for feeding sample to the device 200 and/or for
removing sample and/or cells from the device 200.
[0140] According to various exemplary embodiments, exemplary steps
for using the device 200 of FIG. 8 to identify and sort particles
of interest are shown in FIGS. 9A-9H. In FIGS. 9A-9H, the particles
that are desired to be collected are referred to as target
particles and are labeled T. The other particles in the sample are
referred to as nontarget particles and are labeled N. To begin, as
depicted in FIG. 9A, valves 122 and 132 are open, while valve 142
remains closed. A sample 150, such as a suspension containing
target particles T and nontarget particles N is introduced into the
chamber 110. By way of example, a microfluidic pump (not shown) may
be placed in flow communication with the chamber 110 in order to
supply the sample 150 to the chamber 110. Once the chamber 110 has
been filled with the sample 150, valves 122 and 132 are closed and
an elongated light beam 800 (e.g., a light beam that spans the
width of the chamber) scans across the chamber 110 in the direction
of the arrow, as shown in FIG. 9B.
[0141] In the exemplary step of FIG. 9B, the target particles T
contained in the sample 150 within the chamber 110 are identified,
for example, in accordance with the exemplary embodiment for
particle identification described with reference to FIGS. 4C.
According to various exemplary aspects, the cell identification
step of FIG. 9B may occur by using image processing software and a
CCD camera, for example, to take a snapshot of the particles in the
chamber 110 before and after scanning. Based on information
regarding the dielectrophoretic movement characteristics (e.g.,
displacement) that various particle types exhibit in response to
predetermined light beam scanning speeds and other operational
parameters of the chamber 110, the target particles T may be
identified by comparing measured information resulting from the
scanning step of FIG. 9B with stored information or otherwise known
information.
[0142] After the target particles T in the manipulation chamber 110
have been identified in the exemplary identification step of FIG.
9B, the scanning light beam may be turned off and focused light
sources 810 may be illuminated on the identified target particles
T. By way of example, a focused ring of light like that described
with reference to FIG. 6C may be used if the target particles T
have a negative Clausius-Mosotti value. Alternatively, a focused
solid circle of light like that described with reference to FIG. 5C
may be used if the target particles T have a positive
Clausius-Mosotti value.
[0143] The focused light sources 810 may then be moved so as to
route the target particles T through the outlet 140 and collection
channel 144 and into the collection reservoir 144, as illustrated
in FIGS. 9C and 9D.
[0144] According to various exemplary embodiments, additional
identification and sorting steps may be performed as desired. For
example, if additional sorting is desired, valves 122 and 132 may
again be opened and a new sample containing target particles T and
nontarget particles N may be introduced into the manipulation
chamber 110, as illustrated in the exemplary embodiment of FIG. 9E.
As described with reference to FIGS. 9B-9D, the valves 122 and 132
may be closed, optoelectronic scanning may be performed to identify
target particles T in the manipulation chamber 110, and then
focused light sources 810 may be used to illuminate the target
particles T and move those additional target particles T through
the channel 144 and into the collection reservoir 146, as shown in
FIG. 9F.
[0145] Once the desired number of target particles T have been
collected in the collection reservoir 146 and/or the desired number
of identification/sorting/collection iterations have been
performed, valves 122 and 132 may again be opened and the
manipulation chamber 110 may be cleared of the sample 150.
According to various exemplary embodiments, and as shown in FIG.
9G, the manipulation chamber 110 may be washed by flushing the
chamber with a medium 160. The medium may be the same as the medium
used to suspend the cells. Other mediums may also be used, for
example, buffer solutions, such as a phosphate buffer saline
containing 1% BSA, that are compatible with the cells may be
used.
[0146] After the manipulation chamber has been flushed, valve 132
may be closed while valves 122 and 142 are opened, as shown in FIG.
9H. The target cells T in the reservoir 146 may be moved from the
collection reservoir 144 and the sorting device 200 through outlet
148 to another location and/or instrumentation for further
processing and the like of the target particles T. To remove the
target cells T, an aqueous medium 170 (e.g., a solution or a
solvent) may be introduced into the chamber 110 and drawn through
the channel 144 and collection reservoir 146 to suspend the target
particles T and remove them from the sorting device 200. A
microfluidic pump or other device suitable for removing the target
particles T may be placed in flow communication with the sorting
device 200 to accomplish removal of the target particles T from the
device. Media that may be suitable to flush the chamber include,
but are not limited to, the same media that may be used to prepare
the suspension of the particles. Thus, suitable media include
buffers, such as, for example, phosphate buffers, phosphate buffer
salines (which may contain 1% BSA), a solution of sodium- or
potassium-chloride, or other buffers. The flushing medium also may
in the form of an organic solvent, for example, a solution of
ethanol or acetonitrile. As a further example, if the target
particles T are cells that may be used and propagated after
collection, e.g., stem cells, it may be desirable to use a cell
growth medium, such as, for example, Eagle's, RPMI, Fischer's,
Ham's F10, or other suitable cell growth media, to remove and
collect the target particles T.
[0147] With reference to FIGS. 10A and 10B, two exemplary
embodiments of a multi-manipulation chamber sorting device are
illustrated. The multi-chamber sorting devices of FIGS. 10A and 10B
may be used, in a manner similar to that described with reference
to FIGS. 9A-9H, to identify, sort, and collect two or more target
particle types.
[0148] The sorting device 300 shown in FIG. 10A is a serial
multi-chamber sorting device comprising a first manipulation
chamber 310 and a second manipulation chamber 320. The first and
second manipulation chambers 310 and 320 may be configured so as to
be capable of performing optoelectronic manipulation, including
optoelectronic scanning, to identify and segregate differing
particle types as has been described herein. The first chamber 310
and the second chamber 320 may be in flow communication with each
other via a channel 330 and a valve 332. The first chamber 310 may
also be provided with an inlet 320 and an associated valve 322,
which may be similar to the inlet 120 and valve 122 described above
with reference to FIG. 8. The second chamber 320 may be provided
with an outlet 340 and a valve 342. Each of the chambers 310 and
320 may further be in flow communication with respective outlets
350 and 360 leading to collection channels 354 and 364 which in
turn are in flow communication with respective collection
reservoirs 356 and 366. Each of the collection reservoirs 356 and
366 may be provided with a valve 352 and 362 and an outlet channel
358 and 368 configured to direct collected target particles out of
the sorting device 300.
[0149] Various modes of operation may be envisioned for the serial
sorting device 300 of FIG. 10A and, based on the teachings provided
herein, would be understood by one of ordinary skill in the art.
According to various exemplary embodiments, the first manipulation
chamber 310 may be filled with a sample containing a plurality of
particle types, including a first target particle type, a second
target particle type, and other nontarget particle types. The first
manipulation chamber 310 may be filled with the sample while the
valve 332 is closed such that the manipulation chamber 320 remains
free of sample. A scanning light beam similar to that described
with reference to FIG. 9B may be used to scan the manipulation
chamber 310 and thus create a DEP force acting on the various
particles in the chamber 310. By controlling the scanning speed of
the light source and capturing images of the chamber so as to
determine the dielectrophoretic movement characteristics (e.g.,
displacement) of the various particles as a result of the scanning,
particles of the first target particle type may be identified and
moved into the collection reservoir 356 by using focused light
beams in a manner similar to that described with reference to FIG.
9C.
[0150] After the particles of the first target particle type have
been collected in collection reservoir 356, the sample now
containing the second target particles type and nontarget
particles, and from which the first target particle type has been
removed, may be moved into the second manipulation chamber 320, by
opening at least valve 332. By way of example, a pump (e.g., a
microfluidic pump) and/or other fluid handling equipment, which may
be in flow communication with the manipulation chamber 320 (for
example, via the inlet 320 or outlet 340) may be used to move the
sample from the first manipulation chamber 310 into the second
manipulation chamber 320. Those having skill in the art would
understand that various methods may be used to cause the sample in
chamber 310 to flow to chamber 320 and the corresponding positions
of the valves 322, 332, 342 may be determined based on the desired
flow and devices used to create that flow.
[0151] Optoelectronic scanning, identification, and collection of
particles of the second target particle type can again be performed
in manipulation chamber 320, consistent with the principles
discussed herein and as described with reference to FIG. 9B-9D, and
the particles of the second target particle type can be segregated
and moved into the collection reservoir 366. The collected target
particles of the first and second target particle type can then be
removed from the sorting device 300 by passing through the
respective outlet channels 358 and 368 with the valves 352 and 362
in the open position. In an exemplary aspect, the target particles
may be removed via flushing, pumps, or other fluid handling
mechanisms, for example, in a manner similar to that described with
reference to FIGS. 9H. It should also be understood that flushing
of the manipulation chambers 310 and 320 may occur as desired
during the sorting/collection process described with reference to
FIG. 10A.
[0152] In an alternative exemplary aspect, the serial sorting
device 300 of FIG. 10A may be used to accomplish simultaneous
sorting of a first target particle type and a second target
particle type by filling both chambers 310 and 320 with a sample
containing one or more particles of the first target particle type,
one or more particles of the second target particle type, and one
or more nontarget particles. Once sample fills both chambers 310
and 320, two differing scanning light beams (not shown) may be used
to respectively scan each chamber 310 and 320. Using the various
principles described herein, particles of the first target particle
type may be identified in chamber 310 and collected in collection
reservoir 356 and particles of the second target particle type may
be identified in chamber 320 and collection in collection reservoir
366. Once the first and second target particle types are collected
in chambers 356 and 366, respectively, they may be removed from the
sorting device 300 for further processing and/or manipulation.
[0153] As with the embodiment of FIGS. 9A-9H, multiple iterations
of sample filling, scanning, collecting, flushing, etc. may be
performed as desired in the sorting device embodiment of FIG. 10A.
Moreover, although FIG. 10A shows two manipulation chambers and
corresponding collection reservoirs, it is envisioned that the
device could include any number of chambers and collection
reservoirs, depending, for example, on the number of differing
target particle types it is desired to collection, the total number
of particles it is desired to collect, and other factors.
[0154] Referring now to FIG. 10B, an exemplary embodiment of a
parallel multi-manipulation chamber sorting device is schematically
depicted. In this embodiment, the sorting device 400 comprises a
manipulation chamber 410 having two outlets 440 and 450 leading to
collection channels 444 and 454 and ultimately to two respective
collection reservoirs 446 and 456. Each collection reservoir 446
and 456 may have an outlet 448 and 458 and corresponding valve 442
and 452, which may be a microfluidic valve, for example. Similar to
the manipulation chamber 110 described with reference to FIGS.
9A-9H, the manipulation chamber 410 also may have an inlet 420 for
sample introduction and an outlet 430 for waste removal, with
corresponding valves (e.g., microfluidic valves) 422 and 432.
[0155] According to various exemplary aspects, the sorting device
400 may be used for parallel optoelectronic identification,
sorting, and collecting. For example, a sample containing a first
target particle type, a second target particle type, and nontarget
particles may be introduced so as to fill the manipulation chamber
410 by opening valves 422 and 432, while valves 442 and 452 remain
closed. The sample in the manipulation chamber 410 may be scanned
via a scanning light beam (not shown) in a manner similar to that
described with reference to FIGS. 9A-9H, for example. After
scanning, identification of particles of the first and second
target particle types in the sample may occur in accordance with
various principles described herein. Such identification may occur
as explained with reference to FIG. 4C, for example. Once the
particles of the first and second target particle types have been
identified and located, focused light beams, such as, for example,
rings of light or solid circles of light, may be used to capture
the target particles and move them via respective collection
channels 444 and 454 into respective collection reservoirs 446 and
456. For example, the first target particle type may be moved into
the collection reservoir 446 and the second target particle type
may be moved into the collection reservoir 456, or vice versa. The
sorting device 400 may be flushed and numerous iterations of sample
addition, scanning, particle collection, and/or flushing may occur
as desired.
[0156] Further, although FIG. 10B depicts two collection channels
and collection reservoirs in flow communication with the
manipulation chamber 410, it should be understood that any number
of collection channels and collection reservoirs may be used
depending, for example, on the number of differing target particle
types it may be desired to sort and collect.
[0157] The velocity of particle movement via optoelectronic
scanning or other optoelectronic particle manipulation may be
determined by the size and dielectric constant of the particle, the
intensity of the light, the applied potential bias, the frequency
of the AC current, the medium in which the particles are suspended,
and other factors that affect the DEP force acting on the particle.
In some circumstances, such as, for example, in some sorting
processes where it may be necessary to carefully sort a specific
particle type or types from among others, it may be desirable to
have a relatively slow velocity at which the particle or particles
move during manipulation. A relatively slow manipulation velocity
may also be used to capture and reroute particles having a
relatively weak DEP force (e.g., a relatively low threshold or
escape velocity) in order to prevent such particles from escaping.
In other situations, however, it may be desirable to move particles
at a faster speed. For example, for particles having a relatively
strong DEP force, a relatively high velocity may be used for
capturing and rerouting the particles, which may in turn increase
sorting throughput. As discussed above, it may be possible to alter
the DEP force acting on a particle by changing various
parameters.
[0158] The exemplary embodiment of FIGS. 11A-11C schematically
depicts an optoelectronic sorting device and technique in which the
particle manipulation speed (e.g., the speed at which nontarget
particles N are removed from the chamber) may rely on the flow of
the medium rather than the speed of the optoelectronic (DEP)
manipulation of the particles. In this case, as will be explained
further below, the nontarget particles may be separated from target
particles T and removed from the chamber by flushing the chamber
510 while the target particles T are held stationary by focused
incident light 581 and 582 and a corresponding DEP force, as shown
in FIG. 11C. Thus, collection of the target particles T in the
chamber 510 occurs substantially at the flow speed at which the
nontarget particles N are removed from the chamber, rather than the
speed of DEP manipulation out of the chamber, as described, for
example, with reference to FIGS. 9C and 9D. After removal of the
nontarget particles T, the target particles T may also be moved
into the collection reservoir 546 via flowing the target particles
T into the chamber. In this way, a higher sorting throughput may be
achieved since several particles at a time may be moved from the
chamber via flushing, as opposed to moving smaller amounts or
individual particles from the chamber via optoelectronic
manipulation. The speed at which the particles T and/or N are
removed from the chamber depends on a flow speed rather than the
speed of optoelectronic manipulation. The flow can be induced, for
example, by pressure, electro-osmosis, or other flow inducing
mechanisms. In an exemplary aspect, pressure may be used to flush
the particles from the chamber may occur. Further, the process of
the exemplary embodiment of FIGS. 11A-11C, which will be described
below, may comprise an optoelectronic scanning step, a capturing
step (via optoelectronic dielectrophoresis), and two flushing steps
(one for flushing nontarget particles and one for flushing target
particles for collection). Thus, the sorting aspect of the process
relies in large part on the speed at which the flushing occurs.
[0159] As depicted in the exemplary embodiment of FIGS. 11A-11C, a
sample 550 containing one or more particles of a target particle
type T and one or more particles of a nontarget particle type N may
be introduced into a manipulation chamber 510 of a sorting device
500 which may be similar in structure to the sorting device 200
described with reference to FIGS. 9A-9H. That is, the manipulation
chamber 510 may include an inlet 520 and corresponding valve 522 to
allow for introduction of sample and/or flushing medium into the
manipulation chamber 510. The manipulation chamber 510 also may
comprise an outlet 530 and corresponding valve 532 to permit
removal of sample and/or flushing medium out of the chamber 510.
