U.S. patent application number 14/882143 was filed with the patent office on 2016-02-04 for enhanced microfluidic electromagnetic measurements.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Laurent Giovangrandi, Gregory Kovacs, Antonio J. Ricco.
Application Number | 20160033311 14/882143 |
Document ID | / |
Family ID | 45890532 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160033311 |
Kind Code |
A1 |
Giovangrandi; Laurent ; et
al. |
February 4, 2016 |
ENHANCED MICROFLUIDIC ELECTROMAGNETIC MEASUREMENTS
Abstract
Techniques for enhanced microfluidic impedance spectroscopy
include causing a core fluid to flow into a channel between two
sheath flows of one or more sheath fluids different from the core
fluid. Flow in the channel is laminar. A dielectric constant of a
fluid constituting either sheath flow is much less than a
dielectric constant of the core fluid. Electrical impedance is
measured in the channel between at least a first pair of
electrodes. In some embodiments, enhanced optical measurements
include causing a core fluid to flow into a channel between two
sheath flows of one or more sheath fluids different from the core
fluid. An optical index of refraction of a fluid constituting
either sheath flow is much less than an optical index of refraction
of the core fluid. An optical property is measured in the
channel.
Inventors: |
Giovangrandi; Laurent; (Palo
Alto, CA) ; Ricco; Antonio J.; (Los Gatos, CA)
; Kovacs; Gregory; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Palo Alto |
CA |
US |
|
|
Family ID: |
45890532 |
Appl. No.: |
14/882143 |
Filed: |
October 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13250605 |
Sep 30, 2011 |
9170138 |
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14882143 |
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61388916 |
Oct 1, 2010 |
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Current U.S.
Class: |
702/45 ;
73/861.12 |
Current CPC
Class: |
G01F 1/58 20130101; G01F
1/588 20130101; G01F 1/584 20130101; G01F 1/586 20130101 |
International
Class: |
G01F 1/58 20060101
G01F001/58 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with Government support under
Cooperative Agreement No. NNA04CC32A awarded by the National
Aeronautics and Space Administration, and Contract #: 1R21
1EB007390-01A1 awarded by the National Institutes of
Health/National Institute of Biomedical Imaging and Bioengineering.
The Government has certain rights in the invention.
Claims
1. A method comprising: causing a core fluid to flow into a channel
between two sheath flows of one or more sheath fluids different
from the core fluid, wherein a dielectric constant of a fluid
constituting either sheath flow is substantively less than a
dielectric constant of the core fluid, and wherein flow in the
channel is laminar; and determining impedance in the channel based
on measurements at a first pair of one or more pairs of
electrodes.
2. A method as recited in claim 1, further comprising determining
impedance of a particle in the core fluid.
3. A method as recited in claim 2, wherein determining impedance of
the particle in the core fluid further comprises determining a
temporal change in measured impedance in the channel between the
first pair of electrodes.
4. A method as recited in claim 2, wherein determining impedance of
the particle in the core fluid further comprises determining a
difference between a first determined impedance in the channel at
the first pair of electrodes and a second determined impedance at a
different second pair of electrodes disposed along the channel
separately from the first pair of electrodes.
5. A method as recited in claim 2, wherein a largest spatial
dimension of the particle is much less than a narrowest spatial
dimension of the channel.
6. A method as recited in claim 2, wherein a largest spatial
dimension of the particle is not substantively greater than a
narrowest spatial dimension of the core fluid.
7. A method as recited in claim 2 wherein the particle is a
platelet.
8. A method as recited in claim 7, further comprising
distinguishing an activated platelet and resting platelet based, at
least in part, on impedance determined for the particle.
9. A method as recited in claim 1, wherein determining impedance in
the channel at a first pair of electrodes further comprises
determining impedance in the channel at the first pair of
electrodes at a plurality of alternating current frequencies.
10. A method as recited in claim 9, wherein the plurality of
alternating current frequencies span a frequency range greater than
about 100 megahertz (MHz).
11. A method as recited in claim 1, wherein causing the core fluid
to flow into the channel between two sheath flows further comprises
controlling a narrowest spatial dimension of the core by
controlling relative pressure or flow rate of a core fluid compared
to a corresponding pressure or flow rate of one or more of the
sheath fluids.
12. A method as recited in claim 11, wherein controlling relative
pressure or flow rate of the core fluid compared to the
corresponding pressure or flow rate of the one or more of the
sheath fluids further comprises controlling the relative pressure
or flow rate to stabilize a measurement of a property of the
core.
13. A method as recited in claim 12, wherein the measurement of the
property of the core is impedance determined in the channel at the
first pair of electrodes.
14. A method as recited in claim 12, wherein the measurement of the
property of the core is impedance determined in the channel at the
first pair of electrodes when a particle is absent from the channel
between the first pair of electrodes.
15. A method as recited in claim 12, wherein the measurement of the
property of the core is impedance determined in the channel at the
first pair of electrodes at a particular set of one or more
alternating current frequencies.
16. A method as recited in claim 12, wherein the measurement of the
property of the core is the width of the core between the two
sheath flows in the channel in a vicinity of the first pair of
electrodes.
17. A method as recited in claim 12, wherein the measurement of the
property of the core is based on a measurement of a property of an
internal control.
18. A method as recited in claim 17, wherein the internal control
is a plurality of known particle of uniform properties.
19. A method as recited in claim 1, wherein the core fluid
comprises substantively polar molecules and the sheath fluid
comprises substantively non-polar molecules.
20. A method as recited in claim 19, wherein a width of the core
between the sheath flows in the channel is controlled, at least in
part, by a width of a strip of material with affinity for the
polarity of the core fluid in at least one of a top wall or a
bottom wall of the channel.
21. A method as recited in claim 19, wherein a width of the core
between the sheath flows in the channel is controlled, at least in
part, by topographical features on at least one of a top wall or a
bottom wall of the channel, which features extend into the
channel.
22. A method as recited in claim 1, wherein the sheath fluid is
mineral oil and the core fluid is an aqueous mixture.
23. A method as recited in claim 1, wherein the sheath fluid is a
fluorocarbon solvent and the core fluid is an aqueous mixture.
24. A method as recited in claim 1, wherein the sheath fluid is a
gas.
25. A method as recited in claim 24, wherein the gas is air.
26. A method as recited in claim 1, wherein the method further
comprises measuring an optical property in the channel.
27. A method as recited in claim 1, wherein the core fluid has a
higher index of refraction for optical waves than do the one or
more sheath fluids.
28. A method as recited in claim 27, wherein measuring the optical
property further comprises directing incident light to produce
substantively total internal reflection within a core flow that
encompasses the core fluid.
29. A method as recited in claim 1, further comprising determining
impedance in the channel at a plurality of pairs of electrodes
disposed separately along the channel and disposed separately from
the first pair of electrodes.
30. A method as recited in claim 2, wherein: the method further
comprises introducing a plurality of uniform particles of known
impedance value into the core flow as an internal control; and,
determining impedance of the particle in the core fluid further
comprises normalizing features of measured impedance based, at
least in part, on measured value of impedance for at least one
particle of the internal control.
31. A method comprising: causing a core fluid to flow into a
channel between two sheath flows of one or more sheath fluids
different from the core fluid, wherein an optical index of
refraction of a fluid constituting either sheath flow is much less
than an optical index of refraction of the core fluid; and
measuring an optical property in the channel between an optical
source and an optical detector.
32. A method as recited in claim 31, further comprising determining
impedance in the channel based on measurements at a first pair of
one or more pairs of electrodes.
33. A method as recited in claim 31, wherein measuring the optical
property further comprises directing incident light to produce
substantively total internal reflection within a core flow that
encompasses the core fluid.
34. A method as recited in claim 31, further comprising determining
a property of a core flow that encompasses the core fluid based on
the optical property measured.
35. A method as recited in claim 31, further comprising determining
a property of a particle in a core flow that encompasses the core
fluid based on the optical property measured.
36. An apparatus comprising: means for causing a core fluid to flow
into a channel between two sheath flows of one or more sheath
fluids different from the core fluid, wherein a dielectric constant
of a fluid constituting either sheath flow is substantively less
than a dielectric constant of the core fluid, and wherein flow in
the channel is laminar; and means for determining impedance in the
channel based on measurements at a first pair of one or more pairs
of electrodes.
37. A non-transient computer-readable medium carrying one or more
sequences of instructions, wherein execution of the one or more
sequences of instructions by one or more processors causes an
apparatus to: receive first data indicating measurements of
impedance in a channel between a first pair of electrodes, wherein
a core fluid flows into the channel between two sheath flows of one
or more sheath fluids different from the core fluid, wherein a
dielectric constant of a fluid constituting either sheath flow is
much less than a dielectric constant of the core fluid, and wherein
flow in the channel is laminar; and determine impedance of a
particle in the core fluid based at least in part on the first
data.
38. A computer-readable medium as recited in claim 37, wherein the
apparatus is further caused to determine control data that
indicates, at least in part, relative pressure of a source of the
core fluid compared to pressure of a source of one or more of the
sheath fluids.
39. A computer-readable medium as recited in claim 38, wherein to
determine control data further comprises to determine control data
to stabilize a value of the first data.
40. A computer-readable medium as recited in claim 38, wherein: the
apparatus is further caused to receive second data that indicates
measurements of a property of the core fluid; and to determine
control data further comprises to determine control data to
stabilize a value of the second data.
41. A computer-readable medium as recited in claim 40, wherein the
second data indicates measurements of width of the core fluid.
42. An apparatus comprising: means for receiving first data
indicating measurements of impedance in a channel between a first
pair of electrodes, wherein a core fluid flows into the channel
between two sheath flows of one or more sheath fluids different
from the core fluid, wherein a dielectric constant of a fluid
constituting either sheath flow is much less than a dielectric
constant of the core fluid, and wherein flow in the channel is
laminar; and means for determining impedance of a particle in the
core fluid based at least in part on the first data.
43. A method comprising: causing a core fluid to flow into a
channel between two sheath flows of one or more sheath fluids
different from the core fluid, wherein a value of a first
electromagnetic property of a fluid constituting either sheath flow
is substantially different from a value of the first
electromagnetic property of the core fluid, and wherein flow in the
channel is laminar; and measuring a second electromagnetic property
in the channel using an electromagnetic signal that is concentrated
in the core fluid by a difference in the value of the first
electromagnetic property of either sheath flow and the value of the
first electromagnetic property of the core fluid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of Provisional Appln.
61/388,916, filed Oct. 1, 2010, the entire contents of which are
hereby incorporated by reference as if fully set forth herein,
under 35 U.S.C. .sctn.119(e).
BACKGROUND OF THE INVENTION
[0003] An impedance-based microfluidic flow cytometer uses a small
channel in combination with control of the flow of fluid streams in
that channel to guide cells or other particles between two or more
electrodes that measure electrical impedance in the vicinity of
each electrode or between pairs of electrodes. This principle has
been applied in various embodiments, including several published
micro flow cytometers. Note that the term flow cytometer is used
loosely herein, as the apparatus can be used for the
characterization of the impedance of a large variety of particles,
not only cells.
SUMMARY OF THE INVENTION
[0004] Techniques are provided for enhanced electromagnetic (EM)
measurements in low-shear laminar flows in microfluidic channels.
Such techniques allow precise electrical impedance measurements of
delicate or force-sensitive particles.
[0005] According to a first set of embodiments, a method includes
causing a core fluid to flow into a channel between two sheath
flows of one or more sheath fluids different from the core fluid.
Flow in the channel is laminar. A dielectric constant of a fluid
constituting either sheath flow is much less than a dielectric
constant of the core fluid. The method further comprises
determining impedance in the channel between at least a first pair
of electrodes. In some of these embodiments, active feedback is
used to control the width of the flow of the core fluid. For
example, in some embodiments, a method includes controlling
relative pressure or flow rate of a source of the core fluid
compared to pressure or flow rate of a source of one or more of the
sheath fluids by controlling the relative pressure or flow rate to
stabilize a measurement of a property of the core fluid. In some
embodiments, the property of the core fluid includes position or
shape of the core flow in the channel, or both. In some
embodiments, the position or shape of the core flow, or both, is
stabilized based on impedance or optical measurements, e.g., to
maintain a certain width or cross-section or position in the center
of the channel or some combination. In some embodiments, the
position or shape of the core flow, or both, is stabilized based on
measurements of known particles included in the core flow.
