U.S. patent application number 12/936147 was filed with the patent office on 2011-05-12 for ex vivo multi-dimensional system for the separation and isolation of cells, vesicles, nanoparticles and biomarkers.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Dennis Carson, Sadik Esener, Michael Heller, Rajaram Krishnan, Benjamin Sullivan.
Application Number | 20110108422 12/936147 |
Document ID | / |
Family ID | 41377870 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110108422 |
Kind Code |
A1 |
Heller; Michael ; et
al. |
May 12, 2011 |
EX VIVO MULTI-DIMENSIONAL SYSTEM FOR THE SEPARATION AND ISOLATION
OF CELLS, VESICLES, NANOPARTICLES AND BIOMARKERS
Abstract
Devices and techniques are described that involve a combination
of multidimensional electrokinetic, dielectrophoretic,
electrophoretic and fluidic forces and effects for separating
cells, nanovesicles, nanoparticulates and biomarkers (DNA, RNA,
antibodies, proteins) in high conductance (ionic) strength
biological samples and buffers. In disclosed embodiments, a
combination of continuous and/or pulsed dielectrophoretic (DEP)
forces, continuous and/or pulsed field DC electrophoretic forces,
microelectrophoresis and controlled fluidics are utilized with
arrays of electrodes. In particular, the use of chambered DEP
devices and of a properly scaled relatively larger electrode array
devices that combines fluid, electrophoretic and DEP forces enables
both larger and/or clinically relevant volumes of blood, serum,
plasma or other samples to be more directly, rapidly and
efficiently analyzed. The invention enables the creation of
"seamless" sample-to-answer diagnostic systems and devices. The
devices and techniques described can also carry out the assisted
self-assembly of molecules, polymers, nanocomponents and mesoscale
entities into three dimensional higher order structures.
Inventors: |
Heller; Michael; (San Diego,
CA) ; Sullivan; Benjamin; (San Diego, CA) ;
Krishnan; Rajaram; (La jolla, CA) ; Carson;
Dennis; (La Jolla, CA) ; Esener; Sadik;
(Solana Beach, CA) |
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
41377870 |
Appl. No.: |
12/936147 |
Filed: |
April 3, 2009 |
PCT Filed: |
April 3, 2009 |
PCT NO: |
PCT/US09/39565 |
371 Date: |
January 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61042228 |
Apr 3, 2008 |
|
|
|
Current U.S.
Class: |
204/547 ;
204/450; 204/600; 204/643 |
Current CPC
Class: |
B03C 5/005 20130101;
G01N 30/0005 20130101; G01N 33/491 20130101; B03C 2201/26 20130101;
B03C 5/026 20130101; G01N 27/447 20130101 |
Class at
Publication: |
204/547 ;
204/643; 204/450; 204/600 |
International
Class: |
G01N 27/447 20060101
G01N027/447; G01N 27/403 20060101 G01N027/403 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This work was supported by NIH Grant/Contract CA119335. The
Government of the United States of America may have certain rights
in this invention.
Claims
1. A sample processing device for separation of biological material
in a sample fluid, the device comprising: at least one inlet that
receives a flow of the sample fluid and directs the sample fluid
flow to an electrode array; and at least one outlet that receives
the sample fluid flow from the electrode array; wherein the
electrode array comprises a plurality of electrodes configured such
that at least one subsection of the electrodes can be charged
differently from remaining subsections of the electrodes and
thereby establish differently charged dielectrophoresis (DEP) force
regions of the electrodes in accordance with the differently
charged electrodes and initiate separation of the biological
material according to the differently charged DEP force
regions.
2. A sample processing device according to claim 1, wherein the
electrode array includes: a plurality of alternating current (AC)
electrodes that are distributed in a planar region of the device
between the inlet and the outlet; a plurality of direct current
(DC) electrodes that are arranged adjacent to the AC
electrodes.
3. A sample processing device according to claim 2, wherein the DC
electrodes extend outside the planar region of the AC
electrodes.
4. A sample processing device according to claim 2, wherein at
least one subsection of the AC electrodes receives an AC current
having a different current frequency from that of the remaining AC
electrodes.
5. A sample processing device according to claim 1, wherein the
electrodes are constructed of robust materials resistant to
corrosion from electrolysis effects.
6. A sample processing device according to claim 1, wherein an
overlay of porous material is located on the electrodes such that
the porous material is resistant to corrosion from electrolysis
effects.
7. A sample processing device according to claim 1, further
including at least one analyte outlet through which a flow of
analyte is directed out of the device.
8. A system for separation of biological material, the system
comprising: a sample processing device according to claim 1; and a
controller configured to selectively energize the electrodes of the
sample processing device and initiate separation of the biological
material.
9. A system according to claim 8, further comprising an associated
detection system for monitoring the separation process and analysis
of a separated biological component of the biological material.
10. A method of separating biological material, the method
comprising: receiving the biological material into a sample
processing device having an electrode array having a plurality of
electrodes configured such that at least one subsection of the
electrodes can be charged differently from remaining subsections of
the electrodes and thereby establish differently charged
dielectrophoresis (DEP) force regions of the electrodes in
accordance with the differently charged electrodes; selectively
energizing the electrodes and establishing the differently charged
DEP force regions of the electrodes in accordance with the
differently charged electrodes; and separating the biological
material according to the differently charged DEP force regions
into at least one separated analyte component that is received at
an analyte outlet of the sample processing device, and remaining
components of the biological material.
11. A method according to claim 10, further including monitoring a
separation process and analysis of separated biological
components.
12. A method according to claim 10, wherein separating comprises
holding at least one type of biological material at one of the
electrode subsections, the method further including: introducing a
reagent into the sample processing device; and reacting the
introduced reagent with the held type of biological material in the
sample processing device.
13. A method according to claim 12, wherein the reagent comprises a
fluorescent die.
14. A method according to claim 12, wherein the reagent comprises
antibodies.
15. A method according to claim 12, wherein reacting comprises
performing PCR operations on the held type of biological
material.
16. A system for separation of biological materials, the system
comprising: two or more separate electrode chamber structures each
with and electrode, said groups of electrode chamber structures
separated by an inner chamber structure with pores (portals,
holes), which allows an electric field to pass from outer electrode
chambers through the inner chamber, an inlet into the inner chamber
that receives the biological materials, at least one analyte outlet
that receives a separated component of the biological materials
from the inner chamber, and another outlet that receives remaining
components of the biological materials; a controller configured to
selectively energize the electrodes and initiate separation of the
biological material and associated detection system for monitoring
separation process and analysis of separated biological
components.
17. A method of separating biological materials, the method
comprising: receiving the biological materials into an inner
chamber structure with outer electrode chamber structures, an inlet
that receives the biological materials, at least one analyte outlet
that receives a separated component of the biological materials
from the inner chamber, an outlet that receives remaining
components of the biological materials, and an associated detection
system for monitoring separation process and analysis of separated
biological components; selectively energizing the electrodes; and
separating the biological material into at least one separated
analyte component that is received at an analyte outlet of the
inner chamber structure, and remaining components of the biological
material.
18. A method where the systems of any of claims 11 through 15 are
used to carry out the assisted self-assembly of molecules,
polymers, nanocomponents, including DNA and protein derivatized
nanoparticles, quantum dots, nanotubes, and the like, and mesoscale
components into higher order three dimensional structures,
materials, and devices.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/042,228 filed Apr. 3, 2008 entitled "Ex-Vivo
Multi-Dimensional System for the Separation and Isolation of Cells,
Vesicles, Nanoparticles and Biomarkers", the disclosure of which is
incorporated herein by reference in its entirety.
BACKGROUND
[0003] In biomolecular research and clinical diagnostics it is both
important and a challenge to separate and identify rare cells,
bacteria, virus, and biomarkers (e.g. DNA, RNA, antibodies, other
proteins, etc.) in complex fluid samples like blood, plasma, serum,
saliva, and urine. Additionally, the advent of bio-nanotechnology
has led to numerous drug delivery approaches that involve
encapsulation of drugs and imaging agents within nanovesicles and
nanoparticles. Such approaches mean it will now also be important
to identify and separate residual nanovesicles and nanoparticles
that remain in the blood stream. A variety of physical, electronic,
and biological techniques and mechanisms can be used for sample
preparation and isolation of specific cells, nanovesicles, and
biomolecules from complex samples like blood. These techniques and
mechanisms include centrifugation, gel filtration, affinity
binding, DC electrophoresis, and various combinations incorporated
into lab-on-a-chip, microfluidic devices, and sample-to-answer
systems.
[0004] Many of these conventional techniques (or combinations) are
relatively time consuming processes that are not without problems
and limitations. In particular, the isolation of rare cells (cancer
cells, fetal cells, and stem cells), low numbers of bacteria and
virus or very low numbers of specific antibodies, proteins,
enzymes, DNA, and RNA molecules, still remains difficult. In the
case of clinical diagnostics, rare cell and biomarker detection may
also be limited by sample size; i.e., only a relatively small
amount of blood may be drawn from very ill patients, the elderly
and infants. Thus, sample preparation processes that are
inefficient or require high dilution of the original sample often
fail or are unreliable for isolating cells and other
disease-related markers at lower concentration ranges. This is in
particular a problem for early detection of cancer, residual
disease, fetal cells/DNA/RNA in maternal blood, bacteria and virus
in blood (septic infection), and the detection of low numbers of
pathogens (e.g. bacteria, virus, etc.) and bioterror agents in
large volumes of air, water, or in food stuffs.
[0005] Alternating current electrokinetic techniques that involve
the use of AC fields to manipulate cells and nanoparticles offers
some particularly attractive mechanisms for the separation of cells
[see References 2-5], biomarkers (DNA [Ref. 5-8], proteins [Ref.
9], etc.), and ultimately drug delivery nanovesicles [Ref. 10].
These techniques can be broken down into three distinct phenomena:
(1) AC electroosmosis, which is surface fluid flow due to the
surface charge on an electrode; (2) electrothermal flow, which is
bulk flow in solution due to thermal gradients produced by the
electric fields; and (3) dielectrophoresis (DEP), which is an
induced motion of particles produced by the dielectric differences
between the particles and media in an AC electric field [Ref. 10].
Unfortunately, most conventional forms of DEP and related
electrokinetic effects have problems that limit the usefulness of
these technologies for clinically relevant sample preparation and
diagnostics.
[0006] First, efficient DEP separations in terms of speed and
control of selectivity usually have to be carried out at relatively
low conductance on the order of <10-100 mS/m [Ref. 11].
Additionally, the ability to isolate the desired entities/analytes
such as nanoparticles or DNA biomarkers in the positive or DEP high
field regions (usually around or on the electrodes) becomes more
difficult as the solution ionic strength increases and the
conductance becomes greater than 10 mS/m. Thus, biological samples
such as blood or plasma that have ionic strengths in the 100-200 mM
range (conductance .about.500-1000 mS/m) must be significantly
diluted and/or processed before DEP separations can be carried out
[Ref. 13, 14]. This alone often limits the usefulness of DEP for
clinical diagnostics involving the detection of rare cells or low
numbers of biomarkers. In cases where a sample (one ml blood) has
to be diluted 100 to 1000-fold, now means that a very large sample
volume must be processed, which can be prohibitively time
consuming. If cells are first concentrated by physical mechanisms
such as centrifugation or filtration and then are diluted into low
conductance buffers, these processes are not only time consuming,
but also costly and cause considerable perturbation to the sample.
