U.S. patent application number 17/071202 was filed with the patent office on 2021-02-11 for apparatus for pathogen detection.
This patent application is currently assigned to Fluid-Screen, Inc.. The applicant listed for this patent is Fluid-Screen, Inc.. Invention is credited to Siu Lung Lo, Hazael Fabrizio Montanaro Ochoa, Mark A. Reed, Monika Weber, Christopher Daniel Yerino.
Application Number | 20210039099 17/071202 |
Document ID | / |
Family ID | 1000005170140 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210039099 |
Kind Code |
A1 |
Weber; Monika ; et
al. |
February 11, 2021 |
APPARATUS FOR PATHOGEN DETECTION
Abstract
An apparatus for separating an analyte from a test sample, such
as bacteria from blood components, based on their dielectric
properties, localizing or condensing the analyte, flushing
substantially all remaining waste products from the test sample,
and detecting low concentrations of the analyte. The module array
includes a plurality of microfluidic channels with connecting
microfluidic waste channels for directing undesired material away
from the analyte. An electric field is applied causing a positive
dielectrophoretic force to the analyte to capture the analyte. The
electric field is applied to at least one electrode having a
plurality of concentric rings or concentric arcs extending radially
outwards from a center point, electrically connected to a voltage
source such that when voltage is applied to the at least one
electrode, the concentric rings or concentric arcs alternate in
voltage potential.
Inventors: |
Weber; Monika; (Cambridge,
MA) ; Lo; Siu Lung; (Mid-Levels, HK) ;
Montanaro Ochoa; Hazael Fabrizio; (Asuncion, PY) ;
Yerino; Christopher Daniel; (New Haven, CT) ; Reed;
Mark A.; (Monroe, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fluid-Screen, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
Fluid-Screen, Inc.
Boston
MA
|
Family ID: |
1000005170140 |
Appl. No.: |
17/071202 |
Filed: |
October 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14582525 |
Dec 24, 2014 |
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17071202 |
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13664967 |
Oct 31, 2012 |
9120105 |
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14582525 |
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61557654 |
Nov 9, 2011 |
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61553413 |
Oct 31, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2400/0406 20130101;
B03C 2201/26 20130101; G01N 27/4145 20130101; B03C 2201/18
20130101; B01L 2400/0487 20130101; B03C 5/005 20130101; B01L
2300/0874 20130101; B03C 5/024 20130101; B01L 3/502753 20130101;
B01L 2400/0424 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; B03C 5/00 20060101 B03C005/00; B03C 5/02 20060101
B03C005/02 |
Claims
1-15. (canceled)
16. A method of separating a first component from one or more
second components in a test sample, the method comprising:
introducing the test sample into a microfluidic channel of a
microfluidic separator, wherein the microfluidic channel includes
at least one electrode arranged therein, the at least one electrode
comprising a first set of electrically connected concentric rings
or concentric arcs coupled to a first voltage source and a second
set of electrically connected concentric rings or concentric arcs
coupled to a second voltage source, wherein the concentric rings or
concentric arcs of the first set and second set are arranged to
alternate and extend radially outwards from a center point; and
separating the first component from the one or more second
components in the test sample by controlling the first voltage
source and the second voltage source to generate an alternating
current (AC) electric field within the microfluidic channel,
wherein the generated AC electric field produces a
dielectrophoretic force on the test sample when the test sample
traverses the microfluidic channel, the dielectrophoretic force
causing the first component in the test sample to be separated from
the one or more second components in the test sample by aligning
and holding the first component to a curvature of at least some of
the concentric rings or concentric arcs in the first set and/or the
second set of electrically connected concentric rings or concentric
arcs of the at least one electrode while the one or more second
components in the test sample are not attracted to the at least one
electrode.
17. The method of claim 16, further comprising: storing the first
component when separated from the other components in the test
sample; and capturing at least one of the one or more second
components in the test sample once the test sample has passed
through the microfluidic channel and is substantially separated
from the first component; and detecting said at least one of the
one or more second components using a sensor.
18. The method of claim 16, further comprising: storing the first
component when separated from the one or more second components in
the test sample; and capturing at least one of the one or more
second components in the test sample once the test sample has
passed through the microfluidic channel and is substantially
separated from the first component; and detecting said first
component using a sensor.
19. The method of claim 17, further comprising: attracting the at
least one of the one or more second components in the test sample
at a collecting electrode arranged at an inlet of the sensor.
20. The method of claim 17, further comprising: applying, by the
sensor, a confining dielectrophoretic force to trap the at least
one of the one or more second components of the test sample.
21. The method of claim 17, further comprising: applying a
confining dielectrophoretic force to trap the at least one of the
one or more second components of the test sample and imaging the at
least one of the one or more second components of the test sample
with an optical sensor.
22. The method of claim 16, wherein introducing the test sample
into the microfluidic channel comprises pumping the test sample
through the microfluidic channel.
23. The method of claim 16, further comprising transporting at
least one of the one or more second components of the test sample
to a location in a vicinity of a sensor.
24. The method of claim 16, further comprising transporting the
first component away from the one or more second components of the
test sample.
25. The method of claim 16, further comprising: tuning a frequency
of the generated AC electric field such that the first component is
attracted to the at least one electrode and the one or more second
components of the test sample are not attracted to the at least one
electrode.
26. The method of claim 16, further comprising: turning off the AC
electric field to release the first component from the at least one
electrode.
27. The method of claim 16, further comprising: detecting a
concentration of the first component separated from the test
sample.
28. The method of claim 16, wherein controlling the first voltage
source and the second voltage source to generate an alternating
current (AC) electric field within the microfluidic channel
comprises applying a first voltage potential to the first set of
electrically connected concentric rings or concentric arcs and
applying a second voltage potential to the second set of
electrically connected concentric rings or concentric arcs.
29. The method of claim 28, wherein the first voltage potential and
the second voltage potential are of opposite polarity.
30. The method of claim 16, wherein separating the first component
from the one or more second components in the test sample comprises
separating at least 95% of the first component from the one or more
second components in the test sample within 15 seconds of the AC
electric field being generated in the microfluidic channel.
31. The method of claim 16, wherein the first component and/or the
one or more second components comprise a bacteria.
32. The method of claim 16, wherein the first component and/or the
one or more second components comprise a virus.
33. The method of claim 16, wherein the first component and/or the
one or more second components comprise a pathogen.
34. The method of claim 16, wherein the first component and/or the
one or more second components comprise a microbe.
35. The method of claim 16, wherein the first component and/or the
one or more second components comprise mold.
36. The method of claim 16, wherein the first component and/or the
one or more second components comprise yeast.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] The invention relates to an apparatus for pathogen
detection. Specifically, the invention relates to the field of
pathogen detection systems and diagnostic devices and their
micro-component assembly. More specifically, the invention utilizes
an apparatus that includes a dielectrophoretic separator, a
dielectrophoretic condenser, a dielectrophoretic trap, microfluidic
components, and field effect sensor, such as an ion sensitive
sensor, nanowire sensor, or nanoribbon sensor configured as
biosensors, to perform a pathogen detection process. The invention
further relates to a specific electrode design for high yield
pathogen and cell capture and separation.
