The present exemplary embodiment relates to instruments
or devices for collecting particles or samples, particularly from flowing streams.
It finds particular application in conjunction with the detection of biological
agents, and will be described with particular reference thereto. However, it is
to be appreciated that the present exemplary embodiment is also amenable to other
Bio-agents dispersed either in aerosol form or in water
are typically in such low concentrations that they are below the limit of detection
(LOD) of even the most sensitive detection schemes. Yet, the ingestion of even a
single bacterium may lead to fatal consequences. Accordingly, regardless of whether
the sample is derived from aerosol or water collection, there exists a need to further
concentrate the sample prior to detection.
Aerosol collection schemes typically sample large volumes
of air at very high rates (up to 150 kL/min), and use either a cyclone or a virtual
impactor design to collect particles having a size in the threat range and capture
them in a wet sample of 5-10 mL volume. This hydrosol is then used as the test sample
for agent detection. In order to use currently available detection strategies, it
would be desirable to further concentrate the hydrosol by another two orders of
magnitude. For example, this could be achieved by collecting all the bio-particles
in the sample volume within a smaller volume of 50-100 µL.
Contaminants in water are typically treated by several
filtration steps to recover the sample for agent testing. After initial pre-filtration
to remove larger vegetative matter, the sample is further concentrated by two to
three orders of magnitude using ultra-filtration. This method of tangential flow
filtration (TFF) is laborious as it requires multiple sequential steps of TFF; each
step utilizing a filter of lower molecular weight (MW) cut-off, and recycling of
the retentate. The limiting factor for TFF is system loss, where there is a cut-off
below which it may not provide any further improvement in concentration. The supernatant
at the end is approximately a 50 mL volume to be presented to the detector. It would
be particularly desirable to further concentrate the hydrosol by up to another three
orders of magnitude.
Field Flow Fractionation (FFF) is a technique that allows
the separation of particles of different charge to size ratios (q/d) in a flow channel.
This technique is useful in many fields ranging from printing to biomedical and
biochemical applications. Separation is achieved because particles with different
q/d ratios require different times to move across the flow channel, and therefore
travel different distances along the flow channel before arriving at a collection
wall. To obtain well-defined and separated bands of species with different q/d values,
the particles are typically injected through a narrow inlet from the top of the
channel. Total throughput depends on the inlet geometry and flow rate, which in
turn affects the q/d resolution of the system.
FFF relies upon the presence of a field perpendicular to
the direction of separation to control the migration of particles injected into
a flow field. The separated components are eluted one at a time out of the system
based on retention times, and are collected in a sequential manner. The separations
are performed in a low viscosity liquid, typically an aqueous buffer solution, which
is pumped through the separation channel and develops a parabolic velocity profile
typical of Poissieulle flow. The process depends on controlling the relative velocity
of injected particles by adjusting their spacing from the side walls. Particles
with higher electrophoretic mobility or zeta potential will pack closer to the walls
and therefore move slower than those that are nearer the center of the channel.
In effect, particles move at different rates through the system based on zeta potential
and size. Use of different separation mechanisms such as thermal, magnetic, dielectrophoretic,
centrifugation, sedimentation, steric, and orthogonal flow has given rise to a family
of FFF methods. Although satisfactory in many respects, there remains a need for
an improved FFF separation technique.
The present exemplary embodiment contemplates a new and
improved bio-enrichment system, device, cells, and related methods which overcome
the above-referenced problems and others.
In accordance with one aspect of the present exemplary
embodiment, a device adapted for collecting particulates from a flowing medium is
provided. The device comprises a body defining an inlet, an outlet, and opposing
bottom and top walls extending at least partially therebetween and defining an expansion
cavity. The cavity includes a collection wall extending from a downstream region
of the bottom wall. The device also comprises a traveling wave grid disposed along
the bottom wall and adapted to transport particulates proximate to the grid, to
the collection wall.
In one embodiment the device further comprises a planar electrode disposed on the
upper wall of the body.
In accordance with another aspect of the present exemplary
embodiment, a bio-enrichment device is provided. The bio-enrichment device comprises
a cell body defining an inlet, an outlet, an inlet wall, a collection wall opposite
from the inlet wall, a bottom wall extending between the inlet wall and the collection
wall, and a top wall extending between the inlet and the outlet and opposite from
the bottom wall. The inlet wall, the collection wall, the bottom wall, and the top
wall define an expansion cavity. The bio-enrichment device further comprises a first
traveling wave grid disposed on the bottom wall. The bio-enrichment device also
comprises a second traveling wave grid extending along the collection wall. The
cell body further defines at least one sample collection port at a region proximate
one of the first traveling wave grid and the second traveling wave grid. Upon operation
of the device and admittance of a flowing medium containing bio-agents dispersed
therein to the inlet defined in the body, bio-agents are collected at one or more
of the sample collection ports.