[0006] In another set of embodiments, a method comprises causing a
core fluid to flow into a channel between two sheath flows of one
or more sheath fluids different from the core fluid. An optical
index of refraction of a fluid constituting either sheath flow is
much less than an optical index of refraction of the core fluid.
The method further comprises measuring an optical property in the
channel between an optical source and an optical detector.
[0007] In another set of embodiments, a method comprises causing a
core fluid to flow into a channel between two sheath flows of one
or more sheath fluids different from the core fluid. A value of a
first electromagnetic property of a fluid constituting either
sheath flow is substantially different from a value of the first
electromagnetic property of the core fluid. Flow in the channel is
laminar. The method also comprises measuring a second
electromagnetic property in the channel using an electromagnetic
signal that is concentrated in the core fluid by a difference in
the value of the first electromagnetic property of either sheath
flow and the value of the first electromagnetic property of the
core fluid.
[0008] According to various other sets of embodiments, an apparatus
comprises means to perform each step of one of the above methods;
or a computer-readable storage medium is configured to cause an
apparatus to perform one or more steps of one of the above
methods.
[0009] Still other aspects, features, and advantages of the
invention are readily apparent from the following detailed
description, simply by illustrating a number of particular
embodiments and implementations, including the best mode
contemplated for carrying out the invention. The invention is also
capable of other and different embodiments, and its several details
can be modified in various obvious respects, all without departing
from the spirit and scope of the invention. Accordingly, the
drawings and description are to be regarded as illustrative in
nature, and not as restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The present invention is illustrated by way of example, and
not by way of limitation, in the figures of the accompanying
drawings and in which like reference numerals refer to similar
elements and in which
[0011] FIGS. 1A-1C are diagrams that illustrates a microfluidic
flow cytometer, according to an embodiment;
[0012] FIG. 2A and FIG. 2B are flow profile diagrams that
illustrate effect of channel width on shear, according to an
embodiment;
[0013] FIG. 3A and FIG. 3B are diagrams that illustrate an
apparatus for dynamic electromagnetic focusing, according to an
embodiment;
[0014] FIG. 4A and FIG. 4B are diagrams that illustrate
electromagnetic measurement apparatus and data, according to an
embodiment;
[0015] FIG. 5A is a block diagram that illustrates in plan view an
example microfluidic electromagnetic measurement apparatus,
according to an embodiment;
[0016] FIG. 5B is a block diagram that illustrates in partial
cross-section view an example microfluidic impedance measurement
apparatus, according to an embodiment;
[0017] FIG. 5C through FIG. 5G are diagrams that illustrate an
apparatus for dynamic electromagnetic focusing and multiple
electromagnetic measurements, according to another embodiment;
[0018] FIG. 6A is a diagram that illustrates channel engineering to
stabilize two-phase flows, according to an embodiment;
[0019] FIG. 6B and FIG. 6C are block diagrams that illustrates
example channel cross sections for a microfluidic electromagnetic
measurement apparatus, according to various embodiments;
[0020] FIG. 6D and FIG. 6E are micrographs that illustrate core
flow in the presence of example rails and electrodes, respectively,
according to an embodiment;
[0021] FIGS. 7A through 7C are diagrams that illustrate core
modulation controlled by a flow rates or pressures or both;
according to various embodiments;
[0022] FIG. 7D is a graph that illustrates example core width
variability based on alternative flow driving mechanisms, according
to various embodiments;
[0023] FIG. 8 is a diagram that illustrates optical confinement of
light in core, according to an embodiment;
[0024] FIGS. 9A and 9B are micrographs that illustrate resting
platelet and an activated platelet, respectively;
[0025] FIG. 10 and is a graph of simulated data of the impedance
spectrum of a mixed population of activated and resting platelets,
according to an embodiment;
[0026] FIGS. 11A through 11F are graphs of simulated histogram data
of the impedance at various frequencies for platelet populations
activated to various degree with adenosine diphosphate (ADP),
according to an embodiment;
[0027] FIG. 12 is a diagram that illustrates electromagnetic
measurement apparatus for multiple frequencies, according to an
embodiment;
[0028] FIG. 13 is a block diagram that illustrates a computer
system upon which an embodiment of the invention may be
implemented;
[0029] FIG. 14 illustrates a chip set upon which an embodiment of
the invention may be implemented;
[0030] FIG. 15A and FIG. 15B are flow charts that illustrate an
example method for determining condition of a subject based on a
microfluidic device, according to various embodiments;
[0031] FIGS. 16A and 16B are graphs that illustrate example
detection of particles in a core flow, according to an
embodiment;
[0032] FIGS. 17A and 17B are graphs that illustrate example
frequency response of resting and activated platelets, according to
an embodiment;
[0033] FIG. 18 is a graph that illustrates example classification
of resting and activated platelets, according to an embodiment;
[0034] FIG. 19 is graph that illustrates example agreement of
degree of platelet activation with a standard, according to an
embodiment; and
[0035] FIG. 20A and FIG. 20B are graphs that illustrate example
detection of platelets and red blood cells in a blood sample,
according to an embodiment.
DETAILED DESCRIPTION
[0036] A method and apparatus are described for enhanced
microfluidic electrical measurements. In the following description,
for the purposes of explanation, numerous specific details are set
forth in order to provide a thorough understanding of the present
invention. It will be apparent, however, to one skilled in the art
that the present invention may be practiced without these specific
details. In other instances, well-known structures and devices are
shown in block diagram form in order to avoid unnecessarily
obscuring the present invention.
[0037] Some embodiments of the invention are described below in the
context of impedance measurements of platelets in a high dielectric
fluid in microfluidic channels of a particular range of sizes.
However, the invention is not limited to this context. In other
embodiments the same or different electrical properties of the same
or different particles are measured in microfluidic channels of the
same or different sizes. The proposed methods bring a substantial
gain in sensitivity over traditional microfluidic approaches, while
minimizing the drawbacks of small channel size (high shear stress
on cells, channel clogging). These improvements pave the way for
the characterization of very small particles (<1 micron, 1
micron=10.sup.-6 meters), and particles that are sensitive to shear
stress (e.g., platelets).
[0038] A microfluidic channel, also called a microchannel herein,
is a channel for fluid flow with a width and height each less than
1000 microns (also called micrometers, .mu.m). Fluid flows in
microfluidic channels are usually laminar rather than turbulent and
have comparatively low Reynolds number values (<1), both
situations resulting from the microchannel dimensions, the flow
rates, and the fluid properties.
[0039] A used herein, a particle is any single object in motion
within a fluid flow, which is small enough to fit within a
microfluidic channel and large enough to affect an electromagnetic
measurement of a fluid property within the channel. Particles
include cells (including bacteria) and portions thereof, including
platelets, as well as dust, pollen and other organic and inorganic
materials.
1. OVERVIEW
[0040] In this section, an overview of the methods and apparati are
given. More detailed embodiments are described in other sections.
Electrical impedance, represented by the symbol Z, is a well known
measure of opposition to alternating current (AC) and combines the
effects of electrical resistance for direct current (DC) and phase
shifts for (AC). Some detailed embodiments are described for
electric impedance measurements of platelets, including a
description of a prototype apparatus for measuring the electric
impedance of platelets. Each reference cited herein is hereby
incorporated by reference as if fully set forth herein, except so
far as the terminology is inconsistent with the terminology used
herein.
[0041] FIGS. 1A-1C are diagrams that illustrate a microfluidic flow
cytometer, according to an embodiment. FIG. 1A shows a perspective
cutaway view of a microfluidic channel 110 (also called
microchannel herein) formed between two side walls 102, a top wall
104, and a bottom wall 106. For some electromagnetic measurements,
such as impedance, one or more electrodes 120 are included on one
or both opposite walls, e.g., a top wall and bottom wall in FIG.
1A. FIG. 1B shows a cross section across the width of the channel.
FIG. 1C shows a cross section along a portion of the length of the
channel. Impedance measured between the pair of electrodes is
related to the total impedance of the particle 132 and the fluid
media 130 between the electrodes 120. An impedance model 140 for a
cell is depicted, with different contributions from cell wall and
cell body represented by different boxes that represent different
parameters of the model. Values for one or more parameters are
estimated based on the impedance measurements at one or more
alternating current (AC) frequencies.
[0042] To first order, the magnitude of an electromagnetic signal
associated with the passage of a particle (including electrical
impedance and optical interaction) is related to the size of the
particle relative to the size of the channel in which the particle
is flowing. If the particle blocks most of the channel when it is
between the sensors (such as electrodes), the signal due to the
passing particle is comparatively larger, and the property can be
measured with higher resolution. If the particle is small compared
to the channel, then the signal is comparatively weaker, reducing
the resolution. This means that, for small particles, the channel
is preferably small in order to produce a detectable change in
signal. A further disadvantage of large channels is that such large
channels can increase the probability of having more than one
particle (co-occurrence) in the detection region of the sensor,
e.g., between the detection electrodes, which could compromise the
detection.
[0043] Unfortunately, small microfluidic channels (widths or
heights <20 .mu.m) imply large shear forces (e.g., compared to
shear forces in the venous portions of an uncompromised circulatory
system) across particles due to the typical parabolic flow profile
in pressure-driven flows. FIG. 2A and FIG. 2B are flow profile
diagrams that illustrate the effect of channel width on shear,
according to an embodiment. Flow direction 131 is as depicted. Flow
profile shows velocity profiles 151, 152, respectively, as a
function of distance from a boundary, such as a channel wall 102.
Velocity is given by the length of an arrow touching the parabolic
curve. When width is small as in FIG. 2A, shear, a measure in this
context of gradients perpendicular to a wall in velocity, is great.
The same rate of flow through a wider channel, as in FIG. 2B,
produces much smaller shears.
[0044] Small channels also are very prone to occlusion when dealing
with samples having significant particle content (e.g., blood). In
particular, for specific applications where shear rates have to be
minimized (e.g., blood platelets, which are activated by shear),
optimization of the channel size for signal strength (small
channel) conflicts with the desire for a large channel to minimize
shear values.
[0045] To circumvent these limitations, several embodiments use
dielectric-based hydrodynamic focusing in order to allow the use of
a large physical channel (hence reduced shear rates), with the
simultaneous advantage of a small electrically conductive channel
that carries the particles. While conductivity is the primary
parameter defining the field and current path through a structure
at direct current (DC) and low alternating current (AC)
frequencies, the dielectric constant and related permittivity are
the dominant factors at high AC frequencies. The exact frequency at
which dielectric properties start to dominate over conductivity
properties is dependent on a number of factors (e.g., actual
electrical parameters of the materials and structure, geometry,
parasitic capacitance/resistance, etc.), but only an appropriate
control of the dielectric/permittivity characteristics of the
channel would allow the contrast between the physical channel and
the electrically conductive channel to be maintained over an
extended frequency range.
[0046] The dielectric constant is a dimensionless ratio of the
permittivity of a substance to the permittivity of free space. It
is an expression of the extent to which a material concentrates
electric flux. Permittivity is determined by the ability of a
material to polarize in response to the electric field, and thereby
reduce the total electric field inside the material. Thus,
permittivity relates to a material's ability to transmit (or
"permit") an electric field (in particular an alternating electric
field); and is dependent on a number of factors, including AC
frequency. Thus, the electric field between two plates passes
preferentially through a material with high permittivity (large
dielectric constant) and around a material with low permittivity
(small dielectric constant). For example, for impedance
measurements, the particles are included in a core fluid with
relative large dielectric constant and kept apart from the walls of
the channel by a different fluid with a relatively small dielectric
constant. The different fluid forms a sheath around the core fluid
and is called hereinafter the sheath fluid. Therefore the electric
field between the electrodes in opposite walls of the channel
passes predominately through the core fluid rather than the sheath
fluids. In other embodiments, the sheath fluid has different values
of other electromagnetic properties, such as different values for
an index of optical refraction. As used herein the core fluid
refers to the material flowing in a core flow, and the core, or
core flow, refers to the spatial distribution of the core fluid,
having such properties as position, width, height, flow rate (mass
per unit time) and velocity.
