U.S. patent application number 16/507363 was filed with the patent office on 2019-11-14 for cell separation using microchannel having patterned posts.
The applicant listed for this patent is BIOCEPT, INC.. Invention is credited to Ram BHATT, Zhongliang TANG, Pavel TSINBERG.
Application Number | 20190344271 16/507363 |
Document ID | / |
Family ID | 36684420 |
Filed Date | 2019-11-14 |
United States Patent
Application |
20190344271 |
Kind Code |
A1 |
TANG; Zhongliang ; et
al. |
November 14, 2019 |
CELL SEPARATION USING MICROCHANNEL HAVING PATTERNED POSTS
Abstract
A micro flow 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.
The posts are spaced across the width of an expanded collection
chamber region in the flow path, extending between the upper and
lower surfaces thereof; they have rectilinear surfaces, being
curved in cross-sections, e.g. circular or tear-drop shaped, and
are randomly arranged so as to disrupt streamlined flow. The device
is oriented so that its lower surface is aligned at about
45.degree. to the horizontal. Sequestering agents, such as Abs,
which are attached to surfaces of the collection region via a
hydrophilic coating, preferably a permeable hydrogel containing
isocyanate moieties, are highly effective in capturing cells or
other targeted biomolecules while the remainder of the liquid
sample exits horizontally.
Inventors: |
TANG; Zhongliang; (San
Diego, CA) ; BHATT; Ram; (San Diego, CA) ;
TSINBERG; Pavel; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOCEPT, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
36684420 |
Appl. No.: |
16/507363 |
Filed: |
July 10, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15193712 |
Jun 27, 2016 |
10369568 |
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16507363 |
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14937481 |
Nov 10, 2015 |
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15193712 |
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13525225 |
Jun 15, 2012 |
9212977 |
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14937481 |
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11814276 |
Nov 25, 2008 |
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PCT/US2006/000383 |
Jan 5, 2006 |
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13525225 |
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11038920 |
Jan 18, 2005 |
8158410 |
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11814276 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0681 20130101;
G01N 1/405 20130101; B01L 3/502753 20130101; G01N 33/54386
20130101; B01L 2200/141 20130101; G01N 1/40 20130101; B01L
2400/0457 20130101; B01L 2300/0816 20130101; G01N 1/34 20130101;
Y10T 436/25375 20150115; B01L 2200/12 20130101; B01L 2300/16
20130101; B01L 2400/086 20130101; G01N 33/491 20130101; B01L
2200/0647 20130101; B01L 2300/0825 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 33/49 20060101 G01N033/49; G01N 1/34 20060101
G01N001/34; G01N 33/543 20060101 G01N033/543 |
Claims
1. A microflow apparatus for separating biomolecules from a sample
of a bodily fluid or other liquid, which apparatus comprises: a
body having a flow path defined as a cavity in a flat surface
thereof 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 extending between said inlet
and outlet means, which microchannel arrangement includes a
collection region with a plurality of transverse separator posts
located in said region, and closure plate means having a flat
surface that abuts said body flat surface and closes said flow path
cavity, said posts being integral with a base surface of said
collection region and projecting therefrom so as to extend to the
surface of said closure plate means, 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 wherein said
posts are conjugated with sequestering agents that will bind with
target biomolecules, 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 on said surfaces in said collection region.
2. The apparatus according to claim 1, wherein the base surface is
conjugated with sequestering agents that will bind with target
biomolecules.
3. The apparatus according to claim 1, wherein the flat surface of
the closure plate is conjugated with sequestering agents that will
bind with target biomolecules.
4. The apparatus according to claim 1, wherein the microchannel
comprises side walls, and wherein the side walls are conjugated
with sequestering agents that will bind with target
biomolecules.
5. The apparatus according to claim 1, wherein the biomolecules are
cells.
6. The apparatus according to claim 1, wherein said posts have at
least about 3 different cross sectional sizes.
7. The apparatus according to claim 1, wherein said posts are
aligned substantially perpendicular to the base surface of said
microchannel.
8. The apparatus according to claim 1, wherein said sequestering
agents are covalently bound to said posts using polyethylene glycol
(PEG) or polypropylene glycol (PPG).
