U.S. patent application number 12/721510 was filed with the patent office on 2010-07-01 for device for cell separation and analysis and method of using.
This patent application is currently assigned to BIOCEPT INC.. Invention is credited to Zhongliang TANG, Pavel TSINBERG.
Application Number | 20100167337 12/721510 |
Document ID | / |
Family ID | 38233166 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100167337 |
Kind Code |
A1 |
TSINBERG; Pavel ; et
al. |
July 1, 2010 |
DEVICE FOR CELL SEPARATION AND ANALYSIS AND METHOD OF USING
Abstract
A microflow device for separating or isolating cells from a
bodily fluid or other liquid sample uses a flow path where
straight-line flow is interrupted by a pattern of transverse posts
which are arranged across the width of a collection region in an
irregular or set random pattern so as to disrupt streamlined flow.
Sequestering agents, such as Abs, are attached to all surfaces in
the collection region via a hydrophilic permeable hydrogel coating.
The collection region is formed as a cavity in a body molded from
PDMS, which flexible body is sandwiched between a glass slide or
comparable flat plate and a rigid top cap plate, both of which are
pressed into abutting relation with the PDMS body by a heat-shrunk
polymeric sleeve. Following cell separation and washing, cells can
be released from the sequestering agents and the device centrifuged
to force said cells to collect adjacent the hydrogel-coated slide
or plate. Slitting the polymeric sleeve allows the body to then be
peeled from the slide or plate, using an integral tab, to expose
the separated cells on the top surface thereof for ready
microscopic examination.
Inventors: |
TSINBERG; Pavel; (Carlsbad,
CA) ; TANG; Zhongliang; (San Diego, CA) |
Correspondence
Address: |
COOLEY LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
BIOCEPT INC.
San Diego
CA
|
Family ID: |
38233166 |
Appl. No.: |
12/721510 |
Filed: |
March 10, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11331988 |
Jan 12, 2006 |
7695956 |
|
|
12721510 |
|
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Current U.S.
Class: |
435/29 ;
435/283.1 |
Current CPC
Class: |
G01N 1/2813 20130101;
B01L 3/5027 20130101; G01N 1/40 20130101 |
Class at
Publication: |
435/29 ;
435/283.1 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12M 1/00 20060101 C12M001/00 |
Claims
1. A microflow device for separating biomolecules, such as cells,
from a sample of a bodily fluid or other liquid, which device
comprises: a body having a flow path formed therein through which
such a sample containing target biomolecules can be caused to flow,
the flow path having inlet means, outlet means, and a microchannel
arrangement which includes a collection region extending between
said inlet and outlet means, which collection region is formed as a
cavity in a flat bottom surface of said body and includes a
plurality of transverse separator posts that protrude from a base
surface of said cavity, a flat, rigid closure plate having a top
surface that is in abutting contact with said flat bottom surface
of said body and closes said flow path cavity, said posts being
integral with said base surface of said collection region and
extending to the top surface of said closure plate, said posts
being arranged in an irregular pattern extending laterally across
said flow path in said collection region so as to interrupt
straight-line flow and streamlined flow of liquid through said
region, and a polymeric sheet wrap encircling said body and said
flat rigid plate and pressing same into surface to surface contact
with each other to seal said flow path against leakage, whereby
disruption of streamlined flow throughout said collection region as
a result of said irregular pattern of said posts creates effective
capture of target biomolecules via sequestering agents attached to
said surfaces in said collection region, including said top surface
of said flat plate, and whereby removal of said wrap, following
separation of target biomolecules from such a sample, allows smooth
dissociation of said flat rigid closure plate with said
biomolecules exposed on said top surface thereof from said body and
permits ready microscopic examination and/or analysis of the
separated biomolecules.
2. The device according to claim 1 wherein said body is molded from
a flexible polymeric material and wherein said wrap encircles said
body and said plate throughout at least the entire longitudinal
length of said collection region.
3. The device according to claim 1 wherein said body is molded from
a flexible polymeric material in substantially the shape of a
parallelepiped and wherein a flat cap is superimposed atop said
body so that said encircling wrap sandwiches said body between said
flat cap and flat closure plate.
4. The device according to claim 3 wherein said body is molded with
a tab extending from one longitudinal end thereof which facilitates
separation of said body from said flat closure plate after the
separation of target biomolecules has been effected.
5. The device according to claim 3 wherein said cap is formed with
a pair of openings which extend transversely therethrough, which
openings are aligned respectively with said inlet means and said
outlet means in said body.
6. The device according to claim 5 wherein said encircling wrap is
a heat-shrunken sleeve of polymeric material having a pair of
spaced apart, generally circular apertures, which apertures are in
alignment with said openings in said cap.
7. The device according to claim 6 wherein said cap has a width
which is less than the width of said body and has longitudinal
sides that are beveled.
8. The device according to claim 3 wherein said body has a width
equal to between about 50% and 80% of the width of said flat
closure plate.
9. The device according to claim 8 wherein said cap has a width, at
its widest dimension, equal to between about 75% and 100% of the
width of said body.
10. The device according to claim 1 wherein said inlet means and
said outlet means comprise passageways of substantially circular
cross section, which passageways have axes which are respectively
aligned at between 120.degree. and 150.degree. to said flat bottom
surface of said body.
11. The device according to claim 10 wherein said axes lie in a
vertical plane substantially perpendicular to said flat bottom
surface of said body and are angularly aligned at between
80.degree. and 100.degree. to each other.
12. The device according to claim 10 wherein said inlet passageway
is of generally conical shape and wherein a reservoir is received
in said inlet passageway which has an interior volume of at least
about 0.2 ml and serves as a well from which a liquid sample can be
drawn so as to flow through said collection region as a result of
the application of vacuum to said outlet passageway.
13. The device according to claim 1 wherein said posts have at
least about 3 different cross sectional sizes and said posts are
aligned substantially perpendicular to the base surface of said
microchannel.
14. The device according to claim 1 wherein a hydrophilic permeable
hydrogel coating at least about 1 micron thick is formed on all
said surfaces in said collection region from an
isocyanate-functional prepolymer that is a reaction product of PEG,
PPG or a copolymer thereof and polyisocyanates, to which hydrogel
coating said sequestering agents are directly or indirectly
bound.