[0047] This concept is illustrated in FIG. 3A and FIG. 3B, which
are diagrams that illustrate an apparatus for dynamic
electromagnetic focusing, according to an embodiment. FIG. 3A
depicts a plan view of a central channel 310 and two side channels
312. The target of the electromagnetic measurements is one or more
particles in a core fluid introduced into the central channel at
the sample inlet 320. The sheath fluid with the different value of
an electromagnetic property, e.g., a much lower dielectric
constant, is introduced at the sheath flow inlets. The fluids merge
at a junction 314; and the core fluid is confined to a core flow in
a central portion of the channel downstream of the junction, well
away from the channel walls, and, thus, in a portion of the flow
profile where shear is near zero. The sensor for the
electromagnetic measurements, such as a set of electrodes 120 on
top and bottom walls, is also downstream of the junction 314. The
flows leave the device at outlet 316.
[0048] The effect of a low dielectric sheath fluid in the cross
section at the sensor is depicted in FIG. 3B, where the sheath
fluid is light gray, and the core fluid is darker gray, and a
particle 132 of size comparable to the width of the core fluid is
shown as a white oval. Electric field lines 330 between electrodes
120 are depicted as downward pointing arrows, with flux decreasing
as spacing between arrows increases. The electric field flux is
predominately through the core fluid and the particle. The
cross-section emphasizes the localization of the sensing current in
the core (as symbolized by arrow density) due to the dielectric
constant (and thus conductivity) contrast between the core and the
sheath.
[0049] For core and sheath flow of similar viscosities, the flow
profile across the channel will be similar to the classic parabolic
flow of a large channel. Particles or cells traveling in the core
are thus subjected to little shear. However, since the core is more
conductive, the sensing current for the impedance measurement is
confined to the core, mimicking an electrically conductive channel
of the width of the core.
[0050] In the illustrated embodiment, it is very advantageous to
use a sheath fluid with a very low dielectric constant (e.g.,
fluorocarbon solvent or mineral oil), which provides
electromagnetic focusing over a much larger range of AC frequencies
than a water-based sheath most often described in the literature.
For instance, a perfluorocarbon sheath (3M Fluorinert.TM. FC
Series, .di-elect cons..sub.r=1.9, .rho.>10.sup.15 .OMEGA.cm,
where .di-elect cons..sub.r is dimensionless relative permittivity
and .rho. is linear DC resistance in ohm centimeters, .OMEGA.cm,
and 1 cm=10.sup.-2 meters) in a 50.times.50 .mu.m channel (50
microns wide and 50 microns high) with a 10 .mu.m wide core and 10
.mu.m long electrodes, extends the usable AC frequency range for
impedance from 70 megahertz (MHz, 1 MHz=10.sup.6 Hertz, 1 Hz=1
cycle per second) for a deionized water sheath (.di-elect
cons..sub.r=80, .rho..about.10.sup.18 .OMEGA.cm) to more than 360
MHz for the perfluorocarbon sheath.
[0051] In addition, the use of a low dielectric constant liquid
immiscible in water (e.g., fluorocarbon solvents or oils such as
mineral oil) for the sheath fluid provides an abrupt boundary at
the sheath/core interface, as opposed to aqueous-based sheaths,
which are subject to diffusion and `blurring` of the interface.
Such immiscible liquids comprise predominately non-polar molecules
and are said to be hydrophobic. Sheath fluids of different
molecular polarity from the core fluid (called two-phase flows
herein) thus offer the advantage of a sharp boundary and further
offer the advantage of producing small fluid core widths (<10
.mu.m) most useful to measure small particles (<1-5 .mu.m) such
as platelets.
[0052] A disadvantage of two-phase flows is difficulty found in
maintaining a stable flow boundary. An unstable flow boundary leads
to variability in the width of the core and variability in the
electromagnetic measurement that is not related to the passage of
particles. In various embodiments described in more detail below,
adjustments to the apparatus or methods or both are made to
decrease the disadvantageous effects of unstable core width in
two-phase flows.
[0053] While this hydrodynamic focusing is only along one dimension
in the illustrated embodiment, it is enough to reduce the
cross-section of the sensing volume to a size comparable to the
size of the particle to be sensed. The vertical dimension is not
critical with this approach, and can be kept large, thus reducing
shear rates in the vertical direction.
[0054] In some embodiments the particles or core fluid or both are
actively centered in the channel (in the region of lowest shear)
using well-known methods such as dielectrophoresis. Such active
centering further reduces the variability of the measurements and
the shear rates to which the particles are subjected.
[0055] Another benefit of electromagnetic property focusing is the
possibility of achieving the narrow fluid cores (required for high
resolution) in wide channels produced by low-cost techniques, such
as laser-cut channels in pressure-sensitive adhesive. These less
stringent requirements with respect to channel geometry mean
simpler, cheaper fabrication methods can provide measurement
resolution comparable to conventional but expensive,
lithographically-defined microchannels.
[0056] Any method can be used to make the electromagnetic
measurements. For example, in a simple embodiment the electrical
impedance is measured as depicted in FIGS. 4A and 4B. FIG. 4A and
FIG. 4B are diagrams that illustrate electromagnetic measurement
apparatus and data, according to an embodiment. FIG. 4A depicts an
example electrical measurement apparatus using two pairs of
electrodes 402a, 402b. In this embodiment, particle spacing in the
core fluid is controlled so that the typical distance between
particles is greater than the distance between the two pairs of
electrodes. Flow direction is represented by flow 404. Thus when
the particle is in the detection region for the first pair of
electrodes, no particle is in the detection region of the second
pair. The impedance difference between the two pairs is dependent
on the impedance of the particle Rp 412, and independent of the
impedance of the channel, Rc 414.
[0057] The apparatus includes, besides the electrodes 402a, 402b
and the channel carrying the fluids, an excitation module 420, a
differential amplifier 422, a synchronous demodulator 424 and a
computer 426. The AC frequency of the measurement is determined by
the excitation module. Multiple frequencies are usually
superimposed in the excitation signal from the excitation module.
The current measured through the electrodes on opposite walls is
inversely proportional to the impedance. The current difference is
amplified by the differential amplifier and AC phase determined in
the synchronous demodulator. The measurements are sent to a
computer, such as general purpose computer depicted in FIG. 13 or
chip set depicted in FIG. 14, where program instructions are used
to derive the impedance of the cell based on the electrical current
phase and amplitude differences at the two pairs of electrodes. By
using two pairs of electrodes, a differential measurement is
performed, reducing the sensitivity to common-mode noise. FIG. 4B
is graph 450, with time on horizontal axis 452 and impedance change
on vertical axis 454. The impedance data are graphed in FIG. 4B as
trace 460 showing one impedance change as the particle first passes
the first pair of electrodes and then the opposite effect when the
particle passes the second pair of electrodes. In some embodiments,
multiple AC frequencies are summed at the excitation module to
measure impedance at multiple frequencies simultaneously on the
same electrode pairs.
[0058] In some embodiments, enabled by micro-fabrication
techniques, impedance measurements at multiple AC frequencies rely
on the use of multiple sets of electrodes along the channel, each
set including one or more pairs of electrodes and measuring the
same particle as it flows through the set. Such embodiments provide
independent measurements of the impedance at each set, allowing
averaging or other statistical approaches to be used for increasing
the signal-to-noise ratio (SNR). For instance, if the particle is
measured by four sets of independent electrodes, then the averaging
of these four measurements increases the SNR by two. In some
embodiments, using multiple measurements along the channel also
averages out the impedance variation due to possible rotation of a
non-spherical particle within the core. Using other sets of
electrodes as reference also allows adaptive signal processing to
be used, such as for noise removal, in some embodiments.
Furthermore, in some embodiments, the various sets of electrodes
are used to probe different frequencies. Such sequential
measurements of the same particle along the channel are not
obviously available in typical aqueous flows, due to the rapid
diffusion and degradation of the core profile. Such sequential
measurements are much more feasible with two-phase flows, which
conserve a well-defined core over long distance even at low flow
rates.
[0059] The microfluidic flow cytometer can be coupled to other
fluidic functions, such as cell sorting or inline chemical
activation/treatment of cells prior to or after these
electromagnetic measurements.
[0060] As described above, with more details to follow below,
dielectric focusing provides many advantages. When combined with
impedance spectroscopy, these techniques provide increased
signal-to-noise ratio over an extended frequency range (>100
MHz). Low-shear-rate flow cytometry apparatus can still be used to
obtain high impedance resolution. Two-phase (polar/non-polar) flow
further improves dielectric focusing by limiting diffusion and
allowing the use of ultra-low-dielectric-coefficient liquids (e.g.,
fluorocarbon solvents and mineral oil). High-resolution impedance
spectroscopy is therefore possible in even wider,
simpler-to-fabricate channels.
[0061] In an example embodiment, impedance spectroscopy of
platelets is demonstrated in a microfluidic flow cytometer for the
evaluation of platelet activation levels without disturbing
platelet activation state by high shear flows. In some embodiments,
the use of air sheath or hydrophilic tracks in an
otherwise-hydrophobic channel is shown to confine the aqueous core,
thus providing flow with minimal shear rate at the air-water
interface, yet with increased electrical confinement. Precise and
stable core width is achieved in some embodiments through
closed-loop control of sheath and core flows based on continuous
impedance measurement. In some embodiments inner wall surface
features, such as rails, are effectively employed to stabilize
two-phase flows. Such stabilization can be applied to both aqueous
flows and two-phase flows, and could also be used in some
embodiments as a calibration step rather than for real-time
control. Sequential measurements of the same particle along the
channel using multiple independent sets of electrodes allows
separation of AC frequencies or application of signal processing
technique to increase information content (e.g., higher SNR through
averaging, adaptive filtering). Particles with known
electromagnetic properties are also used in some embodiments to
track the actual variability of the core flow.
[0062] Use of low-dielectric sheath fluid (with low refractive
index) is also useful to guide light for optical interrogation of
particles. The low-refractive-index sheath with the
higher-refractive-index core provides in effect an optical
waveguide to focus the light beam onto the particles in the core.
The shape of the core can be assessed by measurement of total
internal reflection. In various embodiments, such optical
measurements with index of refraction focusing are correlated with
simultaneously-acquired electrical (e.g., impedance) measurements,
with or without dielectric focusing. In various embodiments,
optical measurements that do not rely on total internal reflection
are used as well as or instead of optical measurements that do rely
on total internal reflection, with or without electrical
measurements, or with or without dielectric focusing.
2. EXAMPLE APPARATUS EMBODIMENTS
[0063] FIG. 5A is a block diagram that illustrates an example
microfluidic electromagnetic measurement apparatus 500, according
to an embodiment. The apparatus 500 includes microchannels 502
through which a sample input port 504 is connected to a central
microchannel that joins two sheath microchannels each fed by a
sheath fluid input port 506a, 506b. The microchannels from each
input port meet at a junction which connects to an exit port 508
through a portion of the central microchannel. In some embodiments,
additional sample or sheath microchannels are included.
[0064] The sample to be analyzed is introduced at the sample input
port 504, and flows along the central microchannel 502 to the
junction. One or more sheath fluids are introduced at the sheath
fluid input ports 506a, 506b and flows along the side microchannels
502 to the junction. Downstream of the junction the sample fluid
forms a core flow between the sheath fluid flows and all fluids
exit at the exit port 508.