9. The apparatus according to claim 1, wherein said sequestering
agents are coupled to said posts through bifunctional coupling
agents.
10. The apparatus according to claim 1, wherein the closure plate
and base surface are fabricated of plastic.
11. The apparatus according to claim 1, wherein the sequestering
agents are selected from the group consisting of antibodies,
avidin, and streptavidin.
12. The apparatus according to claim 1, wherein fluid flow through
the microchannel is driven using a pump.
13. The apparatus according to claim 12, wherein the pump is a
peristaltic pump.
14. The apparatus according to claim 1, wherein the inlet means
comprises a fluid reservoir.
15. The apparatus according to claim 1, wherein the biomolecule is
recovered by cleaving the sequestering agent.
16. The apparatus according to claim 1, wherein a linker attaches
the target biomolecule to the sequestering agent.
17. The apparatus according to claim 16, wherein the linker is a
cleavable linker, and wherein the biomolecule is recovered by
cleaving the linker.
18. The apparatus according to claim 1, wherein the flow through
for capturing biological substances within the microchannel is at a
rate of 0.1 to 2 mm/sec.
19. The apparatus according to claim 10, wherein the plastic is a
moldable plastic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 15/193,712, filed Jun. 27, 2016, which is a Continuation
of U.S. patent application Ser. No. 14/937,481, filed Nov. 10, 2015
(now abandoned), which is a Continuation of U.S. patent application
Ser. No. 13/525,225 filed Jun. 15, 2012 (now U.S. Pat. No.
9,212,977, issued Dec. 15, 2015), which is a continuation of U.S.
patent application Ser. No. 11/814,276, filed Nov. 25, 2008 (now
abandoned), which is a National Stage Application filed under 35
U.S.C. .sctn. 371 for PCT Application No. PCT/US2006/000383, filed
Jan. 5, 2006, which also claims priority to Provisional Patent
Application 60/678,004, filed May 4, 2005; and which is also a
Continuation-in-Part of U.S. patent application Ser. No.
11/038,920, filed Jan. 18, 2005, (now U.S. Pat. No. 8,158,410,
issued Apr. 17, 2012), all of which are incorporated herein by
reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention relates to separation or isolation of target
biomolecules from feed liquids and more particularly to methods and
apparatus for separating desired target human cells from bodily
fluids or the like.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] Cell separation is a rapidly growing area of biomedical and
clinical development, and improved methods of separating a desired
cell subset from a complex population will permit a broader study
and use of cells that have relatively uniform and defined
characteristics. Cell separation is also widely used in research,
e.g. to determine the effect of a drug or treatment on a targeted
cell population, to investigate biological pathways, to isolate and
study transformed or otherwise modified cell populations; etc.
Present clinical uses include, for example, the isolation of
hematopoietic stem cells for reconstitution of blood cells,
particularly in combination with ablative chemo- and radiation
therapy.
[0005] 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.
[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., J. Nanobiotechnology, 2, 5 (13 Jun.
2004) (6 pp) 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 waste
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 that contains cells may 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] The foregoing, briefly described references provide evidence
that there is continuing searching for improved separation methods
for isolating cells or other biomaterials from bodily fluids or the
like.
SUMMARY OF THE INVENTION
[0009] The invention provides a microflow apparatus for recovering
rare cells or other target biomolecules from relatively small
amounts of bodily fluids or the like which incorporates at least
one specially constructed microchannel device. Such device is
constructed using a substrate that is formed with a channel-like
flow, path which incorporates a plurality of transverse fixed posts
in a collection region; these posts are integral with the substrate
and extend between the upper and lower surfaces of the channel. 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 and eddies in a
bodily fluid or other liquid that is being caused to flow along
this flow path through the collection region. The posts vary in
size, e.g. cross-sectional diameter. Sequestering agents which are
selected to capture the desired target biomolecules and thereby
collect them within the collection region of the microchannel are
appropriately attached to the surfaces of the transverse posts and
all other surfaces throughout the entire collection region.
Preferably a supply well is provided at a location upstream of the
microchannel leading to the collection region. It has been found
that, by orienting the microflow apparatus so that it is inclined
to the horizontal, more complete cell separation can be achieved as
a result of force vectors created by gravity.