In the case where DEP might be used for stem cell separations,
dilution into low ionic strength, less physiological-type buffers
may result in perturbation of sensitive stem cells and may affect
their further differentiation. The isolation of DNA, RNA, and
protein biomarkers from blood is also important for future clinical
diagnostics, in particular for monitoring cancer chemotherapy [Ref.
15], residual disease [Ref. 16], and early detection of cancer
[Ref. 17].
[0007] While DEP has been used for the isolation of DNA and
proteins, problems and limitations do exist in using DEP to carry
out the detection of DNA in blood. The first problem again is the
need to dilute and/or process the blood sample before DEP analysis.
In the case of clinically relevant cell-free circulating DNA and
RNA biomarkers in blood, finding and measuring the amount of
DNA/RNA, its size and base composition (mutations and
polymorphisms) is important [Ref. 17-19]. Sample processing that
involves or requires centrifugation, filtration, and washing
procedures can cause the release of DNA molecules by normal cells
that are damaged or lysed in the process, as well as shear the
clinically relevant DNA into smaller fragments. The release of
extraneous DNA fragments and processing damage to the clinically
relevant DNA greatly compromises and limits the diagnostic value of
using such procedures. Such sample processing is also highly
inefficient, and up to 60% of the DNA and over 90% of the RNA in
the blood can be lost during the procedure [17].
[0008] A second problem area is that most DEP separation devices
that have been used for DNA, protein, and nanoparticle separations
use either polynomial gold microelectrodes created with a very
small separation (6 .mu.m or less) between them to serve as
particle traps; or use castellated gold microelectrode arrays with
6-8 microns or less separation between them [Ref. 18-19]. These
gold microelectrode array devices are usually fabricated by
sputtering gold unto a glass substrate material. There are also a
number of DEP approaches involving the use of nanoelectrodes [20].
The problem with these approaches are that the arrays have
intrinsically low throughput, since the actual space to capture DNA
or other biomolecules is relatively small and the electric field
effect is significantly reduced when distance from the
nanoelectrode increases (e.g. >10 nm). If this type of device is
scaled for sample preparation (e.g., to process 1-10 ml of blood),
the actual sample area that can be interrogated by the limited DEP
field near the electrodes means that most of the DNA will be
missed, or an extremely long sample processing time would be
required. If the device is designed to constrict the liquid flow so
as to pass within ten's of nanometers of the nanoelectrodes, then
the processing time is again extremely long or a massively large
(x-y dimension) device would be required. A variety of other
problems exist including uncontrolled fluidic eddy currents due to
other electrokinetic effects and osmotic forces. In other DEP
applications, arrays that utilize circular platinum microelectrodes
(50 .mu.m to-80 .mu.m diameter) with about 200 .mu.m spacing and
over-coated with a porous hydrogel have also been used to carry out
the DEP separation of bacteria from blood, and for the separation
of cancer cells [Ref. 13, 14]. Again, for these DEP separations,
the blood sample was centrifuged and a small fraction of the cells
were re-suspended in a low ionic strength buffer [Ref. 13, 14,
24-26].
[0009] A third general problem for AC electrokinetic techniques is
often that the resulting sensitivity versus specificity ratios are
not sufficiently high for carrying out important or clinically
relevant separations. For cell separations using dielectrophoresis
(DEP), carrying out efficient rare cell separations with ratios of
one in a million is difficult. Because many early disease
diagnostics require rare cell or low level biomarker detection, it
is important to be able to improve sensitivity versus specificity
ratios as much as possible. In general, most DEP devices are not
scaled properly to deal with the clinical reality of rare cell or
low level biomarker isolation and detection, where a relatively
large sample of from 1-10 ml of blood might be necessary for simple
statistical reasons. When DEP device are designed for large
samples, they are usually inefficient and unable to operate at high
conductance conditions, and thus require further sample
dilution.
[0010] A fourth problem for AC electrokinetic techniques is
carrying out efficient (low loss) and highly selective separation
processes in complex biological samples (e.g. blood, plasma, serum,
etc.) for analytes and biomarkers which include; rare cells,
bacteria, virus, DNA, RNA and proteins where all the entities might
have 2-3 orders of magnitude difference in size range, and it is
still necessary to achieve an efficient separation between entities
that are more similar in size and composition. One important
example is the separation of DNA nanoparticulates (20-50 kb), high
molecular weight DNA (5-20 kb), intermediate molecular weight DNA
(1-5 kb), and lower molecular weight DNA (0.1-1 kb).
[0011] The final and most serious problem for AC electrokinetic
(DEP) devices and techniques is the introduction of
electrochemistry that becomes more pronounced in higher conductance
solutions (>100 mS/m), at lower AC frequencies (<20 kHz) and
at higher voltages (>20 volts pt-pt). As will be shown in the
Detailed Description section of this document, such
electrochemistry can cause a number of adverse effects including
bubbling, heating, fluidic turbulence, electrode degradation, and
destruction of labile analytes. These adverse effects greatly limit
the overall DEP device performance, prevent the accumulation,
isolation, and detection of specific entities (cells,
nanoparticles, DNA and proteins) from occurring in the DEP high
field regions, and interfere with the isolation of cells and
analytes into the DEP low field regions.
[0012] Other types of AC electrokinetic devices have been used to
separate cells and nanoparticles, but have not proved viable in
high conductance solutions. One of the most convincing arguments
for the non-viability of AC electrokinetic and DEP devices is the
fact that unlike DC electrophoresis, which has widespread use in
biological research and clinical diagnostics, DEP has not been used
for any practical applications. It would be desirable to perform
dielectrophoresis with high performance characteristics that allow
separations in high conductance biological samples and buffers.
SUMMARY
[0013] Embodiments of the present invention relate to novel sample
preparation, sample-to-answer and point-of-care systems, devices,
methods, and techniques that involve unique combinations of
multidimensional AC electrokinetic and dielectrophoretic (DEP), DC
electrophoretic, on-device microelectrophoresis and fluidic
techniques for separating and identifying rare cells, bacteria,
virus, drug delivery nanovesicles and nanoparticles, cellular
organelles and structures (nuclei, mitochondria, vacuoles,
chloroplasts, cylomicrons, etc.), cell-free circulating DNA/RNA
biomarkers and other disease-related cellular nanoparticulates
(e.g. partially degraded cellular components which are released
into the blood, lymph or organs by cancerous, diseased or damaged
cells), antibodies, antibody complexes, proteins, enzymes, and
drugs and therapeutics directly in blood or other biological
samples or buffers. In the disclosed embodiments, a combination of
continuous and/or pulsed electrokinetic/dielectrophoretic (DEP)
forces, continuous and/or pulsed field DC electrophoretic forces,
on-device microelectrophoresis size separation, and controlled
fluid flow (externally pumped and/or DC/AC electrokinetic driven)
are utilized via novel chambered devices and other devices that
incorporate arrays of robust electrodes (micro and/or macro sized)
with over-layered porous structures which are used to carry out
complex sample preparation, biomolecule separations, and diagnostic
analyses.
[0014] This specification first discloses novel electrokinetic DEP
devices and systems in which the electrodes are placed into
separate chambers and positive DEP regions and negative DEP regions
are created within an inner chamber by passage of the AC DEP field
through pore or hole structures. Various geometries can be used to
form the desired positive DEP (high field) regions and DEP negative
(low field) regions for carrying cell, nanoparticle, and biomarker
separations. Such pore or hole structures can contain (or be filled
with) porous material (hydrogels) or can be covered with porous
membrane type structures. By segregating the electrodes into
separate chambers, such pore/hole structure DEP devices basically
eliminate any electrochemistry effects, heating, or chaotic fluidic
movement from occurring in the inner separation chamber during the
DEP process (see FIG. 1 and FIG. 2).
[0015] The specification also discloses the use of scaled sectioned
(x-y dimensional) arrays of robust electrodes and strategically
placed (x-y-z dimensional) arrangements of auxiliary electrodes
that combine DEP, electrophoretic, and fluidic forces so that
clinically relevant volumes of blood, serum, plasma, or other
samples may be more directly analyzed under higher ionic
strength/conductance conditions. This specification discloses the
overlaying of robust electrode structures (e.g. platinum,
palladium, gold, etc.) with one or more porous layers of materials
(natural or synthetic porous hydrogels, membranes, controlled
nanopore materials, and thin dielectric layered materials) to
reduce the effects of any electrochemistry (electrolysis)
reactions, heating, and chaotic fluid movement that occur on or
near the electrodes, and still allow the effective separation of
cells, bacteria, virus, nanoparticles, DNA, and other biomolecules
to be carried out (FIGS. 3-8). In addition to using AC frequency
cross-over points to achieve higher resolution separations,
on-device (on-array) DC microelectrophoresis can also be used for
the secondary separations. For example, the separation of DNA
nanoparticulates (20-50 kb), high molecular weight DNA (5-20 kb),
intermediate molecular weight DNA (1-5 kb), and lower molecular
weight DNA (0.1-1 kb) fragments (FIGS. 9-12). The fact that the
device can be sub-sectioned means concurrent separations of
different blood cells, bacteria and virus, and DNA can be carried
out simultaneously on such a device (FIGS. 13-16).
[0016] Embodiments of the present invention also relate to the use
of temperature control to provide more selective and efficient cell
separations (e.g. of cancer and stem cells). Embodiments of the
invention thus relate in one aspect to ex-vivo sample preparation,
seamless sample-to-answer, lab-on-a chip and point of care (POC)
diagnostic systems that can be used to monitor and/or analyze blood
for cancer cells, bacteria, virus, nanovesicles (drug delivery),
nanoparticles, high molecular weight DNA nanoparticulates, cellular
organelles, proteins, antibodies and antibody complexes, and a
variety of other clinically relevant biomarkers of disease and
metabolic state. Such ex-vivo systems and devices can monitor or
scan the blood by AC electric fields, separating, isolating, highly
concentrating, and detecting and analytes and clinically relevant
entities. Systems can be used to selectively collect such entities
for more complex analysis including but not limited to
immunochemistry; DNA/RNA probe hybridization; polymerase chain
reaction (PCR), rolling circle amplification (RCA), strand
displacement amplification (SDA) and other techniques for
genotyping, sequence analysis, gene expression all within the same
sample chamber (seamless sample to answer), or via associated
analytical devices and/or collection systems. A novel device
constructed in accordance with the invention could be a
point-of-care (POC) seamless sample-to-answer system that allows
rapid molecular diagnostics to be rapidly carried out on an
undiluted blood sample. Another novel device in accordance with the
invention could be an ex-vivo cancer chemotherapy monitoring system
that would allow blood to be shunted from the patient, rapidly
analyzed (measure biomarker DNA, drug or drug delivery nanovesicle
levels and isolate cancer cells), and then returned to the patient
(via closed loop) with minimal dilution or physical/chemical
perturbation to the sample. Such ex-vivo systems could also be used
for monitoring other therapeutics, diseases, and patient
dispositions, particularly in critical care situations.