[0065] The apparatus further comprises one or more pressure
actuators (such as pressure controllers or pressure regulators) to
apply fluid pressure at one or more input or exit ports. For
example, in the illustrated embodiment, apparatus 500 includes a
sample pressure actuator 514 that acts at sample input port 504,
and two sheath fluid pressure actuators 516a, 516b that act at
sheath input ports 506a, 506b, respectively. Any method may be used
to apply pressure at the various input ports, for example a servo
motor controlling a syringe can be used, compressed air exerting a
pneumatic pressure on the sample and sheath fluid in a reservoir,
hydrostatic pressure due to a fluid reservoir at fixed elevation
above the main apparatus, or a diaphragm controlled by hydraulics.
In an experimental embodiment, these pressures are applied to the
sample and sheath inlets, with the outlet remaining at atmospheric
pressure. In various embodiments the same or different pressure is
applied at each input port. In some embodiments, some or all of the
actuators are on the chip with the microchannels. In other
embodiments, the actuators are external to the chip that includes
the microchannels. In some embodiments, one or more pressure
actuators 514, 516a, 516b are omitted.
[0066] The apparatus 500 includes a processor/controller 520, such
as a microprocessor or general purpose computer with zero or more
application specific integrated circuits (ASICs) programmed to
control various components of the apparatus. In some embodiments,
the processor/controller 520 controls the pressure actuators 514,
516a and 516b. In some embodiments some or all of the
processor/controller is on the chip with the microchannels. In
other embodiments, the entire processor/controller is external to
the chip that includes the microchannels. In an experimental
embodiment, pressures was either generated by stand-alone syringe
pumps (Cole-Parmer), manually set to a fixed flow rate, height
(hydrostatic pressure from manually raised sample and sheath fluid
reservoir), or pneumatically (compressed air), in which case the
pressure was regulated by a combination of pressure sensor and
proportional pneumatic valve in a closed-loop system
(microcontroller).
[0067] The apparatus 500 includes one or more sample conditioning
components 530 to condition the sample fluid before it becomes the
core fluid downstream of the junction. For example, reservoirs of
one or more reagents are included in conditioning components 530,
such as reagents that stimulate activation of platelets. In some
embodiments, chemical or mechanical filters, mixers or buffers are
included in the sample conditioning components 530, such as
mechanical filters for separating platelets from red blood cells.
In some embodiments, the sample conditioning components include a
source of known particles 540 that are used to determine or correct
for variability in core width, or both, as described in more detail
in a later section. In an experimental embodiment, sample
preparation (dilution, reagent mixing, addition of calibration
particles) was performed manually in separate containers, then
connected to the apparatus 500 for analysis.] In some embodiments
some or all of the sample conditioning components 530 are on the
chip with the microchannels. In other embodiments, all the sample
conditioning components 530 are external to the chip that includes
the microchannels. In some embodiments, the sample conditioning
components 530 are omitted; and, any known particles 540 are
included in the sample fluid at the input port 504. In some
embodiments, the sample conditioning components 530 are controlled,
in whole or in part, by the processor/controller 520.
[0068] The apparatus 500 includes one or more centering components
to center particles in the sample fluid in the core flow. For
example, in the illustrated embodiment, the apparatus 500 includes
dielectrophoresis components 550, e.g., to drive particles toward
the center of the core flow. In some embodiments the centering
components, such as dielectrophoresis components 550, are omitted.
In some embodiments, the centering components, such as
dielectrophoresis components 550 are controlled, in whole or in
part, by the processor/controller 520.
[0069] The apparatus includes one or more core fluid sensors 560,
such as electrodes, as described above, or optical sensors. The
core fluid sensors 560 produce or detect, or both, electromagnetic
signals that, owing to the preferential passage of the
electromagnetic signals through the core (e.g., by dielectric
focusing), are more sensitive to perturbations that occur in the
core fluid, such as the passage of a particle. In some embodiments,
the core fluid sensors 560 are monitored or controlled, in whole or
in part, by the processor/controller 520.
[0070] FIG. 5B is a block diagram that illustrates in partial
cross-section view an example microfluidic impedance measurement
apparatus, according to an embodiment. In this embodiment, a custom
impedance spectroscopy system is composed of a lock-in detector
(SR830 DSP, Stanford Research Systems, Sunnyvale, Calif.) 564, a
custom differential current-voltage converter 566, and a
computer-controlled acquisition system 520. This system gave
satisfactory results; however, it was limited in frequency to 100
kHz. For measurements at higher frequencies, a multi-frequency
impedance spectroscopy system from Zurich Instruments was used, and
greatly improved measurement capabilities in other embodiments.
This system was capable of simultaneously measuring six frequencies
up to 50 MHz (10 MHz effectively once parasitic capacitance of
various chip components was taken into account). All platelet
characterizations, described in a later section, were carried out
using this system. Typical excitation voltages were kept under 1
Volt (V) per excitation frequency to avoid heating of the sample.
Sampling speed was 7.8 kHz in order to fully capture the transient
impedance change.
2.1 Multiple Sets of Detection Electrodes
[0071] FIG. 5C through FIG. 5G are diagrams that illustrate an
apparatus 570 for dynamic electromagnetic focusing and multiple
electromagnetic measurements, according to another embodiment. The
example microfluidic impedance cytometer uses a fluidic layer
fabricated from 50-.mu.m-thick, double-sided adhesive film
(Adhesives Research, Glen Rock, Pa.) using a CO.sub.2 laser
engraving and cutting system (Universal Laser Systems, Scottsdale,
Ariz.). The device 570 includes sample inlet 571 and two sheath
inlets 572a, 572b and focusing electrodes 573 and outlet 578. Three
sets of electrodes 574 (for single or repeated measurements), each
comprising two pairs of 20-.mu.m-wide platinum electrodes,
separated by 100 .mu.m, patterned on Borofloat.RTM. glass using
standard photolithography and sputtering, were used for
differential measurements. The fluidic layer was sandwiched between
two glass chips 582 (see FIG. 5D) and bonded using a hot plate at
70.degree. C. and a hand roller. Fluidic connections were
fabricated from polydimethylsiloxane (PDMS) and bonded to the top
glass chip with the aid of air plasma that was used to chemically
activate the surface to improve adhesion.
[0072] FIG. 5C shows a plan view of the channel layer with two
sheath inlet channels 572a, 572b flanking a central sample inlet
channel 571 that meet at a common junction beyond which is a common
channel ending in an outlet 578. The inlet channels are about
10-millimeter long (mm, 1 mm=10.sup.-3 meters), as is the common
channel. In the common channel downstream of the junction are
focusing electrodes 573 that cause the particles to flow near or at
the center of the common channel using dielectrophoresis. Farther
downstream are three sets of detection electrodes 574, each set
comprising two pair of electrodes for comparative measurements of
each particle. The common channel narrows appreciably in the
vicinity of the detection electrodes from 1000 .mu.m to 350 .mu.m
in order to facilitate the focusing of narrow cores.
[0073] FIG. 5D shows a cross sectional view in a plane
perpendicular the long dimension of the channel. The channel is 50
.mu.m high, 350 .mu.m wide with a high conductive and high
dielectric constant core fluid 583 occupying a core flow only about
20 .mu.m wide at the center of the channel and flanked by
dielectric sheath fluids 585. The top and bottom walls are glass
wafers 582 upon which one or more of the electrodes are
supported.
[0074] FIG. 5E depicts three dimensional (3D) computed aided design
(CAD) drawings of the microfluidic chip exploded by layers 590a,
590b, 590c. FIG. 5F is a photograph of bonded chip 592. FIG. 5G is
a photograph of a complete example microfluidic cartridge 594 with
ports 596 for fluidic connections and electrical contacts.
2.2 Channels for Two-Phase Flows
[0075] In some embodiments, as described above, two-phase flows are
used. For example, in some embodiments an aqueous core fluid (polar
water molecules) is used with sheath fluids comprising non-polar
molecules. FIG. 6D and FIG. 6E are micrographs that illustrate core
flow in the presence of example rails and electrodes, respectively,
according to an embodiment. FIG. 6E shows an example of such a
flow, with a plan view of electrodes 678 and a core fluid 680
flowing between immiscible sheath fluids 684. A 100 micron scale
bar 694 is depicted. However, achieving stable, reproducible
two-phase flows in microchannels has been notoriously difficult,
and most of the solutions found in literature require the use of
high-viscosity oils as sheath fluid, generating large shear at the
interface core/sheath. In order to allow the use of less viscous
fluid (such as fluorocarbon solvents with very low dielectric
constants), a method based on work by Zhao, Moore and Beebe
(Analytical Chemistry, Vol. 74, No. 16, Aug. 15, 2002) using
surface energy patterning is used in some embodiments, as depicted
in FIG. 6A.
[0076] FIG. 6A is a diagram that illustrates channel engineering to
stabilize two-phase flows, according to an embodiment. In channel
formed by side walls 602a, 602b, top wall 604 and bottom wall 606,
an aqueous core fluid 610 is guided by high-surface-energy
(hydrophilic, .gamma..sub.2) tracks 622 in a low-surface-energy
(hydrophobic .gamma..sub.1) coated 624 channel. In some
embodiments, these tracks are patterned by prior hydrodynamic
focusing with fluids containing surface modification chemicals,
resulting in a self-aligned process. In some embodiments, the
sheath fluid is simply air (or another gas), simplifying the
fluidics and reducing shear rates at the core/sheath interface. It
is believed that the application of such a gas as a sheath fluid to
provide reduced shear rates has not been suggested yet.
[0077] For example, in some embodiments, the channel surface
modification are self-aligned using hydrodynamically-focused
laminar flows containing hydrophobic (non-polar) molecules in a
sheath flow and hydrophilic molecules in a core flow. These
molecules are selected so that they attach to the surface as they
flow through the channel. Once the surface has been modified, the
channel is flushed and the fluids and particles of interest for
measurement are introduced into the device. The hydrophilic `lane`
then stabilizes and guides the aqueous core in its desired path
along the center of the channel. The guiding effect provided by the
patterned surface energies can also be augmented by topographical
cues (slight height difference between the high and low surface
energy coatings). Including such surface patterning provides a
geometrically stable and spatially reproducible core.
[0078] Experiments were performed using this surface chemistry
approach. In some embodiments, standard silane chemistries
(combinations of fluorosilanes and aminosilanes), as well as Bovine
Serum Albumin (BSA) or Pluronic F68 (an amphiphilic copolymer from
BASF, Ludwigshafen, Germany) adsorption were used. In some
embodiments, a micro-stamping method was used as a way to define
the hydrophobic/hydrophilic track.
[0079] In some embodiments, topographical cues were used, alone or
in combination with surface chemistry. FIG. 6B and FIG. 6C are
block diagrams that illustrates example channel cross sections for
a microfluidic electromagnetic measurement apparatus, according to
various embodiment. FIG. 6B shows a single rail 670 surface
topography on both top and bottom walls. In this embodiment, a
boundary between a core fluid 680 and each immiscible sheath fluid
684 is expected to follow an edge of the single rail 670. FIG. 6C
shows multiple rail 672 surface topography. In this embodiment, a
boundary between a core fluid 680 and each immiscible sheath fluid
684 is expected to follow an edge of one rail of the multiple rails
672. For example, tracks and ridges were defined in photoresist or
SU-8 along the channel to physically guide the core. These methods
were successful in bare channels. Thus rails were observed to
provide an advantage in stabilizing two-phase flows. In some
embodiments, the rails are added to only one of the top and bottom
walls. FIG. 6D shows a plan view of a example rail 670 and a core
fluid 680 flowing between immiscible sheath fluids 684, according
to an embodiment. A 50 micron scale bar 692 is depicted.
[0080] However, the addition of electrodes on the surface
noticeably disrupted the flow. Ultimately, a combination of
topographical and surface modification is anticipated to achieve a
reliable, stable two-phase flow at those dimensions and flow
rates.