[0010] In one particular aspect, the invention provides a method
for separating biomolecules, such as cells, from a sample of bodily
fluid or other liquid, which method comprises causing such a sample
containing target biomolecules to flow downstream along a flow path
in a microflow device from an inlet to an outlet, which flow path
comprises a microchannel arrangement that includes a collection
region of expanded cross section, while orienting the device so
that said flow path collection region is aligned at an angle of
about 30.degree. to about 60.degree. to the horizontal, separating
target biomolecules from the flowing sample by (a) interrupting
straight-line flow of the liquid through said collection region, as
a result of blocking such flow with a plurality of separator posts
located in said region, which posts are integral with an upper or
lower surface of said microchannel and extend therefrom to the
opposite surface thereof, said posts extending transverse to said
flow path and being located in an irregular pattern that extends
laterally across said collection region and prevents straight-line
flow and streamlined flow therethrough, and all surfaces of said
collection region including said posts having sequestering agents
carried thereupon, and (b) capturing target biomolecules found in
the flowing liquid sample on surfaces in the collection region by
binding the target molecules to the sequestering agents as a result
of flow disruption by said irregular posts and force vectors that
result from gravity, which vectors are aligned at an acute angle to
said lower surface of said collection region, and discharging the
remainder of the liquid sample through the outlet.
[0011] In another particular aspect, the invention provides a
microflow apparatus for separating biomolecules, such as cells,
from a sample of bodily fluid or other liquid, which apparatus
includes a device that comprises a body having a flow path defined
therein through which such a sample containing target biomolecules
can be caused to flow, the body having inlet passageway to said
flow path, an outlet passageway therefrom, and a microchannel
arrangement extending between said inlet and outlet passageways,
and a closure plate, said microchannel arrangement includes a
collection region having upper and lower surfaces, one of which is
provided by said closure plate, and a plurality of transverse
separator posts, said posts being integral with one of said upper
and lower surfaces of said collection region and extending
laterally across said flow path to the other of said surfaces
provided by said closure plate, said posts being located in an
irregular pattern so as to interrupt straight-line flow and
streamlined flow of liquid through said region, said surfaces of
said collection region, including said posts, carrying sequestering
agents that will bind with target biomolecules, and said inlet
being aligned at an angle of between about 120.degree. to about
150.degree. to said flow path through said collection region,
whereby a sample can be fed substantially vertically downward
through said inlet while said body is aligned with said flow path
at about an angle of 30.degree. to 60.degree. to the horizontal and
whereby said irregular pattern of said posts and force vectors
resultant from gravity cause effective capture of target
biomolecules in said collection region, particularly upon the lower
surface thereof.
[0012] In a further particular aspect, the invention provides a
microflow apparatus for separating biomolecules, such as cells,
from a sample of a bodily fluid or other liquid, which apparatus
comprises a body having a flow path defined as a cavity in a flat
surface thereof 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 extending
between said inlet and outlet means, which microchannel arrangement
includes a collection region with a plurality of transverse
separator posts located in said region, and closure plate means
having a flat surface that abuts said body flat surface and closes
said flow path cavity, said posts being integral with a base
surface of said collection region and projecting therefrom so as to
extend to the surface of said closure plate means, 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 all said surfaces of said collection region including
said posts being coated with a hydrophilic permeable hydrogel and
carrying sequestering agents that will bind with target
biomolecules, 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 on said
surfaces in said collection region.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a perspective view of a substrate for a microflow
device wherein there is fabricated a simplified post-containing
collection region in a microchannel.
[0014] FIG. 2 is an enlarged fragmentary view showing a portion of
the collection region of FIG. 1 where the patterned posts are
located.
[0015] FIG. 3 is a front cross-sectional view of the substrate of
FIG. 1 taken along the line 3-3 with a cover plate attached to its
bottom surface.
[0016] FIG. 4 is a schematic perspective view of an apparatus that
incorporates two valves with a substrate as generally shown in FIG.
1 through the inclusion of an intermediate plate.
[0017] FIG. 5 is a cross-sectional view taken along line 5-5 of
FIG. 4.
[0018] FIG. 6 is a schematic plan view showing a substrate of the
type shown in FIG. 1, wherein pumps are fabricated as part of the
microflow apparatus.