[0017] The disclosed systems, devices, methods, and techniques
embodying the invention allow the separation of cells,
nanoparticles, and biomarker entities to now be carried out under
higher conductance (>100 mS/m) ionic strength conditions, at
lower AC (DEP) frequencies (<20 kHz), and at higher field
strengths (>20 voltages pk-pk) than those used for most previous
DEP separations. More specifically, DEP separations can be carried
out not only under higher ionic strength conditions, but also
directly in complex biological samples including blood, plasma,
serum, and undiluted buffers where now nanoscale (500 nm to 5 nm)
analytes and entities can be isolated in the DEP high field
regions, while the larger entities (cells, micron particles, etc.)
can isolated in the DEP low-field regions between the
electrodes.
[0018] The new devices ameliorate the electrochemistry, heating,
and chaotic fluidic effects that occur with the use of castellated
DEP electrode arrays, which are currently a preferred method to
separate nanoparticles and biomolecules. In another aspect, devices
and processes can use more macroscopically scaled arrangements of
robust multiple electrodes in sectioned arrays, which not only
allows larger sample volumes to be more rapidly and efficiently
interrogated, but essentially allows a very small number of cancer
cells, bacteria, virus, nanoparticles, and nanoparticulates and
very low concentrations of DNA, RNA biomarkers, and antibody
complexes to be isolated from complex samples containing very large
numbers of normal cells, i.e. blood. Essentially, the use of a
"properly scaled" macroscopic system of electrodes changes the
processes of finding one specific cell (or other entity) in a
million, to finding one specific cell in one thousand, i.e., the
sample is spread out over many subgroups of electrodes, creating a
parallel hierarchical sorting mechanism. This separation process
can be applied to treat blood, and will remove smaller-size DNA,
RNA, and higher molecular weight DNA from proteins as well as
cells. As a result of the size of the electrodes (10-100 micron
diameter, with 20-100 micron separation) and ability to use less
diluted samples, the separation process can now be completed in a
rapid and high-throughput manner on scaled array devices, which
have from 2-100 array sections, each section of which might contain
from 100-1000 individual electrodes. The device would also
incorporate strategically placed auxiliary electrodes in the x-y-z
dimensions.
[0019] Embodiments described herein show that the disclosed array
devices and systems can be used to separate out nanoparticles and
cellular nanoparticulates in lower frequency ranges (10-50 kHz)
from entities of larger sizes (cells and micron-size particles)
based off of the Clausius Mossotti factor effects (along with other
AC Electrokinetic phenomena) inherent in every nanoparticulate less
than or equal to about 500 nm in diameter. This specification also
discloses that when AC electrokinetics effects are used in
conjunction fluid flow, the process will relieve excess heat
build-up. This specification further discloses that when fluid flow
and DC electrophoresis are combined with AC electrokinetics
effects, both cells and proteins can be effectively moved
downstream to the lower array section of the illustrated devices,
while the highly negatively charged DNA nanoparticulates and DNA
molecules can be concentrated upstream in the upper array section
of the devices. Thus, the different array sections of the
illustrated devices can now be used to carry a more selective
separation process such as: multiplexing with red blood cell, white
blood cell, cancer cell separations, and protein removal on the
lower array section; bacteria, virus, nanoparticles and
nanovesicles in the middle array section; and DNA nanoparticulates
and DNA molecules on the upper array section of the devices.
[0020] Finally, this specification also discloses devices with
separate electrode chambers and pore/hole structures leading to an
isolated separation chamber, as well as robust electrode array
devices that are over-layered with nanoporous materials (from one
nanometer to one millimeter in thickness) that can be used to carry
out simultaneous or subsequent secondary size-separation processes.
For example, if the upper array section of an illustrated device
can be used to concentrate a complex mixture of DNA components,
then a combination of AC electrokinetics effects and DC
electrophoretic forces can be used to achieve the secondary
separation of DNA nanoparticulates from high molecular weight DNA
(5-50 kb), intermediate molecular weight DNA (1-5 kb), and lower
molecular weight DNA (0.1-1 kb). In addition, the illustrated
embodiments permit DC microelectrophoresis within the nanoporous
layers to be used to carry out the size separation of the various
DNA fragments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows a new electrokinetic DEP device in which
electrodes have been placed into separate chambers and DEP fields
are created within an inner chamber by passage through pore
structures.
[0022] FIG. 2 shows surface pore/hole geometry for new
electrokinetic DEP device shown in FIG. 1.
[0023] FIG. 3 shows an electrode arrangement constructed in
accordance with the invention, with an exemplar fluid flow and
sample indicated.
[0024] FIG. 4 illustrates the electrode arrangement of FIG. 1 with
electrode pulsing in accordance with the invention.
[0025] FIG. 5 shows the electrode arrangement of FIG. 1 with
selective activation of electrodes to achieve to achieve better
separation results.
[0026] FIG. 6 shows more detailed scheme of blood sample separation
process, before combined pulsed AC DEP/DC
electrophoresis/controlled fluidic flow are applied.
[0027] FIG. 7 shows blood sample at initial stage of combined
pulsed AC DEP/DC electrophoresis/controlled fluidic flow.
[0028] FIG. 8 shows blood sample at final stage of combined pulsed
AC DEP/DC electrophoresis/controlled fluidic flow
[0029] FIG. 9 shows the combined pulsed AC DEP and DC
electrophoresis of Fluorescent stained DNA nanoparticulates, very
high molecular weight DNA and intermediate-lower molecular weight
DNA selection and separation on the upper array section.
[0030] FIG. 10 shows the initial combined pulsed AC DEP and DC
electrophoresis of fluorescent stained DNA nanoparticulates, vh MW
DNA and intermediate-lower MW DNA selection and separation on upper
array section.
[0031] FIG. 11 shows the final combined pulsed AC DEP and DC
electrophoresis of fluorescent stained
DNAnanopariculates,vh-MW-DNA-and-intermediate-10werMWDNA-selection
and separation on upper array sections
[0032] FIG. 12 shows the removal of DNA nanoparticulates and very
high MW DNA and on-array DC electrophoretic size separation of the
intermediate and low MW DNA fragments.
[0033] FIG. 13 shows the initial pulsed AC DEP applied to red and
white blood cells on lower array section of device.
[0034] FIG. 14 shows the final pulsed AC DEP applied to red and
white blood cells on the lower array section of device.
[0035] FIG. 15 shows the initial pulsed AC DEP for separation of
bacteria, virus and nanovesicles on the middle array section of the
device.
[0036] FIG. 16 shows the final pulsed AC DEP for separation of
bacteria, virus and nanovesicles on the middle array section of the
device.
[0037] FIG. 17A-H shows DEP separation of 60 nm and 200 nm
nanoparticles under intermediate and high conductance
conditions.
[0038] FIG. 18A-D shows DEP separation of 200 nm nanoparticles
under intermediate and high conductance conditions.
[0039] FIG. 19A-H shows 3D fluorescent intensity images for the DEP
separation of 60 nm and 200 nm nanoparticles under intermediate and
high conductance conditions.
[0040] FIG. 20A-D shows real image and 3D intensity images for the
DEP separation of 60 nm nanoparticles high conductance
conditions.
[0041] FIG. 21A-B shows graphs of nanoparticle fluorescent
intensity increase versus increasing conductance for 60 nm and 200
nm nanoparticles.
[0042] FIG. 22 shows graph of the experimental results versus
theoretical DEP crossover curves for 60 nm and 200 nm nanoparticles
as function of conductance.
[0043] FIG. 23A-H shows both real images and 3D intensity images
for the DEP separation of 200 nm nanoparticles on un-coated and
hydrogel over-coated platinum electrodes at increasing conductances
(shows electrode darkening).
[0044] FIG. 24A-F shows light microscope and SEM images of
electrode damage following high conductance DEP without
nanoparticles present.
[0045] FIG. 25A-H shows SEM images of electrode damage and fusion
of 200 nm nanoparticles following high conductance DEP.
[0046] FIG. 26A-C shows fluorescent and SEM images of 60 nm
nanoparticles and electrode damage following high conductance
DEP.
[0047] FIG. 27 shows a seamless sample-to-answer hmw-DNA process in
blood comprising step 1.
[0048] FIG. 28 shows a seamless sample-to-answer hmw-DNA process in
blood comprising step 2.
[0049] FIG. 29 shows a seamless sample-to-answer hmw-DNA process in
blood comprising step 3.
[0050] FIG. 30 shows a seamless sample-to-answer hmw-DNA process in
blood comprising step 4.
[0051] FIG. 31 shows a seamless sample-to-answer hmw-DNA process in
blood comprising step 5.
[0052] FIG. 32 shows a seamless sample-to-answer process with
complex sample.
[0053] FIG. 33 shows a seamless sample-to-answer complex sample
process with PCR and immunoassay analyses.
[0054] FIG. 34 shows a seamless sample-to-answer complex sample
process with PCR and immunoassay analyses and detection.
DETAILED DESCRIPTION
[0055] This document teaches novel sample separation and
sample-to-answer systems, devices, methods, and techniques that
combine multi-dimensional AC electrokinetics, including
dielectrophoresis (DEP), DC electrophoretics, microelectrophoresis,
and fluidics in unique ways that can be used to separate and
identify cells, nanovesicles and nanoparticulates, bacteria and/or
viruses, as well as a host of other clinically relevant biomarkers
of disease from relevant volumes of high conductance (ionic
strength) biological and clinical samples and buffers including but
not limited to blood, plasma, serum, urine, lymph fluid, saliva,
biopsied samples, cell cultures (stem cells), bacterial, and
fermentation cultures. While disclosed embodiments of the invention
enable the DEP separation of cells, nanoparticles, and other
analytes to be carried out directly in undiluted samples (blood,
plasma, biological buffers), the embodiments do not preclude the
use of the disclosed devices and methods for partially diluted
samples or buffers, or for samples that have gone through other
sample preparation procedures.
[0056] Using novel multi-chambered devices and electrode array
devices with robust electrodes of defined diameter and separation
distances allows viable DEP to be carried out in high conductance
(ionic strength) solutions. These novel DEP devices are designed in
such a manner that bubbling, heating, and other adverse effects due
to the increased electrochemistry that occurs under high
conductance conditions does not reduce the efficiency or prevent
the separation, concentration, and detection of specific analytes
or entities from complex biological samples and high ionic strength
buffers. Carrying out DEP separations under high conductance
conditions has been a major problem and limitation for most
problematic AC electrokinetic and dielectrophoretic separation
devices [Ref. 1-28]. Even when some degree of high conductance DEP
separations could be achieved for a short period of time using
microarray devices with hydrogel over-coating the electrodes, such
devices were not viable as a sample separation tool and diagnostic
device [Ref. 13, 14, 24-28].
[0057] In order to better demonstrate this DEP conductance
limitation, an initial first example described herein shows the DEP
separation of nanoparticles in a low conductivity buffer. This
example involves separating 60 nm DNA derivatized nanoparticles
from 10 .mu.m particles in MilliQ water (5.5 .mu.S/m). The
separation was carried out at 10 kHz AC at 10 volts peak to peak
(pk-pk). FIG. 17a shows the initial conditions under white light
before the AC electric field is applied with a random distribution
of the 10 .mu.m particles over the microelectrode array. The
initial conditions under red fluorescence detection show a red
fluorescent haze across the microarray as would be expected from
the 60 nm DNA derivatized fluorescent nanoparticles (see FIG. 17b).