2. Description of Related Art
[0002] Bacterial infections cause thousands of diseases in humans
and animals every year. Recent deadly outbreaks of E.coli,
Salmonella, and Listeria have highlighted the urgent need for more
effective methods of detection, identification, and
characterization of pathogens, and their origin and proliferation.
Conventional detection methods have proven inadequate because they
suffer from long incubation periods, high cost, and require highly
trained personnel to operate. There remains a strong need for a
reliable, time-efficient apparatus and method for specific
detection of bacteria in low concentration.
[0003] Conventional methods rely on bacterial culture growth, which
require highly qualified personnel and time, both contributing to
higher costs for the procedure. The most widely used method for
bacterial detection, the standard plate count, takes from 24 to 48
hours due to the time needed for bacteria to grow detectable
colonies, and requires a stocked microbiology lab. Although faster
methods, such as PCR (Polymerase Chain Reaction) Plates or labeled
detection and fluorescent imaging, can reduce the response time to
one hour, these require complex sample preparation, highly trained
personnel, high cost per test, and have limited portability.
[0004] The major challenge in automated sample preparation for
detection from blood or other unprocessed liquids using
microstructures is efficient separation of the analyte of interest
(bacteria, cells, or particles) from large blood components. Red
blood cells (RBC) and white blood cells (WBC) range between 6
.mu.m-21 .mu.m in size and constitute over 50% of the whole blood
volume. RBC and WBC presence obstructs the detection of bacteria,
cells, or particles. The present invention is a miniaturized device
for rapid pathogen screening that overcomes these obstacles.
[0005] Dielectrophoresis ("DEP") is a separation method based on
size and dielectric properties and has been described in literature
as for example in Pohl et al, Science 1966, and Sher Nature 1968,
Voldman, Annual Review Of Biomedical Engineering, 2006. The use of
DEP to manipulate particles and cells has been previously
described, as for example, in H. Pohl, I. Hawk, "Separation Of
Living And Dead Cells By Dielectrophoresis," Science, 152, 3722
(1966); Y. Huang, R. Holzel, R. Pethig, X. Wang, "Differences In
The Ac Electrodynamics Of Viable And Non-Viable Yeast Cells
Determined Through Combined Dielectrophoresis And Electrorotation
Studies," Phys. Med. Bid., 37, 7 (1992); S. Chang, Y. Cho, "A
Continuous Size-Dependent Particle Separator Using A Negative
Dielectrophoretic Virtual Pillar Array," Lab Chip, 8, 1930-1936
(2008); and J. Yang, Y. Huang, X. Wang, F. Becker, P. Gascoyne,
"Differential Analysis Of Human Leukocytes By Dielectrophoretic
Field-Flow-Fractionation," Biophysical Journal, 78, 2680-2689
(2000). However, effective methods for cell/pathogen separation on
a micro-scale from fluids containing pollutants of comparable size
are still unattainable.
[0006] High-frequency electric fields when applied to an
electrically neutral object cause polarization. A high-frequency
non-uniform electric field gives rise to a dielectrophoretic force
(DEP) F.sub.DEP which acts on the object.
[0007] A spherical object of a given electrical permittivity .sub.p
placed in a medium of a different permittivity .sub.m in a
spatially varying electric field E(x,.omega.) is subjected to a
dielectrophoretic force, F.sub.DEP. The dielectrophoretic force is
given by:
F.sub.DEP=2.pi. .sub.m r.sup.3 Re{CM(.omega.)}.gradient.E.sup.2
[0008] where [0009] CM(.omega.) is the Clausius-Mossotti factor=(
{tilde over (.sub.p)}- {tilde over (.sub.m)})/( {tilde over
(.sub.p)}+2 {tilde over (.sub.m)}) [0010] {tilde over ( )}=
+.sigma./i.omega.; [0011] Re{CM(.omega.)} is the real part of the
CM(.omega.), which can be a complex number; [0012] .sub.p is the
particle permittivity; [0013] .sub.m is the permittivity of the
liquid medium; [0014] r is the particle radius; [0015] {tilde over
( )} is the complex permittivity (complex dielectric function);
[0016] .sigma. is the conductivity; [0017] .omega. is the angular
frequency; and [0018] .gradient.E is the gradient of the electric
field.
[0019] Depending on the respective permeability ( {tilde over ( )})
and conductivity (.sigma.) of the object and the medium, the force
can be attractive (positive dielectrophoresis (pDEP)), or repulsive
(negative dielectrophoresis (nDEP)). If Re{CM(.omega.)} is
positive, then the particle experiences a positive
dielectrophoretic force, and if Re{CM(.omega.)} is negative, then
the particle experiences a negative dielectrophoretic force.
Different species have different dielectric properties. The
dielectric functions .sub.m, .sub.p depend on the frequency of the
external electric field. The permittivity of the medium affects the
CM(.omega.) factor and the value of Re{CM(.omega.)}. Importantly,
if the signs of Re{CM(.omega.)} for different species are opposite
then the species are subject to forces acting in opposite
directions and separation occurs.
[0020] There is a cross-over frequency, .omega..sub.co, that occurs
when the Re{CM(.omega.)} goes to zero. Critical to separation is
that .omega..sub.co is uniquely different for different cells and
bacteria. Separation procedures for stained cells have been
described in U.S. Pat. No. 7,153,648 entitled "Dielectrophoretic
Separation Of Stained Cells," where appropriate frequency and
amplitude are applied via a function generator, and red blood cells
are attracted to electrodes by positive dielectrophoresis force,
while stained white blood cells are repelled to the area with
weakest electric field by negative dielectrophoresis force. The
differential behavior and separation of E. coli cells from human
blood cells on electrodes under applied electric field has been
described in patent U.S. Pat. No. 6,989,086 entitled "Channel-less
separation of bioparticles on an Electronic Chip by
Dielectrophoresis."
[0021] The dielectrophoretic force is affected both by the geometry
of the electrodes (gradient of the electric field), the
Re{CM(.omega.)} factor, and depends on the dielectric constant of
the medium .sub.m.
[0022] Using the aforementioned prior art techniques for
dielectrophoresis, the separation of bacteria from blood may
achieve, at best, an efficiency of approximately 30%.
SUMMARY OF THE INVENTION
[0023] Bearing in mind the problems and deficiencies of the prior
art, it is therefore an object of the present invention to provide
a filtration system for pathogen detection that utilizes a
plurality of dielectrophoretic modules with distinctive
functionality and geometry to obtain separation performance which
cannot be obtained using the single module apparatus of the prior
art.
[0024] It is another object of the present invention to provide a
pathogen detection system that includes a capture/release mechanism
for solution exchange without cell loss to enhance pathogen
detection at low concentrations.
[0025] It is yet another object of the present invention to provide
a method for separating a low concentration of bacteria (or other
pathogens/particles) from a high volume of blood (or other fluids)
which is based on the dielectric properties of the products. The
species separation being enhanced and promoted by dielectrophoretic
forces acting on the test sample in a plurality of microfluidic
channels.