Another outlet 540 may lead to a collection channel 544 for the
routing and collection of target particles T in a collection
reservoir 546 that is in flow communication with the channel 544.
The collection reservoir 546 may comprise an outlet 548 and
corresponding valve 542. As was described with reference to the
sorting device and technique of FIGS. 9A-9H, a variety of fluid
handling devices and other instrumentation, such as, for example,
microfluidic pumps, electro-osmosis fluid handling devices, and
various other flow control mechanisms, including valves (e.g.,
microfluidic valves) may be provided in connection with the sorting
device 500 of FIGS. 11A-11C so as to control flow therethrough.
[0160] By way of example, use of the sorting device 500 for sorting
target particles T having a negative Clausius-Mosotti value from
nontarget particles N having a negative Clausius-Mosotti value will
be described. It should be understood, however, that the sorting
device 500 could also be used to sort target and nontarget
particles which both have positive Clausius-Mosotti values or to
sort target particles of negative Clausius-Mosotti value and
nontarget particles of positive Clausius-Mosotti value or vice
versa.
[0161] Referring to FIG. 11A, with valves 522 and 532 open and
valve 542 closed, sample 550 may be introduced into the
manipulation chamber 510. Once the manipulation chamber 510 has
been filled with the sample 550, the valves 522 and 532 may be
closed and a light beam 580 (e.g., a light beam that spans the
width of the chamber 510) may be used to scan across the length of
the manipulation chamber 510, for example, in the direction of the
arrow shown in FIG. 11B. After the light beam 580 has scanned the
manipulation chamber 510, the respective displacements (or other
dielectrophoretic movement characteristics) of the particles T and
N may be measured, for example, via a CCD camera and image
processing software. From those displacement measurements, the
target particles T may be identified, for example, as described
with reference to FIG. 4C or by otherwise correlating the measured
displacement of the various particles with individual particle
types.
[0162] With the scanning light beam 580 turned off, the identified
target particles T in the sample 550 may then be captured by using
a focused light beam to individually trap each identified target
particle T (or possibly clusters of particles T) in the sample. For
the example of FIGS. 11A-11C wherein the target particle type T has
a negative Clausius-Mosotti value, the focused light beams used to
trap the particles T may have a substantially ring-like
configuration, as shown by focused light beams 581 and 582 in FIG.
11C. If, however, the target particle type had a positive
Clausius-Mosotti value, the focused light beams used to trap the
target particles could have a solid substantially circular
configuration such that the target particles would be trapped
within the light surrounding the particles.
[0163] With the target particles T captured (e.g., trapped) by the
focused light beams 581 and 582, the valves 522 and 532 may be
opened and the chamber 501 may be flushed with a flushing medium
560 (e.g., an aqueous buffer solution) to remove the nontarget
particles N, as depicted in FIG. 11C. Because the target particles
T are trapped (captured) by the negative DEP force created by the
light beams 581 and 582, the target particles T are not removed
from the manipulation chamber 510 with the flushing medium. In
order to hold the target particles T within the focused light 581
and 582 while the nontarget particles N are being flushed from the
chamber, the target particles T should have a relatively strong DEP
force. In other words, the DEP force acting on the target particles
T should be sufficient to prevent the particles from escaping the
focused light 581 and 582 and being flushed out of the chamber with
the nontarget particles N.
[0164] Once the chamber 510 has been flushed and rid of all the
nontarget particles N, the trapped target particles T may be routed
through outlet 540, channel 544, and into collection reservoir 546.
The routing of the target particles T may be accomplished via a
variety of fluid handling techniques, including, but not limited
to, pressure-induced flow, for example, pumping, electro-osmotic
flow. Alternatively, as with other embodiments described herein,
the target particles T could be removed from the chamber 510 via
optoelectronic manipulation.
[0165] FIGS. 30-32 illustrate yet another exemplary embodiment of a
device and technique for identifying differing particle types,
including, for example, particles of interest, in a sample
containing a plurality of differing particle types, separating the
differing particle types, and collecting the differing particle
types utilizing the principles of optoelectronic manipulation
(e.g., optoelectronic scanning) described herein. As explained
further below, the exemplary embodiment of FIGS. 30-32 may achieve
a relatively high throughput of particles (e.g., cells) because the
optoelectronic scanning occurs over a flowing stream of particles
rather than over a stationary solution of particles. Moreover, as
compared to conventional sorting techniques such as, flow cytometry
and/or techniques relying on patterned, deposited electrodes and/or
high-power lasers, for example, the embodiment of FIGS. 30-32 may
be easier and less costly to fabricate, may be simpler to operate
without the need for chemical labels, an analysis region, and/or
may provide operational flexibility by changing scanning
parameters.
[0166] In various exemplary embodiments, a device 3010 for sorting,
separating and collecting small particles utilizing optoelectronic
scanning may have a configuration similar to a conventional flow
cytometer, as depicted, for example, in FIG. 30. The device 3010,
however, may include an optoelectronic manipulation chamber 3100 in
place of the analysis and sorting regions typically found in
conventional flow cytometers. FIG. 30A, shows the optoelectronic
manipulation chamber 3100 portion of the device 3010 in greater
detail in the cross-sectional view taken through line 30A-30A. By
way of example and not limitation, the chamber 3100 may have a
configuration substantially the same as the chamber 100 depicted
and described with reference to FIG. 1. Like parts of the chamber
3100 are represented by the same reference labels as the chamber
100 in FIG. 1 except in the 3100 series. The spacers 3140 in FIG.
30A may be configured so as to form a microfluidic channel 3045
through which the flow of particles may pass during sorting and
separating. The spacers 3140 may have a variety of forms as would
be understood by those skilled in the art. According to an
exemplary embodiment, the spacers 3140 may be a pressure sensitive
adhesive layer that is die-cut to form channels and chambers. Those
having ordinary skill in the art would also understand that the
chamber 3100 may have a variety of configurations consistent with
the teachings herein to perform optoelectronic scanning of
particles.
[0167] The device 3010 also may include a sample input port 3150
for introducing a sample containing a plurality of particles of
differing types into the device 3010 (e.g., a biological sample
containing a suspension of differing types of cells). Ports 3145
may be disposed on either side of the sample input port 3150 for
introducing an aqueous medium, buffer solution, such as, for
example, phosphate buffered saline with 1% bovine serum albumin, or
other suitable buffer medium for suspending the particles as
described herein, into the device 3010. The buffer can be
introduced in a manner that causes a sheath flow similar to that in
conventional flow cytometers. In other words, the flow through the
portions 3145 "pinches" the sample that is introduced into the
portion 3150 so as to cause the particles in the sample to
substantially align and flow through the microfluidic channel 3045
of the optoelectronic scanning chamber 3100.
[0168] A plurality of branch channels 3155 may be positioned on a
side of the chamber 3100 opposite to the ports 3145 and 3150. The
branch channels 3155 may be configured to collect the differing
types of particles that have been sorted and separated in the
chamber 3100. Each branch channel 3155 may be configured to collect
a differing type of cell or, alternatively, waste. Although the
exemplary embodiment of FIGS. 30 and 31 depict three branch
channels 3155, it should be understood that the device 3010 may
include any number of branch channels, depending, for example, on
the number of differing particle types for which collection is
desired. In an exemplary aspect, the number of channels may equal
the number of differing particle types that are being collected
plus an additional channel to collect waste.
[0169] FIG. 31 illustrates an exemplary embodiment of using the
device 3010 of FIG. 30 to sort three differing types of particles
(e.g., cells) in a sample. The differing particle types P1, P2, and
P3 are indicated by different shading in FIG. 31. To begin, a
sample S containing the differing particle types P1, P2, and P3 may
be introduced into the sample introduction port 3150 and a sheath
flow medium M may be introduced into the ports 3145 in the
directions shown by the arrows in FIG. 31. The sheath flow medium M
is directed toward the sample S so as to "pinch" the flow of sample
S and the particles P1, P2, and P3 contained in the sample into a
columnated flow substantially through the microfluidic channel 3045
in the chamber 3010.
[0170] As the sample S containing the particles P1, P2, and P3
flows through the channel 3045, a series of differing scanning
light beams 3700, 3701, and 3702 may scan across the chamber 3100
in the direction of the arrow shown in FIG. 31 (e.g., from right to
left across the chamber 3100). The speed, frequency, and/or
intensity of the light beams 3700, 3701, and 3702 may be selected
so as to entrain and capture the differing particle types P1, P2,
and P3 via the DEP force associated with each light beam 3700,
3701, and 3702, as is described in the teachings herein. The
optoelectronic scanning that occurs as the particles P1, P2, and P3
pass through the optoelectronic scanning portion 3100 of the device
3010 causes the particles P1, P2, and P3 to become sorted and
separated into substantially three distinct columns as they move
down the length of the device 3010. That is, due to the varying
balance of the viscous drag, the DEP force, and the momentum of the
moving stream of sample acting on a particle, as schematically
represented in FIG. 32, the differing particle types P1, P2, and
P3, are sorted and separated in the lateral direction as they move
through the chamber 3100. Thus, by the time the flow of the sample
S and particles P1, P2, and P3 reach the branch channels 3155, they
are sorted into columns that substantially align with each branch
channel 3155. This permits the particles of each particle type P1,
P2, and P3 to be collected in a respective branch channel 3155, as
shown in FIG. 31, and removed from the device and/or moved to a
location for further processing. In various exemplary embodiments,
the branch channels 3155 may be positioned and configured such that
all of the differing particles flow into a single channel 3155 in
the absence of optoelectronic scanning as they pass through the
portion 3100 of the device 3010. Those having skill in the art
would recognize that one of the branch channels 3155 in FIG. 31 may
be used to collect waste and/or particles not of interest, while
the other two are used to collect differing particle types of
interest.
[0171] In various exemplary embodiments, various design parameters
associated with the device 3010, such as, for example, the width W
and the length L of the channel 3045, the rate of flow of the
sample S, the rate of flow of the medium M, the flow pinch ratio,
the scan speed of the light beams, etc., may be determined by
running experiments in an optoelectronic scanning chamber to
determine the amount of displacement that particles of particular
type experience as they are subject to optoelectronic scanning. A
database may be compiled that stores the displacement data for each
particle type along with the various parameters used to achieve the
displacement. The design, configuration, and operational parameters
of the optoelectronic scanning chamber portion 3100 may be selected
accordingly to achieve the desired sorting, separating, and
collection as described above.
[0172] The various sorting device embodiments depicted in FIGS.
8-11 and 30-32 are schematic depictions intended to illustrate
various exemplary steps that may be used to scan, identify, sort,
and/or collect particles of interest from other particles in a
sample. It should be understood that the manipulation chambers, the
inlets and outlets thereto, the collection reservoirs, the light
sources, and the various other elements of the sorting devices
could have a variety of configurations permitting the
implementation of optoelectronic manipulation and other flow
handling as described and/or otherwise taught herein. Further, in
order to provide control over the scanning, identification, and/or
sorting techniques described herein and in particular with
reference to FIGS. 8-11 and 30-32, it may be desirable to alter the
positivity or negativity of the Clausius-Mosotti values of the
target particles and/or the nontarget particles. By way of example,
surface-active agents may be applied to the target and/or nontarget
particles as desired to change the Clausius-Mosotti values of those
particles from positive to negative or vice versa. According to
exemplary aspects, such a change to the particles may be a
reversible change such that the original Clausius-Mosotti value can
be recovered after scanning, identification, sorting, and/or
collection has been performed.
[0173] As described above, DEP (dielectrophoresis) is the motion
imparted on uncharged particles, for example, through a solution,
as a result of polarization induced by nonuniform electric fields,
whereas electrophoresis (EP) is the migration of particles through
a solution under the influence of an applied electric field by
virtue of the particle's charge. It is envisioned that both
optoelectronically induced DEP and EP may be used to manipulate
particles in accordance with various exemplary aspects of the
invention. FIGS. 12A-12D illustrate an exemplary embodiment for
utilizing an optoelectronic manipulation chamber subject
intermittently to an AC current source and a DC current source in
order to manipulate particles in a sample via DEP forces and EP
forces. Utilizing such intermittent AC-DEP and DC-EP manipulation
schemes may achieve enhanced particle separation and sorting.
[0174] In the exemplary embodiment of FIGS. 12A-12D, an
optoelectronic manipulation chamber 810 may be similar in structure
to the manipulation chamber described with reference to FIG. 1. In
accordance with an exemplary aspect, the first substrate 820 of the
chamber 810 may be a glass substrate and may comprise a transparent
electrode layer 822, such as, for example, PEGylated transparent
gold electrode, which will be described in further detail in the
example below. The second substrate 830 may be a glass substrate
and may comprise an electrode layer 832, such as, for example, an
aluminum, gold, or ITO electrode, and a photoconductive layer 834.
The photoconductive layer 834 may be, for example, a PEGylated
photoconductive layer 834. A variety of other materials may be used
for the substrate materials and various layers on the substrate and
could be selected based on the desired operating features of the
manipulation chamber. For various examples of suitable materials,
reference is made to U.S. application Ser. No. 10/979,645,
incorporated by reference herein. The various exemplary materials
for the manipulation chamber 810 set forth above may be
particularly suitable for using the manipulation chamber to perform
both optoelectronic DEP and EP, as will be described below.
[0175] A sample 850 containing a plurality of differing particle
types, for example particle type A and particle type B, may be
provided in the space formed between the substrate 820 and the
substrate 830, as illustrated in FIG. 12A. As with the embodiment
of FIG. 1, a seal 840 may be provided along the edges of the
substrates 820 and 830 to hold the substrates together and to
define a closed cavity configured to receive the sample 850. Though
not shown in FIGS. 12A-12D, it should be understood that the
chamber 810 may be provided with various inlets and outlets so as
to permit addition of materials to the chamber and removal of
materials from the chamber as desired based on, for example, the
manipulation processes that may occur in the chamber.
[0176] A light beam 880, which may extend in a direction into the
drawing sheet so as to span across a width of the manipulation
chamber 810, may be used to illuminate the photoconductive surface
834 and, in conjunction with the applied power source 860 (AC or
DC), as will be described, create a DEP or EP force for
manipulating the particles A and B contained in the sample 50.
[0177] The particles in the sample 850 may have either positive or
negative Clausius-Mosotti values, respectively. In the exemplary
embodiment shown in FIGS. 12A-12D, particle type B is negatively
charged. The negative charge may be imparted to the particles of
type B, for example, by a surface-active modification agent and/or
a ligand, or may be the inherent charge of the particle type B. The
negative charge on particle type B is depicted by the negative sign
(-) in the circle attached to the particles.
[0178] Optoelectronic scanning may be initiated and a DEP force
created to act on the particles by applying an AC current from the
power source 860 and by moving the light beam 880 along the
manipulation chamber 810 in the direction of the dark arrow in FIG.
12A. When the light beam 880 scans along the manipulation chamber
810 in the direction shown in FIG. 12A, the DEP force pushes and
concentrates all of the particles to the right side of the chamber
810, as illustrated in FIG. 12B. That is, assuming the scanning
speed of the light beam has been selected appropriately, the light
beam scans the manipulation chamber, particles with negative
Clausius-Mosotti values will be repelled by the light and thus be
expelled in front of the light beam and toward the end of the
manipulation chamber at the right hand side of the drawing sheet.