In one embodiment the first and second traveling wave grids are oriented perpendicular
to each other.
In a further embodiment the concentration of bio-agents in the medium as measured
at the at least one sample collection port is greater than the concentration of
bio-agents in the medium as measured at the inlet of the body, by a factor of about
100 to about 1000.
In a further embodiment the the inlet includes an angled bend of about 90 degrees.
In a further embodiment the bio-enrichment device further comprises a fin extending
within the expansion chamber and adapted to reduce initial dispersion of the bio-agents
upon entering the expansion cavity.
In a further embodiment the bio-enrichment device further comprises a planar electrode
disposed on the top wall.
In accordance with yet another aspect of the present exemplary
embodiment, a method is provided for collecting and concentrating bio-agents from
a flowing medium. The method comprises providing a hybrid flow cell including (i)
a body defining an inlet, an outlet, opposing bottom and top walls extending at
least partially therebetween and defining an expansion cavity, the cavity including
a collection wall extending from a downstream region of the bottom wall, and (ii)
a traveling wave grid disposed along the bottom wall and adapted to transport particulates
proximate to the grid to a destination location. The method also comprises introducing
the flowing medium containing bio-agents to the inlet of the flow cell. The method
further comprises activating the traveling wave grid disposed on the bottom wall
to thereby collect bio-agents from the flowing medium and transport the collected
bio-agents to the destination location. The concentration of bio-agents as measured
at the destination location is greater than the concentration of bio-agents as measured
at the inlet.
BRIEF DESCRIPTION OF THE DRAWINGS
In one embodiment the concentration increases by a factor within the range of from
about 100 to about 1000.
In a further embodiment the hybrid flow cell further includes (iii) a second traveling
wave grid extending along the collection wall, and the destination location to which
collected bio-agents are transported is the collection wall, the method further
comprising: activating the second traveling wave grid to thereby transport collected
bio-agents to a second destination location.
FIGURE 1 is a schematic of an exemplary embodiment bio-enrichment
FIGURE 2 is a schematic planar view of an exemplary embodiment
field flow fractionation and traveling wave assembly hybrid bio-enrichment cell.
FIGURE 3 is a schematic side elevational view of the cell
taken along line 3-3 in FIGURE 2.
FIGURE 4 is a schematic end view of the cell taken along
line 4-4 in FIGURE 2.
FIGURE 5 is a perspective schematic illustration illustrating
flow in another exemplary embodiment bio-enrichment cell.
FIGURE 6 is a perspective schematic view illustrating flow
within another exemplary embodiment bio-enrichment cell.
FIGURE 7 is a schematic of another exemplary embodiment
FIGURE 8 is a schematic side view taken along line 8-8
in FIGURE 7 illustrating flow through the cell.
FIGURE 9 illustrates laminar flow through the cell of FIGURES
7 and 8.
FIGURE 10 is a graph illustrating free flow fractionation
and flow velocity profiles in a typical exemplary embodiment cell.
FIGURE 11 is a side view illustrating typical flow lines
through a simulated exemplary embodiment cell.
FIGURE 12 is a cross-sectional schematic side view of another
exemplary embodiment cell illustrating flow therethrough.
FIGURE 13 is a top planar schematic view of the flow cell
depicted in FIGURE 12.
FIGURE 14 is an end view of the flow cell of FIGURES 12
FIGURE 15 is a side schematic view of another exemplary
embodiment flow cell illustrating the use of a bias field.
FIGURE 16 is a side view illustrating flow lines through
yet another exemplary embodiment flow cell.
FIGURE 17 is a side view illustrating flow lines through
another exemplary embodiment flow cell.
FIGURE 18 is a side elevational schematic view of another
exemplary embodiment flow cell depicted in FIGURE 20, illustrating flow through
FIGURE 19 is an end view illustrating the flow cell depicted
in FIGURE 20.
FIGURE 20 is a top planar view depicting the exemplary
embodiment flow cell shown in FIGURES 18 and 19.
FIGURE 21 is a side view illustrating flow lines through
another exemplary embodiment flow cell.
FIGURE 22 is a graph illustrating the concentration profile
at the bottom of a cell, the flow in which is depicted in FIGURE 21.
The exemplary embodiment relates to bio-enrichment systems,
devices, cells and methods that can perform initial separation (by charge/diameter,
herein designated as q/d) of a sample mixture, followed by a concentration step.
The exemplary embodiment can serve as either the back-end to collection strategies
or the front-end to detection strategies. Specifically, the exemplary embodiment
provides a system 100 as shown in FIGURE 1 for sample concentration. FIGURE 1 collectively
depicts two separate systems, as follows. The system 100 concentrates a retentate
by another two to three orders of magnitude. The system 100 comprises a flow conduit
110 through which a flow containing a sample to be detected travels. The flow is
sampled and that portion is directed to a pre-filtration unit 120 which removes
relatively large particles, contaminants, or other undesirables. The filtered sample
is then directed to an ultra-filtration unit 130 which performs another filtering
operation. The retentate from the ultra-filtration unit 130 is directed to an exemplary
embodiment bio-enrichment cell 140 as described herein. The output of the cell 140
can then be directed to a conventional detector unit 150. Exemplary volume reductions
and thus degrees of concentration for this sampling configuration are as follows.