[0026] It is another aspect of the present invention to provide a
filtration system for pathogen detection that can accommodate high
and low throughput, capable of processing test sample volumes
significantly greater than micro- or pico-liters, yet capable of
processing the minute test sample volumes as well.
[0027] It is yet another object of the present invention to provide
an electrode design that can perform high yield pathogen and cell
capture, and enhance separation.
It is another object of the present invention to provide pathogen
detection in liquid media, especially for use with food and
agricultural products to improve health standards at the consumer
level.
[0028] It is another object of the present invention to provide
time sensitive pathogen detection for point-of-care
diagnostics.
[0029] Still other objects and advantages of the invention will in
part be obvious and will in part be apparent from the
specification.
[0030] The above and other objects, which will be apparent to those
skilled in the art, are achieved in the present invention which is
directed to an apparatus for pathogen detection comprising: a first
chamber for storing a test sample including product to be analyzed
and microscaled components to be separated from said product to be
analyzed; a second chamber for storing a reference solution; a pump
for pumping said test sample and said reference solution; a
microfluidic separator separating said product to be analyzed from
said microscaled components, said microfluidic separator including
a plurality of microfluidic channels, each microfluidic channel
including: at least one electrode for producing a dielectrophoretic
force on said test sample when said test sample is pumped through
said microfluidic channel to perform a dielectrophoresis-based
separation, said at least one electrode comprising: a plurality of
concentric rings or concentric arcs extending radially outwards
from a center point, electrically connected to a voltage source
such that when voltage is applied to said at least one electrode,
said concentric rings or concentric arcs alternate in voltage
potential, wherein each odd numbered ring or arc counted from said
center point is held to a first voltage potential, and each even
numbered ring or arc is held to a second voltage potential, said
first voltage potential different from said second voltage
potential in magnitude, phase, polarity, or any combination
thereof; and channels for transporting said microscaled components
away from said product to be analyzed.
[0031] The apparatus further includes a third chamber for storing
said microscaled components when separated from said product to be
analyzed by said plurality of microfluidic channels; a condenser
for capturing said product to be analyzed once said product has
passed through said microfluidic channels and is substantially
separated from said microscaled components; and a sensor for
detecting said product to be analyzed.
[0032] The plurality of microfluidic channels may be assembled in
an array, each microfluidic channel having at least one electrode
on an internal wall for delivering the dielectrophoretic force to
the test sample traversing through the microfluidic channel.
[0033] The microfluidic channels preferably comprise a plurality of
plates, such that each microfluidic channel represents an elongated
pathway for the test sample capable of providing a
dielectrophoretic force or force arising from AC electric field in
fluids to the test sample as the test sample traverses the
microfluidic channel.
[0034] A collecting electrode is used to attract the product to be
analyzed at an inlet of the sensor. The sensor includes a field
effect based sensor, nanowire sensor, nanoribbon sensor, or ion
sensitive field effect transistor, and is capable of applying a
confining dielectrophoretic force, trapping the product to be
analyzed.
[0035] The pump may be a micro-pump operating in tandem with
micro-valves to achieve a fully automated pathogen detection
filtration system capable of miniaturization to a chip-scale
design.
[0036] The apparatus may also include a microfluidic transport
module for transporting the product to be analyzed to a location in
the vicinity of the sensor.
[0037] The electrode design may include even numbered concentric
rings or arcs in electrical communication with one another, and odd
numbered concentric rings or arcs are in electrical communication
with one another.
[0038] In a second aspect, the present invention is directed to an
apparatus for pathogen detection comprising: a microfluidic
assembly including a plurality of microfluidic channels forming an
array, each of said microfluidic channels including: at least one
electrode for establishing dielectrophoretic forces on a test
sample separating portions of said test sample into an analyte and
a waste product, said at least one electrode comprising a plurality
of concentric rings or concentric arcs extending radially outwards
from a center point, electrically connected to a voltage source
such that when voltage is applied to said at least one electrode,
said concentric rings or concentric arcs alternate in voltage
potential, wherein each odd numbered ring or arc counted from said
center point is held to a first voltage potential, and each even
numbered ring or arc is held to a second voltage potential, said
first voltage potential different from said second voltage
potential in magnitude, phase, polarity, or any combination
thereof; adjacent microchannels for receiving said waste product
attracted by a dielectrophoretic force, removing said waste product
from said analyte; a condenser including an electrode for
localizing said analyte for sensing; and a sensor for detecting
said analyte.
[0039] A pharmaceutical or other substance may be introduced which
pierces membranes of an alive analyte component in the reference
solution at a predetermined frequency, but not membranes of other
analyte components or dead analyte components, and differentiating
the alive component from other analyte components and dead analyte
components through dielectrophoretic forces.
[0040] The pH or conductivity of the test sample may be adjusted
for control of voltage and frequency dependence for the
Clausius-Mossotti factor cross-over frequency.
[0041] Change of the Clausius-Mossotti factor cross-over frequency
may be induced for the analyte by adding or mixing an additional
fluid.
[0042] The step of detecting low amounts of analyte using a
microfluidic sensor includes using an electric field at
predetermined flow conditions, to immobilize the analyte on the
surface of a field effect based sensor, nanowire sensor, nanoribbon
sensor, or ion sensitive field effect transistor sensor.
[0043] Super positioning or tuning of frequency components,
waveform shapes, and waveform tunings, or any combination thereof,
may be performed to maximize separation, differentiation, capture,
or release of the analyte.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The features of the invention believed to be novel and the
elements characteristic of the invention are set forth with
particularity in the appended claims. The figures are for
illustration purposes only and are not drawn to scale. The
invention itself, however, both as to organization and method of
operation, may best be understood by reference to the detailed
description which follows taken in conjunction with the
accompanying drawings in which:
[0045] FIG. 1 depicts a filtration system 10 of the present
invention for pathogen detection;
[0046] FIG. 2 is a schematic view of a microchannel assembly having
a plurality of individual microchannels;
[0047] FIG. 3 is a schematic view of an array of microfluidic
microchannel assemblies of the present invention used for
separation;
[0048] FIG. 4 depicts a cross-section of microchannel with ground
electrodes and waste collecting electrode;
[0049] FIG. 5 depicts a computer generated model of the resulting
dielectrophoretic forces FDEP acting on bacteria in microfluidic
channels;
[0050] FIG. 6 depicts a computer generated model of the resulting
dielectrophoretic forces acting on red blood cells in microfluidic
channels;
[0051] FIG. 7 is a cross-sectional top view of a microchannel
showing the trajectories of the test sample components.