Particles with positive Clausius-Mosotti values will be attracted
to the light beam as it scans and thus also will wind up at the end
of the manipulation chamber toward the right hand side of the
drawing sheet as they move toward the scanning light beam 880.
[0179] Once the particles have been pushed to the end of the
manipulation chamber 810, as illustrated in FIG. 12B, the direction
of scanning of the light beam 880 may be reversed, for example,
such that the light beam 880 moves along the chamber 810 in the
direction indicated by the dark arrow in FIG. 12C. At the same
time, the power source 860 may be switched such that a DC signal is
supplied instead of the AC signal. The DC power source may be
controlled such that the electrode 822 becomes negatively charged
and the portions corresponding to the regions of the
photoconductive layer 834 that are illuminated by the light beam
880 become positively charged. The respective charges are
illustrated by a plus sign (+) in a circle and a minus sign (-) in
a circle in FIGS. 12C and 12D. As the light beam 880 moves along
the manipulation chamber 810 in a direction toward the end of the
chamber toward the left hand side of the drawing sheet, the
negatively charged particles of particle type B move toward the
scanning light beam 880 as a result of the EP force acting thereon.
The negatively charged particles of particle type B move away from
the neutral particles of particle type A, which are not under the
action of the induced EP force. As illustrated in FIG. 12D, the
particles of type B may be moved a relatively large distance away
from the particle type B particles by continuing the scanning of
the light beam 880 toward the end of the manipulation chamber 810
at the left hand side of the drawing sheet. Thus, due to the
ability to move the particles of particle type B away from the
particles of particle type A, substantially without moving the
particle type A particles, enhanced separation of the particles may
be possible.
[0180] The scanning steps of FIGS. 12A-12D may be repeated as many
times as desired in order to achieve the desired separation and/or
other manipulation of the particles. Those skilled in the art would
understand that positively charged particles may also be separated
from other neutral particles by altering the polarity of the
applied DC source.
[0181] In various exemplary embodiments, it may be useful to rely
on both DEP and EP forces, either simultaneously or separately, to
cause particles to move in two dimensions, such as, for example,
and/or to enhance particle sorting. FIG. 13 illustrates an
exemplary embodiment of a manipulation chamber 910 that utilizes
both DEP and EP forces to enhance sorting.
[0182] The manipulation chamber 910 may comprise a first substrate
920 with a transparent electrode 922 and a second substrate 930
with an electrode layer 932 and a photoconductive layer 934. The
photoconductive layer 934 may have a transparent protective layer
provided thereon, as was described with reference to FIG. 1.
According to an exemplary aspect, the first and second substrates
and various layers provided thereon may be made of the materials
set forth in the Example below, however other suitable materials
also may be used, as has been described above with respect to the
exemplary embodiment of the manipulation chamber of FIG. 1.
[0183] As illustrated in FIG. 13, the manipulation chamber 910 also
may comprise an inlet 901 configured to permit introduction of
material to the chamber 910, and an outlet 902 configured to permit
removal of material from the chamber. Vents 903 and 904 also may be
provided to release pressure that may develop in the chamber, for
example, due to hydrolysis of water in the chamber during
application of the DC power source. The number of inlets, outlets,
and vents illustrated in FIG. 13 is exemplary only and it should be
understood by those having skill in the art that any number of
inlets, outlets and vents may be provided as desired depending on
the application of the manipulation chamber. It also should be
understood, that the manipulation chamber 910 could be in flow
communication or otherwise coupled to a variety of other
instrumentation, reservoirs, or the like for further fluid
handling, manipulation, and/or processing of the sample and/or
particles introduced to the chamber. Such instrumentation may
include, for example, microfluidic pumps, valves, additional
manipulation chambers, and/or other fluid handling devices.
[0184] Similar to the exemplary embodiment of FIG. 1, the
manipulation chamber 910 may comprise an AC power source 960
connected between the electrodes 922 and 932. The power source 960
may be closed to complete the circuit between the electrodes 922
and 932 when optoelectronic scanning occurs. Thus, as was described
above for various embodiments of optoelectronic scanning, a
scanning light beam 980 may be used in conjunction with the applied
AC signal to modulate a nonuniform electric field in the
manipulation chamber and corresponding DEP force acting on
particles A and B contained in an aqueous medium 950 supplied to
the chamber 910.
[0185] The manipulation chamber 910 may further comprise electrodes
connected to a device that imposes and controls a potential DC
bias. For example, as illustrated in the exemplary embodiment of
FIG. 13, electrodes 940 may be positioned at opposite ends of the
chamber 910 so as to face one another. For example, the electrodes
940 may be positioned toward the left hand and right hand side of
the chamber 910, as illustrated in FIG. 13. The electrodes 940 may
be connected to a DC power source 965. In this manner, in addition
to the DEP force created by the electrodes 922 and 932, the AC
source, and the scanning light beam 980, the incorporation of an
orthogonal DC potential bias across the length of the manipulation
chamber 910 enables the particles to be separated in a second
dimension via EP forces.
[0186] According to various exemplary aspects, the movement of a
particle by virtue of the EP force may be the same or opposite to
that of the OET scanning. For example, scanning the chamber from
left to right creates a DEP force which concentrates the particles
to the right hand side of the chamber shown in FIG. 13. If a DC
bias is applied, either during or after scanning, such that the
electrode 940 on the left hand side of the chamber 910 is positive,
the drag force for a negatively charged particle, such as particle
type B illustrated in FIG. 12, may be artificially increased by
adding an EP force to the particle in the same direction as the
drag force, which will facilitate its escape from the scanning
light beam and cause the negatively charged particles to migrate
further to the left hand side of the chamber 910 in FIG. 13. The
additional migration of the negatively charged particles may result
in enhanced separation. Those having ordinary skill in the art
would understand how to alter the DC polarity of electrodes 940 in
order to improve separation of positively or negatively charged
particles.
[0187] According to various exemplary aspects, the manipulation
chamber 910 may be repeatedly scanned with the light beam 980 and
an electro-osmotic flow in the medium may be induced in a direction
opposite the scan via the electrodes 940. In such circumstances,
particles having sufficient positive DEP force acting on them may
be collected to the right hand side of the chamber in the direction
of scanning, for example, while other cells would be swept to the
left hand side of the chamber 910 in the direction of the
electro-osmotic flow, for example. A series of scans may be
implemented with the electro-osmotic flow being relatively strong
initially so as to collect cells experiencing a relatively strong
positive DEP force. Those cells may then be diverted and collected
in a collection area (e.g., reservoir) outside of the manipulation
chamber. The electro-osmotic flow may then be progressively reduced
in subsequent scans to collect cells of decreasing dielectric
potential (e.g., decreasing DEP force acting thereon).
[0188] When compared to other fluid flow mechanisms, such as,
pumping, for example, electro-osmotic flow may produce a plug flow
profile as compared to a parabolic flow profile. This plug flow
profile results in a flow velocity that is uniform throughout the
manipulation chamber such that cell velocity is not reduced close
to the walls of the manipulation chamber.
[0189] As described above, therefore, particle sorting may be
achieved by subjecting the particles to competing forces, e.g., DEP
force and fluid flow (electro-osmosis). An EP force also may be
established, for example, by the electrodes 940 and DC power source
965 of the embodiment of FIG. 13. According to an exemplary aspect,
the inner walls of the manipulation chamber 910 may be coated with
substances designed to eliminate and/or reduce electro-osmotic flow
such that the EP force becomes the dominant force outweighing (or
replacing) the fluid flow forces moving particles in the
description above.
[0190] In order to control and/or facilitate electrophoresis of one
or more particles, surface active agents and/or ligands may be
applied to the particles to introduce an artificial electric charge
to the particles, such as, for example, as was described with
reference to FIGS. 12A-12D. Additional examples of surface
modification of particles can be found in U.S. application Ser. No.
10/979,645, incorporated by reference herein.
[0191] According to various exemplary embodiments, as discussed
above, the EP forces created by the electrodes 940 also may induce
electro-osmotic flow of the medium containing the particles. Again,
by controlling the polarity of the DC bias of electrodes 940, the
EP force acting on the medium may be altered and thus the flow
direction may be the same as or opposite to the direction of
scanning of the light beam 980. The electrophoresis and
dielectrophoresis movement of particles and/or medium may take
place concurrently, intermittently, or sequentially, depending on
the desired movement and/or application.
[0192] Aside from positioning the electrodes 940 within the chamber
910 at opposite ends thereof as depicted in FIG. 13, it is
envisioned that electrodes could be placed within reservoirs
located at either end of the chamber 910. Such reservoirs could be
disposed, for example, so as to be in flow communication with inlet
901 and/or outlet 902.
[0193] The electrodes used to create the EP force may have a
configuration chosen from wires, flags, plates, dots, buttons,
rods, tubes, thin layer coatings, arrays, and/or other suitable
electrode configurations and combinations thereof. The electrodes
may be made of a variety of materials including, but not limited
to, noble metals, such as, for example, gold and platinum;
non-ferrous metals, such as, for example, aluminum; alloys, such
as, for example, stainless steel; and oxides, such as, for example,
indium tin oxide; or combinations thereof. A variety of techniques
may be used to fabricate the electrodes 940 including, but not
limited to, sputtering, vapor deposition, ink-jet printing,
electroplating, welding, or other suitable fabrication techniques.
According to an exemplary aspect, it may be desirable to fabricate
electrodes on a surface with robust adhesion characteristics and
those skilled in the art would be familiar with how to accomplish
such fabrication.
[0194] As discussed, the various substrates, electrode layers,
photoconductive layer, and surfaces facing the cavity configured to
receive the sample containing particles to be manipulated may be
made of a variety of materials and/or have various configurations.
According to various exemplary embodiments, it may be desirable to
provide the electrode that is disposed underneath the
photoconductive layer as a noncontinuous electrode rather than a
continuous surface underneath the photoconductive layer. An
exemplary embodiment of a manipulation chamber having a
noncontinuous electrode in association with the second substrate
1130 is illustrated in FIGS. 14A and 14B.
[0195] FIG. 14A depicts a side view of an exemplary embodiment of a
manipulation chamber 1110 having a noncontinuous electrode
underneath the photoconductive layer, and FIG. 14B depicts a top
view of the bottom substrate layers of the manipulation chamber
1110 of FIG. 14A. For sake of clarity in FIG. 14B, the glass
substrate 1130 itself is not shown. As shown in the figures, the
manipulation chamber 1110 may have a configuration similar to other
manipulation chambers described herein including a first substrate
1120 (e.g., a glass substrate) having a transparent electrode layer
1122 deposited thereon (e.g., layer 1122 may be ITO or a PEGylated
gold layer) and a second substrate 1130 (e.g., a glass substrate)
having an electrode layer 1132 (e.g. a transparent or
nontransparent metal electrode layer such as, for example,
aluminum, gold, or ITO) and a photoconductive layer 1134. An
additional protective layer (e.g., surface) may be provided on the
photoconductive layer 1134 as discussed above. By way of example
only, the photoconductive layer 1134 may be provided with a
PEGylated SiO.sub.2.
[0196] As shown in FIG. 14A, a power source 1160, such as for
example an AC power source, may be provided so as to complete the
circuit between the electrode 1122 and 1132. Moreover, an incident
light beam 1180 may be provided so as to scan or otherwise induce
the nonuniform electric field and consequent DEP forces acting on
the sample within the cavity of the manipulation chamber 1110, as
has been described herein.
[0197] As shown in the side view of the manipulation chamber 1110
in FIG. 14B, the electrode layer 1132 comprises two individual
electrodes 1133 and 1135 rather than a continuous electrode
surface. The individual electrodes 1133 and 1135 may be deposited
as two elongate strips along the edges of the glass substrate 1130.
The electrodes 1133 and 1135 may be deposited by a variety of
techniques, including, for example, vapor deposition, sputtering,
electroless plating, electroplating, inkjet printing, screen
printing of conductive paint, or other suitable deposition
techniques. It should be noted that only one of the electrode
strips 1133 is illustrated in FIG. 14A, as the other electrode
strip 1135, shown in FIG. 14B, is deposited along the edge of the
substrate 1130 opposite to the edge shown in FIG. 14A and into the
drawing sheet.
[0198] According to an exemplary aspect, the photoconductive layer
1134 may be illuminated by a light pattern, such as, for example,
by a plurality of individual light beams 1190, such that a
plurality of strips on the surface of the photoconductive layer
1134 are illuminated so as to form virtual (e.g., photo-activated)
electrodes 1136. The term virtual (or photo-activated) electrode
should be understood to refer to an illuminated region of the
photoconductive layer 1134 such that the electric field proximate
that illuminated region is modulated (e.g., strengthened). The
virtual electrodes 1136 may be established via illumination such
that adjacent electrodes 1136 are each placed in contact with a
different electrode strip 1133 and 1135, as illustrated in FIG.
14B, and such that each electrode 1136 is placed in contact with
only one of electrode strips 1133 and 1135.
[0199] In accordance with an exemplary aspect, the photo-activated
electrodes 1136 may extend from the respective electrode strip 1133
and 1135 in which they are in contact in a direction substantially
perpendicular to the direction in which the electrode strips 1133
and 1135 extend. For example, a plurality of individual
photo-activated virtual electrodes 1136 may be positioned so as to
alternatively contact one of the electrode strips 1133 and 1135 and
not the other of the electrode strips 1133 and 1135, as shown in
FIG. 14B. Each virtual electrode 1136 may extend in a direction
substantially perpendicular to the direction in which the electrode
strips 1133, 1135 extend.
[0200] It should be understood that the dimensions, position, and
number of virtual electrodes that are activated by illuminating the
photoconductive layer 1134 may be altered as desired by modifying
the light pattern which is mapped onto the photoconductive layer
1134. Moreover, with reference to the description of FIGS. 26A and
26B below, the intensity of the light illuminating the
photoconductive layer may also be varied to modulate the strength
of the electric field in the vicinity of illuminated regions of the
photoconductive layer 1134. Thus, the electric field strength
corresponding to the virtual electrodes 1136 may be controlled as
desired by respectively varying the intensity of the light
illuminating each respective virtual electrode 1136.
[0201] A power source 1170, which may be, for example, an AC power
source, may be provided so as to electrically couple (e.g., bias)
the electrode strips 1133 and 1135. When the strips 1133 and 1135
are biased with an electric potential and when light beams 1190 are
activated to individually illuminate each of areas of the
photoconductive layer 1134 so as to create virtual electrodes 1136,
a nonuniform electric field may be generated between the virtual
electrodes 1136, thereby creating DEP forces to act on the
particles in the manipulation chamber 1110. It should be understood
that the light beams 1190 are depicted schematically and that a
footprint of light that illuminates substantially the entire area
corresponding to a virtual electrode 1136 shown in FIG. 14B may be
created from each respective beam 1190.
[0202] Thus, the exemplary embodiment of the manipulation chamber
1110 of FIGS. 14A and 14B, may permit nonuniform electric fields,
and resulting DEP forces, to be created between virtual electrodes
within the same plane (e.g., virtual electrodes 1136) or between
electrodes in differing planes (e.g., electrode 1122 and
electrode/photoconductor 1132, 1134). A switch, which may be
programmable, may be provided to activate either the power source
1160 electrically coupling the electrodes 1122 and 1132 or the
power source 1170 electrically coupling the electrodes 1133 and
1135. Moreover, it is envisioned that only one power source may be
used and switched so as to apply a potential to either electrodes
1122 and 1132 or 1133 and 1135. In this latter case, DEP could only
occur in the direction corresponding to the coupled electrodes. On
the other hand, if two power sources are used, it may be possible
to apply a potential simultaneously to the electrodes 1122 and 1132
and the electrodes 1133 and 1135, which may permit manipulation of
the particles in two dimensions.