15. A method of separating and examining biomolecules, such as
cells, from a sample of a bodily fluid or other liquid, which
method comprises the steps of: a. causing a sample containing
target biomolecules to flow along a flow path in a body of a
device, the flow path including a microchannel arrangement which
includes a collection region formed as a cavity in a flat bottom
surface of said body wherein a plurality of transverse separator
posts protrude from a base surface of said cavity, said posts being
arranged in an irregular pattern extending laterally across said
flow path in said collection region so as to interrupt
straight-line flow and streamlined flow of liquid through said
region, said body having inlet means and outlet means connected to
said microchannel arrangement, said device including a flat, rigid
closure plate having a top surface that is in abutting contact with
said flat bottom surface of said body and closes said flow path
cavity, and said device also having a polymeric sheet wrap
encircling said body and said flat rigid plate and pressing same
into surface to surface contact with each other to seal said flow
path against leakage, whereby disruption of streamlined flow
throughout said collection region occurs as a result of said
irregular pattern of said posts and creates effective capture of
target biomolecules via sequestering agents attached to said
surfaces in said collection region, including said top surface of
said flat plate, b. removing said wrap following separation of
target biomolecules from such a sample, c. smoothly dissociating
said flat rigid closure plate, having said biomolecules exposed on
said top surface thereof, from said body, and d. subjecting the
separated biomolecules on said closure plate to microscopic
examination and/or analysis.
16. The method according to claim 15 wherein microchannel
arrangement containing said captured biomolecules is washed prior
to said dissociation.
17. The method according to claim 16 wherein said collection region
is treated with a chemical reagent to release said captured
biomolecules from said surfaces following washing and prior to said
dissociation.
18. The method according to claim 17 wherein, following said
release, said device is subjected to centrifugal force to cause
said released biomolecules to collect upon said closure plate
surface prior to said dissociation.
19. The method according to claim 15 wherein said body is made of
flexible polymeric material and is peeled from said plate using a
tab provided at one longitudinal end thereof.
20. The method according to claim 15 wherein said sample is caused
to flow through said collection region at an average liquid flow
rate of about 0.2 to about 1 mm/sec.
Description
[0001] This invention relates to separation or isolation of target
biomolecules from feed liquids and more particularly to an improved
device for separating desired target human cells from bodily fluids
or the like.
BACKGROUND OF THE INVENTION
[0002] Effective isolation and collection of rare cells from a
heterogeneous cell population remains of high interest, due to the
increasing demand for isolated cell populations for use in disease
diagnosis and treatment, e.g. gene therapy, as well as for basic
scientific research. For example, pathologically changed cells,
such as cancerous cells, can be separated from a larger normal cell
population, and the cleaned cell populations may then be
transplanted back into the patient.
[0003] One prominent demand is for the isolation of particular
fetal cells from heterogeneous maternal cell populations to permit
early fetus diagnosis, such as early screening of potential
chromosomal disorders during pregnancy. Fetal cells have been
obtained by methods such as amniocentesis and chorionic villus
sampling; however, such methods can pose risks, especially to the
fetus. Some fetal cells are also present in circulating maternal
blood, as these cells pass from fetus to the maternal bloodstream
in very low numbers; however, the ratio of fetal cells to maternal
cells is on the order of only a few ppm. Thus, there are
significant challenges associated with the isolation and collection
of rare fetal cells from the major population of maternity cells in
maternal blood. These challenges also exist in separating fetal
cells from cervical mucus, and they may also be common to other
rare cell recoveries from bodily fluids or the like, as well as to
the separation and isolation of other biomolecules present in only
minute quantities.
[0004] Cell separation is often achieved by targeting molecules on
the cell surface with specific affinity ligands in order to achieve
selective, reversible attachment of a target cell population to a
solid phase. Nonspecifically adsorbed cells are subsequently
removed by washing, and the release of the target cells for
analysis may follow. Such specific affinity ligands may be
antibodies, lectins, receptors, or other ligands that bind
proteins, hormones, carbohydrates, or other such molecules having
biological activity.
[0005] In addition to column separation, other methods have also
now been developed for separating target cells from a diverse
population of cells such as may be found in bodily fluid or the
like. Published U.S. Patent Application No. 2004/038315 attaches
releasable linkers to the interior luminal surfaces of capillary
tubing, with the desired bound cells subsequently being released
via a cleavage reagent and recovered. U.S. Published Patent
Application No. 2002/132316 uses microchannel devices to separate
cell populations through the use of a moving optical gradient
field. U.S. Pat. No. 6,074,827 discloses the use of microfluidic
devices that are constructed to have "enrichment channels" wherein
electrophoresis is used to separate and identify particular nucleic
acids from samples. Also mentioned is the optional use of
antibodies or other binding fragments to retain a desired target
biomaterial. U.S. Pat. No. 6,432,630 discloses a microflow system
for guiding the flow of a fluid containing bioparticles through
channels where selective deflection is employed, and it indicates
that such systems may be used to separate fetal cells from maternal
blood samples. The disclosure of these patents and published
applications are incorporated herein by reference.
[0006] U.S. Pat. No. 6,454,924 discloses microfluidic devices
wherein analyte-containing liquids are caused to flow generally
downward past sample surfaces disposed atop upstanding pillars on
which capture agents are attached, with the side surfaces of such
pillars having been rendered hydrophobic so as to facilitate flow
in channels that they define.
[0007] K. Takahashi et al., in J. Nanobiotechnology, 2, 5 (13 Jun.
2004) (6 pp) (incorporated herein by reference), disclose on-chip
cell sorting systems wherein multiple microfluidic inlet
passageways lead to a central cell-sorting region fashioned in a
PDMS plate (made in a master mold created in photoresist epoxy
resin) that is bonded to a glass plate. Agar gel electrodes are
provided in the PDMS plate which facilitate the separation of
undesired cells by the application of electrostatic forces, that
direct these cells into a parallel, continuous stream of buffer,
during their flow through a short, cell-sorting region of
confluence. A pre-filter which uses posts to physically trap large
dust particles is also shown. Published International Application
WO 2004/029221 discloses a similarly constructed microfluidic
device that can be used for cell separation, such as separating
fetal RBCs from maternal blood by selective lysis of maternal RBCs.
A sample containing cells may also be introduced into a
microfluidic channel device which separates whole cells; it
contains a plurality of cylindrical obstacles, with the surfaces of
the obstacles having binding moieties, e.g., antibodies, suitably
coupled thereto, which moieties will bind to cells in the sample.