For a 50 L volume from unit 120 to 130, the retentate from unit 130 to the cell
140 is about 50 ml, and the supernatant volume from the cell 140 to the detector
150 is about 50 to 100 µl.
As previously noted, the exemplary embodiment also provides
systems for concentrating an aerosol sample. This strategy is also depicted in FIGURE
1 in which an aerosol collector 160 receives a sample from a suitable source. The
output of the collector is then directed to the bio-enrichment cell 140, the output
of which can then be directed to the detector 150 as previously explained. Exemplary
volume reductions and thus degrees of concentration for this sampling configuration
are as follows. For a 5 to 10 ml volume of hydrosol exiting the aerosol collector
160, the output of the bio-enrichment cell is about 50 to 100 µl.
The bio-enrichment device of the exemplary embodiment uses
field flow fractionation (FFF) to initially deposit bio-matter onto a lower surface
patterned with a planar inter-digitated traveling wave electrode grid as a function
of q/d. The inter-digitated electrodes are driven in 4-phases (or n phases with
n>2) with a traveling wave (TW) of voltage pulses to move the deposited bio-matter
to an edge or region on the grid where another orthogonal TW array collapses the
edge into a sample well. The resulting concentration is achieved by collecting bio-particles
of the same q/d range within a much smaller volume of fluid. This technique will
work for all material with a net charge or zeta potential. As a front-end to detection,
the separation function increases selectivity, while the concentration function
increases sensitivity. The concentrated sample may be transferred by micro-pipette
for immunoassay. For compact and potentially autonomous operation, the bio-enrichment
cell may be directly connected to a microfluidic channel into which analytes may
be selectively metered, sorted, and transported with hydraulic and electro-osmotic
flow (EOF) pumps through a series of orthogonal hybrid detectors for interrogation
and agent identification.
The term "traveling wave grid" as used herein, collectively
refers to a substrate, a plurality of electrodes to which a voltage waveform is
applied to generate the traveling wave(s), and one or more busses, vias, and electrical
contact pads to distribute the electrical signals (or voltage potentials) throughout
the grid. The term also collectively refers to one or more sources of electrical
power, which provides the multi-phase electrical signal for operating the grid.
The traveling wave grids may be in nearly any form, such as for example a flat planar
form, or a non-planar form. The non-planar form can be, for example, in the form
of an arcuate region extending along the outer wall of a cylinder. The non-planar
grid could be in the form of an annular grid defined within an interior region of
a tube. Traveling wave grids, their use, and manufacture are generally described
in U.S. patents 6,351,623; 6,290,342; 6,272,296; 6,246,855; 6,219,515; 6,137,979;
6,134,412; 5,893,015; and 4,896,174, all of which are hereby incorporated by reference.
The bio-enrichment cell uses both field flow fractionation
(FFF) and traveling wave (TW) mechanisms. Various aspects of an exemplary embodiment
bio-enrichment cell are shown in FIGURES 2-4 which cell includes thin inlet and
outlet channels and a recessed expansion cavity. The roof of the cavity provides
a continuous indium tin oxide (ITO) surface while the bottom provides a TW electrode
array. A DC bias is maintained to provide an electric E field orthogonal to the
direction of fluid flow. Purely laminar flow is required in the cavity for uniform
separation. In FFF, bio-particles are deflected (as such occurs in a mass spectrometer)
downwards due to their q/d or zeta potential, with the higher q/d particles depositing
first or closest to the lead edge of the cavity. A 90 degree or angled bend at the
inlet is utilized to defuse the flow to result in a more laminar flow field over
the expansion cavity area where angled flow impingement would be undesirable. The
expansion into the cavity slows down the flow to lower the requirement for bias
deflection voltage and in turn a shorter TW dimension. It also allows more time
for the bio-particles to respond to the applied electric field. The recessed cavity
also acts to trap bio-particles, especially in the vortex or recirculation area
created at the bottom right corner of the flow cell thus keeping the bio-particles
tightly focused. TW voltages are then used to move the deposited bio-particles as
desired. In one embodiment, the TW moves bio-particles to a collection wall where
an orthogonal TW grid as shown in FIGURE 5 further re-directs the bio-particles
to one corner, thus further concentrating them.