After the AC DEP field was applied for only 30 seconds, most of the
10 .mu.m particles have concentrated in very orderly arrangements
into the negative DEP low field regions (see FIG. 17c). After a
1-minute application of the AC field, the 60 nm DNA derivatized
nanoparticles have concentrated onto the positive DEP high field
regions over the microelectrodes (see FIG. 17d). The high
fluorescent intensity on the microelectrodes together with the
decrease of fluorescent intensity in the surrounding areas
indicates that most of the nanoparticles have concentrated into the
high field regions. The next example shows the DEP separation of
200 nm nanoparticles mixed with 10 .mu.m particles in
0.01.times.TBE (1.81 mS/m) carried out at 3 kHz AC at 10 volts
pk-pk. The initial white light view shows a random distribution of
the 10 .mu.m particles before the field is applied (FIG. 17e), and
the green fluorescence view shows no accumulation of the 200 nm
nanoparticles in the high field regions (FIG. 17f). In less than 10
minutes, the 10 .mu.m particles are concentrated into the low field
regions (FIG. 17g), and the 200 nm nanoparticles are highly
concentrated into the positive DEP high field regions (FIG. 17h).
These low conductivity DEP results are generally consistent with
other low conductivity DEP nanoparticle separations cited in the
literature, and expected from classical DEP theory [Ref.
11-14].
[0058] The next set of DEP examples shows the separations of 60 nm
DNA derivatized nanoparticles, 200 nm nanoparticles, and 10 .mu.m
particles in buffer solutions with conductivities greater than 100
mS/m. For 1.times.TBE (0.109 S/m), after the AC field was applied
for 20 minutes, the separation between 200 nm nanoparticles and 10
.mu.m particles in under white light conditions showed the 10 .mu.m
particles concentrated in the low field regions (FIG. 18a). Under
green fluorescence, the 200 nm nanoparticles were concentrated in
the positive DEP high field regions on top of the microelectrodes
(FIG. 18b). For DEP experiments carried out in 1.times.PBS (1.68
S/m), after 20 minutes the 10 .mu.m particles are concentrated into
the low field regions (FIG. 18c). The green fluorescence 20 minute
image for the high conductance 1.times.PBS buffer experiment was
taken after removal of some small bubbles and at an increased gain
(FIG. 18d). The image shows that the 200 nm nanoparticles have
concentrated into the positive DEP high field regions of four
microelectrodes. The microelectrodes, however, now show significant
darkening and two of the microelectrodes had bubbled. The
observation that the 200 nm nanoparticles have predominantly
concentrated on these four microelectrodes is consistent with the
fact that they produce slightly higher fields.
[0059] The high conductance experiments in 1.times.PBS buffer that
were carried out using 60 nm DNA derivatized nanoparticles also
yielded similar results, i.e., in that the 60 nm nanoparticles were
still observed to concentrate in the positive DEP high field
regions over three of the microelectrodes. Further analysis of the
fluorescence images was performed in a mathematical model using
MATLAB to produce three-dimensional peaks, which better demonstrate
the concentration of the fluorescent nanoparticles over the high
field regions. For the 1.times.TBE experiments with 60 nm DNA
derivatized nanoparticles, the 3D fluorescent data showed a
significant increase from time points 0 minutes (FIG. 19a), 2
minutes (FIG. 19b), 8 minutes(FIG. 19c), and 16 minutes (FIG. 19d).
Similarly, the 3D fluorescent data for the 200 nm nanoparticles in
1.times.PBS also shows an increase from time points at 0 minutes
(FIG. 19e), 8 minutes (FIG. 19f), 16 minutes (FIG. 19g), and after
20 minutes (FIG. 19h).
[0060] For the 60 nm DNA derivatized nanoparticles in 1.times.PBS,
there is still concentration as is seen in as seen in the
fluorescent image (FIG. 20a). The 3D fluorescent image data also
shows a similar fluorescence increase from 0 minutes (FIGS. 20b),
to 8 minutes (FIGS. 20c) and to 20 minutes (FIG. 20d). Due to the
in-activation of one of the microelectrodes (third row, second
column) shown in FIG. 20a, the electric field pattern is slightly
altered. The overall fluorescence data was compiled using MATLAB
for experiments in buffers of 1.times.TBE, 0.1.times.PBS (0.177
S/m) and 1.times.PBS at the time points of 0, 0.5, 1, 2, 4, 8, 16,
and 20 minutes. The results for the 60 nm DNA derivatized
nanoparticles are shown in graph (FIG. 21a), and the results for
the 200 nm nanoparticles are shown in graph (FIG. 21 b). These
examples show an increase in concentration of the fluorescent
nanoparticles over time. More importantly, these examples also show
a significant decrease in overall concentration of the fluorescent
nanoparticles as the conductivity of the buffers increases, i.e., a
much longer time is needed to concentrate entities at the higher
conductance conditions.
[0061] FIG. 22 now shows the theoretical curves and the ranges for
the experimental results for the real part of the Clausius-Mossotti
factor (Re(K(.omega.))) versus conductivity for the 60 nm DNA
derivatized nanoparticles and the 200 nm nanoparticles. The graph
indicates that the theoretical Re(K(.omega.)) values should be
negative for the conductivities used in these examples, and
therefore the nanoparticles should have accumulated in the low
field regions. Nevertheless, the actual results show that
accumulation of nanoparticles continues in the high field region.
Unfortunately, under these high conductance conditions, (>100
mS/M) bubbles, electrode darkening, and electrode failures occur,
and much longer DEP times are required which produce relatively
inefficient separations.
[0062] It has been discovered that these DEP-related adverse
effects are due to the increased electrochemical activity that
occurs when using higher ionic strength buffers that contained
sodium (Na.sup.+) and chloride (Cl.sup.-) electrolytes [Ref.
29-30]. A better understanding of these effects was necessary to
develop more viable and robust DEP devices for molecular diagnostic
applications. Further examples that clearly demonstrate the
microelectrode/nanoparticle/electrolyte adverse interactions under
high conductance conditions are now shown in the examples described
herein. These examples involved carrying out the separation and
detection of 200 nm yellow-green fluorescent polystyrene
nanoparticles from 10 micron spheres under different conductance
(ionic strength) conditions, on platinum microelectrode structures
with hydrogels (FIG. 23 A-F), and without a hydrogel layers (FIG.
23 G-H). The results for all buffers (0.01.times.TBE, 1.times.TBE,
1.times.PBS) show the separation and concentration of the green
fluorescent 200 nm nanoparticles into the DEP high field regions
over the microelectrodes, and the concentration of the 10 .mu.m
spheres into the low field regions between the microelectrodes.
Again, the concentration of 200 nm nanoparticles appears highest
for 0.01.times.TBE, and decreases as the buffer ionic strength
increase to 1.times.PBS (see FIGS. 23B, 23D, 23F, and 23H). The
concentration of nanoparticles occurs more at the center of
microelectrodes with hydrogels, and at the outside perimeter for
the uncoated microelectrodes (FIGS. 23A, 23C, 23E, 23G). At the
highest buffer conductance (1.times.PBS), both the hydrogel
over-coated microelectrodes (FIG. 23E) and the uncoated
microelectrodes show significant darkening of the electrodes (FIG.
23G).
[0063] While not shown in these drawing figures, increased
micro-bubbling was also observed in 1.times.PBS buffer on both the
hydrogel over-coated and the uncoated microelectrodes after four
minutes of DEP. Nevertheless, the micro-bubbling appeared more
pronounced on the uncoated microelectrodes. Also, in a 1.times.PBS
buffer, chaotic bubbling occurs over almost all the electrodes when
the AC voltage is increased above 20 volts pt-pt. While
nanoparticle concentration and darkening could be observed on both
the hydrogel overcoated and the uncoated platinum microelectrodes,
the uncoated microelectrodes provided an opportunity to use
scanning electron microscopy (SEM) to analyze the electrochemical
effects and to verify nanoparticle concentration and adhesion.
[0064] In the next set of examples, DEP was carried out in high
conductivity 1.times.PBS buffer on uncoated microelectrodes with no
nanoparticles present. The microelectrode array was washed, dried,
and then imaged using SEM. FIG. 24A first shows the light
microscope images of an un-activated control microelectrode, and an
activated microelectrode (FIG. 24 B) after 10 minutes of DEP at
3000 Hz, 10 volts pk-pk. Significant darkening of the activated
microelectrode is clearly observed. FIGS. 24C and 24D now show the
SEM images of the same un-activated and activated microelectrodes.
Significant damage and degradation of the activated microelectrode
is clearly observed in the SEM image. FIGS. 24E and 24F are higher
magnification SEM images of the microelectrodes, and show even more
clearly the degradation of the platinum layer that has occurred
around the microelectrode perimeter (FIG. 24F).
[0065] Similar DEP examples were carried out in high conductance
1.times.PBS buffer with the 200 nm nanoparticles present. FIG. 25A
first shows SEM images of the un-activated control microelectrode
after two minutes of DEP at 3000 Hz, 10 volts pk-pk in high
conductivity 1.times.PBS buffer. The control microelectrode with no
activation shows only a few 200 nm nanoparticles randomly
distributed over the structure. FIG. 25B shows a higher
magnification SEM image of the edge of a control microelectrode,
where some nanoparticles appear randomly trapped in the area
between the edge of the platinum microelectrode. FIG. 25C shows the
SEM image of a microelectrode, which was activated for 2 minutes
with 200 nm nanoparticles present. A large number of nanoparticles
have concentrated and adhered to the microelectrode, especially at
the edges. The close-up image (FIG. 25D) shows much better the
concentrated clusters of nanoparticles and indicates some
degradation of the platinum at the edge of the microelectrode.
FIGS. 25E and 25F now show images of an activated microelectrode
after 5 minutes of DEP with 200 nm nanoparticles. Again
concentration and clustering of the 200 nm nanoparticles is clearly
observed, but the platinum microelectrode structure now appears
more severely damaged and degraded. FIG. 25G is a higher
magnification SEM image of the edge of the microelectrode from FIG.
25D, again showing clustering of the nanoparticles. Finally, FIG.
25H is a higher magnification image of the degraded microelectrode
(seen in FIG. 25F), showing the nanoparticle clusters interspersed
with fused or melted clusters of nanoparticles. These fused
nanoparticle clusters are the results from the aggressive
electrochemical activity (heat, H.sup.+ and OH.sup.-) at the longer
DEP activation times.
[0066] Another set of examples involved carrying out the DEP
separation and detection of 40 nm red fluorescent nanoparticles
from 10 micron spheres in high conductance 1.times.PBS buffer on
microelectrode structures without a hydrogel. FIG. 26A is a red
fluorescent image of the microelectrode before DEP activation
showing no concentration of the 40 nm nanoparticles. FIG. 26B is
the red fluorescent image of microelectrode after DEP activation
for 4 minutes at 10,000 Hz, 10 volts pk-pk, which now clearly shows
the concentration of 40 nm nanoparticles on the perimeter of the
microelectrodes. FIG. 26C is an SEM high magnification image
showing the damaged microelectrode and clustering of the 40 nm
nanoparticles.