[0019] FIG. 7 is a schematic view of a portion of a substrate in
which a micro-mixer is incorporated into the supply region.
[0020] FIG. 8 is a schematic representation of antibodies attached
throughout a collection region via the application of a hydrophilic
coating.
[0021] FIGS. 9 and 10 are schematic representations of chemistry
that may be used to covalently attach sequestering agents, e.g.,
antibodies, throughout a collection region using a hydrophilic
coating, along with depiction of subsequent capture of desired
target cells.
[0022] FIG. 11 is a flow sheet illustrating the steps of a cell
recovery operation utilizing such a patterned post, cell separation
device.
[0023] FIG. 12 is a perspective view of an alternative embodiment
of a microflow apparatus wherein there is fabricated a
post-containing collection region in a microchannel device designed
for operation at an incline to the horizontal.
[0024] FIG. 13 is a bottom view of the body portion of the
microflow device of FIG. 12.
[0025] FIG. 14 is a front cross-sectional view, enlarged in size,
taken along the line 14-14 of FIG. 12.
[0026] FIG. 15 is a schematic view showing the apparatus of FIG. 12
in use.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] Basically an apparatus is provided which includes a device
having a body or substrate 11 that has a flow path defined therein
that includes at least one microchannel 13 having a collection
region 17, which flow path is linked to a sample inlet 15 and a
liquid outlet 19. As mentioned hereinafter, the flow path may
include several microchannels, arranged in series, each of which
has one such collection region. Alternatively, a microchannel may
have more than one collection region, arranged in series, and there
may also be more than one inlet and more than one outlet, all as
well known in this art. Moreover it can be a part of an integrated
microfluidic apparatus constructed on a chip, a disk or the like;
in such an apparatus, substantially all of the MEMS
(micro-electro-mechanical systems) or components needed to carry
out cell recovery and/or diagnosis of biomolecules isolated from a
sample may be incorporated as part of a single, compact, easily
handled unit.
[0028] FIG. 1 is a perspective view of the body or substrate 11
which is formed with a flow path that includes a microchannel 13 to
which sample liquid is to be supplied through an opening or well 15
that serves as an entrance or inlet and an opening 19 that serves
as an outlet. The cross-section of the collection region 17 is
greater than that of an inlet section 18 that leads thereinto from
the inlet opening 15. The inlet section contains a pair of axially
aligned divider/supports 21 just upstream of where it widens at the
end of the region 18 to enter the collection region 17. These
central dividers break the flow into two paths and serve to
distribute the flow of liquid more evenly as it is delivered to the
entrance end of the collection region 17. The collection region 17
contains a plurality of upstanding posts 23 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 is
such that there can be no straight-line flow through the collection
region and that streamlined flow streams are disrupted, assuring
there is good contact between the liquid being caused to flow along
the flow path and the surfaces of the posts. The posts are integral
with the flat base 22 of the collection region 17 and extend
perpendicular thereto, presenting surfaces that are vertical
relative to a horizontal path of liquid being caused to flow
through the flow channel of the substrate 11. Preferably they
extend to and are affixed at their free end surfaces as by bonding
to the surface of a facing flat closure plate 27 which is parallel
to the base surface 22 and which closes the flow channel, as is
described in detail hereinafter. Inlet and outlet holes 24a and 24b
may be drilled through such a closure plate, but they are
preferably formed in the substrate 11. Another flow divider/support
21a is located at the exit from the collection region.
[0029] As is well known in this art, a substrate may be knitted
with a flow path that includes a pair of parallel microchannels,
each of which has a collection region. Such could be used in a
series flow arrangement, or they could be used in parallel flow
operation. Flow may be achieved by pumping, e.g. using a syringe
pump or the like, or by vacuum that would draw liquid through from
a reservoir at an inlet well provided by a large diameter inlet
hole 24a. Preferably such a well is included which has a capacity
to hold about 50 pi to about 500 .mu.l of liquid sample.
[0030] 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 at a rate of about
0.05 to 5 min 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 throughout the
collection region. 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.