[0203] By way of example only, in a first mode of operation, the AC
power source 1160 may be used to apply an electric potential
between electrode 1122 and electrodes 1133 and 1135, as shown in
FIG. 14A. In this mode of operation, the light source 1180 may be
used to illuminate the photoconductive layer 1134, such as, for
example, via scanning or focused light beams, and the manipulation
chamber 1110 may operate so as to induce optoelectronic
manipulation (e.g., optoelectronically induced DEP) in a manner
similar to various other manipulation chamber embodiments described
herein, such as, for example, the embodiment of FIG. 1. A second
mode of operation may include disabling power source 1160 and
enabling power source 1170. In this mode, an electric potential
from an AC power source may be applied between electrodes 1133 and
1135 and virtual electrodes 1136 may be activated by illuminating
corresponding regions of the photoconductive layer 1134 with
incident light, for example, in the pattern shown by 1190 in FIG.
14B. As a result of DEP forces created in the second mode of
operation, manipulation of particles may occur between the
photoactivated electrodes 1136. In yet another mode of operation,
the power source of FIG. 14B may be a DC power source instead of an
AC power source. In this case, the photoactivated electrodes 1136
will cause EP forces to be generated and thus the electrodes 1136
may be used to manipulate charged particles in the manipulation
chamber 1110. The various modes of operation may occur
concurrently, sequentially, intermittently, or any combination
thereof in order to achieve desired separation, or other
manipulation of particles in the manipulation chamber 1110. Those
having ordinary skill in the art would understand how the coupling
of the various electrodes and applied power sources may be altered
in order to achieve desired DEP and/or EP manipulation of
particles.
[0204] A manipulation chamber, such as, for example, manipulation
chamber 1110, which permits modulation of a nonuniform electric
field and DEP forces between virtual electrodes in the same plane
may provide advantages for DEP application such as, for example,
applications for which cell levitation is desired. For example, by
applying an AC bias between electrodes 1133 and 1135 of FIG. 14, a
negative DEP force that declines with distance away from the
electrodes (e.g., toward upper substrate 1120) may be created.
Thus, particles in the chamber 1110 having negative
Clausius-Mosotti values will be repelled to a height in the chamber
(as measured from photoconductive surface 1134) at which the DEP
force is balanced by the gravitational force. Using an array of
interdigitated virtual electrodes, such as electrodes 1136, where
an AC bias is imposed between adjacent electrodes may levitate all
the particles experiencing negative DEP above the array. The final
levitation height of a particle depends on the balance of the DEP
and gravitational forces acting on that particle. The negative DEP
force is not uniform above the array, but is strongest near an
electrode edge and weaker over the center of individual electrodes
and in the gaps between electrodes.
[0205] Virtual electrodes such as electrodes 1136 of FIG. 14B may
thus be used in a manner similar to patterned electrodes to achieve
cell levitation, which has been described in Das et al.,
"Dielectrophoretic Segregation of Different Cell Types on
Microscope Slides," Anal. Chem. May 1, 2005, vol. 77, pp.
2708-2719, incorporated by reference herein. Various degrees of
control over the cell levitation may be obtained by altering the
positions and shapes of the virtual electrodes, and/or the
intensity of the light illuminating the photoconductive layer to
create the virtual electrodes. Advantages to using virtual
electrodes for cell levitation, rather than patterned electrodes,
may include reduction in fabrication costs and ability to observe a
majority of the manipulation chamber.
[0206] According to yet another exemplary aspect, a series of
parallel, stationary virtual electrodes could be formed by
illuminating respective regions of the photoconductive layer 1134
and an AC bias could be applied between the electrode 1122 and the
virtual electrodes such that the electric field is modulated
between each virtual electrode and the electrode 1122. When using
this configuration, it may be desirable to minimize the gap between
the virtual electrodes in order to minimize potential dead regions
corresponding to locations without electric field lines where
particles may become trapped.
[0207] Although in the description of the various optoelectronic
manipulation devices and techniques above, the light source used to
illuminate the photoconductive surface and thereby generate the
electric field and corresponding DEP force may be a light source of
substantially uniform intensity, it is envisioned that light
sources having varying intensity may be used. In this manner,
assuming the conductivity of the photoconductor depends on the
applied light intensity, it may be possible to vary the generated
electric field along the surface of the photoconductor, and thus
the resulting DEP force, by varying the light intensity that
illuminates any particular location of the photoconductive surface.
Reference may be made to Chiou et al., Sensors and Actuators, vol.
104, pp. 222-228, 2003, which is incorporated by reference herein,
for information regarding light intensity modulated
conductivity
[0208] By way of example, as the light scans across the
manipulation chamber, the intensity of the light source may be
varied (e.g., in the direction of scanning) so as to alter the
resulting electric field and DEP forces that are generated. The
intensity of the light source may also vary in a direction
perpendicular to the scanning direction (e.g. in the Y-direction
depicted in FIG. 2). Thus, as the intensity of the projected light
beam may vary in both the direction of scanning and in a direction
perpendicular to the scanning direction, modification of the
electric field generated by the illumination of the photoconductive
surface can occur in two dimensions, as desired.
[0209] In contrast to existing techniques for sorting cells that
rely on DEP forces created by patterned electrodes on a substrate
surface subject to an increasing applied electric field in one
direction along the substrate surface, use of the optoelectronic
techniques with varying light intensity described herein permit
variation of the generated electric field, and resulting DEP
forces, in two dimensions as opposed to just one dimension. Thus,
the optoelectronically-induced DEP may provide increased
flexibility and control over the modulation of electric fields, in
terms of both strength and location. Further, the optoelectronic
methodology does not require complex and/or costly fabrication of
patterned electrodes (e.g., microfabrication) and/or high voltage
power sources. Rather, the technique relies on variation of light
intensity. Moreover, due to the transparent configuration of the
manipulation chambers used in optoelectronic techniques, subsequent
cell analysis may be facilitated by enabling the entire
manipulation chamber to be observed. In contrast, in techniques
using surfaces which have patterned electrodes, a relatively large
portion of the patterned surface may not be viewable due to the
presence of the electrodes.
[0210] Thus, various advantages may be achieved by an
optoelectronic manipulation chamber configured such that the light
intensity illuminating the photoconductive surface is variable,
either in a direction of scanning, a direction perpendicular to
scanning, or both. Such advantages include, but are not limited to,
the ability to generate numerous virtual electrode configurations
by controlling location and/or intensity of illumination on the
photoconductive surface, the ability to move the location of such
virtual electrodes within the same device by controlling the
location and/or intensity of illumination, and relatively
inexpensive and simplified device configurations. It should be
further noted that according to various embodiments, optoelectronic
manipulation chambers disclosed herein can achieve most, if not
all, configurations (e.g., location, shape, field strength, etc.)
of patterned electrodes, as well as some configurations which
patterned electrodes cannot achieve.
[0211] FIGS. 26A and 26B schematically depict an exemplary
optoelectronic sorting technique that relies on varying the
intensity of the incident light during a scanning process. The
chambers 2610 of FIGS. 26A and 26B may have a configuration in
accordance with various exemplary embodiments of the invention as
disclosed herein. In FIG. 26A, the light beam 2680 may initially
have a relatively high intensity which may produce a relatively
strong electric field at locations of illumination of the
photoconductive surface of the manipulation chamber 2610. The
intensity and scanning speed of the light beam 2680 may be selected
so as to be sufficient to overcome the viscous drag force on all
particles in the chamber 2610 or of particles of interest in the
chamber. The intensity, and thus electric field, may be reduced
during the scanning process, for example, as the beam 2680 moves
from left to right across the chamber 2610 as shown in FIG. 26A. As
the intensity is reduced, particles with higher viscous drag as
compared to DEP force, such as particle type A labeled in FIG. 26A,
will escape from the beam 2680 before particles with lower viscous
drag as compared to DEP force, such as particle type B labeled in
FIG. 26A. This results in differing displacements
(dielectrophoretic movement) of particles of particle type A as
compared to particles of particle type B, as illustrated in FIG.
26A, thus enabling particle type A particles to be separated from
particle type B particles.
[0212] In another exemplary technique illustrated in FIG. 26B, a
plurality of scanning beams 2680a and 2680b may be utilized with
each having an intensity that differs from the other. For example,
in FIG. 26B, beam 2680a may have a relatively low intensity and
beam 2680b may have a relatively high intensity. In this case,
beams 2680a may be utilized to collect particles having relatively
low viscous drag as compared to DEP force acting thereon. The beam
2680b of higher intensity, and thus higher DEP force, may then be
used to trap particles having relatively higher viscous drag acting
thereon. Those skilled in the art would understand that a series of
any number of scanning beams of differing intensities could be used
to separate various particle types. The series of beams could be
used as a filter to collect particles of a particular type in each
respective beam and those collected particles could be moved to
collection areas within or outside the chamber by moving the
respective beam trapping the particles and/or using various
collection techniques described above. Further, although the
embodiment of FIGS. 26A and 26B uses positive DEP force to move and
sort cells, those skilled in the art would understand, based on the
various teachings provided herein, how to utilize negative DEP
forces for similar manipulation and sorting of particles. In some
cases, negative DEP may reduce cell adhesion and damage.
[0213] Such optoelectronic scanning utilizing scanning beams of
differing DEP strengths was used by the inventors to separate Hela
and Jurkat cells from one another. Jurkat cells were labeled with
CellTracker (Invitrogen) according to the manufacturer's
recommendations. Hela and labeled Jurkat cells were washed in an
isotonic buffer (8.5% sucrose, 0.3% dextrose) and respuspended in
the same buffer. The cells were mixed and the conductivity of the
mixture was adjusted to about 1.7 mS/m with growth media
(Dulbecco's Modified Eagle's Media). The mixture was supplied to an
optoelectronic scanning chamber and subjected to optoelectronic
scanning with 2 beams of 633 nm light scanned at differing speeds
ranging from about 3 micrometers/sec to about 11 micrometers/sec.
The leading beam was about 15 micrometers wide and the trailing
beam was about 23 micrometers wide. Lines of differing widths
resulted in differing DEP forces experienced by the cells. At
scanning speeds of about 8.7 .mu.m/sec, Jurkat cells followed the
leading beam, whereas Hela cells were dropped by the leading beam.
Dropped Hela cells were then trapped by and followed the thicker
trailing beam. Thus, the scanning beams of differing DEP strengths
were generated in the optoelectronic scanning chamber and the beams
were used to separate cells of different types.
[0214] FIGS. 29A-29D illustrate another exemplary embodiment of a
scanning technique in which particle sorting may be achieved by a
series of beams of differing intensities. In the exemplary
embodiment of FIGS. 29A-29D, scanning may be performed within a
continuous pattern such as a "race-track" pattern, as shown. Such a
continuous, race-track pattern of scanning may be useful in
circumstances where the distance and/or time required for particles
to escape from a scanning light beam, for example, a light beam of
lower intensity that does not overcome the viscous drag acting on a
subset of particles, varies and may not be predictable. Providing a
continuous scanning loop (e.g., race track), may enable the desired
separation and sorting of all particles since the light beams can
repeatedly scan around the loop until the desired separation of the
particles occurs, as will be explained in further detail below.
[0215] In various exemplary embodiments, a plurality of differing
types of particles A, B, C, and D, (e.g., cells) may be introduced
into an optoelectronic scanning chamber 2910 at an input area, as
shown in a top view of the chamber 2910 in FIG. 29A. An initial
scanning light beam 2980a may begin to move across the particles A,
B, C, and D. As shown in FIG. 29A, the frequency of the initial
scanning light beam 2980a may be selected so as to impose a cut off
wherein undesired particles D do not enter the portion of the
scanning loop 2920 past the input area 2915. Such a "cut off"
frequency for the initial light beam is optional and in an
alternative all of the particles may be initially entrained by the
light beam 2980a so as to be swept into the scanning loop 2920. The
initial scanning light beam 2980a may be followed by a series
(e.g., two shown in FIGS. 29B-29C) of scanning light beams 2980b
and 2980c of differing (e.g., progressively increasing) field
intensities. Although FIGS. 29B and 29C depict a series of three
scanning light beams 2980a, 2980b, and 2980c, those having skill in
the art would understand that the number of light beams may vary.
For example, any plurality of light beams may be used and chosen
based on, for example, the number of particle types it is desired
to sort and trap.
[0216] Thus, in the exemplary embodiment of FIGS. 29A-29D, three
particle types A, B, and C, are sorted and respectively trapped via
the DEP force created by the three scanning light beams of
differing field intensities 2980a, 2980b, and 2980c. The particles
A, B, and C, are "trapped" by one of the scanning light beams
2980a, 2980b, and 2980c when the field produced at the location of
the virtual electrode created by the light beam 2980a, 2980b, and
2980c generates a DEP force sufficient to overcome the viscous drag
associated with each particle type A, B, or C. At the beginning of
the scanning, as shown in FIG. 29B, for example, particles of
differing types may be moved together by one of the scanning beams
2980a, 2980b, or 2980c. For example, FIG. 29B depicts particles of
types A and B being trapped by the light beam 2980a of lowest
intensity and particles of types B and C being trapped by the light
beam 2980b of medium intensity compared to light beams 2980a and
2980c. This may occur due to the variation in time and/or distance
required for the particle types that experience higher viscous drag
to escape the scanning light beam having a lower field intensity.
The loop 2920 that the scanning light beams 2980a, 2980b, and 2980c
travel around permits the light beams to continuously scan until
sufficient distance and time has occurred to permit the desired
separation between the differing particle types A, B, and C. Thus,
as shown in FIG. 29C, the scanning light beams 2980a, 2980b, and
2980c may continue around the loop 2920 in the direction of the
arrows shown until the desired separation of the differing particle
types A, B, and C, is accomplished and each particle type A, B, and
C is entrained in the appropriate respective light beam 2980a,
2980b, 2980c. Upon achieving this desired separation and
entrainment, the scanning light beams 2980a, 2980b, and 2980c, with
their respective entrained particle types A, B, and C may be moved
out of the loop 2920 and to an exit area 2930, as illustrated in
FIG. 29D, such that the particles A, B, and C, may be further
collected and/or otherwise processed or analyzed.
[0217] Varying the frequency of the applied electric field may also
vary the DEP force on particles, as the permittivity of both the
particles and the medium depend on frequency and thus so does the
Clausius-Mosotti value. At a frequency called the cross-over
frequency, which is different for each particle type, the induced
dipole on a particle is zero and F.sub.DEP is zero. Particles may
experience negative or positive DEP at frequencies lower or higher
than the cross-over frequency. Thus, both magnitude and direction
of the DEP force experienced by a particular particle type may be
altered by varying the applied frequency.