[0067] These examples clearly show that increased electrochemical
activity is occurring when DEP is carried out under high
conductance conditions (>100 mS/m). This very aggressive
electrochemistry causes micro-bubbling and darkening of the
microelectrodes. More importantly, it shows that significant
microelectrode degradation is occurring, which ultimately leads to
electrode failure, and it shows that this microelectrode
destruction increases as DEP activation time increases. The fact
that fusion of the polystyrene nanoparticles was observed on the
degraded microelectrode structures suggests that significant
heating is occurring, in spite of DEP being an AC electrokinetic
process. These results can be attributed to DC electrolysis
reactions which would produce O.sub.2, H.sub.2, H.sup.+, OH.sup.-,
heat and bubbles. The presence of sodium (Na.sup.+), potassium
(K.sup.+), and chloride (Cl.sup.-) electrolytes in the 1.times.PBS
buffer may also contribute to overall corrosive conditions present
on the microelectrode surfaces during DEP. In addition to high
conductance, most biological and clinical samples and buffers have
relatively high concentrations of sodium (Na.sup.+), potassium
(K.sup.+), and chloride (Cl.sup.-). These results immediately make
it clear as to why classical DEP, which utilizes less robust
sputtered gold electrodes, could only be carried out at low
conductance conditions (Ref. 29, 30), i.e., the electrodes would be
destroyed in seconds.
[0068] While the hydrogel overcoated platinum microelectrodes do
allow separation of nanoparticles at high conductance conditions,
they are nevertheless still unsuitable for any practical
applications for the following reasons. First, pronounced random
bubbling and electrode failure would make the device itself
unreliable for any type sample to answer molecular diagnostics
using blood. In further experiments involving the separation of
nanoparticles from buffy coat and whole blood, bubbling, electrode
darkening, and electrode failure were observed. While nanoparticles
could be isolated into the high field regions, they were difficult
to remove by fluidic washing, indicating adverse heating may have
fused them to the array surface. Second, for biological sample
separations and subsequent molecular analyses (e.g. PCR,
immunoassay, etc.) this heating and aggressive electrochemistry
would be severely damaging to cells, DNA, proteins, and most other
analytes. Third, in order to improve the separation efficiency
(increase the total amount of analyte concentrated), longer DEP
times would be required, which would produce even more adverse
effects. Fourth, if higher AC voltages (e.g. 20 volts pt-pt) are
used to increase concentration speed, this would also cause even
more bubbling and electrochemistry effects. This discovery of the
underlying reasons for classical DEP device and conductance
limitations now provides the opportunity to create more viable DEP
sample preparation devices and novel "seamless" sample-to-answer
diagnostic systems. These novel DEP devices will allow rare cells,
nanoparticles, and a variety important disease biomarkers to be
directly isolated, concentrated, and detected in blood, plasma,
serum, and most other biological samples and buffers.
[0069] This description next discloses a combination of continuous
and/or pulsed electrokinetic/dielectrophoretic (DEP) forces,
continuous and/or pulsed DC electrophoretic forces, on-device
(on-array) microelectrophoretic size separation, and controlled
fluid flow (externally pumped and/or DC/AC electrokinetic driven)
together with the novel devices of this invention that can be used
to carry out complex sample preparation, leading to specific
analyte separation and concentration, and subsequent molecular
diagnostic analyses and detection. This can include but is not
limited to (1) both the DEP separation and detection of labeled
analytes and/or the subsequent detection of unlabeled analytes
after DEP separation, using immunochemistry and ligand binding
techniques that include fluorescent antibodies, non-fluorescent
antibodies, antibody derivatized nanoparticles, antibody
derivatized microspheres, antibody derivatized surfaces (specific
sites on the DEP device), biotin/streptavidin, and various lectins;
(2) the pre-DEP or post-DEP use of general and/or specific color
stains, fluorescent dyes, fluorescent nanoparticles, quantum dots
for detecting specific cells, bacteria, virus, DNA, RNA, nuclei,
membranes, cellular organelles, and cellular nanoparticulates (it
is important to keep in mind that DEP is intrinsically a "label
less" technique and that cells, nanoparticles, and other analytes
can be identified by their cross-over frequencies; labeling is used
to increase detection sensitivity, identify individual entities,
and carry out more detailed analysis); and (3) the post analysis of
cells, nuclei, DNA, and RNA by fluorescent probe in-situ
hybridization (FISH, etc.); and (4) use of well-known molecular
analysis methods for cells, nuclei, DNA, and RNA including but not
limited to PCR, RCA, SDA, and other genotyping, sequencing, and
gene expression techniques--all of which can be carried out in the
same chambered compartment in which the DEP separation has
occurred.
[0070] The above examples do not exclude carrying out subsequent
analyses in another separate chamber of the device or moving the
analytes to a sample collection tube(s) for off-device analyses,
storage, or archiving of samples. Additionally, other types of
detection techniques that can be used for analysis include, but are
not limited to, radioisotopes, colorometric, chemiluminescence,
electrochemical, or other methods for biosensing or nanosensing of
the analytes, biomolecules, and cells once they have been isolated.
The devices and processes described herein can be considered a
truly "seamless" sample-to-answer diagnostic system that can be
used directly with undiluted blood or other complex clinical or
biological samples. The seamless sample-to-answer process using the
exemplary DEP devices herein are described below in more
detail.
[0071] FIG. 27 shows the first step in seamless sample-to-answer
diagnostics where a blood sample is applied directly to the device
and DEP is used to carry out, in this case, the separation of a
very low concentration of high molecular weight (hmw) DNA and/or
RNA from the un-diluted whole blood sample. It should be noted,
however, that almost any analyte including but not limited to rare
cells, nanoparticles, cellular nanoparticulates, antibodies,
immunocomplexes, proteins, and RNA could be separated,
concentrated, and detected; and samples could include but are not
limited to plasma, serum, urine, and saliva.
[0072] FIG. 28 shows the second step in a sample-to-answer
diagnostics process where the DEP field is now applied at the
proper AC frequency and voltage that causes the blood cells (red
and white) to move to negative (DEP) low field regions, and the hmw
DNA (RNA) to concentrate into the positive (DEP) high field regions
(in the drawing, dome structures represent the DEP high field
strength areas).
[0073] FIG. 29 shows the third step, where a simple fluidic wash is
used to remove the blood cells from the DEP array device, while the
hmw-DNA (RNA) remains highly concentrated in the DEP high field
regions. It is also within the scope of this disclosure to use a
continuous, pulsed, or intermittent fluidic flow across the DEP
device in order to process a larger sample volume, and as a
mechanism to reduce heating, which is more pronounced in higher
conductance solutions, at lower AC frequencies (<20 kHz), and at
high voltages (>20 volts pt-pt).
[0074] FIG. 30 shows the next step in the process, which involves
the in-situ labeling of the DNA (RNA) by addition of a DNA (RNA)
specific fluorescent dye (e.g. CyberGreen, OliGreen, ethiduim
bromide, TOTO, YOYO, acridine orange, etc.). In this process, a
solution of the appropriate fluorescent dye is flushed across the
DEP device to stain the DNA or RNA. The DNA/RNA can be held in
place by maintaining the DEP field while staining is in progress.
The fluorescent stained DNA/RNA can now be detected and quantified
by using an epifluorescent detection system (FIG. 31). Fluorescent
detection systems and devices are well-known in the art of
molecular biology and clinical diagnostics for analysis of
microarray devices, and a variety of systems are commercially
available.
[0075] It is also within the scope of this disclosure to: (1) use
other molecular analysis detection methods and techniques including
but not limited to PCR, real time PCR with molecular beacons, RCA,
and SDA; (2) to hybridize the sample DNA/RNA during the DEP
separation process to capture probes (DNA, RNA, pNA, etc)
immobilized to specific sites on the DEP device for subsequent
analysis/detection using fluorescent reporter probes; (3) to
release the DNA/RNA from the DEP concentration sites, amplify it
using PCR, RT-PCR, RCA, or SDA, denature and then use
site-selective DC electrophoresis to re-hybridize the amplicons to
capture probes immobilized on the DEP device for subsequent
analysis/detection using fluorescent reporter probes; (4) to use
fluorescent in-situ hybridization with sequence specific
DNA/RNA/pNA probes; (5) to release the DNA/RNA and transport it
either by DEP or electrophoretically (DC fields) to another
specific location on the device; and (6) to release the DNA/RNA and
move it (by fluid flow) to another chamber of the device or to a
sample collection tube for further analysis or storage.
[0076] FIG. 32 shows how the sample-to-answer device can be used to
carry out more multiplex DEP separation of rare cells, bacteria,
virus, cellular nanoparticulates, or GNPs (cellular membrane,
nuclei, hmw-DNA, hmw-RNA, vacuoles, endoplasmic reticulum,
mitochondria, etc.), proteins, antibody complexes, and other
biomarkers from whole blood. FIG. 33 shows the molecular analyses
methods that can now be used to identify the specific analytes that
have been concentrated onto the device; these methods include but
are not limited fluorescent staining, fluorescent immunoassay,
FISH, and PCR, RCA, and SDA procedures. Finally, FIG. 34 shows the
final detection of cells, bacteria, virus, CNPs, and antibody
complexes using well-known fluorescent and other detection
techniques.
[0077] This disclosure further describes unique methods that can be
used to enhance the analytical and diagnostic capability of the
sample-to-answer devices and systems described herein. In the case
of DNA and RNA isolation and detection, while DEP can be used to
efficiently isolate and concentrate hmw-DNA/RNA, lower molecular
weight DNA and RNA (<10 kb) are more difficult to isolate by
DEP. In this case, double-stranded ds-DNA specific antibodies and
single-stranded ss-DNA specific antibodies are available that can
be used to label the lower molecular weight DNA and RNA, creating
larger nanostructures (>5 nm). These larger DNA-antibody
complexes can be more efficiently isolated and concentrated by
DEP.
[0078] Additionally, a variety of new antibody tests can be enabled
using the devices described herein. More specifically, the ability
of DEP to separate single antibodies form larger antibody complexes
means that numerous single and double antibody assays can be
developed in which the formation of the larger antibody-antigen
complex can be separated from the clinical sample by DEP. In these
cases, fluorescent antibodies and/or secondary antibodies could be
added directly to the sample, DEP is applied, and only the
fluorescent labeled antibody-antigen complexes would be
concentrated into the DEP high field regions for subsequent
detection. Such DEP based antibody assays can be used for small
molecule antigens including but not limited to drugs, hormones,
metabolites, and peptides; as well as for larger antigens including
but not limited to proteins, enzymes, and other antibodies. It is
also in the scope of this description to enable many other similar
DEP assays that are based on the formation of larger complexes,
including but not limited to detection of bacteria, virus,
bacteriophage, nanoparticles, CNP's using selective ligand binding
with antibodies, biotin/streptavidin, lectins, proteins, enzymes,
peptides, dendrimers, apatamers, quantum dots, fluorescent
nanoparticles, carbon nanotubes, and other nanoentities designed
for selective labeling an detection purposes. Finally, In addition
to attaching or immobilizing DNA/RNA/pNA capture probes on the DEP
device, a variety of other binding entities can be also be attached
to the DEP device, including but not limited to antibodies,
biotin/streptavidin, lectins, proteins, enzymes, peptides,
dendrimers, and apatamers. Such immobilized ligands will provide
for selective binding of analytes to the DEP device after the DEP
field has been turned off.