[0052] FIG. 8 depicts a table (Table I) providing values of the
coefficient .alpha. for RBC, WBC, and bacteria at 10 MHz in blood
serum;
[0053] FIG. 9 depicts a computer generated model of bacteria and
red blood cell trajectories upon applied dielectrophoretic force in
a microfluidic channel;
[0054] FIG. 10 depicts a table (Table II) providing values for the
real and imaginary permeability as well as the particle radius and
conductivity of E. coli and Micrococcus in a reference solution and
blood serum;
[0055] FIG. 11A depicts a graph of a simulation of the
dielectrophoretic force on E. coli inside a microfluidic channel as
a function of channel position and travel time;
[0056] FIG. 11B depicts a graph of a simulation of the
dielectrophoretic force on RBCs inside a microfluidic channel as a
function of channel position and travel time;
[0057] FIG. 12 depicts a set of electrodes and their respective
geometry in a cross-section of a microfluidic channel for capturing
and immobilizing bacteria;
[0058] FIG. 13 depicts a force diagram of the resultant electrodes
of FIG. 12 showing the direction of the dielectrophoretic force
acting on bacteria;
[0059] FIG. 14 depicts a flow velocity profile with inflow from
left and the dielectrophoresis force directing/pushing bacteria to
the sensor at the bottom of a microfluidic channel (overcoming
diffusion limitations);
[0060] FIG. 15A depicts fabrication levels or steps of integration
of the present invention on an integrated circuit chip;
[0061] FIG. 15B depicts an expanded assembly drawing of the layers
representing the fabrication steps of FIG. 15A;
[0062] FIG. 16 is an expanded assembly drawing of the layers of a
microfluidic separator;
[0063] FIG. 17 depicts electrical connections and microfluidic
connections between components provided in embedded layers of an
integrated circuit device of the present invention;
[0064] FIG. 18 depicts an embodiment of the electrode design 200
for use within the confines of a microchannel;
[0065] FIG. 19 depicts an electrode design having a set of adjacent
concentric rings;
[0066] FIG. 20 depicts the electrode of FIG. 19 delineating
alternating polarities associated with a positive voltage source
connected to one set of electrode rings, and a negative voltage
source connected to another set of electrode rings, where each ring
of each set alternates radially outwards from the center point;
[0067] FIG. 21 depicts a typical pathogen contaminated fluid inside
a microchannel of the present invention in the absence of an
electric field to the electrode;
[0068] FIG. 22 depicts the microchannel containing contaminated
fluid of FIG. 22 with the electrode's electric field ON;
[0069] FIG. 23 depicts the alignment of pathogens when a positive
dielectrophoresis force (pDEP) of 3 volts at 10 MHz is applied to
the electrode; and
[0070] FIG. 24 depicts the absence of pathogen alignment when a
negative dielectrophoresis force (nDEP) of 3 volts at 200 MHz is
applied to the electrode.
DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
[0071] In describing the preferred embodiment of the present
invention, reference will be made herein to FIGS. 1-24 of the
drawings in which like numerals refer to like features of the
invention.
[0072] The filtration system of the present invention performs
pathogen detection using a plurality of dielectrophoretic modules
of microfluidic channels with distinctive functionality and
geometry to obtain separation performance which cannot be obtained
in the prior art. Additionally, the present invention integrates a
nano-scaled sensor with the filtration system. Advantageously, all
components of the filtration system may be embedded, forming an
integrated electronic-microfluidic circuit.
[0073] The assembled filtration system automatically transports,
separates, condenses, and detects low amounts of particles, cells,
and bacteria, or the like, from liquids in a portable configuration
that minimizes false positives and negatives.
[0074] The present invention defines a robust method for separating
bacteria from blood components based on their dielectric
properties, localizing the bacteria, flushing substantially all
remaining by-products from the bacteria, which generally are on the
order of micro-sized or microscaled components, and detecting low
concentrations of the bacteria. The separation is fast and reliable
as species movement is caused by a module array imparting opposing
forces. The module array includes a plurality of microfluidic
channels with connecting microfluidic waste channels for directing
undesired material away from bacteria. The process enables
separation of low concentration of bacteria or other pathogens or
particles from blood or other fluids, which then enables detection
of the low concentration of these species. This is of great
importance for medical diagnostics and determining food safety.
[0075] In a preferred embodiment, the invention includes an
electronic device capable of detecting of a low number of bacteria
or other pathogens or particles from milliliter or larger volumes
of different liquid media on a minute time scale by integrating a
plurality of modules of microfluidic channels capable of performing
a dielectrophoresis-based separation, and incorporating a unique
capture system using field-effect-transistor based biosensors.
[0076] Bacteria present in a sample even of different types will be
subject to a dielectrophoretic force in one direction, whereas all
large blood components red blood cells and white blood cells will
be subject to a dielectrophoretic force in another direction.
Effective separation improves the detection of pathogens. Without
separation, blood components which constitute a vast majority of
micro-sized particles in blood, would clog active sensor sites and
prevent detection of low concentrations of bacteria present in the
same sample.
[0077] Furthermore, many types of sensors cannot operate in high
ionic solutions such as blood plasma. Consequently, the solution
containing the sample to be sensed, most likely bacteria, has to be
changed to a more suitable reference solution, such as a buffer.
Often a pre-concentration step is required. The capture/release
mechanism presented by the present invention provides an excellent
method for solution exchange without cell loss.
[0078] Electric field cell capture, release, and separation based
on forces acting in opposite directions allow precise control of
cell separation without risk of cell loss or contamination.
Advantageously, the present invention may be applied for separating
any species of comparable size in any liquid medium; however,
bacteria separation from blood components, white blood cells and
red blood cells, is illustrated for exemplary purposes, and
represents a predominate utilization of the present invention.
[0079] FIG. 1 depicts a filtration system 10 of the present
invention for pathogen detection. The system includes two injection
chambers: a first injection chamber 12 containing a sample to be
tested, such as blood, and a second injection chamber 14 containing
a reference solution, buffer, or other liquid for flushing at a
later point in the process, generally referred to as the by-product
or waste-product. Injection chambers 12, 14 are connected to
micro-pump inlets which connect to a microfluidic separator 16.
Fluids will be pumped into microfluidic separator 16 in a
controlled manner by pump 18 such that the test sample (e.g.,
blood) will be pumped in first. As will be discussed in further
detail below, the microfluidic separator includes a plurality of
microfluidic channels assembled in an array fashion that uses
dielectrophoretic forces to separate continually during transport
components of the test sample from one another. In the illustrative
example, bacteria may be separated from red and white blood cells.
The separated analyte (bacteria) is then condensed by a localized
electric field, and reference solution, such as a buffer fluid, is
used to replace and dilute or change the chemical composition of
the blood serum that reaches condenser 20.
[0080] Unlike the prior art, the microfluidic separator 16 of the
present invention includes a plurality of channels that apply
dielectrophoretic forces that are exerted on the particles, cells,
bacteria, and/or micro-scale components as they flow through the
channels. The dielectrophoretic field is carefully chosen such that
the components of interest that flow through will experience an
opposite force as compared to the rest of the components or
waste-products that are desired to be separated out. The
waste-products are drained throughout the process from the
plurality of microfluidic channels to waste chamber 22 through
multiple microfluidic channel outlets.
[0081] In this manner, the filtration system of the present
invention is composed of modules/segments each tuned such that the
class of objects under study (i.e., the analyte) has the same
response. For example, all bacterial have an nDEP, which is in the
middle of the response spectrum. An additional "filter" is then
applied for increased accuracy of targeting the analyte. The
assembly of independent modules for this application is comprised
of multiple, but not necessarily continuous, wires. Microchannel
outlets, waste channels, break the continuous wire configuration.
Using this geometry, surface electrode configurations may be
employed, moving away from continuous conducting wire of the prior
art.