[0031] Generally the substrate 11 can be made from any suitable
laboratory-acceptable material, such as silicon, fused silica,
glass and polymeric materials. It may be desirable to use a
material that is optically transparent, particularly when a
diagnosis function is desired to be optionally employed. In its
simplest embodiment, the substrate carrying the fabricated
microchannel is sealed with a plate 27 having a flat surface that
will abut the facing surface of the substrate 11 as depicted in
FIG. 3. Such plate may be fabricated from the same material or may
simply be a solid cover plate made of glass; however, an
intermediate flow regulation plate 25 may be included as explained
hereinafter. Suitable solid impermeable 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. Such patterned substrates may be fabricated using
any convenient method such as those selected from among
conventional molding and casting techniques.
[0032] Substrates 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.
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 patterned
post substrates in PDMS or other suitable polymeric resin.
[0033] 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 substrate 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 substrate
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.
[0034] The layout and the dimensions of the microchannel 13 and of
patterned posts 23 in the collection region 17 are determined by
the mask used in exposure step of the fabrication of the master
mold. The depth of the microchannel 13 is controlled by the
thickness of the SU-8 layer of the master mold, which is determined
by spin-coating conditions. FIG. 2 provides a top view of the
microchannel 13 showing an enlargement of the posts 23 in the
collection region 17 in a preferred generally random
arrangement.
[0035] In alternative embodiments, holes 24 could be drilled into
or otherwise created in the flat, unbroken surface of a released
PDMS replica substrate or in the cover plate to provide for inlet
and outlet connections. In the former instance, it could be mated
with a simple microscope cover slip or other suitable flat plate,
such as a thin flat piece of PDMS, that would provide an
imperforate cover or base plate for the substrate. After subjecting
the two components to plasma-cleaning for two minutes, the two
cleaned surfaces are immediately placed in surface contact, without
touching the facing surfaces, which then become sealed by surface
reaction as well known in this art, forming a permanent seal and
closing the microfluidic flow path.
[0036] Should it be desired to integrate on-chip flow management
into such an apparatus, a separate SU-8 molding master
incorporating cavities for flow regulation features, such as
pneumatic valves and the like, may be similarly fabricated. A flow
regulation plate or layer 25 produced from such a master mold would
first be laminated to the microchannel substrate 11 (see FIGS. 4
and 5), and it would in turn be laminated to a flat closure plate
27. The employment of such flow-regulating components and other
MEMS in a microflow apparatus is shown in U.S. Pat. Nos. 6,074,827
and 6,454,924, the disclosures of which are incorporated herein by
reference. By carefully aligning such a flow regulation plate 25
with a microchannel carrying substrate 11 and then annealing
overnight at 80.degree. C., a composite structure is fabricated.
Thereafter, cavities in the flow regulation plate 25 are closed by
a flat plate or glass slide 27 using the same technique described
earlier. As a further option, a second flow regulation plate might
be laminated to the first plate 25, employing the same technique,
should it be desired to incorporate still more sophisticated
controls and optional processing.
[0037] For example, on-chip flow regulation mechanisms could be
provided in a multichannel system formed in a substrate 11 by
disposing them in a flow regulation layer 25 that would be sealed
to the substrate. A simple system is illustrated in FIGS. 4 and 5
where passageways 24a and 24b lead to the inlet and exit. Air
supply to pneumatic valves 29 may be via drilled or otherwise
suitably formed holes 30 that extend through the substrate 11 into
the plate 25. The flow regulation plate 25 or the substrate 11
could optionally contain alternative supply passageways that could
deliver liquid to the inlet 15 and also might include an
alternative exit or removal passageway as well known in this
art.
[0038] As mentioned, an arrangement wherein two series-connected
collection regions are provided, lends itself to different methods
of operation and use. For example, when a sample liquid is to be
treated that potentially contains two different subpopulations of
target biomolecules or cells of interest, one type of sequestering
agent can be attached to the posts in one collection region or
chamber, and a different type of sequestering agent can be attached
to posts in a downstream collection chamber. Alternatively, in an
instance where the target cells are extremely rare, it might be
desirable to attach the same sequestering agents to the posts in
both collection chambers so as to enhance the likelihood of being
able to capture nearly 100% of the cells in the liquid sample.
[0039] From a constructional standpoint, some additional components
that might optionally be incorporated into such an apparatus are
illustrated in FIGS. 6 and 7. FIG. 6 shows a microchannel similar
to that depicted in FIG. 1 in which peristaltic-type pump
arrangements are incorporated into an inlet passageway region and
an outlet passageway region flanking the collection chamber.