[0079] It should be noted that the novel DEP devices described
herein now enable all these methods by the fact that these new DEP
devices eliminate or greatly reduce the adverse bubbling, heating,
and electrochemistry effects that would otherwise damage or destroy
most of the biomolecules (e.g. DNA, RNA, antibodies, proteins, etc)
that are used for immobilization, as well as the analytes and
biomarkers being isolated and concentrated on specific DEP high
field sites on the device for detection and analyses.
[0080] This first specification discloses in more detail novel
electrokinetic DEP devices and systems in which the electrodes are
placed into separate chambers and positive DEP regions and negative
DEP regions are created within an inner sample chamber by passage
of the AC DEP field through pore or hole structures. Various
geometries can be used to form the desired positive DEP (high
field) regions and DEP negative (low field) regions for carrying
cell, nanoparticle and biomarker separations with the sample
chamber. Such pore or hole structures can be filled with a porous
material (agarose or polyacrylamiide hydrogels) or be covered with
porous membrane type structures (paper, cellulose, nylon, etc).
Such porous membrane overlaying structures can have thicknesses
from one micron to one millimeter, but more preferably form 10
microns to 100 microns; and pore sizes that range from one
nanometer to 100 microns, but more preferably from 10 nanometers to
one micron. By segregating the electrodes into separate chambers,
these unique DEP devices basically eliminate any electrochemistry
effects, heating or chaotic fluidic movement from influencing the
analyte separations that are occurring in the inner sample chamber
during the DEP process. These chambered devices can be operated at
very high AC voltages (>100 volts pt-pt), and in addition to DEP
they could also be used to carry out DC electrophoretic transport
and electrophoresis in sample chamber. In general these devices and
systems can be operated in the AC frequency range of from 1000 Hz
to 100 mHz, at voltages which could range from 1 volt to 2000 volts
pt-pt; and DC voltages from 1 volt to 1000 volts, at flow rates of
from 10 microliters per minute to 10 milliliter per minute and in
temperature ranges from 1.degree. C. to 100.degree. C. The
chambered devices are shown in FIG. 1 and FIG. 2. Such devices can
be created with a variety of pore and/or hole structures
(nanoscale, microscale and even macroscale) and may contain
membranes, gels or filtering materials which can control, confine
or prevent cells, nanoparticles or other entities from diffusing or
being transported into the inner chambers. However, the AC/DC
electric fields, solute molecules, buffer and other small molecules
can pass through the chambers.
[0081] FIGS. 1 and 2 represents a most basic version of the
chambered devices that can be constructed in accordance with the
invention. A variety of configurations are envisioned for the
devices in accordance with the invention. Such devices include, but
are not limited to, multiplexed electrode and chambered devices,
devices that allow reconfigurable electric field patterns to be
created, devices that combine DC electrophoretic and fluidic
processes; sample preparation devices, sample preparation and
diagnostic devices that include subsequent detection and analysis,
lab-on-chip devices, point-of-care and other clinical diagnostic
systems or versions. FIG. 1 is a schematic diagram of a sample
processing device constructed in accordance with the teachings
herein, and shows that the device 100 includes a plurality of
electrodes 102 and electrode-containing chambers 104 within a
housing 106. A controller 108 of the device independently controls
the electrodes 102, as described further herein.
[0082] FIG. 2 shows a top view of a the device 100 which is
illustrated with six electrode chambers 104, each of which has at
least one robust platinum electrode. FIG. 2 shows the device
configured with one main central separation chamber 110, which has
an arrangement of eighteen pore/hole structures 112 of varying size
that are filled with a hydro-gel (the inner chamber could also have
a porous membrane covering the pores or holes). The pore/hole
structures are arranged in three groups of six pore/hole
structures. While the upper part of the separation chamber 110 has
no physical separations, the lower portion is divided into nine
separate compartments (indicated by the light dashed line). Each of
these compartments is in fluidic contact with an electrode chamber,
but not with each other. When an AC DEP field is applied to the
electrodes, the field passes through the pores 112, creating
positive DEP high field regions on top of the pore structures and
negative DEP low field regions between the pore structures. Samples
can be added and removed from the device via the inlet 220 and
outlet 222. The device may additional inlets 224 and outlets 226.
The device shown in FIGS. 1 and 2 represents just one form of a
high conductance DEP chambered device; it should be understood that
a large number of different types of devices with larger numbers of
pores/holes and different geometries can be created.
[0083] Another device embodiment involves using electrode arrays
with robust electrodes of defined diameter and separation distances
that will allow for less electrochemical effects and heating, which
is a problem in current electrokinetic and dielectrophoretic
separation devices. Proper construction and overcoating of robust
electrodes (e.g. platinum, palladium and gold) can reduce adverse
effects of electrochemistry products on the separation process, and
allow much higher voltages to be applied, which can greatly improve
separation times. Also, current devices are relatively low
throughput and this embodiments described herein have overcome that
problem by providing a system that uses multiplexed parallel
section arrays and that allows the device to be used as one large
separation zone and then switched to separately controlled
separations zones, resulting in increased sensitivity and
selectivity of the overall system. A third problem seen in other
conventional systems is the inability to separate sample components
that are relatively similar in size and composition. This problem
is overcome in accordance with the description herein by providing
a device that can carry out secondary separation processes, such
microelectrophoresis, directly on the DEP array device itself.
[0084] Negative dielectrophoretic (DEP) forces are relatively
weaker than positive DEP forces; thus entities that experience
negative DEP can be moved by fluid flow, while positive DEP
experiencing entities will remain in place. In the presently
described embodiments, by using both fluid and DC electrophoretic
forces in opposite directions, DNA fragments and highly charged DNA
nanoparticulates can be separated from cells and proteins in blood
and other samples. In this way, using multiple AC frequencies,
pulsed DC electrophoresis, and micro-electrophoresis, a more
complete size separation of DNA nanoparticulates and DNA fragments
can be accomplished.
[0085] Commercial uses of such novel systems and devices that now
allow DEP to be carried out under high conductance conditions
(blood, plasma, serum, etc.) will likely include numerous research
and clinical diagnostic applications, such as point of care
diagnostics, therapeutics and drug monitoring, environmental and
water supply monitoring, and bioterror agent detection. Numerous
analytes and entities such as rare cells (cancer cells, fetal
cells, hematopoietic stem cells), bacteria, virus, DNA/RNA, and DNA
nanoparticulate biomarkers, drug delivery nanovesicles, as well as
normal or aberrant proteins, might be detected using such a
system.
[0086] An experimental AC DEP and DC electrophoretic separation
system (a laboratory bench-top version described further in the
Experimental Section below) has been built and experiments were
conducted to refine the new prototype devices. The results obtained
on these devices (which are described above) lead to the important
discovery as to why classical DEP has been limited to low
conductance solutions.
[0087] Now new devices that use planar, parallel, and robust
platinum electrode arrays with electrodes of roughly about 1-1000
micron in diameters with 10 to 5000 micron separation distance and
overcoated with a 5-100 micron thick hydrogel (agarose,
polyacylamide) or porous membrane layer(s), allows for less heating
and electrochemistry issues, as the electric field lines are not as
highly concentrated as they are in other classical conventional DEP
systems, and more importantly the DEP high field accumulation
region is actually now some distance from the actual electrode
surface. One significant difference from previous electrode designs
is not using sputtered platinum or gold electrodes, which are
easily degraded and destroyed by electrochemistry, particularly at
higher field strengths and high solution conductance. The
electrodes for the new devices will be constructed from solid
platinum or gold materials, including wires or rods. A second
difference is that the separation efficiency for isolating one
unique entity in a million relatively similar entities (cells,
nanoparticles, biomarkers) can be improved by changing the problem
of one large separation to that of many separate separations which
are much more controllable. The devices described herein accomplish
this by using multiplexed sectioned arrays and a controlled
parallel sorting process. This is achieved by using individually
controlled array subsets of 10 to 100 or more electrodes in a large
array device that allows a complex biological sample (blood) to be
distributed across the array device, separating the components into
smaller separation sections (areas) for further separation and
isolation of the desired analytes or entities. Breaking the complex
sample separation problem down into smaller parts holds the most
promise for solving the issue of sensitivity versus specificity,
i.e., the process allows both rapid and higher overall sample
throughput, as well as relatively longer interrogation (separation)
times for isolating and identifying unique cells or other entities
in the sample. Finally, the last problem can be overcome by
creating a multi-dimensional hierarchical sorting device. This
solution relies on the fact that negative DEP is a weaker force
than positive DEP and cells or other entities experiencing negative
DEP can be moved by controlled fluid flow, whereas the positive DEP
experiencing analytes or entities will stay concentrated in the DEP
high field areas. Through the use of controlled fluid flow and
pulsed DC electrophoresis in opposite directions, DNA/RNA and
charged nanoparticulates can be separated from cells and proteins
in a complex biological sample (this is in addition to the
intrinsic ability of DEP to separate cells and DNA).
[0088] Combining controlled fluid flow and pulsed DC
electrophoresis with using multiple AC frequencies, i.e. low
frequency to trap the CNPs and hmw-DNA/RNA nanoparticulates in on
the initial electrodes array subset, and higher AC frequencies on
other electrode array subsets to trap cells progressively larger
particles (bacteria and virus) a complete separation of most of the
cells and entities by size can be obtained. If desired the
electrodes can be switched to different frequencies for finer
separation to occur locally while globally the overall size
separation is maintained.
[0089] We describe a separation system involving a device with
planar, robust, platinum electrode array structures and auxiliary
electrodes, into which a complex biological sample (blood, plasma,
serum) is directly applied, such that controlled AC signals from
one or more function generators produce dielectrophoretic forces,
and a controlled DC power supply produces electrophoretic forces.
The inlet and outlets of the device also allow for the controlled
passage of fluids (water, buffers, etc.) through the system at a
controlled flow rate. The system also includes an
optical/epifluorescent microscope and digital camera for
monitoring, detecting, quantifying, and recording the separation
processes that are occurring on the device (visual and
fluorescent). The device is ultimately a multiplexing, parallel
hierarchical sorting system that is enabled by controlling
electrokinetic effects, dielectrophoretic forces, electrophoretic
forces, microelectrophoresis, and fluid flow. It should be noted
that such novel multiplex sample-to-answer processes are made
possible by the fact that the new DEP devices eliminate or greatly
reduce the adverse bubbling, heating, and electrochemistry effects
experienced by conventional devices.