Specifically, referring to FIGURES 2-4, an exemplary embodiment
bio-enrichment cell 200 is depicted. The cell 200 comprises a body 210 defining
an inlet 212, an outlet 214, and an interior hollow region 216 generally extending
therebetween. It is preferred that the inlet 216 include an angled region or bend,
and ideally a 90 degree bend, such as bend 213. The cell body 210 also defines a
sample discharge port 215. The interior hollow region 216 or expansion cavity is
defined between an upper wall 220, and a lower wall 222. Disposed on the upper wall
220 and directed toward the interior region 216 is a planar electrode 230, which
as previously noted, can be formed from a thin layer of ITO. A plurality of closely
spaced TW electrodes forming a primary grid 240 are disposed on the lower wall 222.
The cell 200 also comprises a collection wall 250 generally extending at right angles
with respect to flow within the cell, and positioned along the downstream side of
the TW grid 240. The wall 250 is in the form of a plurality of closely spaced TW
electrodes 252. Defined at an opposite end of the region 216, from the collection
wall 250, is an inlet wall 217.
Referring further to FIGURES 2-4, a flow stream containing
sample to be collected enters the cell 200 through the inlet 212 as shown by arrow
A. The stream enters the interior hollow region 216 or expansion cavity of the cell.
The bend 213 within or proximate the inlet 212 serves to promote laminar flow once
the flow stream enters the expansion region 216. Once the flow stream enters region
216, the velocity of the stream decreases. Concurrently, the TW grid 240 provides
a bias deflection voltage or electric field that attracts or otherwise deflects
the flow of sample in the flowstream. Exemplary flow lines a1, a2,
and a3 depict flow lines for three particles having different q/d ratios.
Particles having relatively high q/d ratios will exhibit greater deflections, and
so be displaced toward the grid 240 sooner. In contrast, particles having lower
q/d ratios will be directed toward the grid 240 proximate the collection wall 250.
The TW grid is operated to transport collected particles or sample in the direction
of arrow C. Collected sample is transported along the wall 250 toward the sample
discharge port 215. The exiting flow travels out of the cell 200 through the outlet
214 and as shown by arrow B.
The configuration of the exemplary embodiment cell 200
provides several significant advantages. Due to the flows through the cell, normal
or perpendicular impingement of sample with the TW grid 240 is avoided. The cell
200 achieves generally laminar flow within its interior with negligible in-plane
velocity on the TW grid 240. The recessed nature of the expansion region 216, with
regard to the inlet 212, serves to reduce velocity of the sample and promote collection
of the sample. In addition, the cell 200 enables incremental processing of a partial
volume of a greater flow, such as that from which flow A originates.
The bio-particles distributed in the expansion region are
sequentially pushed onto a surface, e.g. the collection wall, then collapsed into
an edge and finally concentrated into a much smaller volume for sample collection.
An estimate of the concentration factor as the particles undergo these operations
is the ratio of the initial volume to the final volume into which most of the bio-particles
are collected. Typical initial volumes may be 5-50 mL so if the final volume is
50-100 mL, concentration factors of 100X-1000X are theoretically possible. The limit
to the linear volume dimension is backdiffusion to counter electrodynamic drift.
A simple estimate is given by E(w/2)=kT/q, where E, w, k, T, q are respectively,
E field, width of the final sample volume, Boltzmann constant, temperature, and
particle charge, respectively. By increasing the local E field by an order of magnitude,
the band may be compacted by up to 10X. The diffusion length, R, over a time, t,
is given by R2 = (kT/phr)t, where h is viscosity and r is the particle radius. For
bio-particles in the 1-10 µm range, the diffusion distance is only a few microns
per second. Smaller protein-sized particles may experience more diffusion and therefore
require continuous TW operation and the optimal selection of TW pitch to contain
the diffusion. Alternatively, small bio-particles can be concentrated into a higher-viscosity
medium, such as a gel, to reduce diffusion.
In another exemplary embodiment bio-enrichment cell 300,
a TW grid moves bio-particles to a collector wall where another orthogonal TW grid
as shown in FIGURE 5 further re-directs the bio-particles to one corner, thus concentrating
them. Operations can be sequenced so that after separation into bands, each band
may be moved in turn against a lateral edge where it can either be concentrated
into the sample well for detection, or purged by reversing the direction of the
traveling wave. Therefore, only the particular band of interest (or q/d range of
interest) is moved into the detection zone. Other uninteresting bands may be purged
Specifically, the cell 300 comprises a cell body 310, in
which an expansion region is defined between an inlet wall 317, a collection wall
350 which is in the form of a TW grid 352, and a primary TW grid 340 extending along
a lower wall 322. It will be understood that the body 310 includes an upper wall
(not shown) having an appropriate inlet and outlet. An incoming flow stream D enters
the expansion region at which particles in the stream are drawn toward the grid
340 and collected therein. The grid 340 is operated to transport the particles toward
the second TW grid 350 which is preferably oriented transversely with respect to
the grid 340 and disposed at a location furthest downstream within the expansion
cavity. As particles collect on or near the second grid 350, that grid is operated
to transport particles to a desired location along the grid 350, such as at location
E at which the collected particles can be transported for subsequent detection or
analysis. As previously explained with regard to the cell 200, in the cell 300,
particles having a relatively high q/d ratio will deposit on the grid 340 closer
to the wall 317.