[0082] In this example, isolated bacteria flows into a condenser
chamber 24, which has a collecting electrode to attract the
bacteria to the inlet of a microfluidic sensor 26 containing sensor
arrays. Movement of bacteria to the field-effect-transistor based
sensor is enhanced using the electric field and the
dielectrophoretic force to overcome the diffusion limitation of the
motion. Furthermore, the present invention is capable of tuning the
electric field such that only the particle of interest gets
through, the remaining product is eliminated. Thus, detection is
label-free; it does not require sensor functionalization with
specific antibodies.
[0083] The method is based on dielectrophoretic separation followed
by dielectrophoretic concentration, and replacement or partial
replacement or dilution of the original liquid with a reference
solution. The next step is dielectrophoretic manipulation of
bacteria to the sensor surface to overcome the diffusion limitation
and enable bacteria contact with the sensor surface for
detection.
[0084] The device operation and automated sample preparation is
described in some detail below. First, a test sample injection is
distributed into the system. This is performed by a pump that
causes the automated distribution of the test sample, placing the
test sample in a plurality microfluidic channels (microchannels)
via capillary forces (porous media) and pump-pressure driven flow.
Each of the plurality of microfluidic channels are lined with
electrode geometry capable of establishing an electric field and a
dielectrophoretic force on the test sample. Separation within the
microfluidic channels is then performed by the dielectrophoretic
force. In order to achieve adequate and efficient separation,
waveform tuning of the electric field is selected with the
intention that two types of species are subjected to forces acting
in opposite directions. The separation occurs within microfluidic
separator 16. The unwanted micro-scaled components and blood cells
(waste-product) separated from the analyte
(bacteria/cells/particles) are collected in waste chamber 22. The
separated analyte is then collected on condenser 20. An electrode
immobilizes the desired analyte material.
[0085] In order to remove the waste-product, extraneous serum and
other unwanted blood products, the remaining analyte is exposed to
a reference solution while held by the condenser electrode. In this
manner the unwanted blood products are flushed away, replaced or at
least partially replaced, and/or diluted by the reference solution.
The remaining analyte is localized to a sensor surface.
Dielectrophoretic manipulation of bacteria is used at the sensor
surface to overcome the diffusion limitation and enable bacteria
contact with the sensor surface for detection.
[0086] Preferably the sensor is of nanowire or nanoribbon
technology, which enables the filtration system of the present
invention to be integrated on a semiconductor chipset. Once the
final analyte is interrogated, the output may be digitized for
automated data processing and readout.
[0087] In a preferred embodiment, a multi-step approach to
filtration for pathogen detection is achieved using a plurality of
dielectrophoretic modules including a plurality of microfluidic
channels in an array fashion. The microfluidic separator 16
separates the test sample components of interest (e.g., bacteria)
from pollutants (e.g., blood cells and blood serum). In a
subsequent process step, the surrounding medium is then exchanged
or diluted with a reference solution more suitable for
comprehensive electronic detection applications.
[0088] The process introduces condensation of the analyte onto a
concentrating electrode using a dielectrophoretic (pDEP) force.
Once all remaining analyte from the sample is collected on the
condensing electrode and the remaining waste-product has been
exchanged with the reference solution, the frequency of the applied
electric field is then changed (generally from high to low) so that
the dielectrophoretic force changes sign and becomes repulsive, and
the analyte is then released into a small (.about.1 .mu.l) volume
of the reference solution. Next, the analyte is transported to the
sensor chamber and restricted in the vicinity of the sensor.
Detection is then performed by sensor arrays, selectively
functionalized for the target analyte (bacteria) of interest.
[0089] Microfluidic separator 16 is comprised of a high throughput
system of multiple microchannels, preferably an array 10.times.100
microchannels although any number of microchannels may be utilized
with varying degrees of efficiency. FIG. 2 is a schematic view of a
microchannel assembly 30 having a plurality of individual
microchannels 32. Each microchannel assembly 30 has multiple-outlet
linear microchannels 32 with copper/metal sets of electrodes 34
deposited on the microchannel walls. In a preferred embodiment,
microfluidic microchannel assembly 30 includes plates 36 patterned
with metal electrodes 34, such as copper and the like, on each
side, generally having a preferred geometry of 5 .mu.m.times.10
mm.times.1 .mu.m, sandwiching an internal channel structure or
"tree" 38 outlined with copper electrodes 34. When plate 36 comes
in contact with internal channel structure 38, multiple
microfluidic channels are formed. Plates 36 and internal channel
structure 38 may comprise plastic material or other light, durable
material capable of securing metal electrodes, and containing the
test sample without degradation.
[0090] An additional electrode design is introduced for high yield
pathogen (bacteria and cell) capture, and which lends itself to
enhanced and reliable separation using an alternating electric
field.
[0091] The capture of pathogens is generally from blood, water, and
other fluids, and the separation includes the removal of pathogens
from, for example, large blood components, red blood cells, white
blood cells, and the like.
[0092] The purpose of optimizing the electrode design for this
application is to maximize the yield for a given applied voltage.
FIG. 18 depicts an embodiment of the electrode design 200 for use
within the confines of a microchannel. Electrode 200 is composed of
a set of densely packed conductive rings fragments 204, 206. The
rings may be connected separately to a voltage source or multiple
voltage sources, or interconnected and attached to a single voltage
source. Electrode 200 may include concentric rings, connected
rings, or a spiral configuration. An embodiment depicting a set of
adjacent concentric rings is shown in FIG. 19. In this embodiment,
the rings are coaxial, extending radially outwards from a center
point 202. The innermost or first ring 204a may be electrically
connected to the third ring 204b, which may be electrically
connected to the fifth ring 204c, and so on for the odd numbered
rings extending radially from center point 202. Each of the odd
numbered rings are either electrically connected together to have a
same first voltage and polarity, for example V+, or connected
separately to at least one voltage source having the same voltage
and polarity. In a similar fashion, the even numbered rings extend
radially from center point 202 and may be electrically connected
together. As depicted in FIG. 18, second ring 206a may be
electrically connected to the fourth ring 206b, which are connected
to a single voltage source having a second polarity, for example
V-, the second voltage being different from the first voltage in
either magnitude, polarity, or both. Each of the even numbered
rings are either electrically connected together to have a same
second voltage and polarity, or connected separately to at least
one voltage source having a second voltage and polarity.
[0093] An alternating current (AC) electric field is applied to the
electrode at a predetermined frequency or frequencies in the range
of 1 kHz to 400 MHz, such that the resulting force acting on the
different species allows for a differential response. The method
can be applied for separation of similar size particles also from
fluids other than blood.
[0094] FIG. 20 depicts the alternating polarities associated with a
positive voltage source connected to one set of electrode rings,
and a negative voltage source connected to another set of electrode
rings, where each ring of each set is concentric and alternates
radially outwards from the center point, one set representing the
odd numbered rings and the other set representing the even numbered
rings.
[0095] FIG. 21 depicts a typical pathogen contaminated fluid inside
a microchannel of the present invention. Red blood cells 210 and E.
coli 212 are present in the fluid. FIG. 22 depicts the same
microchannel containing contaminated fluid with the electric field
ON. As can be seen, the E. coli 212 aligns with the curvature of
the electrodes under the dielectrophoresis force. This force
"holds" or redirects the pathogen while the carrier fluid (blood)
traverses through the microchannel.