U.S. Pat. No. 5,637,469 discloses microfluidic devices having a
plurality of channels of a depth of 100 microns or less wherein
binding moieties, such as antibodies, are immobilized on surfaces
to capture biomolecules of interest which can be analyzed in situ.
U.S. Pat. No. 5,147,607 teaches the use of devices for carrying out
immunoassays, such as sandwich assays, where antibodies are
mobilized in microchannels. A recessed area can be provided in the
microchannel that contains a group of protrusions which extend
upward from the bottom surface of the channel and to which the
antibodies are immobilized.
[0008] Copending U.S. patent application Ser. Nos. 11/038,920 and
60/678,004, the disclosures of which are incorporated herein by
reference, disclose microfluidic devices that can be used for cell
separation, such as separating fetal red blood cells from maternal
blood or trophoblasts from cervical mucus. A microfluidic channel
contains a set irregular pattern of transverse posts which are
strategically positioned to disrupt straight-line and streamlined
flow and thereby effectively capture target cells with sequestering
agents, e.g., antibodies, suitably coupled to the surfaces of the
region containing the posts. Although the foregoing briefly
described two applications provide improved separation methods for
isolating cells or other biomaterials from bodily fluids or the
like, this art is considered to be in its infancy, and the search
continues for further improvements.
SUMMARY OF THE INVENTION
[0009] The invention provides a microflow device for recovering
rare cells or other target biomolecules from relatively small
amounts of bodily fluids or the like, which device incorporates a
microchannel arrangement wherein there is a collection region that
is formed with a plurality of transverse fixed posts extending from
a base surface of the collection region. The posts are arranged in
a particular irregular array pattern to disrupt straight-line flow
therethrough and importantly to break-up regular streamlined flow
through the array, thereby assuring collisions with the posts and
promoting swirling eddies in a bodily fluid or other liquid that is
being caused to travel along this flow path through the collection
region. Sequestering agents for the desired target biomolecules are
appropriately attached to the surfaces of the transverse posts and
throughout the entire collection region.
[0010] The collection region is preferably molded as a part of a
cavity in the bottom surface of a body wherein a flow path is
provided having an inlet and an outlet. The cavity is closed by a
flat bottom plate of rigid material, and the body preferably
contains a tab extending from one longitudinal end thereof which
facilitates separation, i.e. peeling, of the body from the plate
after the target biomolecules have been recovered from the sample.
A top cap having openings leading to the inlet and outlet is
disposed atop the body, and a polymeric wrap, preferably a
heat-shrunk sleeve, sandwiches the body between the two plates and
assures fluidtight sealing between the bottom surface of the body
and the top surface of the plate. Slitting of the polymeric sleeve
allows the body to be readily peeled from the bottom plate and
exposes the captured target biomolecules on the upper surface of
the plate for ready microscopic observation and/or analysis.
[0011] In one particular aspect, the invention provides a microflow
device for separating biomolecules, such as cells, from a sample of
a bodily fluid or other liquid, which device comprises a body
having a flow path formed therein through which such a sample
containing target biomolecules can be caused to flow, the flow path
having inlet means, outlet means, and a microchannel arrangement
which includes a collection region extending between said inlet and
outlet means, which collection region is formed as a cavity in a
flat bottom surface of said body and includes a plurality of
transverse separator posts that protrude from a base surface of
said cavity, a flat, rigid closure plate having a top surface that
is in abutting contact with said flat bottom surface of said body
and closes said flow path cavity, said posts being integral with
said base surface of said collection region and extending to the
top surface of said closure plate, said posts being arranged in an
irregular pattern extending laterally across said flow path in said
collection region so as to interrupt straight-line flow and
streamlined flow of liquid through said region, and a polymeric
sheet wrap encircling said body and said flat rigid plate and
pressing same into surface to surface contact with each other to
seal said flow path against leakage, whereby disruption of
streamlined flow throughout said collection region as a result of
said irregular pattern of said posts creates effective capture of
target biomolecules via sequestering agents attached to said
surfaces in said collection region, including said top surface of
said flat plate, and whereby removal of said wrap, following
separation of target biomolecules from such a sample, allows smooth
disassociation of said flat rigid closure plate with said
biomolecules exposed on said top surface thereof from said body and
permits ready microscopic examination and/or analysis of the
separated biomolecules.
[0012] In another particular aspect, the invention provides a
method of separating and examining biomolecules, such as cells,
from a sample of a bodily fluid or other liquid, which method
comprises the steps of: a. causing a sample containing target
biomolecules to flow along a flow path in a body of a device, the
flow path including a microchannel arrangement which includes a
collection region formed as a cavity in a flat bottom surface of
said body wherein a plurality of transverse separator posts
protrude from a base surface of said cavity, said posts being
arranged in an irregular pattern extending laterally across said
flow path in said collection region so as to interrupt
straight-line flow and streamlined flow of liquid through said
region, said body having inlet means and outlet means connected to
said microchannel arrangement, said device including a flat, rigid
closure plate having a top surface that is in abutting contact with
said flat bottom surface of said body and closes said flow path
cavity, and said device also having a polymeric sheet wrap
encircling said body and said flat rigid plate and pressing same
into surface to surface contact with each other to seal said flow
path against leakage, whereby disruption of streamlined flow
throughout said collection region occurs as a result of said
irregular pattern of said posts and creates effective capture of
target biomolecules via sequestering agents attached to said
surfaces in said collection region, including said top surface of
said flat plate, b. removing said wrap following separation of
target biomolecules from such a sample, c. smoothly dissociating
said flat rigid closure plate, having said biomolecules exposed on
said top surface thereof, from said body, and d. subjecting the
separated biomolecules on said closure plate to microscopic
examination and/or analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a bottom perspective view of a main body for a
microflow device embodying various features of the invention
wherein there is fabricated an irregular post-containing collection
region in a microchannel pathway.
[0014] FIG. 2 is an enlarged fragmentary view showing a portion of
the collection region of FIG. 1 where the irregular pattern of
posts is located.
[0015] FIG. 3 is a bottom view of the body of FIG. 1.
[0016] FIG. 4 is an exploded perspective view of a device that
incorporates the body shown in FIG. 1 in combination with upper and
lower plates and a tubular sleeve.
[0017] FIG. 5 is a cross-sectional view taken along line 5-5 of
FIG. 4 showing the assembled device without the sleeve.
[0018] FIG. 6 is an end view showing the assembled device of in
FIG. 5, with the sleeve loosely encircling it.