Another exemplary embodiment bio-enrichment cell 400 is
shown in FIGURE 6 where separated bands may be concentrated in parallel against
a side wall. The orientation of the TW grid is perpendicular to the previous embodiment
depicted in FIGURE 5, and should have little effect on separation since use of an
electrode pitch of 40 mm is much less than a typical cell height of 1.5 mm. By adding
another TW grid at the side wall where the different bands are concentrated, one
can move each band sequentially into one corner where it can either be concentrated
into the sample well for detection, or purged by reversing the direction of the
Specifically, the cell 400 includes a cell body 410 in
which an expansion region is defined between an inlet wall 417, a collection wall
450, and a TW grid 440 extending along a lower wall 422. It will be understood that
the body 410 includes an upper wall (not shown) having an appropriate inlet and
outlet. An incoming flow stream E enters the expansion region at which particles
in the stream are drawn toward the grid 440 and collected thereon. The electrodes
of the grid extend parallel to the direction of flow of stream E in contrast to
the configuration of the cell 300 in FIGURE 5. Upon collection or deposition of
particles on the grid 440 in the cell 400, the grid 440 is operated to transport
bands of particles toward a desired location on or relative to the grid 440. Specifically,
a collection of discrete locations or discharge ports can be defined along a side
of the grid 440. A plurality of sample collection ports can be defined in one or
more side walls of the cell body. Collected bands of particles on the grid 440 can
be transported to one or more of the desired locations, while the particles are
essentially maintained in their various separated populations. For example, a band
of particles collected nearest the inlet wall 417, and so indicative of those particles
having a relatively high q/d ratio, can be transported to location F by suitable
operation of the grid 440. Conversely, a band of particles having a relatively low
q/d ratio can be transported and collected at location I. Similar bands of particles
having q/d ratios within these populations having high and low q/d ratios, can be
collected at locations G and H.
As will be appreciated, the TW grid extending along the
collection wall is generally oriented at right angles to the TW grid disposed on
the bottom wall of the cell body. However, the exemplary embodiment includes other
configurations in which the TW grids are not transversely oriented. Additionally,
the exemplary embodiment includes the use of a point electrode TW grid for either
or both grids, and particularly as the TW grid disposed along the bottom wall. The
use of a point electrode grid facilitates the passing of traveling waves in nearly
any direction or path along the grid.
The exemplary embodiment bio-enrichment devices provide
excellent front-end processing to optical detection of bio-agents. Extension of
sample concentrators to incorporate enrichment capabilities described herein provides
a significant step towards allowing reagentless (for example specific binding, tagging,
labeling, dyes or stains) identification of bio-agents. The exemplary embodiment
bio-enrichment cell performs sample separation into bands according to q/d to increase
selectivity, and can further concentrate the bands into sample wells to increase
sensitivity. Interfacing of the sample wells with microfluidic channels further
allows sequential interrogation of the sample analyte by a hybrid collection of
detection schemes. FIGURE 7 shows an exemplary embodiment cell incorporating a singular
micro-fluidic channel connecting a sequential series of detection schemes which
may comprise separate capabilities including: Coulter counter or MIE scattering;
spectra from intrinsic UV fluorescent sources of possibly several excitation wavelengths
(e.g. 280 nm and 350 nm); UV, visible and Far-IR absorption; and Raman spectroscopy.
Specifically, FIGURES 7 and 8 depict another exemplary
embodiment bio-enrichment cell 500 comprising a cell body 510 having an inlet 512
for receiving a flow stream K, an outlet 514, an interior hollow expansion region
516 which generally extends between an upper wall 520, a lower wall 522, an inlet
wall 517 and an oppositely disposed collection wall 550. A primary TW grid 540 is
disposed on the lower wall 522 and a secondary TW grid 552 extends along the collection
wall 550. A sample discharge port J is defined along a lateral location relative
to the grids. As will be appreciated, upon operation of the cell, particles or sample
are collected on the grid 540 and transported in the direction of arrow M toward
the secondary grid 552. An exiting flow stream L passes through the outlet 514.
Disposed proximate the discharge port J is a detector 560. The detector 560 is adapted
to detect or otherwise analyze particles or sample collected and transported to
the port J. Although a wide array of detectors 560 can be utilized, generally, the
employed detector will include a purge 562 and utilize one or more detector arrays
564, 565, 566, and 567. The arrays can use any appropriate technology, however,
it is contemplated to use Raman, IT absorption, UV fluorescence, MIE Scattering,
Coulter Counters, and/or combinations of these techniques. It is often preferred,
in certain applications to utilize a microfluidic channel having a serpentine configuration
with multiple detector arrays constituting the detection unit 560.