[0218] FIGS. 27A and 27B schematically depict exemplary
optoelectronic sorting techniques that rely on varying the
frequency of the applied electric field between the electrodes in a
manipulation chamber in order to separate particles of differing
particle types, such as A and B in FIGS. 27A and 27B. Initially, as
shown in FIG. 27A, a light beam 2780 of an optoelectronic
manipulation chamber 2710 may be operated at relatively high
frequency, for example, greater than about 300 kHz such that a
relatively strong electric field occurs in the proximity of the
light beam 2780. Due to the relatively strong electric field, as
shown in FIG. 27A, all of the particle types A and B may be dragged
along in a group with the light beam 2780, assuming positive
Clausius-Mosotti values in this example. As scanning continues, the
frequency of the applied electric potential may be varied, for
example, progressively decreased, as shown in FIG. 27B. As the
frequency reaches the cross-over frequency for each particle type,
that particle type will cease to migrate and as the frequency
further declines, that particle type will experience a repelling,
negative DEP force. Particle types experiencing negative DEP as a
result of the variation in the frequency, may escape through gaps
provided down the length of the scanning light beam 2780. That is,
the light beam 2780 could be configured as a plurality of
individual illuminated portions separated by gaps of unilluminated
portions between adjacent illuminated portions down the length of
the beam 2780. Alternatively, for a solid light beam without such
gaps, as shown in FIGS. 27A and 27B, and scanning at a sufficiently
high speed, the viscous drag on the particles of negative or weaker
DEP may overcome the DEP force near the particles' threshold
velocity such that the particles escape the scanning light
beam.
[0219] Thus, as shown in FIG. 27B, differing particle types A and B
may be collected in groupings based on cross-over frequency by
altering the frequency of the applied electric field during
scanning. By correlating the position along the chamber of the
scanning beam 2780 with the applied frequency, the cross-over
frequency of the differing particle types A and B also may be
determined.
[0220] It should be understood that varying the intensity of the
light and the frequency of the applied field either simultaneously
or sequentially, with or without scanning, in an optoelectronic
manipulation chamber may be utilized to potentially gain additional
separation of differing particle types. Moreover, a two-dimensional
separation may be achieved, for example, wherein an initial
particle population is confined to a predetermined area, such as,
for example, a corner of the chamber. Separation using field
(intensity) modulation scanning could be used in a first dimension
separation/sorting scheme while frequency modulation scanning could
be used in a second dimension. In an exemplary aspect, pulsing
light may be used so as to alter the intensity.
[0221] According to various exemplary embodiments, one or more
interior surfaces of an optoelectronic manipulation chamber may be
provided with surface modification treatments such that portions of
the surface become non-selectively or selectively adsorptive. In an
exemplary aspect, different areas of the chamber surface associated
with the substrate comprising the photoconductive material may be
treated with different modifiers (e.g., ligands, antibodies, smart
polymers, lectins, etc.) so as to cause different types of
particles to bind to the different areas after separation of the
particle types via optoelectronic DEP. Examples of suitable
materials that may be used to modify the surfaces of the chamber
are set forth in U.S. patent application Ser. No. 10/979,645,
incorporated by reference herein.
[0222] In yet another exemplary aspect, an optoelectronic
manipulation chamber according to exemplary aspects of the
invention may include electric field concentrators. For example,
the chamber may incorporate insulating material, such as, for
example, patterned insulating obstacles configured to produce
electric field concentrations or otherwise alter the electric field
within the chamber. Those with skill in the art would understand a
variety of materials and techniques that may be used to form such
insulating structures within the chamber in order to alter the
electric field therein.
[0223] As discussed above, a variety of materials for the various
components of optoelectronic manipulation chambers suitable for a
variety of applications, including, for example, scanning,
identifying, sorting, collecting, and/or otherwise manipulating
small particles, such as, for example cells, including stem cells,
DNA, and/or other biological material. An embodiment of an
optoelectronic manipulation chamber that may provide advantages
over other optoelectronic manipulation chamber embodiments is set
forth below in the following example. In addition to the
manipulation chamber materials, various methods of fabrication and
data obtained by performing various tests on manipulation chamber
components are described. It should be understood that the
optoelectronic manipulation chamber described in the following
example could be used for any of the applications described
herein.
EXAMPLE
[0224] FIG. 15 is a side view of a manipulation chamber comprising
a first substrate 1520 made of glass and having a PEGylated
transparent gold electrode 1522 thereon. The manipulation chamber
1510 further comprises a second substrate 1530 made of glass with a
metal electrode 1532 made of ITO deposited thereon in a relatively
thin layer, followed by a PEGylated SiO.sub.2 photoconductor
1534.
[0225] To obtain the PEGylated gold electrode layer 1522, a gold
electrode was first deposited on the substrate 1520 via vapor
deposition in a vacuum chamber. It is envisioned, however, that
other suitable deposition techniques may be employed as well. The
gold electrode may be deposited in a relatively thin layer having
an average thickness ranging from about 30 angstroms to about 200
angstroms, for example, about 50 angstroms to about 100 angstroms.
For the chamber of the present example, the gold electrode layer
ranged from about 70 angstroms to about 80 angstroms thick.
[0226] As those skilled in the art will appreciate, deposition of
the gold electrode layer does not result in a layer of uniform
thickness, but rather a noncontinuous layer that is interconnected,
for example, like a series of islands on the surface of the glass
substrate 1510. By way of example, the gold electrode layer may
permit about 10% to about 80% of light to pass through the layer.
As will be discussed further below, the gold electrode layer formed
in accordance with this example, and as described below with
reference to the process illustrated in FIG. 16, permitted
approximately 60% of transmitted light to pass therethrough.
[0227] Following deposition of the transparent gold electrode (TGE)
on the glass substrate 1520, the gold electrode may be subject to a
PEGylation process in which a PEG group is chemically bonded (e.g.,
covalently bonded) to the surface of the deposited gold layer. The
surface of gold is relatively easily modified by such a PEGylation
process. The term "PEGylation" is used herein to refer to a process
or processes that covalently bond poly(ethylene glycol) onto a
surface. The resulting PEGylated transparent gold electrode 1522
results in an electrode in which nonspecific adsorption of
biomolecules may be reduced (e.g., minimized).
[0228] The use of a transparent gold electrode layer may permit
operation of the manipulation chamber at a lower AC frequency
and/or with a DC power source. Other electrode materials, such as,
for example, ITO, may become oxidized and thereby reduced to an
insulator when operated at relatively low AC frequencies or under
DC current if the DC power source is coupled incorrectly. Thus,
manipulation chambers using ITO electrodes are typically operated
at relatively high AC frequency. Because metallic gold is highly
conductive and it cannot be reduced any further, it may be used in
conjunction with relatively low AC frequency and/or DC power
sources, in addition to being used with high AC frequency power
sources.
[0229] FIG. 16 illustrates steps which were used to fabricate the
PEGylated transparent gold electrode 1522 on the glass substrate
1520. Referring to FIG. 16, the glass substrate 1520 was first
cleaned so as to remove impurities, such as, for example, organic
impurities. A Piranah solution was used to clean the glass
substrate 1520,
[0230] The Piranah solution enhances surface density of silanol
groups with OH groups bonded to the glass substrate surface, as
shown in step 16(i) in FIG. 16. After cleaning the glass substrate
1520, a silylation process was used to treat the surface of the
substrate 1520 on which the gold electrode layer is deposited. As
shown in step 16(ii), the mercapto-containing silylating agent
(obtained from Gelest, Inc.) reacts with the OH groups on the
surface of the glass, resulting in the incorporation of surface
hydrogen sulfide groups (HS). The silyation process ultimately
provided good adhesion of the gold to the glass substrate because
the formed HS groups (e.g., mercapto groups) react and form
chemical bonds with gold.
[0231] After the silylation process, the gold was deposited on the
glass substrate via a vapor deposition process. As shown in step
16(iii) of FIG. 16, the gold was deposited so as to achieve a
transparent gold electrode layer (TGE). The mercapto functional
groups also resulted in strong adhesion of the transparent gold
layer to the glass surface, as shown by the resulting covalent bond
formed between the deposited TGE and the sulfur (S) in step
16(iii).
[0232] The resulting glass substrate with a TGE deposited layer in
accordance with the exemplary steps above demonstrated good
adhesion of the TGE to the glass substrate. For example, the
resulting substrate with a TGE deposited layer, as shown in step
16(iii) of FIG. 16 passed a standard ASTM D3359-02 "Scotch tape"
test. In various produced samples, the thickness of the TGE ranged
from about 71 angstroms to about 78 angstroms, and the sheet
resistance was about 60 omhs/sq, representing relatively high
conductivity.
[0233] FIGS. 17A and 17B show perspective views of a 10 micron by
10 micron portion of a glass substrate (e.g., slide) after being
subject to the silylation process and vapor deposition process of
FIG. 16. That is, FIG. 17A shows a perspective view of a portion of
the glass substrate resulting from the cleaning and silylation
process as illustrated in step 16(ii) of FIG. 16, and FIG. 17B
shows a perspective view of the portion of the glass substrate
after the cleaning, silylation, and deposition processes as
illustrated in step 16(iii) of FIG. 16. The pictures in FIGS.
17A-17C were obtained via Atomic Force Microscopy (AFM).
[0234] Referring again to FIG. 16, after the transparent gold
electrode (TGE) was deposited on the surface of the glass substrate
1520, the deposited gold layer was subject to a PEGylation process,
which may protect the surface facing the interior of the
manipulation chamber against nonspecific passive adsorption of
biomolecules. To perform the PEGylation, the gold surface was
exposed to an aqueous tetrahydrofuran (THF) containing a
mercapto-functionalized poly(ethylene glycol) (molecular weight
5723 Da, obtained from Nektar), with x equal to about 126, as shown
by reference number 1521 in FIG. 16. In various exemplary aspects,
the PEGylation of the gold surface may be achieved with x in FIG.
16 ranging from 5 to 1000, for example, from 10 to 300, or, for
example, from 20 to 200. Those skilled in the art would be able to
determine the value of x to achieve desired surface features. As
with step 16(ii), the mercapto groups form a strong covalent bond
with the gold electrode layer via the sulfur (S) bond, as shown in
step 16(iv). The resulting gold electrode layer in step 16(iv) of
FIG. 16 has poly(ethylene glycol) groups (PEG) bonded to the gold.
The resulting PEGylated TGE layer 1522 resulted in a sheet
resistance of about 20 ohms/sq.
[0235] FIG. 17C shows a perspective view of the portion of the
glass substrate of FIGS. 17A and 17B after being subject to the
cleaning, silylation, vapor deposition, and PEGylation process of
FIG. 16. Thus, FIG. 17C shows resulting substrate of step 16(iv) of
FIG. 16. When compared with FIG. 17B, the PEGylated TGE has a
smoother surface.
[0236] FIGS. 18-22 present data and results of various tests that
were performed on glass substrates processed in accordance with
FIG. 16 so as to obtain a PEGylated TGE layer deposited thereon.
FIG. 18 shows a perspective view of such a processed substrate
after exposure of the PEGylated TGE surface to a 10.times. bovine
serum albumin (BSA) (e.g., a concentrate solution containing 1
mg/mL of BSA, whereas 1.times.BSA contains 0.1 mg/mL of BSA). As
BSA is a protein, this BSA test may be used to observe the
nonspecific passive adsorption characteristics of the surface.
[0237] FIG. 19 shows a comparison of the surface roughness that
resulted from subjecting the glass substrate to various treatment
processes, including the resulting surfaces corresponding the
various steps 16(ii)-16(iv) shown in FIG. 16. The surface roughness
was measured by Atomic Force Microscopy (AFM) and is the root mean
square (RMS) in nanometers. The results are taken over a 10 micron
by 10 micron area of the surface of the treated substrate. Thus,
the first bar to the left in FIG. 19 (labeled HS-silylated glass in
FIG. 19), shows the resulting roughness of the substrate surface
corresponding to step 16(ii) of FIG. 16, that is, after the
substrate has been cleaned and subject to the silylation process in
which the HS groups are bonded thereto. The second bar from the
left in FIG. 19 shows the surface roughness of the substrate
surface corresponding to step 16(iii) of FIG. 16, after the TGE
layer has been deposited thereon (labeled TGE in FIG. 19), and the
third bar from the left shows the surface roughness of the
PEGylated TGE substrate surface corresponding to step 16(iv) of
FIG. 16 (labeled PEG-TGE in FIG. 19). As can be seen by a
comparison of the second and third bars, the PEGylation processing
of the TGE results in a smoother surface (lower RMS surface
roughness) than the TGE surface alone. The fourth and fifth bars
from the left in FIG. 19 correspond to the exposure of the
PEGylated TGE layer to 1.times.- and 10.times.-BSA, respectively
(labeled 1.times.BSA PEG-TGE and 10.times.BSA PEG-TGE,
respectively, in FIG. 19).
[0238] The slight decrease in roughness of the last two samples
(i.e., the 1.times.BSA PEG-TGE and 10.times.BSA PEG-TGE samples,
respectively, suggests a low degree of passive adsorption of BSA on
the surface, resulting in some leveling (e.g., smoothing) effect on
the surface.
[0239] FIG. 20 shows a comparison of surface wettability that
resulted from subjecting the glass substrate to various treatment
processes, including the resulting surfaces corresponding the
various steps 16(ii)-16(iv) shown in FIG. 16. The results in FIG.
20 show measurements of contact angle for water applied to the
various surfaces, with error bars for each also shown. In general,
lower contact angles correspond to higher
hydrophilicity/wettability and lower nonspecific adsorption due to
hydrophobic-hydrophobic interaction. From left to right in FIG. 20,
the bars correspond to the wettability measured for a glass
substrate surface corresponding to step 16(ii) in FIG. 16 (labeled
HS-silylated glass in FIG. 20); the glass substrate surface
corresponding to step 16(iii) in FIG. 16 (labeled TGE in FIG. 20);
the glass substrate surface corresponding to step 16(iii) in FIG.
16 in combination with an exposure to 1.times.BSA thereon (labeled
1.times.BSA on TGE in FIG. 20); the glass substrate surface
corresponding to step 16(iii) in FIG. 16 in combination with an
exposure to 10.times.BSA thereon (labeled 10.times.BSA on TGE in
FIG. 20); the glass substrate surface corresponding to step 16(iv)
in FIG. 16 (labeled PEG-TGE in FIG. 20); the glass substrate
surface corresponding to step 16(iv) in FIG. 16 in combination with
an exposure to 1.times.BSA thereon (labeled 1.times.BSA on PEG-TGE
in FIG. 20); and the glass substrate surface corresponding to step
16(iv) in FIG. 16 in combination with an exposure to 10.times.BSA
thereon (labeled 10.times.BSA on PEG-TGE in FIG. 20).
[0240] As can be seen from the results shown in FIG. 20, using a
PEGylated TGE on the glass substrate yielded a surface having a
relatively high wettability. The water contact angle for the
PEGylated TGE surface (PEG-TGE) was significantly less than that of
the TGE surface alone (TGE). In turn, nonspecific adsorption of the
PEGylated TGE was significantly reduced in comparison to the TGE
surface. The reduction in nonspecific adsorption is demonstrated by
the 1.times. and 10.times.BSA on TGE and on PEG-TGE results shown
in FIG. 20.
[0241] FIG. 21 shows results of measuring the transparency of the
glass substrate after various surface treatment processes,
including the resulting surfaces corresponding the various steps
16(ii)-16(iv) shown in FIG. 16. From left to right in FIG. 21, the
bars correspond to the transparency measured for a glass substrate
surface corresponding to step 16(ii) in FIG. 16 (labeled
HS-silylated glass in FIG. 21); the glass substrate surface
corresponding to step 16(iii) in FIG. 16 (labeled TGE in FIG. 21);
the glass substrate surface corresponding to step 16(iii) in FIG.