[0096] As depicted by FIG. 23, a positive dielectrophoresis force
(pDEP) of 3 volts at 10 MHz was shown to align the pathogens
effectively and efficiently. This is in stark contrast to a
negative dielectrophoresis force (nDEP) of 3 volts at 200 MHz as
depicted in FIG. 24. This illustrates the frequency dependence of
establishing pathogen attraction or repulsions.
[0097] Preferably, voltages of opposite polarity are applied to
adjacent rings, or a different voltage level is applied to adjacent
rings, such that there exists a potential difference between
adjacent rings. Thus, either V.sub.1=-V.sub.2, or there is a phase
difference between the two voltage sources (V1=Vsin (.omega.t);
V2=Vsin (.omega.t+.pi.)), or the magnitude of V.sub.1 is not equal
to the magnitude of V.sub.2.
[0098] FIG. 3 is a schematic view of an array 40 of microfluidic
microchannel assemblies 30 of the present invention used for
separation. The invention provides for an assembly of layers with
defined components of multiple microchannels. Microchannel
assemblies 30 are stacked such that array 40 comprises an
"m.times.n" microchannel array. Each microchannel 32 has a height h
and width w. In a preferred embodiment, each microchannel 32 is
approximately 100 .mu.m wide and 100 .mu.m high. The preferred
length of each microchannel 32 is 10 mm. Other dimensions may be
pre-determined for particular efficiencies and for specific test
samples. The advantage of an array of microfluidic channels is the
ability to transport and separate the test sample in an extremely
small package--on the order of an integrated circuit. The chip-set
size of the filtration system promotes reliability, portability,
and discrete packaging.
[0099] In a preferred embodiment, array 40 is composed of multiple
plates 36 sandwiching internal channel structures 38 such that when
stacked they form an array of 10 (horizontal).times.100 (vertical)
microfluidic channels. The 11 layers are aligned to the edges and
thermally bonded.
[0100] In an assembled microfluidic separator 16, laminar flow
conditions are provided for separation. Under preferred operating
conditions, flow velocity, v, is approximately 100 .mu.m/s, channel
length, L, is about 1 cm, the total flow time through a single
channel, t.sub.flow, is on average about 100s, and the flow rate
per channel is about 1 nL/s. Thus, in total 1 cc is pumped very
quickly through 1000 microchannels.
[0101] The test sample throughput may be "tuned" by increasing or
decreasing the number of parallel microchannels, increasing or
decreasing the parallel stacked microchannel assemblies 30, and
changing the flow velocity by setting the pumping speed.
[0102] The invention utilizes the frequency dependence of the sign
of the CM factor between different contaminant/blood species for
separation in a series of custom designed dielectrophoretic
modules.
[0103] For the purposes of the present invention, a coefficient
.alpha. will be defined as follows: [0104] .alpha.=2.pi. .sub.m
r.sup.3 Re{CM(.omega.)}; and [0105]
F.sub.DEP=.alpha..gradient.E.sup.2; [0106] where CM(.omega.) is as
defined previously.
[0107] The .alpha. coefficient accounts for the particle size r and
the dielectric properties of the particle itself .sub.p and the
surrounding medium .sub.m.
[0108] Thus, the invention provides for separation of species based
on the different signs of the Re{CM(.omega.)} factor and which
follows the different signs of the .alpha. coefficient at a chosen
operating frequency. The separation method of two select groups of
the components of interest, i.e.,
pathogens/cells/bacteria/particles (group 1) and blood cells (group
2), is based on tuning the electric field frequency such that the
Re{CM(.omega.)} factor is positive for one group and negative for
the other group. This causes component movements in different
directions, which leads to separation. Unlike the prior art, the
microfluidic separator is uniquely designed to complete separation,
and the subsequent condensing and flushing process steps result in
pure isolation of the bacteria, cells, or particles of
interest.
[0109] A description for bacteria in blood is provided below for
the separation, capture, and release mechanisms, and for flow
conditions inside a microfluidic channel. Figures of force fields
are generated from program runs using COMSOL Multiphysics
software.
[0110] An electric field gradient is generated by an electrical
waveform applied to sets of electrodes on the plurality of
microchannel walls. FIG. 4 depicts a cross-section of microchannel
32 with ground electrodes 42 and waste collecting electrode 44. In
this example, 20 volts are applied to waste collecting electrode
44. The resulting dielectrophoretic forces F.sub.DEP acting on
bacteria are depicted by arrows 46 in FIG. 5, while the
dielectrophoretic force acting on red blood cells are shown by
arrows 48 in FIG. 6. These forces indicate that F.sub.DEP for
bacteria has an opposite direction from F.sub.DEP for red blood
cells.
[0111] FIG. 7 is a cross-sectional top view of a microchannel 32
showing the trajectories of the test sample components.
Trajectories of bacteria 50 and red blood cells 52 in the direction
of flow 54 are depicted for pre-determined electrode geometry
inside one of the many microfluidic channels 32 that comprise
microfluidic separator 16. Under the applied wave form, bacteria
and red blood cells are pushed towards opposite ends of the
microchannels.
[0112] As an illustrative example, the values of .alpha. are
selected for E. coli bacteria, red blood cells, and white blood
cells based on pre-determined permittivity data (real and imaginary
permittivity .sub.p, .sub.m, particle radius r, and conductivity
.sigma.).
[0113] The dielectrophoretic force acting on E. coli bacteria, red
blood cells (RBC), and white blood cells (WBC) is generated by
applying a voltage, which in the preferred embodiment is
approximately 20V, on the electrodes at different operating
frequencies. The provided values of the coefficient .alpha. for
RBC, WBC, and bacteria at 10 MHz in blood serum are shown in Table
I identified in FIG. 8.
[0114] In Table I (FIG. 8), the real and imaginary part of the
complex dielectric function, conductivity, coefficient .alpha., and
the real part of the Clausius-Mossotti factor are calculated at 10
MHz for two different types of bacteria (E. coli and Micrococcus),
white blood cells (T lymphocytes, monocytes, B lymphocytes, and
granulocytes), and red blood cells.
[0115] In this example, .alpha. is negative for bacteria and
positive for the blood components, thus effecting separation under
dielectrophoretic force. At an electric field frequency of 10 MHz,
and using blood serum as a surrounding medium, bacteria (E. coli
and Micrococcus), experience a negative dielectrophoretic force,
while at the same operating conditions the blood components, WBC
and RBC experience a positive dielectrophoretic force. Bacteria and
red blood cell trajectories upon applied dielectrophoretic force in
the microchannel are depicted in FIG. 9. Arrows denote the
direction of the dielectrophoretic force acting on red blood cells
and bacteria. The time based trajectory of motion for different
initial positions of red blood cells is depicted in boxes a, b, and
c. The time based trajectory of motion for different initial
positions of bacteria is depicted in boxes d, e, and f. As shown,
red blood cells initially positioned near microchannel walls far
from the blood cell collecting electrode reach the blood cell
collecting electrode in less than one hundred seconds. In the same
electric field, bacteria are pushed away from the blood cell
collecting electrode and directed towards the channel medium.