The exemplary embodiment bio-enrichment cell as shown in
FIGURES 7 and 8 can be operated to collect a specific separated band of sample or
particles, transport that band to a particle collection port, thereby greatly increase
the concentration of the collected sample or band, and then perform one or more
analytical operations upon the collected band. In FIGURE 7, a band of particles
disposed on grid 540 denoted as band N is transported to the secondary TW grid 550.
As will be appreciated, the band N may for example represent particles having a
narrow range of q/d ratios. Upon transport to the grid 550, the particles formerly
constituting band N are transported to port J at which they are introduced into
the multi-array detector 560.
A hybrid flow cell of 4.5 mL capacity, designed to handle
sample volumes up to a liter, was fabricated using a vertically stacked configuration.
Due to the low concentration involved and the slow flow regime, the Navier-Stoke's
equation may be simplified to a more tractable viscous Stoke's model for the fluidics
with velocity profiles dictated by Poisseuille flow. Bio-particle trajectories subjected
to both hydrodynamic and electric forces were predicted. Gravity may be sufficient
to maintain the slow velocities, rendering a pump unnecessary. FIGURE 9 shows laminar
flow streamlines and FIGURE 10 shows Poisseuille velocity profiles at the inlet
and mid-channel locations of such a flow cell.
Table 1 set forth below, shows various parameters assuming
a 1 liter sample volume and a handling capacity of 4.5 mL. Clearly parameters may
be optimized based on initial specification for sample volume and process time.
Interdependent parameters, such as for example voltage, range of bio matter q/d
or zeta potential, and cell dimension are selected to meet desired specifications.
Total process time is the summation of flow separation (tFFF), and concentration
(tTW), given by:
where m is the electrophoretic mobility, h is the FFF cell height, L is the FFF
cell length, s is the spacing between TW traces, VFFF is the separation
voltage, TW is the TW voltage, and a (~0.25) is a coefficient to represent the effective
tangential E field within a spacing thickness above the plane of the TW traces.
A design for an integrated 3-layer TW module was fabricated
and proof of concept was demonstrated by moving Bacillus thurengiensis in
tap water. An electro-hydrodynamic particle model has been developed to simulate
and predict the performance of the device.
The various exemplary embodiment bio-enrichment devices
can perform initial separation (by charge/diameter, i.e. q/d) of a sample mixture,
followed by a concentration operation. These various devices can serve as either
the back-end to collection schemes or the front-end to detection strategies. These
devices fill the niche for sample concentration currently addressed by ultra-filtration,
and extends it by further concentrating the retentate by about another 100X-1000X.
The various exemplary embodiment bio-enrichment cells utilize
a combination of hydrodynamic and electric forces to both separate and concentrate
bio-agents (or any charged particles) from a fluid into a small volume element in
continuous flow. In certain exemplary embodiments, a TW grid moves bio-particles
to a collection wall where another orthogonal TW grid as shown in FIGURES 2, 3,
5, and 7 further re-directs the bio-particles to one corner, thus concentrating
them. Operations can be sequenced so that after separation into bands, each band
may be moved in turn against the collection wall where it can either be concentrated
into a sample well for detection, or purged by reversing the direction of the traveling
wave. Therefore, only the particular band of interest (or q/d range of interest)
is moved into the detection zone. Other uninteresting bands may be purged as desired.
As noted, the exemplary embodiment cells can be used as a bio-enrichment device
as a front-end to detection by integrating a micro-fluidic interface to transport
samples in a controlled manner as illustrated in FIGURE 5.
The exemplary embodiment bio-enrichment cells exhibit numerous
benefits, features, and advantages. The cells employ step-wise separation and concentration
which allows increased flexibility in sample preparation. The exemplary embodiment
bio-enrichment cells are integrable with one or more collection steps to attain
about 100X - 1000X increases in sensitivity. The exemplary embodiment bio-enrichment
cells are integrable with detection steps to prepare samples with increased selectivity
and about 100X-1000X increases in sensitivity. The exemplary embodiment bio-enrichment
cells feature no mechanical moving parts, low voltage and power operation, are highly
portable, and are easily optimized for prescribed sample volume and process time.
The exemplary embodiment devices or cells can be in the form of a compact, low power,
portable device for bio-enrichment. The exemplary embodiment devices or cells provide
increased selectivity by initial separation into bands according to q/d range and
increased sensitivity with subsequent concentration of the previously separated
bands. The devices or cells may be integrated as front-end to detection systems
for 100X-1000X increase in sensitivity. The devices or cells may be integrated as
back-end to collection systems for 1 00X-1000X increase in sensitivity. The devices
or cells may be operated without flow to concentrate (without separation) a sample
volume equal to the cavity size. The devices or cells may be dimensioned for various
ranges of sample volume, and extended to re-circulating operation to ensure all
biological matter is deposited onto the lower collection plate. Continuous TW operation
sets up a compression force against the collection wall of the sample well to prevent
back-diffusion of the concentrate and hence prevent band broadening.