[0019] FIG. 7 is a view similar to FIG. 6 in which the sleeve is
shrunken onto the assembled device.
[0020] FIG. 8 is a schematic representation of use of the device of
FIG. 7 in a cell recovery method.
[0021] FIG. 9 is a perspective view of the device of FIG. 7
following flow of a sample through the device, removal of
connections and the insertion of plugs in the inlet and outlet of
the device.
[0022] FIG. 10 is a perspective view, similar to FIG. 9, where the
plugs have been removed and a slit is being created in the
heat-shrunk sleeve.
[0023] FIG. 11 is a view similar to FIG. 10 showing the device
following completion of the slit and partial unwrapping of the
heat-shrunk sleeve together with displacement of the top plate from
the upper surface of the body.
[0024] FIG. 12 is a view similar to FIG. 11 following complete
removal of the slit sleeve and the top plate and showing the body
being peeled upward from the rigid lower plate.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] A microflow device 11 is provided which includes a body 13
that has a flow path defined therein that includes a microchannel
arrangement having an inlet 15 leading to a collection region 17
and an outlet 19 exiting therefrom. As well known in this art, the
device can be a part of an integrated microfluidic apparatus
constructed on a chip, a disk or the like in the field now being
referred to as MEMS (micro-electro-mechanical systems); however,
diagnosis of biomolecules isolated from a bodily fluid sample is
preferably performed following disassembly of the device 11.
[0026] FIGS. 1 and 3 are bottom views of the body 13 in which a
flow path is formed through which sample liquid is caused to flow.
The flow path comprises obliquely oriented inlet and outlet
passageways 15, 19 that respectively lead to and from a cavity 21
that is provided in the flat bottom surface 23 of the body 13. The
passageways 15, 19 preferably have centerlines that lie in the same
vertical plane. The cavity 21 may include an enlarged entrance
section 25 that can serve as a well for a liquid sample and a short
discharge section 27 that leads to the oblique outlet passageway
19. The inlet and outlet passageways both terminate in the
opposite, upper flat surface 29 (FIG. 4) of the body 13. The
collection region 17 contains a plurality of upstanding posts 31
that are aligned transverse to the liquid flow path and arranged in
an irregular, generally random pattern across the entire width of
the collection region portion of the flow channel. The pattern of
the posts 31 is such that there can be no straight-line flow
through the collection region 17 and such that streamlined flow
streams are disrupted, assuring there is good contact between the
liquid being caused to travel along the flow path and the surfaces
of the posts. The posts are integral with a flat base 33 of the
collection region 17 which base is parallel to the bottom surface
23, and they extend perpendicular thereto, presenting surfaces that
are perpendicular to the flat bottom surface 23 of the body 13. A
flat closure plate 35 abuts the bottom surface 23 and closes the
flow channel, as is described in detail hereinafter. Flow dividers
37 are located adjacent the entrance to and the exit from the
collection region 17 in the flow path. These dividers serve to
distribute the flow of liquid more evenly as it is delivered to the
entrance end of the collection region 17 and discharged
therefrom.
[0027] Flow through the device may be achieved by pumping, e.g.
using a syringe pump or the like, but it is preferably achieved by
vacuum that draws liquid through from a conical reservoir 39 (FIG.
8) installed in the inlet passageway 15 leading to the well 25.
Preferably such a reservoir/well combination has a capacity to hold
about 50 .mu.l to about 500 .mu.l of liquid sample, and preferably
at least about 200 .mu.l.
[0028] The design of the flow channels is such that, at flow rates
through the device within a reasonable range, e.g. injection of
maternal blood using a standard Harvard Apparatus infusion syringe
pump to create a flow in the collection region 17 at a rate of
about 0.05 to 5 nm per second, there is substantial disruption of
streamlined flow through the region without creating turbulence;
this results from the random arrangement of posts of different
sizes and the relative spacing of the posts 31 throughout the
collection region 17. Relatively smooth, non-streamlined flow
without dead spots is achieved at a preferred average liquid flow
rate of between about 0.1 to 2 mm/sec, and more preferably the
average flow rate is maintained between about 0.2 and 1 mm/sec and
is achieved by suction from an inlet well of defined size.
[0029] Although the body 11 might be made from any suitable
laboratory-acceptable material, such as silicon, fused silica,
glass and polymeric materials, it is desirable to use a polymeric
material, preferably one that is optically transparent and at least
somewhat flexible. Suitable plastics which may be used include
polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA),
polycarbonate, polystyrene, polyethylene teraphthalate, as well as
other polymeric resins well known for acceptable laboratory
material usage. PDMS, which is flexible, is preferred. Such bodies
having patterned cavities may be fabricated using any convenient
method such as those selected from among conventional molding and
casting techniques. A flexible body 13 carrying the microchannel
arrangement facilitates sealing by tight abutment with the flat top
surface of the plate 35 without being bonded thereto. Such plate 35
may be fabricated from a stiff or rigid polymeric material or may
simply be a cover plate made of glass, e.g. a microscope glass
slide. Depending upon the composition of the plate, it may be
desirable to bond a thin PDMS film thereto.
[0030] The body 13 may be conveniently fabricated from polymeric
materials using a master or negative mold structure, which can be
created in a thick negative photoresist, using optical lithography,
as well known in this art and described in the J. Nanobiotechnology
article, the disclosure of which is incorporated herein by
reference. For example, the construction layer can be formed from a
mixture of commercially available, standard grade epoxy resin (EPON
SU-8) photoresist and hardener (SU-8 2025), which may be spun onto
silicon wafer substrates at 2000 rpm to provide, for example, a 40
or 50 .mu.m thick film of such photoresist. The thickness
determines the height of the flow path in the collection region 17.
The film is subjected to pre-exposure baking for 3 minutes at
60.degree. C. and then 7 minutes at 95.degree. C. on a precisely
level hot plate to assure even thickness throughout, and the
resultant samples are cooled to room temperature. A Karl Suss
Contact Mask Aligner is used to expose a film with the desired
pattern for the flow path in the ultimate device. The film is then
post-baked at 65.degree. C. for 2 minutes and then at 95.degree. C.
for 5 minutes before it is developed in a commercial SU-8 developer
for 5 minutes, with light stirring being applied during developing.
This creates a negative pattern mold in the epoxy resin photoresist
that is then used as a molding master for replication of the
patterned post body in PDMS or other suitable polymeric resin.