16 in combination with an exposure to 1.times.BSA thereon (labeled
1.times.BSA on TGE in FIG. 21); the glass substrate surface
corresponding to step 16(iii) in FIG. 16 in combination with an
exposure to 10.times.BSA thereon (labeled 10.times.BSA on TGE in
FIG. 21); the glass substrate corresponding to step 16(iv) in FIG.
16 (labeled PEG-TGE in FIG. 21); the glass substrate surface
corresponding to step 16(iv) in FIG. 16 in combination with an
exposure to 1.times.BSA treatment thereon (labeled 1.times.BSA on
PEG-TGE in FIG. 21); and the glass substrate surface corresponding
to step 16(iv) in FIG. 16 in combination with an exposure to
10.times.BSA thereon (labeled 10.times.BSA on PEG-TGE in FIG.
21).
[0242] The transparency was measured using a He--Ne laser light
beam of 633 nm and 1.15 mW output. The intensity of the light
passing through the HS-silylated glass slide was measured by a
spectrophotometer and is considered to be 100% transparent. The
results of the transparency measurements for each surface treatment
shown in FIG. 21 are normalized with respect to the transparency
measured for the HS-silylated glass. Thus, the HS-silylated glass
is considered 100% transparent and the remaining results in FIG. 21
show transparency measurements as a percentage of the HS-silylated
glass. As shown by the results of FIG. 21, the TGE surface and the
various treatments to that surface (e.g., PEGylation and/or BSA
treatments) yielded transparencies ranging from greater than about
55% of the HS-silylated glass to about just over 60% of the
HS-silylated glass. As discussed above, for the various
manipulation chambers and applications described herein, it may be
desirable for the upper glass substrate and corresponding electrode
surface thereon to have a transparency ranging from about 40% to
about 80%. That is, in comparison with the HS-silylated, it may be
desirable for the upper, first glass substrate provided with an
electrode surface thereon (e.g., the nonphotoconductive substrate)
to transmit about 40% to about 80% of incident light.
[0243] With reference to FIGS. 22A-22D, snapshots of real-time
results of optoelectronic manipulation of polystyrene beads of
about 20 microns in diameter suspended in a potassium chloride
(KCl) solution prepared with deionized water and having a
conductivity of 10 mS/m are shown. The optoelectronic manipulation
chamber for which the results of FIG. 22 were obtained comprised a
first, upper glass substrate having a PEGylated TGE thereon made
according to the process of FIG. 16 and a second, bottom glass
substrate having an ITO electrode layer, a .alpha.-Si:H
photoconductive layer over the electrode layer, and a protective
layer of silicon nitride over the photoconductive layer. It should
be noted that for the results of FIGS. 22A-22D, the bottom
substrate has the configuration disclosed in, for example, Chiou et
al., "Massively parallel manipulation of single cells and
microparticles using optical images," Nature, vol 436, July 2005,
and not the configuration described above in the Example and which
will be further described below with reference to FIGS. 24A and
24B.
[0244] The power source used for the manipulation of the
polystyrene beads shown in FIGS. 22A-22D applied 18 volts at 90 kHz
between the two electrodes of the chamber. An incident He--Ne laser
light beam of 633 nm and 1.15 mW output was mapped onto the
manipulation chamber in a series of moving consecutive rings,
labeled A-G in FIGS. 22A-22D. The images shown in FIGS. 22A-22D
were captured using a CCD camera, as discussed in Chiou et al.,
"Massively parallel manipulation of single cells and microparticles
using optical images," Nature, vol 436, July 2005, incorporated by
reference herein.
[0245] Thus, as shown in FIG. 22A, at the beginning of the
optoelectronic manipulation of the beads (e.g., time=0 sec),
concentric rings of light A-E are illustrated. As time progresses,
for example at 20 seconds into the manipulation, rings A and B have
disappeared and ring C has moved to become the innermost light
ring, as shown in FIG. 22B. After 40 seconds, as shown in FIG. 22C,
light ring E moves to the innermost position with rings A-D having
disappeared. At 60 seconds into the manipulation process, as shown
in FIG. 22D, all of the original light rings A-E shown in FIG. 22A
have disappeared.
[0246] The polystyrene beads present in the aqueous medium in the
manipulation chamber are labeled P.sub.n in FIGS. 22A-22D. As can
be seen by the movement of the various labeled particles P.sub.n in
FIGS. 22A-22D, by encircling the particles in continuously
decreasing rings, the particles were moved from outer regions of
the manipulation chamber so as to be collected within the innermost
ring. Thus, the results of FIGS. 22A-22D demonstrate that a
manipulation chamber wherein the first substrate is provided with a
PEGylated transparent gold electrode, instead of ITO for example,
may be used to manipulate particles via optoelectronically induced
DEP.
[0247] FIG. 23 shows a comparison of the manipulation speed of the
polystyrene beads in a manipulation chambers using various
electrode configurations for the first, upper substrate (e.g., the
nonphotoconductor substrate). In particular the results for using
an ITO electrode are shown by the squares, the results for using a
transparent gold electrode (TGE) are shown by the circles, and the
results for using a PEGylated transparent gold electrode (PEG-TGE)
are shown by the triangles. The second, bottom substrate (i.e.,
with the photoconductor thereon) has the configuration described
above with reference to the results in FIG. 22.
[0248] The results of FIG. 23 show manipulation speed as a function
of applied frequency to a manipulation chamber filled with a 10
mS/m KCl solution in deionized water containing polystyrene beads
having a diameter of about 20 microns. Velocity was measured by
calculating the linear distance in pixels traveled by a bead over a
fixed time period. Each data point in FIG. 23 represents an average
of six velocity measurements take from two manipulation
chambers.
[0249] As demonstrated by the results in FIG. 23, the peak
manipulation speed corresponding to a manipulation chamber wherein
the first substrate electrode was a TGE was about the same as that
for a manipulation chamber wherein the first substrate electrode
was an ITO electrode. The PEGylated TGE substrate surface resulted
in a decrease in manipulation speed.
[0250] With reference now to FIGS. 24A and 24B, two exemplary
approaches for fabricating a PEGylated photoconductive surface,
such as layer 1534 shown in FIG. 15, are shown. In FIG. 24A, a
glass substrate, e.g., substrate 1530, was provided with an
electrode layer such as an ITO electrode layer 1532, which in turn
was provided with a photoconductive layer thereon. In an
alternative, the electrode layer can be an aluminum or gold
electrode layer. Further, the electrode may be transparent or
nontransparent.
[0251] In the exemplary approach shown in FIG. 24A, the
photoconductive layer comprised a n.sup.+ .alpha.-Si:H (doped
amorphous silicon) photoconductor with a .alpha.-Si:H (amorphous
silicon hydride) protective layer deposited thereon. A Piranha
solution was applied to the surface of the substrate of step 24A(i)
in FIG. 24A, which resulted in growing a skin (protective) layer of
silicon dioxide with silanol hydroxy groups (OH) attached to the
surface, as depicted in step 24A(ii). Silicon dioxide was then
subjected to a PEGylation process using
2-methoxy(polyethyleneoxy)propyltrimethoxysilane obtained from
Gelest, Inc., which yielded the configuration depicted in step
24A(iii). It should be noted that n in the PEG groups in step
24A(iii) can range from 2 to 100, however, in the examples made and
tested n ranged from 6 to 9. As n increases, nonspecific adsorption
may be reduced.
[0252] A second exemplary approach for achieving PEGylation of the
photoconductor layer of the second substrate is illustrated in FIG.
24B. In this approach, cleaning and enhancing (e.g., PEGylating)
the photoconductive surface may occur in a single step within a
vacuum chamber. Thus, the glass substrate with the electrode and
photoconductor layers thereon may be substantially the same as
described with reference to step 24A(i) above. Within a single
vacuum chamber process, the substrate shown in FIG. 24B(i) was
subject to an oxygen plasma treatment. The oxygen plasma cleans the
surface of the amorphous silicon hydride (.alpha.-Si:H) and at the
same time grows a skin layer of silicon dioxide on the surface. The
resulting skin layer of silicon dioxide was then subjected to a
mercaptosilanization with 3-(mercaptopropyl)methyldimethoxysilane
(obtained from Gelest, Inc.), as shown in step 24B(ii), implanting
surface mercapto groups thereon. The mercapto groups further
reacted with poly(ethylene glycol) methyl ether acrylate, PEO
acrylate (obtained from Aldrich Chemical), to yield a PEGylated
photoconductive layer, as depicted in FIG. 24B(iii). As with FIG.
24A, n in the PEG groups in step 24B(iii) can range from about 2 to
100, and as n increases, nonspecific adsorption may be reduced. In
the example made and tested n was about 8.
[0253] Based on preliminary tests of the PEGylated photoconductive
surface, subjecting the manipulation chamber to a 10V AC bias and a
light source of 16 W/mm.sup.2, surface adsorption of HeLa cells was
less than about 10% using the PEGylation treatment approach of FIG.
24B and was less than about 20% using the PEGylation treatment
approach of FIG. 24A, with n about 8 in both approaches. Using a
substrate and photoconductive surface as described in, for example,
Chiou et al., "Massively parallel manipulation of single cells and
microparticles using optical images," Nature, vol 436, July 2005,
incorporated by reference herein, the adsorption of HeLa cells
under similar conditions was greater than about 80%. Thus, it
appears that a PEGylated silicon dioxide photoconductive surface
may reduce nonspecific adsorption.
[0254] FIG. 25 shows a comparison of the optoelectronic
manipulation speed of polystyrene beads in a manipulation chamber
comprising a first, upper substrate (e.g., the nonphotoconductor
substrate) having a non-PEGylated transparent ITO electrode
deposited thereon, such as for example, the nonphotoconductive
substrate described in Chiou et al., "Massively parallel
manipulation of single cells and microparticles using optical
images," Nature, vol 436, July 2005, incorporated by reference
herein, and various second, lower, photoconductive substrates. For
example, tests measuring manipulation speed were performed using a
bottom, photoconductive substrate having a configuration like that
described in Chiou et al., "Massively parallel manipulation of
single cells and microparticles using optical images," Nature, vol
436, July 2005, incorporated by reference herein (shown by the
diamonds in FIG. 25), a PEGylated photoconductive surface like that
resulting from the process of FIG. 24A (shown by squares in FIG.
25), and a PEGylated photoconductive surface like that resulting
from the process of FIG. 24B (shown by triangles in FIG. 25). The
other experiment conditions described with reference to FIG. 23
were also used for the results shown in FIG. 25. As illustrated in
the results of FIG. 25, the peak optoelectronic manipulation speed
for the three different photoconductive substrate configurations is
approximately the same.
[0255] It is envisioned that in various exemplary embodiments, the
optoelectronic techniques and devices described herein could be
used in combination and as part of an accessory to a portable
medical device or to a microscope in order to visualize, observe,
and/or collect data about the manipulation of the various particles
within the manipulation chamber. Moreover, it is envisioned that in
various exemplary embodiments, the optoelectronic manipulation
chambers in accordance with the invention could be used in
combination with existing mechanical manipulation and analysis
mechanisms, such as laser pressure catapulting, laser
microdissection, laser microinjection, eletroporation,
microcapillaries, microdissection, microinjection,
micromanipulation, piezoelectric microdissection, patch clamp
electrodes, and/or drug interaction for cell response measurement.
When using optoelectronic manipulation chambers in conjunction with
other manipulation tools, it may be desirable to provide the
manipulation chambers with ports and/or other openings to permit
access to the interior of the chamber by such manipulation
tools.
[0256] Further, it is envisioned that real time visualization
and/or data collection and analysis of particle sorting and/or
other particle manipulation using an optoelectronic manipulation
chamber according to aspects of the invention may occur.
[0257] FIGS. 33-34 show exemplary embodiments of how optoelectronic
sorting devices, including, for example, those depicted in FIGS.
8-11, may be integrated as part of a microcard, chip assembly,
and/or other disposable cartridge assembly.
[0258] FIGS. 33, 33A, and 33B illustrate an exemplary embodiment of
such a cartridge assembly 3300. FIG. 33 represents a top view of
the assembly 3300, while FIGS. 33A and 33B represent isometric
views of the assembly 3300 taken from the top and bottom,
respectively. The cartridge assembly 3300 may include a bottom
substrate 3330 and a top substrate 3320 with a spacer 3340
sandwiched therebetween. The bottom substrate 3330 may include the
various layers (not shown) described with reference to substrate 30
of FIG. 1, including an amorphous silicon photoconductive layer
consistent with the teachings herein. The top substrate 3320 may
include the various layers (not shown) described with reference to
substrate 20 of FIG. 1, including a transparent electrode.
[0259] The spacer 3340 may be provided with openings through the
thickness of the spacer 3340 so as to define a series of chambers
and channels, as shown in FIGS. 33, 33A, and 33B. The series of
chambers and channels may have similar configurations as those in
FIGS. 8-11 or other configurations, as would be understood by those
with ordinary skill in the art. The series of chambers and channels
defined by the openings in the spacer 3340 thus may serve as
optoelectronic scanning chambers disposed between the top and
bottom substrates 3320 and 3330, as is described with reference to
various embodiments herein. Thus, when secured together, elements
3320, 3340, and 3330 define an optoelectronic scanning component
configured to identify, sort, and/or collect particles via
optoelectronic scanning.
[0260] By way of example, in the embodiment shown in FIGS. 33, 33A,
and 33B, the spacer 3340 defines a first chamber 3350 in flow
communication with an inlet channel and an outlet channel and a
second chamber 3346 in flow communication with another inlet
channel and outlet channel. The first and second chambers 3350 and
3346 also are in flow communication with each other via a small
channel. The chamber 3350 may be configured as a
sorting/identification chamber, for example, similar to chambers
110, 310, 410, and 510 described with reference to FIGS. 8-11. The
chamber 3346 may be configured as a target collection chamber, for
example, similar to the chambers 146, 356, 446, and 546 described
with reference to FIGS. 8-11. The channel 3354 may be configured to
introduce a sample containing a plurality of differing types of
particles (e.g., a biological sample containing a plurality of
differing cell types) to the chamber 3350 and the channel 3358 may
be configured to lead nontarget particles from the chamber 3350
after optoelectronic sorting and/or identification has occurred in
the chamber 3350, in a manner similar to that described with
reference to FIGS. 8-11, for example. The channel 3344 may be
configured to provide a buffer solution to the chamber 3346 and the
channel 3347 may be configured to lead the buffer solution and
collected particles from the chamber 3346 for further
processing.
[0261] The elements 3320, 3340, and 3330 may be secured via a
suitable adhesive layer 3360, such as, for example, a PSA layer, to
a base 3375 that has various ports (e.g., cups) 3352, 3362, 3342,
and 3348 in flow communication with channels 3354, 3358, 3344, and
3347, respectively, for loading and unloading sample, buffer, and
collected target and/or nontarget particles. The base 3375 also may
define a relatively large opening configured to provide optical
access to the optoelectronic scanning chambers 3350 and 3346. In
various exemplary embodiments, the base 3375 may be made of a
plastic, such as, for example, cyclic olefin polymer (COP). The
adhesive layer 3360 also may be provided with an optical access
opening in alignment with the opening 3380, and the adhesive layer
3360 and substrate 3320 also may define a plurality of openings
respectively aligned with and in flow communication with the
openings 3352, 3362, 3342, and 3348.