[0090] FIG. 3 shows just one version of a planar platinum electrode
array device 300 comprising a housing 302 through which a sample
fluid can flow. The fluid flow pattern through the device is
indicated by the large arrows, representing flow of an idealized
sample, from an inlet end 304 at the top of the drawing to an
outlet end 306 at the bottom, and a lateral analyte outlet 308. The
device includes multiple AC electrodes 310. Only a few of the
electrodes 310 are identified in FIG. 3, for simplicity of
illustration, but it should be understood that all the small open
circles in the drawing figure represent electrodes of similar
construction. One enlarged 3x3 array 312 of the electrodes is
illustrated on the right side of the drawing figure to show a
sample fluid in the device 300. The sample consists of a
combination of micron-sized entities or cells 314 (the largest
filled-in circles shown in the enlarged view), larger
nanoparticulates 316 (the intermediate-sized filled-in circles) and
smaller nanoparticulates or biomolecules 318 (the smallest-sized
circles). The larger nanoparticulates 316 could represent high
molecular weight DNA, nucleosomes, or CNPs or cellular debris
dispersed in the sample. The smaller nanoparticulates 318 could
represent proteins, smaller DNA, RNA and cellular fragments. The
planar electrode array device 300 in the figure is a 60.times.20
electrode array that can be sectioned into three 20.times.20 arrays
that can be separately controlled but operated simultaneously. The
auxiliary DC electrodes 320 at the top of the figure can be
switched on to positive charge, while the DC electrodes 322 at the
bottom of the figure are switched on to negative charge for
electrophoretic purposes. Each of the controlled AC and DC systems
can be used in both a continuous and/or pulsed manner (e.g., each
can be pulsed on and off at relatively short time intervals). The
planar electrode arrays 324 along the sides of the sample flow,
when over-layered with nanoporous materials, can be used to
generate DC electrophoretic forces as well as AC DEP. Additionally,
microelectrophoretic separation processes can be cared out within
the nanopore layers using planar electrodes in the array and/or
auxiliary electrodes in the x-y-z dimensions. In general these
devices and systems can be operated in the AC frequency range of
from 1000 Hz to 100 mHz, at voltages which could range from
approximately 1 volt to 2000 volts pk-pk; at DC voltages from 1
volt to 1000 volts, at flow rates of from 10 microliters per minute
to 10 milliliter per minute, and in temperature ranges from
1.degree. C. to 100.degree. C. The controller 108 (FIG. 1)
independently controls each of the electrodes 310, 320, 322, 324.
The controller may be externally connected to the device 100 such
as by a socket and plug connection (not illustrated), or can be
integrated with the device housing. Electrical lead lines for the
electrodes are not shown in the drawings, for simplicity of
illustration.
[0091] It can be assumed that the cells and particles and other
entities in the sample are evenly distributed throughout the
electrode array, though only the enlarged 3.times.3 electrode
section 312 is shown in the drawing figure. The fluid flow rate is
such that it exerts a force stronger than the negative DEP that the
larger particles experience, but weaker than the positive DEP that
the larger particles experience.
[0092] FIG. 4 shows the top 320 and bottom 322 DC electrodes being
pulsed on and off (one second on followed by one second off),
thereby providing a brief electrophoretic pulse pushing the DNA,
RNA, and small nanoparticulates toward the positive DC electrode
320, which is located at the top of the drawing figure. The
60.times.20 electrode array is visualized as broken into three
distinct sections or sub-arrays that are independently controlled.
The top twenty AC electrode array rows 402 are tuned to a lower
frequency AC field to ensure that the smaller entities, which
generally move toward the electrodes, due to positive DEP and AC
electrokinetic phenomena at lower frequencies, will be trapped at
those electrodes while the larger cells and entities experience
negative DEP at these frequencies, and are therefore moved to the
lower section of the device by the constant fluid flow. The middle
twenty rows 404 of AC electrodes will hold the large sub-micron
particles (e.g. virus) while allowing the micron-sized particles
and cells to flow through. Finally, the last twenty rows 406 of AC
electrodes can be attuned, if desired, to a high AC frequency,
which can then be used to capture desired cells and micron-sized
particles.
[0093] FIG. 5 shows the separation mechanism for isolating "one
cell in a million", i.e., rare cell detection. By using the
complete electrode array, it is possible to multiplex and
parallelize the problem of separation to make it simpler. This can
be achieved by merely activating as much of the electrode array as
necessary to achieve better separation. By effectively splitting
the array into specific separation areas that can be analyzed by
optical detection (i.e., epifluorescence), it should be possible to
separate out one specific cell experiencing positive DEP from all
the cells around it, once all the cells are evenly distributed. In
FIG. 5, the intermediate-sized filled-in circles 502 represent 10
.mu.m cells of one specific kind, such as lymphocytes, red blood
cells, and the like, and the single filled-in circle 504 shown on
the AC electrode 506 of the third section 406 of AC electrodes
represents the lone "one cell in a million" of a type different
from the others 502 in the sample, which is also the only cell that
experiences positive dielectrophoresis and is therefore easily
distinguishable from the other cells. Using only dielectrophoresis,
it should be possible to separate out cells of the lone "one cell
in a million" 504 type from the undifferentiated 502 cell types.
This is more easily accomplished if there are a sufficieint number
of AC electrodes to spread the separation problem into smaller,
more easily separable and analyzable chunks. Once the cell-type
separation problem is spread out in such a manner, if only certain
sections of the electrode array are analyzed at a time, such as the
3.times.3 array shown in FIG. 5, it should be possible to find the
lone particle 504 of interest. Additionally, temperature control
can be effective in allowing more selective and efficient
separation of cells (e.g., separation of cancer and stem
cells).
[0094] FIG. 6 shows a more detailed scheme of a blood sample
separation process, before the application of combined pulsed AC
DEP/DC electrophoresis/controlled fluidic flow. The FIG. 6 diagram
shows some of a wide variety of potential diagnostic and biomarker
entities that would be found in a complex sample such as blood,
which entities may include: red and white blood cells, bacteria,
virus, nanovesicles, DNA/RNA nanoparticulates, an assortment of DNA
and RNA fragments, and proteins. The FIG. 6 diagram also shows the
planar platinum array electrodes 310 covered with an intermediate
density nanopore layer 604, a low density nanopore layer 606, and a
high density nanopore layer 608 directly over the AC electrodes
310.
[0095] FIG. 7 shows the blood sample in the initial stages of
combined pulsed AC DEP/DC electrophoresis/controlled fluidic flow.
In FIG. 7, the whole array device 300 is utilized to carry out an
overall separation process that begins to concentrate different
classes of entities into each of the electrode sub-array sections
402, 404, 406 (upper, middle, lower, respectively).
[0096] FIG. 8 shows the blood sample now in final stages of
combined pulsed AC DEP, DC electrophoresis, and controlled fluidic
flow. In FIG. 8, the different entities have been concentrated into
their appropriate electrode array sections 402, 404, 406. In this
example, DNA nanoparticulates and smaller DNA fragments are shown
in the upper array section 402; bacteria, virus, and nanovesicles
are shown in the middle array section 404; and cells and proteins
are shown in the lower array section 408.
[0097] FIG. 9 shows an enlarged view of the AC electrode array in
which the combined pulsed AC DEP and DC electrophoresis of
fluorescent-stained DNA nanoparticulates, very high molecular
weight DNA and intermediate lower molecular weight DNA selection
and separation on the upper array section 402. Because these
entities have now been concentrated and isolated in the upper array
section they can be selectively stained with appropriate DNA
fluorescent dye reagents, and the secondary separation process can
now be cared out.
[0098] FIG. 10 shows the enlarged view of the AC electrode array
with initial combined pulsed AC DEP and DC electrophoresis of
fluorescent stained DNA nanoparticulates, very high MW DNA and
intermediate-lower MW DNA selection and separation on the upper
array section 402. This initial process will cause the DNA
nanoparticulates to begin to concentrate onto the top of the
intermediate nanopore layer which has a pore size that excludes
these very large DNA entities; while the more intermediate and
lower molecular weight DNA fragments are transported into lower
nanopore density layer.
[0099] FIG. 11 shows the enlarged view of the AC electrode array
with final combined pulsed AC DEP and DC electrophoresis of
fluorescent stained DNA nanoparticulates, very high MW DNA and
intermediate-lower MW DNA selection, and microelectrophoresis
separation on the upper array section 402. At this point in the
operation of the device 300, the DNA nanoparticulates and very high
molecular weight DNA are fully concentrated and isolated on the top
of the intermediate density nanopore layer 604 and the more
intermediate and lower molecular weight DNA fragments are
concentrated within the inner lower density nanopore layer 606.
[0100] FIG. 12 shows the enlarged view of the AC electrode array
402 after removal of DNA nanoparticulates and very high MW DNA and
on-array DC electrophoretic size separation of the intermediate and
low MW DNA fragments. The DNA nanoparticulates and very high
molecular weight DNA can be further analyzed on another part of the
device 300, while the more intermediate and lower molecular weight
DNA fragments can be size-separated by microelectrophoresis within
the nanopore layers 604, 606, 608. FIG. 12 shows that some of the
AC electrodes 310a are positively charged and other AC electrodes
310b are negatively charged.
[0101] FIG. 13 shows an enlarged view of the AC electrode array
with the initial pulsed AC DEP applied to red and white blood cells
on lower array section 406 of the device 300. In this process, the
proteins in the sample can be removed and/or analyzed on another
component of the device, while the cells and other micron-sized
entities can be further separated and differentiated by AC DEP on
the lower array section 406.
[0102] FIG. 14 shows the enlarged view of the electrode array with
the final pulsed AC DEP applied to red and white blood cells on the
lower array section 406 of the device 300. At this point of the
device operation, the red and white blood cells have been separated
in the DEP high and low field regions, subsequently the red cells
can be removed and the white cells further differentiated; i.e.,
begin the process of isolating cancer cells.
[0103] FIG. 15 shows an enlarged view of the AC electrode array
with the initial pulsed AC DEP for separation of bacteria, virus
and nanovesicles on the middle array section 404 of the device 300.
The FIG. 15 drawing is an example of how sub-sections of the array
device 300 that can be independently controlled can be used to
carry out additional important separation processes.
[0104] FIG. 16 shows the enlarged view of the electrode array with
the final pulsed AC DEP for separation of bacteria, virus and
nanovesicles on the middle array section 404 of the device 300.
Again, it should be kept in mind that this separation process on
the middle array section can be run concurrently with, and
independently of, other separation processes that are occurring on
the other array sub-sections; e.g., DNA fragment separations can
take place on the upper array section and cell separations can take
place simultaneously on the other (lower) array sections.
[0105] Lastly, the parallel multiplexed electrode array can be used
in conjunction with hierarchical cell sorting to create defined
areas within the rows of electrodes where specific particles that
are similar in size but have different dielectric properties can be
trapped. A variety of diagnostic and therapeutic applications which
can utilize electrokinetic, dielectrophoretic, electrophoretic and
fluidic forces and effects all in conjunction with each other to
increase separation sensitivity and efficiency in a device. Most
importantly, these high performance and clinically useful
separation processes are achieved only when the electrokinetic,
electrophoretic and fluidic forces and effects are uniquely
combined on a properly scaled and controlled electrode array
device. In addition to this type of separation, dielectrophoresis,
which is a lossless, potentially label less, parallel separation
method, can be used in conjunction with more traditional separation
methods which have far more sample preparation involved as well as
greater loss of the sample, such as field flow fractionation,
fluorescence assisted cell sorting (FACS) or magnetic assisted cell
sorting, to achieve even greater levels of cell and nanoparticle
separation for use in applications.
[0106] With regard to other aspects in the illustrated embodiments,
it should be pointed out that when labeling (optical fluorescent,
luminescent, electrochemical, magnetic, etc.) is added to the
cells, nanoparticles, and biomarkers to be separated, the
multiplexing described herein would likely be even more effective
due to the labels helping to affect the size, conductivity and
detectability of the entities.