[0031] As one example, a PDMS composition is prepared from a
mixture of a PDMS prepolymer and a curing agent (Sylgard 184 kit,
Dow Corning) at a 10:1 ratio by weight. The mixture is subjected to
vacuum to evacuate bubbles that may be formed during mixing, before
being poured over the epoxy resin master mold, which is located in
a cavity of desired depth to create a body of desired thickness.
The master mold may be optionally pre-coated with a thin layer
(.about.50 nm) of a suitable metal (e.g. gold) to improve the
release of the PDMS replica after curing. Curing of PDMS body may
be carried out at 80.degree. C. for 90 minutes; however, by
initially undercuring the PDMS, it may be possible to facilitate
subsequent functionalization of the collection region including the
post surfaces as discussed hereinafter.
[0032] The layout and the dimensions of the microchannel
arrangement and of patterned posts 31 in the collection region 17
are determined by the mask used in the exposure step of the
fabrication of the master mold. The depth of the cavity 21 is
controlled by the thickness of the SU-8 layer of the master mold,
which is determined by spin-coating conditions. FIG. 2 provides an
enlarged fragmentary view into the cavity 21 showing the posts 31
in the collection region 17 as they are arranged in a preferred
generally random orientation.
[0033] As perhaps best seen in FIG. 4, the microflow device 11
comprises four primary components. In addition to the body 13,
which is preferably molded to have the general shape of a
parallelepiped, and the flat bottom closure plate 35, there are
included a flat top cap plate 41 and a surrounding polymeric wrap
43. The cap plate 41 may have a thickness about the same as the
bottom closure plate, and it may be made out of the same stiff or
rigid polymeric material. It has dimensions that are at least
slightly longer and at least slightly wider than the cavity 21 in
the bottom surface of the body 13 that provides the flow path so as
to assure effective sealing between the bottom surface 23 of the
body 13 and a top surface 45 of the closure plate about the entire
perimeter of the cavity. Preferably, the cap plate 41 has a length
just slightly less than the length of the main portion of the body
13, and it has a width, at its widest point, between about 75% and
100% of the width of the body. Longitudinal side edges 47 of the
cap plate 41 are preferably beveled as best seen in FIGS. 4 and 6.
Cutouts or openings 49 are provided at each longitudinal end of the
cap plate to provide access to the inlet and outlet in the upper
surface 29 of the body 13.
[0034] As previously stated, the body 13 is preferably molded from
flexible, polymeric material, such as PDMS, and it preferably has a
tab 51 extending from one longitudinal end and aligned generally
with the top surface 29. The tab 51 facilitates dissociation of the
body 13 and the flat plate 35 following separation of targeted
biomolecules of a sample, as described in more detail hereinafter.
To effect good sealing at the perimeter of the cavity 21 and
subsequent ready dissociation of the body and the flat closure
plate 35, an arrangement is made to press the body 13 against the
top surface 45 of the closure plate in a manner that can be quickly
relieved. A wrap 43 of polymeric sheet material is preferably used
to create such sealing pressure, which may simply be a sleeve of
oriented polymeric sheet which is commercially available as
"shrink-wrap" material. The dimensions of the cap plate 41 are
chosen such that the force which is applied by this wrap in
sandwiching the body between the plates 35 and 41 is spread across
the width of the entire body 13, thus assuring a tight seal at the
entire perimeter of the cavity 21. As seen in FIG. 4, the sleeve 43
is provided with a pair of apertures 53 which are located so as to
be in alignment with the inlet and outlet passageways 15, 19 in the
final assembled device 11.
[0035] Once a subassembly is made of the body 13 atop the closure
plate 35 and with the cap plate 41 in place above it, this
subassembly is inserted into the sleeve 43 as depicted in FIG. 6.
The apertures 49 are aligned with the inlet and outlet passageways
15, 19, and the assembly is then subjected to heating by hot air or
the like which effects thermal shrinkage of the sleeve, causing its
girth to be dramatically reduced and thus applying force which
presses the plates 35, 41 toward each other so as to tightly
sandwich the body 13 therebetween, resulting in the structure shown
in FIG. 7. The substantial width of the cap plate 41 uniformly
spreads this force and assures there is tight sealing along the
entire perimeter of the cavity 21 in the bottom surface 23 of the
body 13.
[0036] The assembled device shown in FIG. 7 is now ready for use in
an operation to recover targeted biomolecules, such as fetal cells
from maternal blood or from cervical mucus. The interior of the
flow path, and particularly all of the surfaces that make up the
collection region 17, are derivatized or preferably provided with a
coating that facilitates the direct or indirect attachment of
sequestering agents specific to the targeted biomolecules of
interest. The coated surfaces of the patterned post collection
region 17 can be derivatized in various ways to enable the
attachment, onto all the surfaces, of sequestering agents that are
specific to the desired target cells or other biomolecules, as
known in this art. Preferably a hydrophilic permeable hydrogel at
least about 1 micron thick is coated onto all of the surfaces from
an aqueous mixture containing an isocyanate-functional prepolymer
that is a reaction product of PEG, PPG or copolymer thereof and
polyisocyanates. Sequestering agents may be directly or indirectly
bound to the isocyanate groups in the hydrogel. Details of such
coating and the attachment of sequestering agents are set forth in
the aforementioned two pending U.S. patent applications, the
disclosures of which are incorporated herein by reference. For
example, for indirect binding, a coupling agent such as avidin may
be included as a part of the aqueous solution of prepolymer used to
effect the coating, in which case avidin will be covalently linked
to such isocyanate groups and then provide the basis for attachment
of desired biotinylated antibodies specific to particular
biomolecules of interest in the separation method for which the
device will then be used.
[0037] The term sequestering agent is used to refer to material
capable of interacting in a specific fashion with a target
biomolecule to physically sequester or bind to the target. These
sequestering agents may include nucleic acids, such as DNA, RNA and
PNA which bind to proteins; generally nonhybridization sequestering
agents are employed comprising biological material, such as
proteins, e.g. receptors, peptides, enzymes, enzyme inhibitors,
enzyme substrates, immunoglobulins (particularly antibodies),
antigens, lectins, modified proteins, modified peptides,
double-stranded DNA, biogenic amines and complex carbohydrates.