The various exemplary embodiments also provide new configurations
and layout of the FFF/TW cell to enhance flexibility of the design and enable parallel
transfer of bands of bio-particles for subsequent bio-agent detection and identification.
In this regard, the exemplary embodiment additionally modifies the traditional use
of FFF to (i) allow deposition of separated bands on a side wall; (ii) introduces
an expansion chamber to reduce the ratio of fluid to drift velocities so that larger
sample volumes may be handled; and (iii) utilizes the use of a fin structure inside
the expanding fractionation chamber, together with an electrode at the lower lip
of the inlet, for field tailoring to enable higher resolution separation and deposition
of narrower bands of similar q/d material. Electrostatic deflection pushes particulates
to the upper flow region away from the lower region with most diverging streamlines.
The trailing edge of the fin is also designed to allow fully developed parabolic
flow with minimal divergence at the trailing edge. The following exemplary embodiment
devices exemplify feature (iii).
The exemplary embodiment provides a unique variant strategy
for FFF systems by incorporating several modifications to the geometry of a flow
cell. Instead of eluting samples as in traditional FFF, the samples are deposited
in the vicinity of a side wall within the channel for further processing. The objective
is to separate particulates by q/d and deposit them into narrow bands in a distribution
along the length of the channel wall. Analogous to mass spectrometry, particulates
with higher q/d have shorter time-of-flight and deposit earlier or closer to the
inlet. Narrower bands would allow easier discrimination in &Dgr;q/d and therefore
correspond to increased resolution for detection or particulate identification.
Deposited particulates on conducting substrates can be used for detection methods
such as surface enhanced Raman scattering (SERS). Particulates in the vicinity of
the wall where fluid velocity is minimal may also be further transported by other
techniques and strategies. A further innovation is the creation of an expansion
chamber to handle larger sample volumes and to reduce the ratio of convective flow
to electrophoretic flow velocities, thus allowing also for lower voltage use. Typical
FFF geometries are straight channels. This expansion introduces divergence of the
flow streamlines at the inlet leading to increased concentration dispersion. A fin
structure is introduced to reduce this initial particle dispersion by field tailoring
of both electrostatic and flow components.
In a straight channel, the flow field and the electric
field are always perpendicular to each other, or generally so, leading to orthogonal
electrophoretic and drift velocities for the particle. In electroosmotic flow both
of these velocities are constant and each particle is traveling in a straight line
until it hits the side wall. In more complicated flows such as pressure driven flow,
the particle trajectories will be more complicated. In order to achieve separation,
particles of different charge to diameter ratio, q/d, released in a certain elevation
&Dgr;y inside the channel have to make contact with the side wall at a lateral
distance &Dgr;x. For any system where the fluid flow is always perpendicular to
the electric field and the cross-section of the channel does not change, the separability
(i.e. the ability to separate two species of different q/d) is independent of the
applied field and flow rate, and depends only on the relative height y0/H
where the particles enter the channel. However, analytic calculations show that
in pressure driven flow separability can be better than for plug flow, if the particles
are released close to the top wall of the channel.
A simulation was performed using an exemplary embodiment
cell. The inlet was 100 µm high and the FFF cell was 1 mm high and 2 to 4 mm
long. The applied bias voltage was 1V, which ensured that particles with q/d<1
pC/cm would reach the cell bottom within the length of channel. At an inlet flow
speed of 1 mm/s the flow was laminar and the stream lines expanded immediately into
the flow cell. A small vortex formed at the inlet bottom corner of the FFF cell,
which increased in size with increasing fluid velocity at the inlet. Microscopic
particles that move along the streamlines followed this expansion, and, after being
exposed to an electric field, deposited into rather broad bands on the channel floor,
as shown in FIGURE 11 by the highlighted cone extending downward from the cell inlet.
From the results derived for FFF in straight channels,
narrow bands can be achieved. If the particles are injected close to the top wall
of the cell very narrow bands can be achieved. Though the particles are initially
close to the top wall in the flow cell shown in FIGURE 11, this advantage is lost
because the micron-sized particles of interest closely follow the stream lines.
The ideal scenario is a case where the particles stay close to the top wall until
after a parabolic flow profile is established in the flow cell, and before exposing
the particles to the fractionation field.
FIGURES 12-15 illustrate another exemplary embodiment cell
featuring the use of a fin structure to modify flow. A thin fin structure is placed
in the FFF cell a short distance beyond the point of flow expansion. The incoming
liquid still expands into the lower part of the cell, as shown in FIGURES 16 and
17, and at the trailing edge of the fin a nearly perfect parabolic profile is established
inside the cell. The fin structure may be supported at the side walls of the chamber
if the dimensions permit a more rigid assembly. The fins may also be attached to
the top lid much like a "spoiler" with a finite number of SU-8 support posts, as
shown in FIGURES 18-20.