[0107] Presently, the DEP separation mechanisms described above are
in an early experimental data stage. A prototype system has been
constructed, utilizing an electrode array structure as described
above that receives biological materials for separation, that
include cells, nanoparticles and hmw-DNA. Selective energizing of
subsets of electrodes in the array structure has now been achieved
with a function generator operating under control of suitable
software programming, which can execute on conventional computers
such as desktop or workstation computers. Individual electrodes can
be controlled using this apparatus. The prototype system has an
associated epifluorescent microscope for monitoring and recording
the separation experiments (see Experimental Section below for
additional description).
[0108] A variety of separation and isolation applications which
include rare cell detection for adult stem cell isolation from
blood, other bodily fluids or any buffers, e.g. hematopoietic
progenitors; gross separation between cells, proteins and DNA/RNA
fragments in blood, other bodily fluids or other buffers for the
purposes of cancer detection and other diagnostics; cancer cell
isolation from blood, other bodily fluids or other buffers for
research, diagnostic as well as therapeutic purposes. Also
envisioned are the uses for environmental monitoring and for the
rapid detection of pathogens and bioterror agents. Finally, also
envisioned are systems, devices and techniques described in this
invention can be used to separate, isolate and purify a variety of
non-biological entities that include in addition to drug
nanoparticles and nanovesicles; quantum dots, metallic
nanoparticles, carbon nanotubes (CNTs), nanowires, and even micron
and submicron CMOS devices and components; basically any
macromolecule or nanocomponent that can be suspended or solubilized
in an aqueous or mixed solvent system can be processed in the
embodiments illustrated and using the techniques described herein.
We also envision that these new devices will now allow directed
self-assembly of DNA and other bioderivatized nanoparticles,
nanocomponents, and mesoscale objects to be carried out. This can
lead to new DNA genotyping and sequencing technologies ($1000
genome) and nano/micro bio/chemsensor applications, including
highly integrated cell-sized "Fantastic Voyage" devices (inspired
by the movie of the same name) that could be placed in the blood
stream to carry out diagnostics, therapeutic delivery in-vitro
microsurgery, i.e., remove clots and plaques, repair
atherosclerotic arteries, etc.; as well as nanoelectronic,
nanophotonic, photovoltaic, fuel cell, batteries, nanomaterials,
and numerous other heterogeneous integration applications.
[0109] Using the devices and techniques described herein, a "sample
to answer" result can be provided, wherein the separation operation
results in holding at least one type of biological material at one
of the electrode subsections, while the remainder of the sample
fluid is washed from the device, so that a reagent into the sample
processing device, followed by reacting the introduced reagent with
the held type of biological material in the sample processing
device. As noted above, the reagent may comprise a fluorescent die,
antibodies, or the like. The sample-to-answer process may be used
to perform a variety of tasks as described above, including PCR
operations and the like.
[0110] The invention has been described above in terms of presently
preferred embodiments so that an understanding of the present
invention can be conveyed. There are, however, many configurations
and permutations of the devices, system and separation mechanisms
not specifically described herein, but to which the present
invention is applicable. The present invention should therefore not
be seen as limited to the particular embodiments described herein,
but rather, it should be understood that the present invention has
wide applicability with respect to biological separation systems
generally. All modifications, variations, or equivalent
arrangements and implementations that are within the scope of the
attached claims should therefore be considered within the scope of
the invention.
EXPERIMENTAL SECTION
Buffers and Conductivity Measurements
[0111] Concentrated 5.times.Tris Borate EDTA (TBE) buffer solution
was obtained from USB Corporation (USB, Cleveland, Ohio, USA), and
was diluted using deionized Milli-Q Ultrapure water (55 nS/cm) to
the following concentrations: 0.01.times.TBE, 0.1.times.TBE and
1.times.TBE. Dulbecco's Phosphate Buffer Saline (1.times.PBS)
solution was obtained from Invitrogen (Invitrogen, Carlsbad,
Calif., USA) and was diluted using Milli-Q water to 0.1.times.PBS.
Conductivity measurements were made with an Accumet Research AR-50
Conductivity meter (Fisher Scientific, Fair Lawn, N.J., USA) using
a 2 cell (range: 10-2000 .mu.S) and a 4 cell (range: 1-200 mS)
electrode and was adjusted with proper conductivity standards. The
following buffer conductivities were measured: 0.01.times.TBE-18.1
.mu.S/cm; 0.1.times.TBE-125 .mu.S/cm; 1.times.TBE-1.09 mS/cm;
0.1.times.PBS-1.77 mS/cm; and 1.times.PBS-16.8 mS/cm.
[0112] Particles, Nanoparticles and DNA Derivatization
[0113] Fluorescent polystyrene nanoparticles (FluoSpheres) with
NeutrAvidin coated surfaces were purchased from Invitrogen
(Invitrogen, San Diego, Calif., USA). The nanoparticle diameters
were 0.04 .mu.m (40 nm) and 0.2 .mu.m (200 nm). The 40 nm
polystyrene nanoparticles were red fluorescent (ex: 585/em: 605)
and the 200 nm polystyrene nanoparticles were yellow-green
fluorescent (ex: 505/em: 515). Larger 10.14 .mu.m carboxylated
polystyrene particles were obtained from Bangs Labs (Bangs Labs,
Fishers, Ind., USA). Biotinylated DNA oligonucleotide sequences
were obtained from Trilink Bio Technologies (Trilink, San Diego,
Calif., USA). The single-stranded 51 mer DNA oligonucleotide used
to derivatize the 40 nm nanoparticles had the
sequence-[5]'-Biotin-TCA GGG CCT CAC CAC CTA CTT CAT CCA CGT TCA
CTC AGG GCC TCA CCA CCT [3]'. A second single-stranded 23 mer DNA
oligonucleotide used had the sequence--[5]'-Biotin-GTA CGG CTG TCA
TCA CTT AGA CC [3]'. The derivatization of the 40 nm NeutrAvidin
nanoparticles with the biotinylated DNA oligonucleotides was
carried out by first suspending the nanoparticles in different
concentrations of Tris Borate EDTA (0.01.times., 0.1.times.,
1.times.TBE) or Phosphate Buffered Saline (0.1.times., 1.times.PBS)
buffers. The ss-DNA oligonucleotide was added to the mixtures in
the amounts of 400:1 (DNA:40 nm nanoparticles) ratio for the 51 mer
ss-DNA sequence, and 6500:1 (DNA:40 nm nanoparticle) ratio for the
23 mer ss-DNA sequence. Once the DNA was added, the solution was
vortexed at high speed for 20 seconds and then allowed to react for
about 20 minutes. For the 40 nm DNA derivatized nanoparticle
experiments, the DNA nanoparticle mixture was made by adding 0.5
.mu.L of the stock solution into 299 .mu.L of the appropriate
buffer. For the 200 nm nanoparticle experiments, 0.5 .mu.L of the
stock solution was added to 299 .mu.L of the appropriate buffer.
Finally, 1 .mu.L of the 10.14 .mu.m polystyrene particle stock
solution was added to the samples, the samples were then slowly
mixed for about 10 seconds. The samples were now ready to be
applied to the microarray cartridge device.
[0114] DEP Microelectrode Array Device
[0115] The microelectrode array devices used for these studies were
obtained from Nanogen (San Diego, Calif., USA, NanoChip.RTM. 100
Cartridges). The circular microelectrodes on the array are 80 .mu.m
in diameter and made of platinum. The microarray is over-coated
with 10 .mu.m thick porous polyacrylamide hydrogel layer. The
microarrays are enclosed in a microfluidic cartridge which forms a
20 .mu.L sample chamber over the array that is covered with a glass
window. Electrical connections to each individual microelectrode
are pinned out to the bottom of the cartridge. Only a 3.times.3
subset of nine microelectrodes was used to carry out DEP.
Alternating current (AC) electric fields were applied to the nine
microelectrodes in a checkerboard addressing pattern. In this
checkerboard pattern of addressing, each microelectrode has the
opposite bias of its nearest neighbor. The corresponding computer
model for the asymmetric electric field distribution created by
this pattern has been discussed previously [27]. This model
indicates that the positive DEP field maxima (high field regions)
exist at (on) the microelectrodes and the negative DEP field minima
(low field regions) exist in the areas between the electrodes. In
general, for DEP in low conductance solutions the 60 nm DNA and 200
nm nanoparticles are expected to concentrate in the positive or
high field regions over the microelectrodes [28] and the 10 micron
particles concentrate in the negative or low field DEP regions [29]
between the microelectrodes. The computations from the previous
model were performed for a 5.times.5 microelectrode set [27].
Before each experiment, the microarray cartridge is flushed 10
times with 200 .mu.L of the appropriate buffer, over a span of 5
minutes. The cartridge is allowed to sit for 5 minutes, and is then
washed two more times with 200 .mu.L of buffer. A total of 150
.mu.L of the sample solution containing the nanoparticle mixture is
then slowly injected into the cartridge, a final sample volume of
about 20 .mu.L remains in the cartridge.
EXPERIMENTAL SETUP, MEASUREMENTS, FLUORESCENCE AND SEM ANALYSIS
[0116] The microarray devices were controlled using a custom made
switching system (designed and constructed in our lab) that allows
for individual control over the voltage being applied to each of
the 100 microelectrodes. The microelectrodes were set to the proper
AC frequency and voltages using an Agilent 33120A Arbitrary
Function Generator (Agilent, Santa Clara, Calif., USA). AC
frequencies ranged from 1000 Hz to 10,000 Hz, at 10 volts peak to
peak (pk-pk). The wave form used for all experiments was
sinusoidal. The experiments were visualized using a 10.times.PL
Fluotar objective in a JenaLumar epifluorescent microscope (Zeiss,
Jena, Germany) employing the appropriate excitation and emission
filters (green fluorescence Ex 505 nm, Em 515 nm; red fluorescence
Ex 585 nm, Em 605 nm. Both back lighted and the fluorescent images
were captured using an Optronics 24-bit RGB CCD camera (Optronics,
Goleta, Calif., USA). The image data was processed using a Canopus
ADVC-55 video capture card (Canopus, San Jose, Calif., USA)
connected to a laptop computer using either Adobe Premiere Pro
(Adobe Systems Inc, San Jose, Calif., USA) or Windows Movie Maker.
The final fluorescence data was analyzed by inputting individual
fluorescent image frames of the video into MATLAB (Mathworks,
Natick, Mass., USA) at 0 minutes, 30 seconds, 1 minute, 2 minutes,
4 minutes, 8 minutes, 16 minutes, and 20 minutes time points. The
graphs were created using Excel from data gathered through MATLAB
analysis of fluorescence intensity readings across the
microelectrode. was created using MATLAB. The following data was
used to create the graph: .sigma..sub.p (for 200 nm)=18 mS,
.sigma..sub.p (for 40 nm+DNA)=50 mS K.sub.s=0.9 nS,
.epsilon..sub.p=2.55.epsilon..sub.0. r=30 nm & r=100 nm. f=3
kHz. After the conclusion of the DEP experiments the FCOS
microarrays had all the fluid removed from their surface and were
visualized using a Phillips XL30 scanning electron microscope
(SEM). The SEM was used to image the final nanoparticle layers on
the surface of the microarray.
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