Synthetic molecules may also be used, e.g. drugs and synthetic
ligands designed to have specific binding activity of this type. By
"modified" proteins or peptides is meant those proteins or peptides
having one or more amino acids within the molecule altered by the
addition of new chemical moieties, by the removal of existing
chemical moieties or by some combination of both removal and
addition. This alteration may include both natural and synthetic
modifications. Natural modifications may include, but are not
limited to, phosphorylation, sulfation, glycosylation, nucleotide
addition, and lipidation. Synthetic modifications may include, but
are not limited to, chemical linkers to facilitate binding to the
hydrogel, and microstructures, nanostructures, e.g. quantum dots,
or other synthetic materials. In addition, modification may include
the removal of existing functional moieties, e.g. hydroxyl,
sulfhydryl or phenyl groups, or the removal or alteration of native
side chains or the polypeptide amide backbone. Examples of complex
carbohydrates include, but are not limited to, natural and
synthetic linear and branched oligosaccharides, modified
polysaccharides, e.g. glycolipids, peptidoglycans,
glycosaminoglycans or acetylated species, as well as heterologous
oligosaccharides, e.g. N-acetylglucosamine or sulfated species.
Examples of naturally-occurring complex carbohydrates are chitin,
hyaluronic acid, keratin sulfate, chondroitan sulfate, heparin,
cellulose and carbohydrate moieties found on modified protein such
as albumin and IgG. Combinations of two or more of such agents
might be immobilized upon the posts, and such combinations might be
added as a mixture of two entities or may be added serially.
[0038] As mentioned above, attachment of the sequestering agents,
such as antibodies, throughout the collection region is facilitated
so that the sequestering agents perform more efficiently by coating
the surfaces therein with a thin layer (at least about 1 .mu.m
thick) of a particular hydrophilic hydrogel substance which is an
isocyanate-functional polymer containing PEG, PPG or a copolymer
thereof of a MW of about 2,000 to 6,000 daltons, that is
polymerized by urethane bonds and that contains reactive isocyanate
groups. Details of the formulation of such coating material are
disclosed in a co-pending U.S. patent application Ser. No.
11/021,304, filed Dec. 23, 2004, which is assigned to the assignee
of this application. Although sequestering agents can be directly
or indirectly attached to the hydrogel coating, indirect
immobilization is preferred and contemplates the employment of an
intermediate agent or substance that is first linked. It may be
desired to use a coupling pair as an intermediate agent; for
example, streptavidin, or an antibody (Ab) directed against another
species antibody, might be attached to the hydrogel coating, which
would thereafter couple to a biotinylated Ab or to an Ab of such
other species. The use of Abs as sequestering agents may be
preferred for cell separation, and their attachment is discussed in
U.S. Pat. No. 5,646,404, the disclosure of which is incorporated
herein by reference. Such antibodies can be effectively bound by
applying the antibody in aqueous solution to a surface that has
been coated with a layer having free isocyanate or equivalent
groups, such as a polyether isocyanate. Particularly preferred is
the use of a hydrophilic permeable polyurethane-based hydrogel
layer having free isocyanate groups; such is disclosed in the
copending two patent applications and is described hereinafter in
an example.
[0039] The sequestering agents chosen are directed toward specific
capture of the biomolecule of interest. These target biomolecules
may be any of a wide variety of cells, as well as proteins,
viruses, carbohydrates and the like. However, the invention is
believed to exhibit particular efficiencies and have particular
advantages in cell separation. Although the term "cell" is used
throughout this application, it should be understood to include
cell fragments and/or remnants that would likewise carry the
surface ligands specific to the sequestering agents. Appropriate
sequestering agents are selected, as known in this art, which would
have high specific affinity in order to achieve such desired
specificity to the target biomolecules.
[0040] When antibodies are used, they are suitably attached,
preferably through such intermediate agents, using any mechanisms
well known in this art. For example, Abs may be treated with
2-aminothiolane to thiolate them, and the resulting thiolated Abs
conjugated with posts that have been treated with PEG-maleimide;
alternatively, the Abs may be directly covalently bonded to an
appropriate hydrophilic coating having reactive isocyanate groups
or thiocyanate groups.
[0041] With the antibodies or other sequestering agents in place
throughout the patterned post collection region 17, the microflow
device 11 is ready for use. A bodily fluid, such as a blood or
urine sample, or some other pretreated liquid containing the target
cell or other biomolecule population, is caused to travel along a
flow path through the collection region 17, as by being discharged
carefully from a standard syringe pump into the inlet passageway 15
or drawn by a vacuum pump or the like therethrough from a sample
reservoir 39, the lower end of which is received in the passageway
15, which reservoir may hold the desired volume of sample for a
test or be periodically refilled. The passageway 15 is preferably
frustoconical and is designed to mate with the end of such a
conical reservoir when such is used. The pump may be operated to
effect a flow between about 0.5-10 .mu.l/min. through the device;
for a device having a volume of about 0.01 cc, a flow rate of about
3 to 5 .mu.l/min may be used. Depending upon the bodily fluid, or
other cell-containing liquid that is to be treated and/or analyzed,
a pretreatment step may be used to reduce its volume and/or to
deplete it of undesired biomolecules, as is known in this art.
[0042] Sequestering agents (e.g. Abs) are attached to the base, the
posts and the sidewalls of the collection region 17 in the body and
to the facing top surface 45 of the closure plate 35 in this
region. Sequestering agents that can assume their native
3-dimensional configurations in or on the permeable hydrogel as a
result of being properly coupled are surprisingly effective.
[0043] The microflow device 11, as illustrated in FIG. 8, is
operated to take advantage of gravity to both create uniform flow
and improve binding contact between the cells or other biomolecules
in the liquid being treated and the surfaces interior of the device
in the collection region 17 as a result of the direction of gravity
force vectors. In this respect, the device 11 is supported on a
base or stand 55 at an angle to the horizontal of between about
30.degree. and 60.degree.; preferably it is inclined at about
45.degree.. It is secured in position in any suitable manner, as by
clamping its upper edge to the oblique surface of the base 55 using
a clamp plate 57 and a pair of screws 59. The plurality of posts 31
in the collection region 17 are thus likewise oriented at an angle
45.degree. to the horizontal. To facilitate connections to the
inlet and outlet passageways, these two passageways are similarly
aligned in constructing the body 13. In this respect, the inlet
passageway 15 is aligned at an angle of between about 120.degree.
and about 150.degree. to the flat bottom surface 23 of the body and
more preferably at an angle of between about 130.degree. and
140.degree., with the angle being most preferably about 135.degree.
when the device is intended to be aligned at 45.degree. to the
horizontal as depicted in FIG. 8. The axis or centerline of the
outlet passageway 19 is similarly aligned. Moreover, axes of the
inlet and outlet passageways preferably lie in a common vertical
plane, which plane is perpendicular to the flat bottom surface 23
of the body 13, and they are preferably oriented at an angle to
each other within that plane of between 80.degree. and 100.degree.
and more preferably, at about 90.degree.. In this preferred
operating orientation, shown in FIG. 8, the inlet passageway 15 is
vertical and the outlet passageway 19 is horizontal, allowing
gravity to assist in the supply of a liquid sample to the device
and the stream to be smoothly exit, as by being withdrawn in a
horizontal direction without needing to travel at all upward as a
part of exit flow.