2.3 Dynamic Modulation of Core Width
[0081] In some embodiments, the width of the core is modulated to
some extent dynamically by varying the differential pressure
between the core and the sheath. In some embodiments, this
principle is used to provide a variable width of the flow of the
core fluid (called the core flow hereinafter). Such embodiments
allow rapid reconfiguration of core flow width for optimally
sensing particles of different sizes, all with the benefits of low
shear and high resolution. In some embodiments, the impedance of
the core, as measured by the detection electrodes, is used to
estimate its width. For example, the impedance of the core absent a
particle, or the average minimum impedance over a time interval
during which one or more particles have passed, is used to
determine width. In some embodiments, a width calibration curve is
generated through experiment. In other embodiments other properties
are measured, such as the optical measurements of core properties
described in the next section.
[0082] In some of these embodiments, the measurements, such as the
impedance or optical measurements are used to control the flows
(such as by controlling the pressure) in a feedback loop to set
precisely the core to a pre-determined width. For example, by
maintaining or adjusting the pressure to stabilize an optical or
impedance measurement at a predetermined value associated with the
desired width. In various embodiments, such control is implemented
in a separate calibration step, or is accomplished in real time
(e.g., determining an average minimum value for a time
interval).
[0083] In some embodiments, the width of the core flow is
calibrated based on the measured impedance of the known particles
540 depicted in FIG. 5A, such as mono-disperse polystyrene beads.
Such beads, of calibrated diameters, generate a control signal that
can be used to estimate core properties, or normalize the signal
(as described in more detail below). This normalization accounts
for varying flow rate, core flow width due to boundary instability
between oil sheath and core fluid, and core fluid conductivity
differences of different samples.
[0084] FIGS. 7A through 7C are diagrams that illustrate core
modulation controlled by flow rates or pressures or both; according
to various embodiments. FIG. 7A shows a wide core flow 710 caused
by a positive core pressure 712(P2) greater than sheath flow
pressure 714 (P1), and an associated impedance value 716 Z1 absent
a particle (e.g., at a minimum measured impedance). FIG. 7B shows a
moderate width core flow 720 caused by a small relative pressure of
core pressure 722 (P2) about equal to sheath flow pressure 724
(P1), and an associated impedance value Z2 726 absent a particle.
FIG. 7C shows a narrow core flow 730 caused by a negative relative
pressure of core pressure 732 (P2) less than sheath flow pressure
734 (P1), and an associated impedance value 736 Z3 absent a
particle. By decreasing the sheath flow pressure (P1) when the
impedance absent a particle is between Z2 and Z3, and increasing
the sheath flow pressure (P1) when the impedance absent a particle
is between Z1 and Z2, the core width can be stabilized to that
shown in FIG. 7B.
[0085] Because width is the narrowest dimension of the core flow,
these embodiments amount to controlling a narrowest spatial
dimension of the core fluid by controlling relative pressure of a
source of the core fluid compared to pressure of a source of one or
more of the sheath fluids. To stabilize the core width, some of
these embodiments involve controlling the relative pressure to
stabilize a measurement of a property of the core fluid, such as
its impedance, its impedance absent a particle (e.g., average
minimum impedance during a time interval) or optical measurements
of width, alone or in some combination.
[0086] Experiments were performed that showed pressure-induced flow
provided an advantage over pulsatile syringe-pump-induced flow used
in some embodiments. As expected, flows generated with syringe
pumps exhibited pulsatile behavior due to the stepping action of
the motors that drive these pumps. Because of the differential
nature of the measurement, the impact of these pulsations was
strongly reduced. However, these pulsations were still contributing
to some residual instabilities in the flow, and likely to some of
the variability in the final data. To improve on this,
pressure-controlled flows were implemented for the sheaths (the
most sensitive to pulsation) in some embodiments. In one
embodiment, hydrostatic pressure was generated by raising the fluid
reservoir (gravity-flow) between 1 and 20 cm above the chip level
(usually by filling a pipette to the desired height). In a second
embodiment, a closed-loop system was used to provide constant fluid
pressure. This system used a proportional pneumatic valve to
pressurize the fluid reservoir with air. A pressure sensor in line
with the fluid connection to the chip was sensing the fluid
pressure, and feeding it to a closed-loop control system regulating
the proportional valve. This system effectively regulated sheath
inlet pressure with a fixed, selectable pressure.
[0087] This approach greatly reduced signal variation, and improved
overall stability of the core. FIG. 7D is a graph 770 that
illustrates example core width variability based on alternative
flow driving mechanisms, according to various embodiments. The
horizontal axis 772 is time in seconds (s); the vertical axis 774
is voltage proportional to deviations in width of the core flow.
Traces are shown for syringe-driven flows that exhibit a large
variability 782 in widths. In contrast, traces are shown for
pressure-driven flows that have much lower variability 784 of
widths. Based on these data, any non-pulsatile, constant-pressure
fluidic system is advantageous for future implementation. In
addition to simplifying sample handling, some implementations of
such an approach have also the merit of being more easily
integrated into a point of care (POC) system design.
2.4 Optical Measurements of Core Flow
[0088] The concept of electromagnetic property focusing can be used
not only to focus electrical currents, but also, or alternatively,
light beams. By using a core with a higher refractive index than
the sheath (a condition readily obtained with an aqueous core and a
fluorocarbon sheath), one can achieve the conditions for total
internal reflection of light inside the core, depending on the
angle of incidence of the light. This optimally focuses the light
within the core, where it can be used to interrogate passing
particles. Any optical measurement can be used in various
embodiments, such as fluorescence, scattering, absorption,
diffraction or other optical interactions, with or without total
internal reflection, with or without impedance measurements, and
with or without dielectric focusing.
[0089] FIG. 8 is a diagram that illustrates optical confinement of
light in the core, according to an embodiment. The index of
refraction in the sheath fluid 824 is n1, in the core 810 is n2,
and in the bottom wall 806 is n3. The core fluid has a higher
refractive index than the sheath fluid (n.sub.2>n.sub.1). With
both n2 and n3 greater than n1 of the sheath, light is reflected
away from the sheath fluid. With n2 and n3 similar in value, light
easily enters the core which acts as wave guide, emitting most of
the light into the top wall 804 with an index of refraction also
close to that of the core fluid. Side walls 802a, 802b can have any
index of refraction. Since refractive index is directly related to
relative permittivity, the conditions for good electrical focusing
(low dielectric sheath) are ideally suited for such optical
confinement, allowing simultaneous optical and electrical
interrogation.
[0090] Optical interrogation can then be coupled to electrical
interrogation to provide enhanced information on the measured
particles. In a similar application to that shown in FIGS. 7A
through 7C, optical interrogation can also be used to evaluate the
width of the core, since internal reflection will be modified by
the angle of the core/sheath interfaces. In some embodiments,
optical focusing in the core is used even without dielectric
focusing in the core.
3. EXAMPLE METHOD
[0091] FIG. 15A and FIG. 15B are flow charts that illustrate an
example method for determining condition of a subject based on a
microfluidic device, according to various embodiments. Although
steps are depicted in FIG. 15A and FIG. 15B as integral steps in a
particular order for purposes of illustration, in other
embodiments, one or more steps, or portions thereof, are performed
in a different order, or overlapping in time, in series or in
parallel, or are omitted, or one or more additional steps are
added, or the method is changed in some combination of ways.
[0092] In step 1501, a sample is collected from a subject. For
example, a blood sample is collected from a patient. In a
calibration embodiment, a sample with known values of one or more
properties, such as known percentage of activated platelets, is
collected during step 1501.
[0093] In step 1503, the sample is prepared prior to introduction
to the apparatus. For example, the sample is diluted or filtered or
prepped in any other manner known in the art. In one embodiment,
the blood sample is centrifuged to separate the platelet rich
plasma (PRP). The platelets are then re-suspended in Tyrode's
buffer with added chemicals (such as bovine serum albumin, acid
citrate dextrose, apyrase) to limit aggregation and activation. In
some calibration embodiment, the prepared platelet sample is used
as-is (resting sample, usually containing more than 90% resting
platelet in healthy subjects), or mixed with a thrombin
receptor-activating peptide (TRAP) to force platelet activation and
generate a calibration sample with a high percentage (80-90%) of
activated platelets (activated sample). In some embodiments, the
known particles 540 are added to the sample, for example at a known
concentration. In some embodiments, one or more sample preparation
steps are performed in the apparatus, as described below with
reference to step 1517, and the preparation performed in step 1503,
if any, is complimentary to preparation performed in the
apparatus.
[0094] In step 1510, the prepared sample is processed in the
apparatus, such as apparatus 500 described above with reference to
FIG. 5A. One or more steps performed in the apparatus are described
in more detail below.
[0095] In step 1590, the condition of the subject from whom the
sample was collected, or a calibration result, is determined based
on the properties derived by the apparatus. For example, the
apparatus is determined to be calibrated, or the patient is
determined to have normal or abnormally elevated numbers of
activated platelets. In some embodiments, the apparatus performs
other ancillary analyses that allow a more comprehensive
determination in step 1590. For example, in some embodiments, based
on the presence of another marker together with the results of the
assessment of platelet population, it is determined that a patient
from whom the sample was drawn is at increased cardiovascular
health risk. In another example, changes in proportions of
activated platelets before and after taking a specific may reflect
the efficacy of this drug, or on the contrary the resistance of the
subject to this drug.
[0096] In an illustrated embodiment, step 1510 includes one or more
of steps 1511 through 1530. In step 1511, the sample is introduced
at an input port, such as sample input port 504. For example a
pipette containing the prepared blood sample is emptied into a
chamber in the apparatus connected to the input port 504. In step
1513 one or more sheath fluids (such as immiscible sheath fluids in
a preferred embodiment) with different electromagnetic properties
from the sample fluid are introduced at two sheath input ports,
such as sheath fluid input ports 506a and 506b. For example, a
reservoir connected to the input ports 506a, 506b, is filled with
mineral oil that has higher electrical impedance and lower
dielectric than the prepared blood sample.
[0097] In step 1515, the fluids at the input ports are subjected to
pressures to induce stable fluid flow of sample fluid through the
central microchannel. For example, the sample fluid chamber and the
reservoir of sheath fluids are pressurized, respectively, by
separate diaphragms actuated by a pressure source, such as a
hydraulic fluid pressurized by a piston or a column of water or
other fluid. In another embodiment, the pressurization may be
effected by application of a regulated air pressure directly to
each fluid. In some embodiments, as described above, stability is
provided in part by dynamically adjusting the pressure applied to
the sample fluid chamber or reservoir of sheath fluid, or both,
based on observed core properties, such as width, detected during
step 1530, described below. As a result of the pressurization, the
sample fluid flows as a core flow between two sheath flows through
a microchannel to the exit port, e.g., through microchannels 502
toward exit port 508.
[0098] In step 1517, any on-chip conditioning is performed, e.g.,
as the sample flows through the sample conditioning components 530.
For example, filtering, buffering, diluting and platelet
activation, alone or in some combination, is performed on the
sample passing through the sample conditioning components 530. In
some embodiments, the conditioning components 530 add known
particles 540 at a known rate into the microchannel carrying the
sample.
[0099] In step 1519, the conditioned sample is caused to be
sheathed by the sheath fluid. For example, the microchannels 502
carrying sheath fluid meet the microchannel carrying the
conditioned sample at a junction.
[0100] In step 1521, particles in the sample are driven toward the
center of the core flow, e.g., using dielectrophoresis induced by
the dielectrophoresis components 550.
[0101] In step 1523, the electromagnetic properties are detected in
the core flow, e.g., using core fluid sensors 560. The detection is
concentrated in the core flow by the difference in the
electromagnetic properties of the sample and sheath fluids. For
example, optical transmission or electrical impedance is measured,
with greater sensitivity to perturbations that occur in the core
flow, as described above.