[0262] In an alternative exemplary embodiment, as depicted in the
top and bottom isometric views of FIGS. 34A and 34B, a cartridge
assembly 3400 may include a transparent electrode layer directly
deposited on the surface of the base 3375 that faces the spacer
3340 rather than being deposited on a separate substrate. By way of
example and not limitation, the transparent electrode layer may be
coated via e-beam evaporation. In an exemplary embodiment, the
electrode layer may be a gold electrode layer of about 7-8
nanometers in thickness. The transparent electrode may be patterned
on the base 3375 using a shadow mask, Kapton tape, or other
suitable patterning mechanism known to those skilled in the art.
Due to the deposition of the transparent electrode directly on the
base 3375, the exemplary embodiment of FIGS. 34A and 34B obviates
the need for the substrate 3320 and adhesive layer 3360 of the
embodiment of FIGS. 33, 33A, and 33B.
[0263] FIGS. 35A and 35B illustrate an exemplary embodiment of an
interface mechanism configured to provide an interface between the
assemblies 3300 and 3400 of FIGS. 33 and 34, and/or other cartridge
assemblies in accordance with the teachings herein, with fluidic
instrumentation (such as, for example, valves, pumps, etc.) in
order to provide fluidic pumping of buffer, sample, etc. through
the assemblies. FIG. 35A is perspective top view of a cartridge
assembly 3300/3400 and an interface mechanisms 3500 and FIG. 35B is
a perspective bottom view of the cartridge assembly 3300/3400 and
interface mechanism 3500. As the various parts of the cartridge
assembly 3300/3400 are discussed in detail above, they are not
discussed in detail with reference to FIGS. 35A and 35B.
[0264] In various exemplary embodiments, an interface mechanism
3500 may be in the form of a card-like device having approximately
the same dimensions as the cartridge assembly base 3375. The
interface mechanism 3500 may include a plurality of ports 3570
configured to align with and provide fluid communication to the
ports 3344, 3352, 3348, and 3362 provided in the cartridge base
3375. According to various exemplary embodiments, the ports 3570
may be 1/428 or barb. As depicted in FIG. 35B, a plurality of
sealing mechanisms 3580, such as, for example, O-rings, may be
secured to the interface mechanism 3500 so as to surround each of
the ports 3570 on a surface of the interface mechanism 3500 facing
the cartridge base 3375. The sealing mechanisms 3580 may be
configured to provide a seal during loading and unloading of the
cartridge assembly 3300/3400 with fluid while the cartridge
assembly 3300/3400 is clamped to the interface mechanism 3500. The
interface mechanism 3500 also may define an opening 3585 configured
to align with the opening 3380 in the cartridge assembly 3300/3400
to provide optical access to the chambers 3350 and 3346. Air
pressure and/or vacuum may be supplied through the ports 3570 to
achieve fluidic pumping. By way of example only, positive pressure
pumping may be used with an air source and a pressure regulator.
The pressure regulator may be directly connected to one or more
ports 3570 and may be electronically controlled.
[0265] Although not shown in the figures, in an exemplary
embodiment, the interface mechanism 3500 may be an element that is
part of the fluidic instrumentation and loading/unloading of the
cartridge assembly 3300/3400 may occur as described below.
Initially, the cartridge assembly 3300/3400 may be primed with a
buffer solution by introducing the buffer solution through port
3344 in the base 3375 so as to fill all of the various chambers and
channels in the cartridge assembly with the buffer solution.
According to various exemplary embodiments, the buffer may be
introduced into the port 3344 via a manual syringe, although other
means of introducing the buffer into the port 3344 also may be
utilized as would be appreciated by those having skill in the art.
After the cartridge assembly 3300/3400 is primed with buffer, a
sample containing a plurality of particles of differing types
(e.g., a biological sample containing a plurality of cells of
differing types) may be introduced into the port 3352 in the base
3375. The port 3352 may serve as a cup to hold the sample until
pressure and/or vacuum is used to move the sample through the
channels and chambers of the spacer 3340 of the cartridge assembly
3300/3400. By way of example, the sample may be introduced into the
port 3352 via a pipette.
[0266] After introducing sample into the port 3352, the cartridge
assembly 3300/3400 may be clamped to the interface mechanism 3500
and the sample may be pumped into the optoelectronic
sorting/identification chamber 3350 via a positive pressure or
vacuum applied to the port 3352. Optoelectronic scanning may then
be performed by scanning a light across the optical access openings
and/or windows 3380, 3360, and 3585 to achieve desired
identification, sorting, and/or collecting of the particles in the
sample. Once particles of interest (e.g., target cells) have been
collected, they may be brought into alignment with the port 3348,
in a manner consistent with the teachings herein (e.g., with
reference to FIGS. 8-11 and 29) and the cartridge assembly
3300/3400 may be released from the interface mechanism 3500. The
collected target cells may be removed from the cartridge assembly
3300/3400 from the port 3348, for example, via pipetting.
[0267] The exemplary embodiments of FIGS. 33-35 may provide several
features. For example, the cartridge assemblies are relatively
simple in design and configuration and allow for relatively simple
operations for loading, unloading, and optoelectronic scanning.
Further, because loading/unloading ports (e.g., cups) are located
in relatively close proximity to the sorting chamber, cell loss due
to long microfluidic channels may be minimized, thereby achieving
high yield and recovery of target cells. Moreover, contamination of
the fluidic instrumentation may be minimized due to the use of
pressure/vacuum forces for loading and unloading the cartridge
assembly.
[0268] The various devices and methods in accordance with exemplary
aspects of the invention provide advantages over existing
techniques when used for identification, sorting, characterization,
collection, and/or other manipulation of small particles, such as,
for example, cells, stem cells, DNA, and other biological
particles. For example, use of at least some of the optoelectronic
techniques described herein permits the manipulation of cells, stem
cells, DNA, and other biological particles without the use of dyes,
high intensity lasers, and/or other mechanisms that may stress
and/or damage the particles being manipulated. Further, devices in
accordance with various exemplary aspects of the invention are
relatively inexpensive to fabricate and may achieve relatively high
sorting throughput. The devices according to various exemplary
embodiments may not require formation of patterned electrodes,
microchannels, and/or other relatively complex structures, and
thereby may promote added flexibility in the capability to modulate
electric fields in a variety of dimensions and strengths. The
techniques and devices described herein in accordance with aspects
of the invention also may be less susceptible to clogging and
reduce nonspecific adsorption of biomolecules. It is envisioned
that groups (e.g., bunches) of particles also may be collected and
routed together.
[0269] It is envisioned that the use of DEP, including
optoelectronically-induced DEP using the embodiments in accordance
with exemplary aspects of the invention may be applied to a variety
of applications related to cell biology. For example,
surface-active agents, including ligands, antibodies,
glycoconjugates, and other agents may be used to differentiate
tumor (e.g., cancer) cells from other cells by altering the
dielectrophoretic properties of such cells and then using DEP,
including optoelectronically-induced DEP, to separate and/or
identify the tumor cells from other cells. Relying on
dielectrophoretic characteristics as the mechanism to separate
and/or distinguish cell types may provide enhanced differentiation,
faster sorting throughput, and other advantages when compared to
conventional techniques, such as, for example, protocols that
involve centrifugation, suspension, and jelled clots.
[0270] Moreover, using DEP and dielectrophoretic characteristics as
a basis for cell differentiation permits physiology of cells to be
utilized as the parameter for distinguishing cells, rather than
surface or cell expression that is used in conventional techniques
relying on markers, for example. Reliance on cell physiology and
DEP for cell differentiation may provide enhanced differentiation
and may provide an improved methodology for cell differentiation,
isolation, and study. Thus, the use of DEP, including
optoelectronically-induced DEP, for example, may provide various
advantages over conventional technologies in the areas of stem
cell, cancer cell, and tumor cell differentiation, isolation, and
research. A further explanation of how DEP may be applied in stem
cell and cancer cell biology applications is provided below.
[0271] Some conventional methods of stem cell (SC) identification
utilize specific monoclonal antibody markers. However, no single
marker is adequate to identify subclasses of SCs. This reflects the
fact that changes in proliferative/differentiative state is not
quantal in nature, but rather a continuum of phenotypes. For
example, CD34 is a cell surface receptor widely used as a
"specific" marker of hematopoetic stem cells, but a multi-marker
phenotype (CD34.sup.+, c-Kit.sup.+, Thy-1.sup.lo, Lin.sup.-) is
generally utilized and even within this set of "stem cells" are
sub-populations of cells with different differentiation potential.
Further, most organ-restricted stem cells (i.e. adult stem cells)
lack unique and specific markers so that even using "good" markers
and sorting by FACS, recovered cells are heterogeneous.
[0272] Hence, conventional approaches to stem cell research may be
limited by the need for these markers and the inherent inadequacies
of this approach for identifying and isolating stem cells. DEP and
optoelectronic scanning in accordance with the teachings herein may
provide an alternative approach to identifying and isolating stem
cells.
[0273] A "side population" of cells has been described in bone
marrow analyzed by FACS analysis of Hoechst-dye stained cells.
Subsequent sorting and characterization revealed this subpopulation
of cells to be rich in stem cells that were, however, heterogeneous
in CD34 expression. Failure to take up Hoechst-dye is
characteristic of cells expressing MDR-related proteins and an MDR
phenotype. This so-called side population of cells has been
confirmed many times as comprising stem cells and this assay is
commonly used to isolate stem cells; in some settings, "side
population" is taken to mean "stem cells." In addition to showing
that hematopoetic stem cells have an MDR phenotype, it has been
shown that the MDR phenotype is independent of CD34 expression,
meaning that "CD34+" and "stem cell" are not synonymous.
[0274] Multidrug resistance (MDR) is a cellular phenomenon in which
cells are resistant to the cytotoxic effects of a variety of small
molecule antineoplastic agents. Overall the mechanisms of drug
resistance are varied but MDR represents a major mechanism that has
been implicated in clinical resistance to cytotoxic drug action.
The MDR phenotype is conferred by expression of one or more of the
49 members of the ABC transporter protein family that function in
part to pump out drugs by an ATP-driven process before they can
reach cellular targets and kill the cell. The presumed "normal"
function of ABC transporters is in part to rid the cell of toxic
xenobiotics, but regulation of intracellular pH is also a likely
role. Cells can be inherently drug resistant via MDR proteins or
the phenotype may be acquired by exposure to cytotoxic agents. MDR
is problematic for small molecule chemotherapeutic agents, and some
protein-based drugs (immunotoxins), and is estimated to be
responsible for as much as 50% of chemotherapy failures.
[0275] The reagent, Hoechst 33242, bisibenzimide, is a fluorescent
dye that binds with very high specificity to DNA. It is unique
among DNA-binding dyes in penetrating live cells and binding
stoichiometrically to cellular DNA (and not RNA). Being the only
dye that does so, it has been very widely utilized in many contexts
for cell cycle analysis by FACS. Hoechst has also been shown to be
a substrate for some ABC transporters and whereas viable non-MDR
cells exposed to Hoechst will take up the dye and stain brightly,
MDR+ cells stain very dimly reflecting the activity of the ABC
transporter(s) in excluding the compound from cells. The "side
population" of cells described above is "side" because the cells do
not take up Hoechst dye. Exclusion of Hoechst (and other dyes)
represents a common functional assay for MDR in mammalian cells and
routinely used in conventional techniques for stem cell
identification.
[0276] It also has been demonstrated that MDR-human leukemia cells
differ 2-fold from their normal drug-sensitive parental cells in
cytoplasmic conductivity and hence can be discriminated from each
other solely on the basis of DEP differences. While these data
represent the only report of a relationship between MDR and DEP, it
is known that MDR proteins act as proton pumps regulating
intra-organellar pH within cells and can impact overall
intracellular pH. The DEP differences may reflect differences in
intracellular ion content or concentration (i.e. cytoplasmic
conductivity), rather than MDR activity at the plasma membrane
level because standard MDR-reversing drugs did not have an impact
on the DEP of the cells while altering dye uptake.
[0277] Dielectrophoresis has been reported to distinguish CD34+
stem cells in peripheral blood and bone marrow stem cell harvests.
Significant dielectrophoretic differences between CD34+ cells and
all other leukocytes have been measured using dielectrophoretic
crossover frequency. These differences have been explained in terms
of cell size (stem cells are small) and cell surface topology (stem
cells have relatively "smooth" surfaces), both factors known to
influence the dielectrophoretic properties of viable cells. To
date, however, it does not appear that a relationship between stem
cells, the MDR phenotype and dielectrophoresis has been
reported.
[0278] The inventors thus believe that the MDR phenotype of
hematopoetic (and potentially other) stem cells, alone or in
combination with cell size and morphology, represents a key element
of the stem cell phenotype that is uniquely suited to cell
characterization and isolation by dielectrophoresis. Therefore, the
inventors believe that stem cells may be studied using
dielectrophoretic potential as the primary means of cell
identification and isolation and cell surface markers as secondary
phenotypes.
[0279] "Markerless" selection of stem cells has been demonstrated.
For example, size-sieving of bone marrow mononuclear cells has been
utilized to isolate mesenchymal stem cells, which lack discrete
markers altogether, and the differentiation potential of
size-sieved cells differed. Counterflow centrifugal elutriation
which separates cells on the basis of size and density has also
been used to isolate stem cells from peripheral blood. Thus, in
these studies, known stem cell markers were not necessary to
separate subsets of stem cells giving rise to different tissue-type
cells.
[0280] Putative cancer stem cells have recently been reported to
occur in breast, brain and hematopoetic tumors. Increasingly, these
cells are becoming the focus of both oncology and pharma because
they may represent a new and significant class of cellular targets
for cancer therapy. The hypothesis is that these cells represent
the source of rapidly proliferating tumor cells and persist in the
tumor after treatment, becoming responsible for tumor re-growth the
unfortunate ultimate outcome for most treatments.
[0281] The convergence of stem cell research and oncology is
providing something of a "paradigm" change in thinking about cancer
and a significant impetus for research into the nature of these
cells and means of specifically targeting them for therapy. The
emergence of drug resistance following therapy historically has
been explained as either the acquisition of a new phenotype by the
treated cells (which can be readily accomplished in-vitro) or
selection for pre-existing resistant cells. To the extent that
clinical drug resistance is explained by an MDR phenotype, tumor
stem cells display an MDR phenotype, drug resistance (MDR), cancer
stem cells and residual disease may represent semantic distinctions
rather than biological ones. By the same argument that DEP may be a
unique parameter with which to characterize stem cells in a
developmental context, it may also be used to address questions
cancer stem cells and drug resistance in tumors.
[0282] As has been discussed above, optoelectronic
dielectrophoretic physiometry can provide a unique technology for
cell identification, characterization, manipulation and sorting
based on the electrical consequences of differences in cellular
structure and physiology. This technology exploits the fact that
cellular structure and physiologic functions and changes therein
impact the key electrical features of cells including but not
limited to internal and external membrane conductivity, capacitance
and permittivity; cytoplasmic conductivity, capacitance and
permittivity, cell size and nucleocytoplasmic ratio and surface
complexity that collectively determine the polarizability
(dielectric potential) of cells in a high frequency AC electric
fields and their response to external AC/DC fields generated by
optoelectronic means in specified suspending media.