[0116] The dielectrophoretic force acting on red blood cells is
directed towards the field maximum, where the waste collecting
electrode is placed. The dielectrophoretic force confines bacteria
within a certain "safe" region of the microchannel as shown in FIG.
9, boxes d, e, f, while it pushes blood cells in the opposite
direction, which is towards the waste collecting electrode and the
waste channels as shown in FIG. 9, boxes a, b, c. In this manner,
separation occurs continuously during test sample transport through
the microfluidic channels, with each microchannel doing its part to
separate test sample components.
[0117] In the current example, utilizing the preferred array
geometry for the microfluidic separator array with lateral and
vertical DEP electrodes, the provided separation efficiency of E.
coli from RBC and WBC components was nearly 95% in about 15
seconds, and 100% for an approximately 100 micron channel length in
a timeframe of approximately one minute. The microfluidic separator
comprising an array of microfluidic channels, each acting to
separate the test sample and direct waste-product towards a waste
chamber.
[0118] Unique to the present invention, a branched microfluidic
design allows for separated components to be discarded as waste,
while the target of interest, for example E. coli, is transferred
to a condenser, flushed, and then localized for pathogen detection
by an electronic sensor. The invention is not dependent upon a
single critical dimension fabrication or alignment, and the
waveform frequencies may be tuned to change the differential sign
of the Re{CM(.omega.)} factor for different components to be
separated. The cross-over frequency varies for different particles,
bacteria, and/or cells in different media.
[0119] The values of the a coefficient for bacteria E. coli and
Micrococcus in buffer solution and blood serum at frequencies 10
MHz and 400 Mhz are provided in Table I of FIG. 8 and Table II of
FIG. 10. In Table II (FIG. 10), the pre-determined values for the
real and imaginary permeability as well as the particle radius and
conductivity are listed. To enhance separation efficiency a
pre-determined waveform containing frequency components tuned for
particular species (particles/bacteria/cells) of interest is
used.
[0120] Continuing with the example above, the a coefficient for E.
coli and Micrococcus is negative and has a different magnitude in
blood serum at 10 MHz, which for E. coli
.alpha.=-0.0044(10.sup.-24) J(m/V).sup.2, and for Micrococcus
.alpha.=-0.0027(10.sup.-24) J(m/V).sup.2, while the .alpha.
coefficient is positive and has a similar magnitude in blood serum
at 400 MHz (E. coli .alpha.=0.0044(10.sup.-24) J(m/V).sup.2,
Micrococcus .alpha.=0.0043(10.sup.-24) J(m/V).sup.2). Micrococcus
and E. coli will experience a very similar force in blood serum at
400 MHz, while they will experience a very different (opposite)
force in the same medium, blood serum, at a frequency of 10
MHz.
[0121] The .alpha. coefficient for T. Lymphocytes is positive
(.alpha.=0.0136(10.sup.-24) J(m/V).sup.2) in blood serum at 10 MHz.
Thus, the DEP force (negative DEP) exerted on bacteria in blood
serum at 10 MHz has an opposite sign then the DEP force (positive
DEP) exerted on T. Lymphocytes in blood serum at 10 MHz.
[0122] Consequently, a waveform applied to the electronic device of
the present invention, containing only a frequency component at 400
MHz will result in a very similar behavior of both E. coli and
Micrococcus, causing similar motion of both products. A waveform
applied to the electronic device containing only a frequency
component at 10 MHz will result in a similar motion of both E. coli
and Micrococcus, and this motion will be in the opposite direction
of T. Lymphocytes.
[0123] A waveform applied to the device containing both frequency
components 10 MHz and 400 MHz will result in a motion of
Micrococcus while the force will cancel for E. coli, resulting in a
lack of motion of E. coli.
[0124] A choice of a waveform in the same medium allows
differentiating and fingerprinting different species. Unique to the
present invention, a sequence of an array of modules with tuned
waveforms would allow selecting species based on their unique
dielectric function.
[0125] After passing through the segments of microfluidic separator
16, the first component of the filtration system, the targets of
interest (e.g., bacteria) are separated from pollutants (e.g.,
blood cells), at which point, the targets of interest are then
condensed by condenser 20.
[0126] In a preferred embodiment, condenser 20 uses the change of
the Re{CM(.omega.)} factor upon the change of the medium
permittivity ( .sub.m) for species capture on a capturing
electrode, to reduce the volume of the sample and condense the
species bacteria, cells, and/or particles in a significantly lower
volume. A collecting electrode attracts the bacteria to the inlet
of a microfluidic sensor 26 containing sensor arrays. Movement of
bacteria to a field-effect-transistor based sensor is enhanced
using the electric field and the dielectrophoretic force to
overcome the diffusion limitation of the motion.
[0127] FIG. 11A depicts a graph of a simulation of the
dielectrophoretic force on E. coli inside a microfluidic channel as
a function of channel position and travel time. FIG. 11B depicts a
graph of a simulation of the dielectrophoretic force on RBCs inside
a microfluidic channel as a function of channel position and travel
time. These simulation results show that bacteria, if placed within
10 .mu.m of the elimination electrode, are repelled towards the
safe zone within 0.007 seconds. For RBCs, the elimination time is
shorter than 82 seconds. Fifty percent (50%) of the RBCs are
filtered out within the first 0.5 seconds. Ninety-five percent
(95%) of the RBCs are filtered out within the first 16 seconds, and
substantially all of the RBCs are filtered out within the first 82
seconds.
[0128] At a matching frequency, the Re{CM(.omega.)} in the medium
surrounding the species is positive, which results in a positive
dielectrophoretic force directed towards a capturing electrode. The
set of electrodes and their geometry in the microchannel
cross-section is shown in FIG. 12 for capturing and immobilizing
bacteria. The direction of the dielectrophoretic force acting on
bacteria is shown in the force diagram of FIG. 13. Arrows 60 show
the direction of the dielectrophoretic force on bacteria, causing
the bacteria to collect on the electrodes. Despite the flow
directed towards the microchannel outlet 63, bacteria are collected
on the collecting electrodes 62 due to a positive dielectrophoretic
force and a positive Re{CM(.omega.)} factor.
[0129] FIG. 14 depicts a flow velocity profile 64 with inflow from
left and the dielectrophoresis force directing/pushing bacteria to
the sensor at the bottom of a microfluidic channel (overcoming
diffusion limitations). Arrows show direction of the
dielectrophoretic force acting on bacteria 68. Despite the flow
directed towards the microchannel outlet, bacteria are collected on
the collecting electrode due to a positive dielectrophoretic force
and a positive Re{CM(.omega.)} factor. When the surrounding medium
is changed by the buffer solution, the value of the Re{CM(.omega.)}
factor becomes negative, and the dielectrophoretic force repels
bacteria from the electrode causing species release.
[0130] To enhance separation, overcome the limitation caused by
high ionic strength of the solution, and obtain functional analyte
(bacteria) response, the initial medium (e.g., blood serum) is
diluted and partially replaced by the buffer solution. As a result,
the dielectric constant of the medium .sub.m changes and the
Re{CM(.omega.)} factor changes resulting in a change of the
magnitude and potentially direction of the force.