[0044] The irregular pattern of posts 31, which should have at
least three different diameters, is shown in FIG. 2 and is
described in more detail in the aforementioned pending U.S.
applications. It has been found that this post pattern prevents any
straight-line flow through the collection region, and it
destabilizes streamlined flow, creating eddies so that cells in
such a liquid sample are subjected to mixed force vectors in these
eddies. It has now been found that the operation at such an angle
to the horizontal, e.g. about 45.degree., appears to have a
synergistic effect in adding additional force vectors to the cells
which have a density greater than that of the aqueous buffer. These
vectors are oriented at about 45.degree. to the center line of the
flow passageway through the collection region, and tend to direct
the cells out of the path of liquid flow and into contact with the
surfaces within the collection region, with a significant resultant
beneficial effect on targeted cell capture.
[0045] Surprisingly, a set random or irregular pattern of posts 31
of different cross sectional sizes, e.g. circular cross section
posts of at least about 3 or 4 different sizes, about 70 to about
130 microns in diameter, in a collection region 17 about 100
microns high and about 2 to 4 mm wide, appears to promote a
particularly effective capture of cells from the flow of a liquid
sample, when the minimum separation spacing between posts is 50 to
70 .mu.m and preferably about 60 .mu.m. It is particularly
preferred that the cross sectional area of the posts, which all
have sidewalls formed by parallel lines that are perpendicular to
the base 33, is such that they occupy between and about 15 to 25%
of the volume of the collection region.
[0046] Following the completion of the passage of a liquid sample
through the device, a major percentage of such targeted cells, if
present in the sample, will have been captured within the
collection region. Washing is then carried out with buffers so as
to remove extraneous biomaterial that had been part of the sample
and that was not strongly captured by the antibodies or other
sequestering agents in the collection region 17, but may have
nonspecifically bound to the hydrogel-coated surfaces. Washing with
effective buffers is expected to purge the region by removing
substantially all nonspecifically bound material and leave only the
target cells attached in the collection region.
[0047] Once washing with buffers has been completed, the collection
region 17 may be filled with a chemical reagent that will cause the
captured cells to be suitably released, preferably with the device
11 now aligned horizontally. Release may be effected by a suitable
method as known in this art, such as chemically (e.g. change in
pH), or through the use of enzymatic cleavage agents or the like.
For example, a reagent may be applied to cleave a sequestering
agent, or to cleave the bond between such an agent and the cells,
in order to release the target cells from linked or coupled
attachment to a solid surface in the collection region. Specific
methods for both attaching Abs or the like and then effectively
removing captured ligands are discussed in U.S. Pat. No. 5,378,624.
For example, if the cells have been sequestered through the use of
antibodies that are specific to surface characteristics of the
target cells, release may be effected by treating with a solution
containing trypsin or another suitable protease, such as Pronase or
Proteinase K. Alternatively, a collagenase may be used to effect
release from other sequestering agents, or a specifically cleavable
linker may be used to attach the sequestering agent. During such
cleavage, the inlet 15 and the outlet 19 from the microchannel are
preferably plugged with simple stoppers 61 (see FIG. 9), and the
device 11 may be subjected to centrifuging following such release.
The centrifuging may be carried out at a speed equal to about 500 g
for about 5 minutes with the stoppers 61 in place and with the
device 11 oriented so that centrifugal force presses the targeted
biomolecules against the flat surface 45 of the closure plate 35
where they collect. At the completion of the centrifuging, the
device is oriented as shown in FIG. 9, and the targeted
biomolecules in the collection region 17 tend to rest upon and
adhere to the upper plate surface 45. Disassembly of the device 11
is then effected.
[0048] As seen in FIG. 7, there is an open triangular cross-section
region 63 just outside each longitudinal side edge of the body 13.
This open region 63 facilitates the slitting of the heat-shrunk
polymeric wrap 43 by a knife or scalpel (see FIG. 10) so that it no
longer encircles the device. After the slit sleeve 43 is unwrapped
(see FIG. 11), the flat upper cap plate 41 is easily removed, if
desired, as there is no physical bond between these two components
once the thermally shrunk sleeve 43 has been slit and unwrapped.
Then, grasping the tab 51 between thumb and forefinger, the body 13
is readily peeled from the top surface 45 of the bottom closure
plate (see FIG. 12) as they had been simply pressed into abutting
contact with each other without any bonding of surface to
surface.
[0049] As a result, the targeted biomolecules are present on the
top surface of the plate 35, and they can be readily subjected to
microscopic examination, as by FISH or by any other appropriate
analysis, while on the flat surface 45 of the plate itself.
Alternatively, they are readily available for analysis by using
molecular diagnostics as known in this art.
[0050] The following examples illustrate effective use of prototype
microflow devices of this type to sequester trophoblast cells from
an extract of cervical mucus. They should, of course, be understood
to be merely illustrative of only certain embodiments of the
invention and not to constitute limitations upon the scope of the
invention which is defined by the claims that are appended at the
end of this description.