[0283] Applications of optoelectronic DEP include but are not
limited to identification, characterization, manipulation and
sorting of stem cell, cancer cells, cancer stem cells, blood cells
and cells of any organ (e.g. pancreas, skeletal muscle, lung,
liver) that can be suspended in medium ex-vivo. The contexts of
cell analysis include but are not limited to clinical and
non-clinical research, diagnostics, theragnostics, drug discovery
and development, and environmental monitoring. Various applications
and embodiments are discussed in more detail below.
[0284] Optoelectronic dielectrophoretic physiometery represents a
significant advance over prior dielectrophoretic systems in that
the DC component of the cell manipulation system is generated by
optically activated electronic materials rather than
specifically-arranged solid electrodes. This approach may provide
increased flexibility in the manner in which cells are identified,
isolated, manipulated and sorted and also may provide many options
for realtime feedback regulation of the manipulation processes, as
well as generation of optical signals from cells that can provide
structural, functional and molecular information pertaining to
their state. Additional forces, e.g. fluid flow, can be used
simultaneously adding flexibility to the system. Additionally,
fabrication and operational costs can be very low relative to
conventional technologies.
[0285] Exemplary applications of this technology in the fields of
stem cells, cancer and immunology are discussed below providing a
common basis for the utility of this technology in diverse
fields.
[0286] The identification, characterization and isolation of immune
cells and cancer cells has historically been based on expression of
cell surface antigens that are tagged with fluorochrome-labeled
monoclonal antibodies specific to these antigens. Many cocktails of
antibodies are in common use, but stem cell definition based on
surface markers is incomplete. A gene-expression pattern defining
"stemness" has not, however, emerged from expression profiling
studies and therefore, the ultimate only reliable criteria of
"stemness" of stem cells remains the proliferative/differentiative
potential of candidate cells. Somewhat analogously, specific gene
expression profiles of tumors have yielded a wealth of genic
information but in the end more data than information has been
generated and cell morphology is typically utilized as the ultimate
criterion of malignancy.
[0287] Recently, the physiology of cancer cells has re-emerged as a
key area of cancer research. In particular, the unique energy
metabolism and cell-growth environment of tumor cells are receiving
attention not as epiphenomena of altered gene expression but
perhaps as causally related to oncogenesis. The capacity of
"unlimited" proliferation is a key similarity between cancer and
stem cells, and both direct and circumstantial evidence suggests
that stem cells share gene expression patterns and may be
metabolically similar to cancer cells. There is growing evidence
that cancer stem cells and stem cells per se are highly overlapping
sets.
[0288] Among the features shared by stem cells and cancer cells is
the expression of so-called MDR genes that reside in both the
plasma and intracellular membranes where they act as transporters
of a variety of small organic molecules, particularly organic ions.
In the case of tissue stem cells, ABCG2 (BCRP, breast cancer
resistance protein) transporter function is defining of very
primitive precursor cells and for cancer, transporter function is
central to drug resistance and also defining of stem cells at least
in some contexts. The function of these transporters is central to
current phenotypic definition of stem cells and to the MDR
phenotype of cancer cells.
[0289] A so-called "side population" of CD34.sup.+ hematopoetic
stem cells has become an important functional marker of
hematopoetic stem cells. The "sideness" of this population of cells
reflects the failure of cells to stain with either of two
flourescent organic dye molecules, bisbenzimide (Hoechst) or
Rhodamine 123, because they express one or more MDR proteins and
therefore pump these molecules out of the cell. Relative to
non-stem cells, these cells are dim when stained with specific
fluorescent dyes. Side populations have subsequently been
identified in cell populations derived from breast cancer, lung
cancer and neural cancer as well as some normal tissues providing
in part, the rationale for the idea of cancer and non-hematopoetic
tissue stem cells.
[0290] The hematopoetic stem cell phenotype described as side
population has been demonstrated to be due to one particular
transporter, ABCG2 (also called BCRP, breast cancer resistance
protein), that is highly expressed on very primitive stem cells and
many cancer cells. In the hematopoetic system, ABCG2 is a marker of
the most primitive stem cells which may or may not express CD34 or
other surface markers.
[0291] MDR proteins represent a large family of membrane-spanning
proteins that actively extrude or efflux a wide variety of organic
molecules in an ATPase-driven manner. As a class they are referred
to as ABC transporters (ATP binding site cassette) and possess
highly conserved ATP-binding and transmembrane domains. The family
is diverse as are the substrates. While these proteins act to
exclude toxic xenobiotics being responsible for cancer cell
resistance to cytotoxic agents, they also are involved in transport
of a wide variety of bulky lipophilic organic anions and cations.
The extent to which ABC proteins show substrate specificity is
somewhat unclear as those responsible for some drug resistance
phenotypes appear to be promiscuous with respect to substrates.
[0292] While there remains some confusion as to the specific ABC
transporter involved (BCRP and/or MRP-1), the side population
phenotype in hematopoetic stem cells is by definition due to ABC
transporter expression. Side populations have been observed in a
variety of tumor (breast, neural, ALL, AML) and normal tissues
(breast, lung, prostate, eye, skin) and in some cases it has been
shown that the phenotype is the result of ABC transporter activity.
Thus it appears ABC transporter expression and activity represents
a key molecular marker for stem cells in general and cancer
cells.
[0293] While ABC transporters do not transport inorganic ions, they
are known to transport organic ions. ABC transporter function and
the drug resistance phenotype have been studied by
dielectrophoresis in cancer cell lines. One such study demonstrated
that cells with an MDR phenotype can be distinguished from non-MDR
cells by DEP. Because this effect was not abolished by blocking ABC
activity with an MDR-reversing drug (verapamil), it was suggested
that ABC transporter function and DEP differences were related to
differences in cytoplasmic conductivity rather than drug pumping
activity per se.
[0294] While it is clear that the resistance of cancer cells and
stem cells to cytotoxic agents reflects at least in part the
activity of ABCG2, the "normal" role of the protein is not entirely
clear. However, some studies suggest that ABCG2 may be involved in
maintaining stem cell viability and proliferative potential under
hypoxic conditions which are known to occur in the developing
embryo of mammals, embryoid bodies in cultured stem cells. Relative
to arterial blood, bone marrow is known to have a lower pO.sub.2
and hematopoetic stem cells in normal bone marrow are localized in
a relatively hypoxic environment. It is known that hypoxia inhibits
stem cell differentiation and sustains stem cell viability and
proliferation and that down-modulation of ABCG2 expression is an
early and rapid event when stem cells differentiate. Taken
together, it appears that ABCG2 is involved in stem cell
maintenance under hypoxic conditions that would damage/kill normal
non-stem cells but are necessary for stem cell survival and
proliferation.
[0295] Tissue hypoxia is a prominent feature of solid tumors. It is
believed that this is due to the rapid growth of tumor cells which
outstrips the rate of angiogenesis. Stimulation of ABC gene
expression has not been shown to occur in hypoxic cancer cells. The
MDR phenotype and hypoxia may characterize solid tumors and
contribute to drug resistance.
[0296] As described below, tumor cells utilize glycolysis for
energy production under both hypoxic and aerobic conditions and in
fact glycolysis is a hallmark of cancer cells. Lactate is the end
product of glycolysis. Both normal and tumor stem cells are
inhibited from differentiating by hypoxia and it is likely that the
glycolytic pathway is also active in them. Tumor cells are
characterized, not unlike stem cells, by their capacity for
"unlimited" proliferation. Central to the aspect of tumor cell
phenotype is the Warburg effect, a shift in energy metabolism from
aerobic respiration to anaerobic glycolysis and consequent
alkalinization of tumor cell cytoplasm and acidification of the
tumor mass itself (for solid tumors). Glycolytic energy metabolism
while generally regarded as an adaptation to hypoxia proceeds in
the presence of normoxic conditions in cancer cells and the Warburg
effect as applied to aerobic glycolysis defines the cancer cell
metabolic phenotype.
[0297] It should be understood that in various exemplary
embodiments, the techniques and devices according to exemplary
aspects of the invention may permit control over the generated DEP
force and consequent particle manipulation. For example, it is
envisioned that the Clausius-Mosotti factor for a particle may be
changed, for example, to being positive or negative, by altering
the medium in which the particle is suspended and/or by altering
the particle itself, such as, for example, by altering the charge,
size, and/or otherwise modifying the surface of the particle so as
to modify a dielectrophoretic characteristic of the particle.
Various ways to alter the Clausius-Mosotti value, the DEP force,
and/or other forces acting on particles during optoelectronic
manipulation have been described herein, and one having ordinary
skill in the art would understand other ways in which to alter the
Clausius-Mosotti value and/or forces acting on the particles by
altering the various properties, etc. so as to alter the variables
in the equations defining those values.
[0298] By way of example, and in addition to those already
discussed herein, the medium characteristics may be altered so as
to adjust the complex permittivity of the medium, .epsilon..sub.m*,
and thus the Clausius-Mosotti value. It may be desirable to adjust
.epsilon..sub.m* while maintaining cell compatibility. Arnold,
"Positioning and Levitation Media for the Separation of Biological
Cells," IEEE Transactions on Industry App., vol. 37(5), pp.
1468-1475 (2001) describes various media used for dielectrophoresis
with a focus on those suitable for negative dielectrophoresis in
order to avoid cell contact with electrodes to minimize sticking
and damage due to high electric field concentration proximate the
electrode. Media optimized for negative DEP may have higher
polarizability than the particles (e.g., cells) contained in the
medium. With respect to polarizability, changes in conductivity
tend to dominate at low or medium applied frequencies, while
permittivity dominates at higher frequencies.
[0299] Water is relatively highly polar and most solutes in water
tend to decrease the permittivity, including, for example, sugars
added to water to promote osmotic stability. Classes of solutes
that may increase permittivity of aqueous media include, for
example, the following:
[0300] 1) Neutral molecules with relatively large dipole moments
(e.g., urea, formamides, organic carbonates). This class may not be
compatible with cells.
[0301] 2) Small zwitterions (dipolar ions), such as, for example,
amino acids. Zwitterionic buffers may be used at pH about 2 units
below pKa to help ensure complete protonation. At higher pH,
charged versions of these molecules may contribute to increases in
conductivity with a decreased contribution to permittivity. By way
of example, .epsilon.-aminocaproic acid may increase permittivity
of a medium.
[0302] 3) Large zwitterions, such as, for example, polypeptides and
proteins. This class may be cost prohibitive.
[0303] 4) Suspensions of charged particles. This class generally
disperses in AC, and contributes to high conductivity.
[0304] Quantitatively, the permittivity of the medium may be
calculated by the following:
.epsilon..sub.m=.epsilon..sub.w+C.differential..sub.1+c.sup.2.differentia-
l..sub.2
[0305] In the above equation, .epsilon..sub.m is the permittivity
of the medium, .epsilon..sub.w is the permittivity of water, c is
the concentration of the solute, and .differential. equals the
molar increments. For a determination of the molar increments for
many possible solutes, reference is made to Arnold, "Positioning
and Levitation Media for the Separation of Biological Cells," IEEE
Transactions on Industry App., vol. 37(5), pp. 1468-1475 (2001) and
Arnold et al., "Dielectric measurements on electro-manipulation
media," Biochem. Biophys. Acta., vol. 1157, pp. 32-44, 1993, the
entire contents of each of which are incorporated by reference
herein. .differential..sub.2 values for HEPES and related ions are
small relative to .differential..sub.1, resulting in a linear
effect on .epsilon..sub.m.
[0306] Dielectric dispersion occurs when the polarization cannot
follow the electric field at high frequencies. Dispersion tends to
decrease the media permittivity and increase its conductivity. For
most sugars reported in Arnold et al., "Dielectric measurements on
electro-manipulation media," Biochem. Biophys. Acta., vol. 1157,
pp. 32-44, 1993, permittivities and conductivities were
substantially unchanged at concentrations less than about 1.2M,
between 200 kHz and 2 MHz and thus dispersion may be minimized.
Dispersion was observed for many high density solutes, such as,
Percoll, Nicodenz, and Metrizamide, at higher frequencies.
[0307] It should be noted that sizes and configurations of various
structural parts and materials used to make the above-mentioned
parts are illustrative and exemplary only. One of ordinary skill in
the art would recognize that those sizes, configurations, and
materials can be changed to produce different effects and/or
desired characteristics. Further, although many of the embodiments
described above have been discussed in conjunction with using
optoelectronic manipulation principles for applications relating to
cellular analysis and other cell biology applications, it should be
understood that the exemplary techniques and devices disclosed
herein may be used for other applications wherein the manipulation
of small particles is desirable, such as, for example, clinical
diagnostics, drug discovery, environmental monitoring of
bioparticles and non-bioparticles (e.g., detection of viral,
bacterial, or protozoan entities in water samples and detection of
non-biological particulates in water samples), characterization
and/or isolation of non-cell microparticles (e.g., microsphere
sizing or chemical/electrical characterization), and/or other
applications wherein manipulation (including identification,
sorting, separating, moving, quantitating, characterizing, etc.) of
small particles may be desired. Yet other applications may include,
but are not limited to, manipulation, including separation, of
dye-labeled DNA, RNA, proteins, lipids, terpenes, glycoconjugates,
and polysaccharides, for example.
[0308] As used in this application, the term "small particles" may
include micro- and/or nano-particles, for example, particles having
dimensions on the order of a few microns or a few nanometers. In
the context of biological fluid analysis and/or handling, the term
small particles may include cells, cell aggregates, cell
organelles, stem cells, nucleic acids, bacteria, protozoans,
viruses, and other biological particles.
[0309] In this application, the use of the singular includes the
plural unless specifically stated otherwise. It is noted that, as
used in this specification and the appended claims, the singular
forms "a," "an," and "the," include plural referents unless
expressly and unequivocally limited to one referent. In this
application, the use of "or" means "and/or" unless stated
otherwise. Furthermore, the use of the term "including", as well as
other forms, such as "includes" and "included", is not limiting. As
used herein, 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. Also, terms such as
"element" or "component" encompass both elements and components
comprising one unit and elements and components that comprise more
than one subunit unless specifically stated otherwise. Wherever
possible, the same reference numbers will be used throughout the
drawings to refer to the same or like parts.
[0310] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. All literature and similar materials
cited in this application, including patents, patent applications,
articles, books, treatises, and internet web pages are expressly
incorporated by reference in their entirety for any purpose. In the
event that one or more of the incorporated literature and similar
materials defines or uses a term in such a way that it contradicts
that term's definition in this application, this application
controls. While the present teachings are described in conjunction
with various exemplary embodiments, it is not intended that the
present teachings be limited to such embodiments. On the contrary,
the present teachings encompass various alternatives,
modifications, and equivalents, as will be appreciated by those of
skill in the art.
[0311] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing quantities,
percentages or proportions, and other numerical values used in the
specification and claims, are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the following
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the application of the doctrine of equivalents to the scope
of the claims, each numerical parameter should at least be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques.
[0312] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all subranges subsumed therein. For example, a
range of "less than 10" includes any and all subranges between (and
including) the minimum value of zero and the maximum value of 10,
that is, any and all subranges having a minimum value of equal to
or greater than zero and a maximum value of equal to or less than
10, e.g., 1 to 5.
[0313] It will be apparent to those skilled in the art that various
modifications and variations can be made to the structure and
methodology of the present invention. Thus, it should be understood
that the invention is not limited to the examples discussed in the
specification. Rather, the present invention is intended to cover
modifications and variations. Other embodiments of the invention
will be apparent to those skilled in the art from consideration of
the specification and practice of the invention disclosed
herein.
* * * * *