[0102] In step 1530, properties of one or more particles in the
sample fluid are derived based on the detected electromagnetic
properties. For example, temporal or spatial variations in the
electromagnetic properties of the core flow are used to
characterize particles carried by the core flow. Variations in the
properties of known particles 540 are used to determine variations
in the core flow, such as core width or core fluid average
properties, and used, in some embodiments, to dynamically adjust
the pressures on the sample chamber or sheath fluid reservoir, or
both, in step 1515. Such dynamic adjustments can be achieved by
ultra-rapid pressure-based flow controller such the Fluigent
Microfluidic Flow Control System (FASTAB Technology, Fluigent,
Paris, France). For example, the percentage of activated platelets
is determined based on the detected electrical impedance properties
of a number (e.g., at least 50) of platelets.
[0103] In some embodiments, step 1530 includes determining
properties of the core flow itself, such as rate or width or both.
If the impedance of the core flow is directly measured with a just
one pair of electrode, then the core width and average properties
can also be detected. This is an advantageous mode of sensing the
core properties in the absence of particles. Also, this
determination can be done anytime--not only when a calibration
particle passes between the electrode, and, thus, can provide the
advantage of a faster real-time control of the core flow
properties.
[0104] In some embodiments, step 1530 includes one or more of steps
1531 to 1551 of FIG. 15B. In step 1531, the magnitude of the
impedance variation is computed at one or more electromagnetic
frequencies. For example, magnitude= (X.sup.2+Y.sup.2), where X is
the in-phase component of the signal and Y is the out-of-phase
component. Baseline wander (due to slow flow fluctuation for
instance) is removed from one or more electromagnetic frequencies,
e.g., using a Savitsky-Golay filter. In an experimental embodiment,
the slow variations (baseline wander) are extracted with a
Savitzky-Golay filter of low order (e.g., 3), and a frame size
longer than the slowest particle events (e.g., 70 ms, or 501
samples at 7.2 kilosamples per second, ksps, where 1 ksps=10.sup.3
samples per second). These baseline wanders are then subtracted
from the original magnitude signals for each frequency to leave
only fast, particle-related events.
[0105] In step 1533, wavelet decomposition is performed at one or
more electromagnetic frequencies of the measured impedance. Due to
the properties of wavelet transforms, wavelet-based algorithms are
inherently well-suited for the detection of transient events (see,
for example, S. Mallat, A Wavelet Tour of Signal Processing, 2 ed.
San Diego, Calif.: Academic, 1999, the entire contents of which are
hereby incorporated by reference as if set forth herein, except so
far as the terminology is inconsistent with that used here). The
algorithm used typically two levels of wavelet decomposition to
increase robustness. In one embodiment, a quadratic spline wavelet
(see, for example, C. Li, C. X. Zheng, and C. F. Tai, Detection of
ECG characteristic points using wavelet transforms, IEEE Trans.
Biomed. Eng., vol. 42, no. 1, pp. 21-28, January 1995, the entire
contents of which are hereby incorporated by reference as if set
forth herein, except so far as the terminology is inconsistent with
that used here) is used to perform a dyadic wavelet transform of
the impedance magnitude up to scale 5. Scales 4 and 5 were found
typically to have the most frequency overlap with particle events.
Triphasic patterns in the wavelet decomposition, characteristic of
the biphasic impedance change measured by the differential
measurement configuration, are then searched in scales 4 and 5,
typically. This search is done by finding peaks above a defined
threshold (set manually or calculated from an estimate of the
signal noise), and interpreting the timings of these peaks (two
maximum close to a minimum are qualified as an event). Events
detected on each scale are then pooled to increase the robustness
of the particle detector to noise and variability in event shapes,
and flexibility to various size and types of particles.
[0106] In step 1535, the presence of a particle is determined using
the WT-based algorithm described above based only on the magnitude
signal at the lowest frequency (found to be the most sensitive to
the passage of particles).
[0107] In step 1537, the real and imaginary part of the impedance
variation at each of one or more electromagnetic frequencies is
determined by extraction of peak amplitudes in in-phase components
(X) and out-of-phase components (Y) for each frequency.
[0108] In step 1538, the magnitudes and phases are then derived. In
some embodiments, only amplitude is used, because experiments
showed that phase was mostly redundant. The particle or platelet is
then characterized by a vector of the relative real components,
imaginary components, magnitudes or phases or any combination of
one or more electromagnetic frequencies and zero or more derived
quantities, such as opacity features (ratio of impedances at two
frequencies).
[0109] In step 1539, amplitudes so detected are normalized by
internal controls, such as the measured impedances and derived
parameters of the known particles 540. For example, because of the
remaining variability of the fluidics (core size, flow, centering
in channel, sample fluid variations among samples, among others),
the best results were obtained when all platelet parameters
(impedances and derived parameters) were divided by the averaged
values measured over the duration of the sample analysis for 10-mm
polystyrene beads added to the solution as an internal control. In
other embodiments, the normalization is performed using the value
of the nearest calibration bead in the sample stream, or
interpolated between two neighboring calibration beads. Beads can
be identified in the data sets based on a simple magnitude
threshold reflecting large size difference between the beads used
as internal controls and target particles. In other embodiments,
other parameters are used to identify the beads.
[0110] In step 1541, the process diverges based on whether
calibration or sample analysis is being performed. If calibration,
the next step is 1543. If the system is already calibrated and is
being used to detect properties of particles in an unknown sample,
such as platelets, then the next step is 1551.
[0111] In step 1543, the vectors associated with known values of
the particle properties are analyzed by cluster analysis to
determine how to classify such particles, e.g., by defining the
center and envelopes of vectors that represent each class. In some
embodiment, the classes are sufficiently separated that a simple
histogram analysis can separate and quantify the various
populations, such as between 10 .mu.m polystyrene beads and 5 .mu.m
polyamide beads, or platelet and red blood cells (as described
below with reference to FIG. 20B). For populations exhibiting more
subtle differences in impedances (e.g., activated vs. resting
platelet, FIGS. 17A and 17B), statistical approaches are employed.
In various embodiments, a principal component and discriminant
analysis were used. For example, discriminant analysis is
performed, which differentiates between activated and resting
platelets within each calibration sample. Measurements at multiple
frequencies are taken from two samples--one containing resting
platelets and 10-.mu.m polystyrene beads, the other containing
activated platelets and the same 10-.mu.m polystyrene beads. Each
sample is typically composed of more than 90% of the dominant
species--resting or activated. First, data from beads are extracted
from the data sets (based on simple magnitude threshold reflecting
large size difference between the beads and platelets). Beads
parameters are averaged over all detected beads and used to
normalize all platelets parameters. Then a discriminant analysis is
performed on the pooled platelet parameters, with all the platelets
coming from the resting sample labeled `resting`, and all platelets
coming from the activated sample labeled `activated`. The
discriminant analysis projects the data sets onto canonical vectors
that have the most discriminating effect on the dataset. These
vectors, and the associated class centroids, can then be used to
re-classify each sample, or applied to new samples as in step
1551.
[0112] In step 1551, the probability of an unknown particle to
belong to the resting platelet or activated platelet class is
determined based on previously-established classification vectors.
Measurement of many platelets will thus characterize the
population, and lead to a number reflective of the degree of
platelet activation of the sample (percentage of activated
platelet). Alternatively, a score based on the unknown platelet's
parameter projections on these calibration vectors can be used to
provide a degree of activation for each platelet, rather than a
bimodal classification.
[0113] In order to validate the dielectric hydrodynamic focusing
concept, experiments with particles (polystyrene particles with a
10 .mu.m diameter) were performed. These experiments enabled the
quantification of the performance improvement brought by the
proposed approach, These techniques were shown to be capable of
simple size discrimination based on the impedance of a particle at
a single electromagnetic frequency (1.2 MHz) in which 10 micron
particles are polystyrene beads, and 5 micron particles are nylon
beads from a blood-mimicking solution (Supertech CIRS 046).
[0114] Important results include the large improvement brought
about by the two-phase flow in both sensitivity and signal-to-noise
ratio (SNR) compared to standard aqueous hydrodynamic focusing. In
the case of aqueous hydrodynamic focusing, the increase in SNR
expected from the reduction of the core is negated somewhat by the
diffusion effects, increasing relatively at smaller core width. In
contrast, two-phase focusing gets the full benefits from the narrow
core, since diffusion is substantively non-existent. Average
detection signal, .DELTA.I/I.sub.baseline, was determined for the
passage of single 10-micron polystyrene beads in a phosphate
buffered saline (PBS) solution with a range of core widths from 27
to 145 microns (all aqueous) and for a two-phase flow (oil sheath,
33 micron core width). For a fixed excitation voltage of 400
milliVolts, root mean square (mVrms, 1 mV=10.sup.-3 volts), a
detection signal of 1.4.+-.0.6% is measured for a 27-micron aqueous
core, whereas the two-phase system has a signal of 5.1.+-.0.5% for
averages of 21 measurements. The signal-to-noise ratio for the
various core sizes showed no significant dependence on core size
for the aqueous cores. For the two-phase system, a 23 dB
improvement compared to the smallest aqueous core is observed.
[0115] Using the core fluid impedance sensors FIG. 5B, traces (real
part of impedance, after baseline-wander removal) of 10 .mu.m
polystyrene particles in oil/water flow were plotted. FIGS. 16A and
16B are graphs that illustrate example detection of particles in a
core flow, according to an embodiment. FIG. 16A depicts a typical
trace of these particles. The horizontal axis 1602 is time in
seconds; and, the vertical axis is impedance difference between
successive electrodes as outputted in volts by the measuring
instrumentation (lock-in amplifier). The trace 1610 is near zero
when there is no particle between either pair of electrodes, and
the trace 1610 spikes both negatively and positively as a particle
passes the core fluid sensors (differential configuration). FIG.
16B depicts a close-up of individual signals from individual
particles, from a separate experiment. The horizontal axis 1622 is
an expanded time axis in milliseconds; the vertical axis 1624
represents the impedance change as outputted in volts by the
measuring instrumentation. As shown in FIG. 16B, each peak includes
a decrease in voltage as the particle passes between the first pair
of electrodes followed by an increase as the particle passes
between the second pair of electrodes.
4. DETERMINING PLATELET ACTIVATION STATE
[0116] According to an example embodiment, the techniques are
applied to determine the number or fraction of platelets in an
active state. These embodiments address a clinically important
application related to the measurement of platelet activation in
blood, and embodies several aspects of the invention, including
measurement of micron-size, shear-sensitive cells.
[0117] FIGS. 9A and 9B are micrographs that illustrate a resting
platelet and an activated platelet, respectively. FIG. 9A is
magnified 10,000 times and 9B only 5000 times. These micrographs
reveal the marked difference in size and morphology that
characterizes activation. Blood platelets are anuclear and discoid,
1.5 to 3.0 .mu.m in diameter. The human body has a limited reserve
of platelets which, therefore, can be depleted rapidly. Platelet
activation has been linked to the presence of disease, and
therefore is an important disease indicator.
[0118] Most previously available tests characterize platelet
function as a consequence of challenge by a chemical or physical
agonist (with variable predictivity) without direct measure of the
endogenous platelet states in blood circulation. Direct indication
of the endogenous activation state is currently only available
through the use of flow cytometry, primarily by measuring the
presence of certain proteins and receptors known to be expressed
(e.g., CD62p, also known as P-selectin) or known to change
conformation (e.g. GPIIb/IIIa) upon activation (Michelson 2000).
These methods involve labeling the relevant proteins with one or
more fluorophores or other labels for optical detection. These
methods are very sensitive, quantitative, and provide information
on individual platelets. They are, however, expensive,
time-consuming, and mostly found in specialized or central
laboratories.
[0119] One of the main challenges with applying the simpler,
cheaper and faster techniques of impedance spectroscopy to
platelets is the small size of the platelets. Small platelets will
generate a very weak impedance signal if the channel in which they
flow is too large. Reducing channel size, to achieve a signal to
noise ratio (SNR) that is acceptable for measurements, generates
shear that is not suitable for platelets, stressing them
sufficiently to damage them or change their activation state.