[0131] The change of the Re{CM(.omega.)} factor upon the change of
the particle/cell/bacteria permittivity ( .sub.p) is used to obtain
a differential functional response.
[0132] This form of .alpha.-screen testing allows for a portable
platform for rapid multiplexed analyte detection, such as bacteria,
from blood samples of ill patients at a point-of-care application.
Doctors would be able to diagnose the bacteria of infection, and
accurately prescribe only the necessary antibiotic, resulting in a
more efficient disease treatment, and limiting
antibiotic-resistance formation.
[0133] Using the apparatus of the present invention, this
.alpha.-screen testing does not require additional laboratory
space, and is low in energy consumption. It may be used with a
sensor network integrated with food processing lines in food
processing plants for continuous food product quality monitoring,
or used in food storage and transport. It may be integrated in a
hand-held unit for rapid Vibrio cholera and E. coli bacteria
detection from water samples to determine water safety.
[0134] By introducing to the medium a reference solution, such as a
buffer, and additional pharmaceuticals, the dielectric constant of
the medium .sub.m changes, the dielectric constants of the
particles/bacteria/cells .sub.p change, and Re{CM(.omega.)} change
for different species, resulting in a change in F.sub.DEP allowing
to distinguish between the analyte components.
[0135] Using the previous values as an illustrative example, the
.alpha. coefficient for E. coli and Micrococcus is negative and has
a different magnitude in blood serum at 10 MHz (E. coli
.alpha.=-0.0044 (10.sup.-24) J(m/V).sup.2,
Micrococcus.alpha.=-0.0027 (10.sup.-24) J(m/V).sup.2), while
.alpha. is positive in a PBS buffer solution at 10 MHz (E. coli
.alpha.=0.0055 (10.sup.-24) J(m/V).sup.2, Micrococcus
.alpha.=0.0106 (10.sup.-24) J(m/V).sup.2}. The force F.sub.DEP on
Micrococcus in serum will have a lower magnitude than on E. coli;
however, in a buffer solution (such as PBS) the force on
Micrococcus will be stronger than on E. coli. Introducing a
pharmaceutical or a substance (antibiotic) which pierces only the
membrane of alive Micrococcus at 10 MHz in PBS, but not the
membrane of E. coli or dead Micrococcus in buffer will allow
differentiating alive from dead Micrococcus and E. coli, since the
dielectrophoretic force depends on the size of the
particle/bacteria/cell, where F.sub.DEP is proportional to
r.sup.3.
[0136] Thus, a tuned chemical modification of the medium allows
differentiating and fingerprinting different species. A sequence of
modules with tuned chemical modifications will allow species
selection.
[0137] In the preferred embodiment, the invention applies an
electrical waveform and a dielectrophoretic force for enclosing the
separated bacteria in a small volume around a sensor to
significantly decrease diffusion time to the sensor. Bacteria
trapping on a nanowire or nanoribbon sensor is a resultant of the
dielectrophoretic trapping mechanism and surface modification of
the sensor for capture. In this manner, a dielectrophoretic force
is used as a confining force for trapping micro-sized blood
components (RBC, WBC, bacteria, and the like).
[0138] The DEP capture mechanism for bacteria decreases the volume
of diffusion of a product of interest (particle, bacteria, and/or
cell) in the sensor chamber, and decreases the time for the product
of interest to diffuse towards the sensor surface, which is
necessary for detection.
[0139] The electronic device that implements this separation may be
miniaturized to an integrated circuit, and does not require trained
personnel--the user only introduces a sample (such as blood or
water) into the inlet chamber, and an automated process performs
sampling, separation, condensation, transport, and detection. Using
dielectrophoresis, the device automatically separates any present
bacteria from the rest of the sample--for example, with blood, the
large blood components (e.g., red and white blood cells). The
separated bacteria are concentrated by a second dielectrophoretic
region, and finally detected using label-free nanosensors which may
be functionalized with bacteria specific antibodies for
selectivity.
[0140] The levels of integration of the present invention on an
integrated circuit chip are generally depicted by the fabrication
steps of FIG. 15A. Fig. Step A depicts a printed circuit board with
an integrated circuit 69, embedded electrode connections 70, 71 and
an embedded sensor 72. The subsequent layers and components
depicted in Steps B-G are stacked consecutively and thermally
and/or chemically bonded to form the device.
[0141] Step B of FIG. 15A provides a structure provided in an
insulator layer 73 bonded on top (or bottom) of the PCB. The
opening 74 is for the sensor chamber, providing access to the
sensor and embedding electric connections. The opening 75 is for
alignment of the microfluidic separator.
[0142] Step C adds insulator layer 76 with openings for the
separator, the condenser chamber, and a microfluidic channel
connecting chambers, e.g. the condenser with the sensor chamber
77.
[0143] Step D depicts the addition of the microfluidic separator
module 78.
[0144] Step E adds an insulator layer forming the walls of the
condenser 79, an electrode 80, and outlet 81 from the sensor
chamber.
[0145] Step F adds insulator layer 82 forming the walls of the test
sample chamber 83, buffer/reference liquid chamber 84, waste
chamber 85, and the insulator layer 86 forming the walls of the
separator.
[0146] Step G adds lid 87 with inlets to the chambers for sample,
liquid storing, inlets 88 to the separator, and outlet 89 from the
sensor chamber and waste chamber.
[0147] FIG. 15B depicts an expanded assembly drawing of the layers
representing the fabrication steps of FIG. 15A.
[0148] FIG. 16 is an expanded assembly drawing of the layers of a
microfluidic separator. The layers include a first layer 90 having
microchannel structures and coated with a planar electrode,
followed by a second layer 91 having discrete waste electrodes.
These layers are stacked in pairs to form a microfluidic separator
module 92. The interconnected waste collecting microchannels 93, 94
are located inside of the insulating layers. The described assembly
provides customizing the number of microchannels on each layer, the
number of stacked layers, and the device throughput.
[0149] In one embodiment the electrical connections and
microfluidic connections between components are provided in
embedded layers as shown in FIG. 17.
[0150] FIG. 17 shows a printed circuit board 95 with embedded
copper connections 96. An integrated circuit sensor 97 is connected
by wire-bonding, BGA, or flip-chip technology to the PCB. A
microfluidic channel or chamber 98 is embedded in layers of
insulator 99 with openings cut to fit the microfluidic structures
98. The layers of insulator 99 are stacked and thermally or
chemically bonded. The openings and holes 100 in the layers of the
insulator 99 align vertically and form microchannels for fluid
transport. The inlets and outlets 101 to the integrated electronic
microfluidic circuit are defined in the top insulator layer
102.
[0151] While the present invention has been particularly described,
in conjunction with a specific preferred embodiment, it is evident
that many alternatives, modifications and variations will be
apparent to those skilled in the art in light of the foregoing
description. It is therefore contemplated that the appended claims
will embrace any such alternatives, modifications and variations as
falling within the true scope and spirit of the present
invention.
[0152] Thus, having described the invention, what is claimed
is:
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