Specifically, FIGURES 12-14 illustrate another exemplary
embodiment bio-enrichment cell 600 comprising a cell body 610 defining an inlet
612, an outlet 614, an upper wall 620 and a lower wall 622. An expansion region
616 is defined therebetween. A planar electrode 630 is disposed along the underside
of the upper wall 620. A primary TW grid (not shown) can be disposed along the upper
surface of the lower wall 622. As will be appreciated from previous descriptions
of alternate exemplary embodiment cells, sample or particles dispersed in an incoming
flow stream O can be collected on the lower wall 622 and transported to a collection
wall 650 which is generally opposite from an inlet wall 617. The particles are transported
in the direction of arrow Q. The exiting flow leaves the cell as shown by arrow
P. The cell 600 features a fin member 670 that serves to promote flow streams to
extend in a direction parallel with the upper wall 620 for a sufficient distance
or time, until after a parabolic flow profile is established in the cell 600, and
prior to exposing the sample or particles to the fractionation field occurring in
the region of expansion 616.
In order to keep the incoming particles or sample above
the fin, a small force can be applied that urges them away from the streamlines
that connect to the lower part of the fin. One strategy for achieving this is to
apply a small bias field between the inlet wall of the FFF cell and the top wall
(V1 in FIGURE 15). In this particular example, the top wall is grounded,
the bias voltage at the inlet wall V1 is positive in the event it is
desired to fractionate particles with a positive q/d value, and the bottom wall
has a negative voltage V3. It has been discovered that a grounded fin
yields best performance. This seems to be due to the fact that with a grounded fin
there is no variation of the electric field in the volume between the fin and the
top wall (see FIGURES 16 and 17), allowing the particles to follow the fluid unencumbered.
FIGURE 16 illustrates the electrical potential around a grounded fin. And, FIGURE
17 illustrates electric potential around an insulated fin.
Note that for higher bias voltages the particles are pushed
towards the top wall and a good anti-adhesion control is necessary to prevent particles
from being retained there. If the bias voltage is too low, some or all particles
will move below the fin and deposit in a broad peak at the bottom wall.
FIGURES 18-20 depict another exemplary embodiment bio-enrichment
cell 700. The cell 700 comprises a cell boy 710 defining an inlet 712, an outlet
714, an upper wall 720 and a lower wall 722. An expansion region 716 is defined
therebetween. A planar electrode 730 is disposed along the underside of the upper
wall 720. A primary TW grid (not shown) can be disposed along the upper surface
of the lower wall 722. As will be appreciated from previous descriptions of alternate
exemplary embodiment cells, sample or particles dispersed in an incoming flow stream
R can be collected on the lower wall 722 and transported to a collection wall 750
which is generally opposite from an inlet wall 717. Transport occurs in the direction
of arrow T. Exiting flow leaves the cell as flow S. The cell 700 features a fin
member 770 that serves to promote flow streams to extend in a direction parallel
with the upper wall 720 for a sufficient distance or time, until after a parabolic
flow profile is established in the cell 700, and prior to exposing the sample or
particles to the fractionation field occurring in the region of expansion 716. The
fin member 770 can be secured to the upper wall 720 by one or more posts 722.
For a maximum inlet velocity of 1 mm/s and particles in
the q/d range about 1.6 pC/cm, a bias voltage of V1=0.5V is optimal,
as can be seen from FIGURES 21 and 22. The particles stay close to the top wall
until after they pass the fin. Once they are within reach of the bias field, they
start moving towards the bottom wall and deposit in a well-defined and narrow peak
(see FIGURE 22).
These modified FFF geometries may be used for in-channel
separation and deposition with narrow q/d bands in a closed system using an expansion
channel. The use of an expansion chamber allows higher volume handling capacity
within a shorter channel length without compromising throughput. Fin structure minimizes
diverging flow dispersion. The separated q/d bands are narrower with the use of
the fin structure, giving higher separation resolution. The deposited particulates
may be in a form ready for SERS detection. Alternatively, the particulates are moved
to a region of slow fluid flow where other forces may be used for further sample
manipulation. Fin structure serves at least two purposes: (1) minimizes diverging
flow dispersion; and (2) in combination with bias field such as V1 in FIGURE 15,
deflects particles to the top flow region away from the area of most diverging flow
and recombines them with the fully developed flow profile for FFF.
The shape of the fin can be further optimized to facilitate
fabrication. For example, existing streamlines form conforming shapes that approximate
cross-sections that may be suitable fin geometries. So larger cross-section shapes
may be considered which do not significantly alter the desired fully developed parabolic
profile near the trailing edge.