[0120] In an illustrated embodiment, integrated fluid flow and
electrical finite-element modeling (FEM) are used to properly
dimension channels and flow rates to limit shear stress, while
achieving optimal signal-to-noise ratios in the impedance spectrum
measurement. For example, the COMSOL Multiphysics software package
for Matlab.RTM. is used to perform integrated fluidic and
electrical finite-element modeling simulations. High-resolution
spectra over a large frequency range are built
frequency-by-frequency, averaging multiple platelets (n=200-500)
for each frequency. Mixed populations of activated and
non-activated platelets are delineated statistically. Indeed, it is
not practical to obtain a population of 100% non-activated or 100%
activated platelets as reference, for various practical reasons,
such as residual activation level and aggregation. Instead, samples
with varying levels of activation are used and the distributions of
impedances is correlated with these levels. This enables the
association of specific impedance distributions with activation
levels for each individual frequency.
[0121] For example, in an example embodiment, measurements are used
to characterize the impedance at 20-30 frequencies, over a
frequency range of 100 Hz-50 MHz. This range should cover three
domains of interest: low frequencies, where the platelet is
basically non-conductive (signal proportional to size), medium
frequencies, where the signal is sensitive to membrane properties
(.beta. dispersion), and high frequencies, where the signal is
related to the cytosol structure and properties. It should be noted
that while the lower limit of 100 Hz might be practically difficult
to achieve (electrode polarization, measurement time), the
information between 100 Hz and 1-10 kHz is not expected to be
critical. Analysis of the high-resolution spectra allows
determination of a subset of frequencies having the best
discriminating power. The measurement setup allows simultaneous
probing of these frequencies, allowing real-time characterization
of individual platelets.
[0122] FIG. 10 is a graph 1000 of simulated data of the impedance
spectrum of a mixed population of activated and resting platelets,
according to an embodiment. The horizontal 1002 axis is frequency
in arbitrary units and the vertical axis 1004 is simulated
impedance in arbitrary units. FIG. 10 shows a simulated spectrum of
impedance measurements 1008 at different AC frequencies. FIGS. 11A
through 11F are graphs of simulated histogram data of the impedance
at various frequencies for platelet populations activated to
various degree with adenosine diphosphate (ADP), according to an
embodiment. Each horizontal axis is 1102 impedance in relative
units; and each vertical axis 1104 is count, in relative units. In
this simulated case, spectra are clearly different between
activated and resting platelets. The histograms on the left (FIG.
11A, FIG. 11C and FIG. 11E) show the relative contributions of
activation states for a low degree of activation at three discrete
AC frequencies (f1, f2, f3). The histograms on the right (FIG. 11B,
FIG. 11D and FIG. 11F) show the relative contributions of
activation states as the degree of activation is chemically
increased with adenosine diphosphate (ADP) for the same three AC
frequencies.
[0123] FIG. 12 is a diagram that illustrates electromagnetic
measurement apparatus for multiple frequencies, according to an
embodiment. This is a particular embodiment of the core fluid
sensors 560. This arrangement includes multiple frequency
excitation 1220, with corresponding multiple frequency
discriminators using network analyzer modules 1224, and a fast
Fourier Transform (FFT) analyzer module 1226. Digital output
describing differential impedance at multiple frequencies is sent
to the computer 426. The data are plotted as in FIG. 10 to select
discriminating frequencies f1, f2, and f3 and generate the
histograms of FIGS. 11A through 11B. The model includes frequency
dependent values of resistance or capacitance in the membrane 1210
(Rm,Cm), cytoplasm 1212 (Rc), particle 1214 (Rp) and fluid 1216
(Rs) as well as the channel 414 Rc. Resistance of the bridge
circuit 1218 Rb is also considered.
[0124] In some embodiments, the histograms serve as calibration
curves to determine the activation state of a population of
platelets in a sample measured using the techniques described
herein. A sample with platelet population in a low activation state
(corresponding to the absence of disease or blood vessel injury)
will better match the histograms on the left side. Conversely, a
sample with platelet population in a high activation state
(corresponding to the presence of disease or blood vessel injury)
will better match the histograms on the right side.
[0125] Actual experiments with platelets indicated the differences
between populations of activated and non-activated (resting)
platelets is more subtle than depicted in FIG. 11A through 11F.
Subsequent to the acquisition of the multi-frequency impedance
analyzer, up to eight frequencies could simultaneously be acquired.
A dynamic range and resolution large enough to capture the main
transition frequencies expected (.alpha. and .beta. relaxation) was
provided by the instrument. Experiments aiming at increasing the
frequency resolution did not appear to add discrimination
power.
[0126] Most characterizations were done on Platelet-Rich Plasma
re-suspended in a citrated tyrode solution with BSA and apyrase to
limit activation and aggregation. Activation was performed with a
thrombin-receptor activating peptide (TRAP). Gold-standard
activation measurement was performed by flow cytometry, using CD61
(platelet marker) and CD62P (P-selectin, marker of activation)
antibodies. Non-treated samples were considered `resting`. At the
dilution and flow rate used, a few thousands platelets were
typically measured in one minute.
[0127] The characterization of impedance spectra for resting
platelets (non-treated, around 5% activated platelets as confirmed
by flow cytometry) and TRAP-activated (around 85% activated
platelets as confirmed by flow cytometry) did not immediately
reveal significant differences, as expected from the subtle changes
in electrical properties upon activation. For example, FIGS. 17A
and 17B are graphs 1700 and 1720, respectively, which illustrate
example frequency response of resting and activated platelets,
according to an embodiment. The horizontal axis 1712 represents
electromagnetic frequency in MegaHertz (MHz, 1 MHz=10.sup.6 hertz).
The vertical axes 1704 and 1724 represent impedance difference (X
representing in-phase/real, and Y representing
90.degree.-phase/imaginary, respectively). Traces 1712 and 1732
indicate non-treated (resting) platelet samples in the two graphs,
respectively; and traces 1714 and 1734 indicate TRAP-activated
platelets in the two graphs, respectively. No significant
difference between resting and activated platelets is noticed. Data
is normalized to internal control (10 micron polystyrene
particles). The larger variability in X at 6.04 MHz appears to be
due to the low impedance of the internal control at this
frequency.
[0128] Even so, classification techniques are able to discriminate
between activated and resting platelets. FIG. 18 is a graph 1800
that illustrates example classification of resting and activated
platelets, according to an embodiment. The horizontal axis 1802
represents the amplitude of one canonical vector (also called a
projection) in arbitrary units; and, the vertical axis 1804
represents the amplitude of a different, second canonical vector in
arbitrary units. The dark data points 1812 come from a platelet
sample that was activated with TRAP (thrombin receptor agonist);
while the gray data points 1814 are from a sample of resting
platelets. The software uses the different data parameters derived
from the impedance spectroscopy and performs a discriminant
analysis that differentiates between the activated and resting
platelets within each sample. In the plot of the first two
projections (that account for the two highest fractions of the
total variance), activated platelets are grouped to the left while
resting platelets are grouped to the right. The ellipses 1822 and
1824 of the activated and resting platelets, respectively,
represent 50% of the population within each species.
[0129] These results led to other embodiments that consider more
advanced statistical analysis on both raw features
(in-phase/out-of-phase parts at various frequencies), and also
opacity features (ratio of impedances at two frequencies). Because
of the remaining variability of the fluidics (core size, flow,
centering in channel, among others), more favorable results were
obtained when the platelet data were normalized to polystyrene
beads added to the solution as an internal control. These results
are shown in FIG. 19, along with calibration data from FACS
measurements.
[0130] FIG. 19 is a graph that illustrates example agreement of
degree of platelet activation with a standard, according to an
embodiment. The horizontal axis 1902 indicates different groups of
samples and the vertical axis 1904 indicates the number of
activated platelets in the group as a percentage. Comparison of
percentages of activation derived from microchannel impedance data,
EIS 1912a 1912b (N=24) and conventional FACS analysis 1910a 1910b
in two types of samples--resting (non-activated platelets) and
activated (TRAP-activated platelets), respectively. Comparable
trends demonstrate the ability of EIS to quantify the level of
activation. Note that exact match between FACS and EIS was not
expected due to the different features measured. EIS Subset 1914a
and 1914b (N=5) represent a subset of data selected for their low
variability in the internal control (polystyrene beads), an overall
indicator of the stability of the fluidics. Stability of the
internal control was assessed by the lack of discrimination of the
control particles in the various samples based on the canonical
vectors trained on the platelets.
[0131] One can see that the percentages of activated platelets
calculated from impedance data are comparable to those obtained by
the standard method of flow cytometry. It is important to note
however that they should not be expected to be exactly equal, as
both methods are measuring different indicators of activation.
Because activation is more a graded state than an on/off state,
different indicators would result in similar, but slightly
different outcomes. This also shows that by down-selecting to only
those datasets where the internal control was highly stable (an
indication of stable measurement conditions), results could be
further improved. This indicates that further refinement of the
microfluidic system will likely increase the performance of the
method (also called an assay in this context).
[0132] It was also verified that the device and measurement process
were not leading to auto-activation of the platelet (through
reactions with surfaces or shear). Flow cytometry measurements of
resting platelets before and after measurements (collected at the
output of the device) showed no significant difference (6.1%
before, 5.9% after), in effect validating the low-shear design.
[0133] This project has led to the successful development of a
microfluidic-based impedance flow cytometer, and the demonstration
of a novel approach to maintain the signal-to-noise ratio of small
channels while keeping large physical dimensions--two normally
opposed parameters, both desirable simultaneously for the analysis
of platelets without inducing activation. Several fluidic issues
were investigated and feasibility demonstrated. Strong evidence
supports the possibility of quantifying the levels of platelet
activation in a sample by electrical impedance spectroscopy in a
microfluidic device. It is anticipated that these techniques
provide a means for the detection of disease and efficacy of
disease treatment, e.g., to monitor platelet interactions with
anti-platelet drugs such as clopidogrel or aspirin.
5. CHARACTERIZATION OF RED BLOOD CELLS (RBCS) AND LEUKOCYTES
[0134] Ideally, measurements should be performed on whole blood,
allowing information on other blood constituents to be measured in
addition to platelet data (e.g. red and white blood cell counts).
The sample preparation would also be greatly simplified. Undiluted
whole blood, however, contains so many cells that it is difficult
to obtain signals from single cells. Alternatives would be to
either dilute the blood or to selectively lyse the red blood cells
as they are the most prevalent cell type in the blood. Diluting
results in a sample that is easier to handle and the ratio between
platelets and other blood cells will remain unchanged if the
dilution is properly performed while a large number of cells will
have to be analyzed in order to get statistically relevant data on
the platelets. Lysing of the red blood cells will create ghost
cells and membrane residues that, without careful washing of the
sample, may interfere with the measurements.
[0135] However, limited experiments with samples prepared by
differential centrifugation (reducing predominantly the
concentration of RBCs) showed that it was possible to get
information on both cell types, and that discrimination between
both might be achievable by simple size discrimination (using
typically the in-phase component of the impedance at low
frequency).
[0136] FIG. 20A and FIG. 20B are graphs that illustrate example
detection of platelets and red blood cells in a blood sample,
according to an embodiment. Depicted is a partly centrifuged whole
blood sample. In FIG. 20A, in graph 2000, the horizontal axis 2002
is time in samples per seconds (sampling rate: 7.2 ksps) and the
vertical axis 2004 is impedance signal in volts, as outputted by
the measuring instrumentation. Trace 2010 is a typical impedance
signal from a mixed population of platelet and RBCs. The trace 2010
is a raw signal for in-phase component of impedance (X), showing
the different signature of red blood cells (RBCs, also called
erythrocytes) and platelets. The RBCs cause the large peaks; and
the platelets cause the smaller peaks.
[0137] FIG. 20B depicts a histogram 2020 of size distributions. The
horizontal axis 2022 represents the X impedance value at low
frequency (size-dependant); and the vertical axis 2024 indicates
the number of peaks (count). The histogram 2030 shows the small
distribution of platelets (low amplitudes) and the overwhelming
distribution of RBCs, even in a partially-centrifuged sample. On
the basis of these results with RBCs, discrimination of large
leukocytes from small platelets based on size appears feasible.