Example 1
[0051] A microflow device for separating biomolecules is
constructed to provide a prototype device as in the drawings. The
body 13 is formed from PDMS, and with a cap plate 41 in place, it
is pressed against a flat glass plate 35 by a heat-shrunken sleeve
43 of dimensionally oriented polymer to close the flow channel. The
interior surfaces throughout the collection region 17 are
derivatized by incubating for 30 minutes at room temperature with a
10 volume % solution of Dow Corning Z-6020 or Z-6011. After washing
with ethanol, they are treated with nonfat milk at room temperature
for about one hour to produce a thin casein coating. Following
washing with 10% ethanol in water, a treatment is effected to coat
all the interior surface with a permeable hydrogel that is based on
isocyanate-capped PEG triols having an average MW of about 6000. A
prepolymer solution is made using 1 part by weight polymer to 6
parts of organic solvent, i.e. acetonitrile and DMF, and it is
mixed with an 1 mg/ml antibody solution in 100 mM sodium borate pH
8.0 containing BSA. The specific coating formulation comprises 100
mg prepolymer in Acn/DMF; 350 .mu.l, of 0.25 mg/ml of an antibody
mix in aqueous borate buffer; and 350 .mu.l, of 1 mg/ml BSA in
aqueous borate buffer. The formulation contains about 2% polymer by
weight. To isolate trophoblasts from a sample of cervical mucus,
for example, the antibody mix contains antibodies to Trop-1 and
Trop-2 which are specific to ligands carried by the exterior
surfaces of trophoblasts that are of fetal origin. The formulation
is left to incubate for 2 hours at 25.degree. C. in microflow
device 11. Following this incubation period, the flow channel is
flushed with 1% BSA/PBS to give antibody-coated surfaces designed
to try to isolate fetal trophoblast cells.
[0052] To test the effectiveness of such an angularly disposed
microflow device, a feed liquid that includes a mixture of BeWo and
Jurkat cells is used. BeWo cells are chosen because they express
Trop-1 and Trop-2 antigens, whereas Jurkat cells express neither
and thus serve as negative control cells.
[0053] Sufficient test feed solution is prepared for three runs; it
contains about 1,500 BeWo cells and about 1,500 Jurkat cells in a
1% BSA/PBS buffer. The feed solution is split into three aliquots,
with each of the aliquots containing about 500 BeWo cells and about
500 Jurkat cells. Three identical microflow devices are oriented at
45.degree. from the vertical, and one aliquot of the mixed cell
feed liquid is caused to flow through each as a result of suction
supplied by a vacuum pump. Different rates of flow are used: flow
rates of 1 .mu.l/min, 3 .mu.l/min, and 5 .mu.l/min.
[0054] Following flow through these test apparatus, washing is
carried out with a PBS buffer, and each device is then examined by
microscopy. Each of the two groups of captured cells is separately
counted manually using microscopy. With respect to the targeted
BeWo cells, it was found that, at the lower rate, about 75% of the
BeWo cells are captured in the collection channel region. This
value rises to about 82% at the middle flow rate of 3 .mu.l/min,
and remains at about 60% at the highest flow rate tested of 5
.mu.l/min. On the other hand, nonspecific binding of the Jurkat
cells in the collection region is relatively high at the lowest
flow rate, i.e. about 45%; however, it drops to only about 15% at 3
.mu.l/min, and to less than 5% at the highest flow rate. The
performance at the 45.degree. orientation is considered excellent,
and calculations show that, by operating at a flow rate which is
equal to a velocity through the collection chamber region of about
0.27 mm/sec, excellent collection of the targeted cells, with
minimum contamination by nonspecifically bound cells, is
obtained.
Example 2
[0055] Another microflow device of the same construction is
similarly coated with a permeable hydrogel which carries Trop-1 and
Trop-2 Abs. Cervical mucus from expectant mothers (8-12 weeks
gestation) is diluted to 10 ml with HAM's media (InVitrogen) and
treated with DNAse (120 units) at 37.degree. C. for 30 minutes.
After filtering through a 100 .mu.m cell strainer, the cells are
spun at 1500 RPM for 30 minutes. The cell pellet is resuspended in
HAM's media (100 .mu.l) and passed through the Trop-1 and Trop-2
coated microflow device by connecting the outlet tubing to a vacuum
pump and supplying about 50 microliters of this cell suspension of
cervical mucus extract to a vertically oriented conical reservoir.
The pump is operated to produce a slow continuous flow of the
sample liquid through the microflow device at room temperature and
preferably at a rate of about 3-5 .mu.l/min. During this period,
the Trop-1 and Trop-2 Abs that are attached to the surfaces in the
collection region, capture trophoblasts that are present in the
sample. After the entire sample is delivered, a slow flushing is
carried out with a 1% BSA/PBS aqueous buffer. About 100 .mu.l of
this aqueous buffer is fed through the device over a period of
about 10 minutes, which removes non-specifically bound biomaterial
from the flow channel in the device. Two additional washings are
carried out, each with about 100 .mu.l of PBS plus 1% BSA, over
periods of about 10 minutes to assure removal.
[0056] Following the completion of washing, the flow path in the
device is flooded with a 0.25% solution of Pronase and the inlet
and outlet to and from the device are blocked with stoppers. The
device is incubated in a horizontal orientation for about 20
minutes at 27.degree. C. At the completion of this time period, the
device is loaded into a centrifuge and spun at 500 g for about 5
minutes, causing the now-detached cells to be forced by centrifugal
force against the surface of the hydrogel-coated flat closure
plate. At the end of centrifuging, the aqueous Pronase solution is
drained from the device. The polymeric heat-shrunk sleeve is slit
at a triangular region along one side edge of the body and
unwrapped, and the top cap plate is then lifted from the body. The
tab is grasped, and the body is carefully peeled from the
underlying flat closure plate. The cells adhering to the surface of
the flat plate are stained with cystokeratin-7 and cytokeratin-17,
which are specific to cells of trophoblast origin. The cells that
are identified as trophoblasts are then easily analyzed using FISH
technology.
[0057] Although the invention has been described with regard to
certain preferred embodiments which constitute the best mode
presently known to the inventor for carrying out this invention, it
should be understood that various changes and modifications as
would be obvious to one having ordinary skill in this art may be
made without departing from the scope of the invention which is
defined in the claims which follow. For example, although certain
preferred materials have been described for the fabrication of the
substrate in which the microchannels are defined, there is a broad
range of structural materials that may be employed as are well
known in this art as being suitable for laboratory devices such as
this. Although the emphasis has generally been upon the separation
of fetal cells from a maternal blood sample or trophoblasts from a
cervical mucus extract, it should be understood that the invention
is useful for isolating a wide variety of blood cells, e.g.
nucleated erythrocytes, lymphocytes and the like, metastatic cancer
cells, stem cells, etc.; moreover, other biological materials, e.g.
proteins, carbohydrates, viruses, etc., might also be separated
from a liquid sample. When a sample contains specific
subpopulations of cells, negative enrichment can be effected by
targeting a group of unwanted cells to be captured.
[0058] The disclosures of all US patents and applications
specifically identified herein are expressly incorporated herein by
reference. Particular features of the invention are emphasized in
the claims which follow.
* * * * *