6. COMPUTATIONAL HARDWARE OVERVIEW
[0138] FIG. 13 is a block diagram that illustrates a computer
system 1300 upon which an embodiment of the invention may be
implemented. Computer system 1300 includes a communication
mechanism such as a bus 1310 for passing information between other
internal and external components of the computer system 1300.
Information is represented as physical signals of a measurable
phenomenon, typically electric voltages, but including, in other
embodiments, such phenomena as magnetic, electromagnetic, pressure,
chemical, molecular atomic and quantum interactions. For example,
north and south magnetic fields, or a zero and non-zero electric
voltage, represent two states (0, 1) of a binary digit (bit).).
Other phenomena can represent digits of a higher base. A
superposition of multiple simultaneous quantum states before
measurement represents a quantum bit (qubit). A sequence of one or
more digits constitutes digital data that is used to represent a
number or code for a character. In some embodiments, information
called analog data is represented by a near continuum of measurable
values within a particular range. Computer system 1300, or a
portion thereof, constitutes a means for performing one or more
steps of one or more methods described herein.
[0139] A sequence of binary digits constitutes digital data that is
used to represent a number or code for a character. A bus 1310
includes many parallel conductors of information so that
information is transferred quickly among devices coupled to the bus
1310. One or more processors 1302 for processing information are
coupled with the bus 1310. A processor 1302 performs a set of
operations on information. The set of operations include bringing
information in from the bus 1310 and placing information on the bus
1310. The set of operations also typically include comparing two or
more units of information, shifting positions of units of
information, and combining two or more units of information, such
as by addition or multiplication. A sequence of operations to be
executed by the processor 1302 constitutes computer
instructions.
[0140] Computer system 1300 also includes a memory 1304 coupled to
bus 1310. The memory 1304, such as a random access memory (RAM) or
other dynamic storage device, stores information including computer
instructions. Dynamic memory allows information stored therein to
be changed by the computer system 1300. RAM allows a unit of
information stored at a location called a memory address to be
stored and retrieved independently of information at neighboring
addresses. The memory 1304 is also used by the processor 1302 to
store temporary values during execution of computer instructions.
The computer system 1300 also includes a read only memory (ROM)
1306 or other static storage device coupled to the bus 1310 for
storing static information, including instructions, that is not
changed by the computer system 1300. Also coupled to bus 1310 is a
non-volatile (persistent) storage device 1308, such as a magnetic
disk or optical disk, for storing information, including
instructions, that persists even when the computer system 1300 is
turned off or otherwise loses power.
[0141] Information, including instructions, is provided to the bus
1310 for use by the processor from an external input device 1312,
such as a keyboard containing alphanumeric keys operated by a human
user, or a sensor. A sensor detects conditions in its vicinity and
transforms those detections into signals compatible with the
signals used to represent information in computer system 1300.
Other external devices coupled to bus 1310, used primarily for
interacting with humans, include a display device 1314, such as a
cathode ray tube (CRT) or a liquid crystal display (LCD), for
presenting images, and a pointing device 1316, such as a mouse or a
trackball or cursor direction keys, for controlling a position of a
small cursor image presented on the display 1314 and issuing
commands associated with graphical elements presented on the
display 1314.
[0142] In the illustrated embodiment, special purpose hardware,
such as an application specific integrated circuit (IC) 1320, is
coupled to bus 1310. The special purpose hardware is configured to
perform operations not performed by processor 1302 quickly enough
for special purposes. Examples of application specific ICs include
graphics accelerator cards for generating images for display 1314,
cryptographic boards for encrypting and decrypting messages sent
over a network, speech recognition, and interfaces to special
external devices, such as robotic arms and medical scanning
equipment that repeatedly perform some complex sequence of
operations that are more efficiently implemented in hardware.
[0143] Computer system 1300 also includes one or more instances of
a communications interface 1370 coupled to bus 1310. Communication
interface 1370 provides a two-way communication coupling to a
variety of external devices that operate with their own processors,
such as printers, scanners and external disks. In general the
coupling is with a network link 1378 that is connected to a local
network 1380 to which a variety of external devices with their own
processors are connected. For example, communication interface 1370
may be a parallel port or a serial port or a universal serial bus
(USB) port on a personal computer. In some embodiments,
communications interface 1370 is an integrated services digital
network (ISDN) card or a digital subscriber line (DSL) card or a
telephone modem that provides an information communication
connection to a corresponding type of telephone line. In some
embodiments, a communication interface 1370 is a cable modem that
converts signals on bus 1310 into signals for a communication
connection over a coaxial cable or into optical signals for a
communication connection over a fiber optic cable. As another
example, communications interface 1370 may be a local area network
(LAN) card to provide a data communication connection to a
compatible LAN, such as Ethernet. Wireless links may also be
implemented. Carrier waves, such as acoustic waves and
electromagnetic waves, including radio, optical and infrared waves
travel through space without wires or cables. Signals include
man-made variations in amplitude, frequency, phase, polarization or
other physical properties of carrier waves. For wireless links, the
communications interface 1370 sends and receives electrical,
acoustic or electromagnetic signals, including infrared and optical
signals, that carry information streams, such as digital data.
[0144] The term computer-readable medium is used herein to refer to
any medium that participates in providing information to processor
1302, including instructions for execution. Such a medium may take
many forms, including, but not limited to, non-volatile media,
volatile media and transmission media. Non-volatile media include,
for example, optical or magnetic disks, such as storage device
1308. Volatile media include, for example, dynamic memory 1304.
Transmission media include, for example, coaxial cables, copper
wire, fiber optic cables, and waves that travel through space
without wires or cables, such as acoustic waves and electromagnetic
waves, including radio, optical and infrared waves. The term
computer-readable storage medium is used herein to refer to any
medium that participates in providing information to processor
1302, except for transmission media.
[0145] Common forms of computer-readable media include, for
example, a floppy disk, a flexible disk, a hard disk, a magnetic
tape, or any other magnetic medium, a compact disk ROM (CD-ROM), a
digital video disk (DVD) or any other optical medium, punch cards,
paper tape, or any other physical medium with patterns of holes, a
RAM, a programmable ROM (PROM), an erasable PROM (EPROM), a
FLASH-EPROM, or any other memory chip or cartridge, a carrier wave,
or any other medium from which a computer can read.
[0146] Logic encoded in one or more tangible media includes one or
both of processor instructions on a computer-readable storage media
and special purpose hardware, such as ASIC 1320.
[0147] Network link 1378 typically provides information
communication through one or more networks to other devices that
use or process the information. For example, network link 1378 may
provide a connection through local network 1380 to a host computer
1382 or to equipment 1384 operated by an Internet Service Provider
(ISP). ISP equipment 1384 in turn provides data communication
services through the public, world-wide packet-switching
communication network of networks now commonly referred to as the
Internet 1390. A computer called a server 1392 connected to the
Internet provides a service in response to information received
over the Internet. For example, server 1392 provides information
representing video data for presentation at display 1314.
[0148] The invention is related to the use of computer system 1300
for implementing the techniques described herein. According to one
embodiment of the invention, those techniques are performed by
computer system 1300 in response to processor 1302 executing one or
more sequences of one or more instructions contained in memory
1304. Such instructions, also called software and program code, may
be read into memory 1304 from another computer-readable medium such
as storage device 1308. Execution of the sequences of instructions
contained in memory 1304 causes processor 1302 to perform the
method steps described herein. In alternative embodiments,
hardware, such as application specific integrated circuit 1320, may
be used in place of or in combination with software to implement
the invention. Thus, embodiments of the invention are not limited
to any specific combination of hardware and software.
[0149] The signals transmitted over network link 1378 and other
networks through communications interface 1370, carry information
to and from computer system 1300. Computer system 1300 can send and
receive information, including program code, through the networks
1380, 1390 among others, through network link 1378 and
communications interface 1370. In an example using the Internet
1390, a server 1392 transmits program code for a particular
application, requested by a message sent from computer 1300,
through Internet 1390, ISP equipment 1384, local network 1380 and
communications interface 1370. The received code may be executed by
processor 1302 as it is received, or may be stored in storage
device 1308 or other non-volatile storage for later execution, or
both. In this manner, computer system 1300 may obtain application
program code in the form of a signal on a carrier wave.
[0150] Various forms of computer readable media may be involved in
carrying one or more sequence of instructions or data or both to
processor 1302 for execution. For example, instructions and data
may initially be carried on a magnetic disk of a remote computer
such as host 1382. The remote computer loads the instructions and
data into its dynamic memory and sends the instructions and data
over a telephone line using a modem. A modem local to the computer
system 1300 receives the instructions and data on a telephone line
and uses an infra-red transmitter to convert the instructions and
data to a signal on an infra-red a carrier wave serving as the
network link 1378. An infrared detector serving as communications
interface 1370 receives the instructions and data carried in the
infrared signal and places information representing the
instructions and data onto bus 1310. Bus 1310 carries the
information to memory 1304 from which processor 1302 retrieves and
executes the instructions using some of the data sent with the
instructions. The instructions and data received in memory 1304 may
optionally be stored on storage device 1308, either before or after
execution by the processor 1302.
[0151] FIG. 14 illustrates a chip set 1400 upon which an embodiment
of the invention may be implemented. Chip set 1400 is programmed to
perform one or more steps of a method described herein and
includes, for instance, the processor and memory components
described with respect to FIG. 13 incorporated in one or more
physical packages (e.g., chips). By way of example, a physical
package includes an arrangement of one or more materials,
components, and/or wires on a structural assembly (e.g., a
baseboard) to provide one or more characteristics such as physical
strength, conservation of size, and/or limitation of electrical
interaction. It is contemplated that in certain embodiments the
chip set can be implemented in a single chip. Chip set 1400, or a
portion thereof, constitutes a means for performing one or more
steps of a method described herein.
[0152] In one embodiment, the chip set 1400 includes a
communication mechanism such as a bus 1401 for passing information
among the components of the chip set 1400. A processor 1403 has
connectivity to the bus 1401 to execute instructions and process
information stored in, for example, a memory 1405. The processor
1403 may include one or more processing cores with each core
configured to perform independently. A multi-core processor enables
multiprocessing within a single physical package. Examples of a
multi-core processor include two, four, eight, or greater numbers
of processing cores. Alternatively or in addition, the processor
1403 may include one or more microprocessors configured in tandem
via the bus 1401 to enable independent execution of instructions,
pipelining, and multithreading. The processor 1403 may also be
accompanied with one or more specialized components to perform
certain processing functions and tasks such as one or more digital
signal processors (DSP) 1407, or one or more application-specific
integrated circuits (ASIC) 1409. A DSP 1407 typically is configured
to process real-world signals (e.g., sound) in real time
independently of the processor 1403. Similarly, an ASIC 1409 can be
configured to performed specialized functions not easily performed
by a general purposed processor. Other specialized components to
aid in performing the inventive functions described herein include
one or more field programmable gate arrays (FPGA) (not shown), one
or more controllers (not shown), or one or more other
special-purpose computer chips.
[0153] The processor 1403 and accompanying components have
connectivity to the memory 1405 via the bus 1401. The memory 1405
includes both dynamic memory (e.g., RAM, magnetic disk, writable
optical disk, etc.) and static memory (e.g., ROM, CD-ROM, etc.) for
storing executable instructions that when executed perform one or
more steps of a method described herein. The memory 1405 also
stores the data associated with or generated by the execution of
one or more steps of the methods described herein.
7. ALTERATIONS AND MODIFICATIONS
[0154] In the foregoing specification, the invention has been
described with reference to specific embodiments thereof. It will,
however, be evident that various modifications and changes may be
made thereto without departing from the broader spirit and scope of
the invention. The specification and drawings are, accordingly, to
be regarded in an illustrative rather than a restrictive sense.
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