Illustrated is a microchannel arrangement 13' that includes an
inlet 15', a collection chamber 17' and an outlet 19' wherein an
integrated pumping arrangement 41 is constructed by the
incorporation of three specially designed membrane valves located
in an entrance passageway 18' leading to the collection chamber.
The schematic representation is of an arrangement similar to that
shown in FIGS. 4 and 5 where the application of air or other high
pressure gas to a passageway 30' leading to the pressure side of
each valve membrane in a flow regulation layer or plate causes that
membrane to expand, squeezing the liquid in the adjacent region of
the microchannel with which it is associated. By programming a
control unit so as to operate the three valves in sequence, from
left to right, a wave movement is set up whereby the liquid in the
entrance region 18' of the microchannel is pumped to the right and
through the collection device 17'. If desired, a similar
peristaltic-type pumping arrangement 43 is also incorporated into
the exit passageway region 45 leading downstream from the
collection chamber 17'.
[0040] As another potential alternative, a micromixing arrangement
is illustrated in FIG. 7. A micromixer 51 is illustrated that
includes a circular pathway 53 that leads to a supply passageway
55, which could be an entrance passageway leading to a collection
chamber in a substrate such as earlier described. A pair of inlet
channels 57a and 57b are provided to supply liquids to the circular
pathway 53, and liquid flow through the pathways 55, 57a and 57b
are controlled via pneumatic valves 59. Three additional pneumatic
valves 61 are positioned in the passageway itself and constitute a
peristaltic-type pump 63 of the type previously described. The
arrangement provides an efficient way of micro-mixing two liquids
in the substrate itself prior to delivery to a collection chamber
or the like. For example, by filling the circular pathway 53 with
some liquid from one inlet channel 57a and with some buffer from
the inlet channel 57b, mixing can then be effected by operating the
three valves 61 in sequence to pump the liquid around the ring
provided in the circular pathway; thus, the liquid can be
thoroughly mixed it before its discharge through a delivery
passageway 55.
[0041] The polymeric surface of the patterned post region 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. For example, after plasma
treatment and closure of the microchannel-carrying substrate, a 1
to 50 volume % solution of an amino-functional silane (e.g. a 10%
solution of Dow Corning Z-6020), or a thio-functional silane, in
ethanol may be injected into the microchannel to fill the region 17
between the openings 15 and 19, and the flooded microchannel 13 may
then be left to incubate for 30 minutes at room temperature.
Derivatization can be performed on a non-fully cured polymer, such
as PDMS, before the closure of the microchannel region with the
plate. In such case, as earlier mentioned, an alternative is to
slightly undercure the PDMS substrate and then complete the curing
after affixing the seal plate and treating with the substituted
silane or other functionalizing reagent. For example, a final
heating step of about 90 minutes at about 50 to 90.degree. C. might
be used to complete the curing after treating with the Z-6020.
Alternatively one or two days at room temperature would also
complete the curing. Such derivatization treatment may also be
performed before the closure of the microchannel region because
derivatization of the facing flat surface is no real consequence.
The flow path is then purged with ethanol, and the microchannel is
ready for attachment of biomolecule sequestering agents.
[0042] The term sequestering agent is used to refer to material
capable of interacting in a specific fashion with a target
biomolecule to physically sequester 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
polypeptides 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, chondroitin 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.
[0043] Sequestering agents can be directly or indirectly
immobilized upon the posts, and the posts may be pre-treated and/or
coated to facilitate attachment. Indirect immobilization is clearly
preferred and contemplates the employment of an intermediate agent
or substance that is first linked to the post; moreover, it may be
desirable to use coupling pairs to link the sequestering agent to
the intermediate agent. For example, streptavidin, or an antibody
directed against another species antibody, might be attached to the
intermediate agent, which would thereafter couple to a biotinylated
Ab or to an Ab of such other species. Such an arrangement permits a
generic production of such microflow devices that might then be
used to capture a variety of cells from different samples or to
effect negative enrichment.
[0044] The use of Abs as sequestering agents may be preferred for
cell separation, and their attachment is discussed in U.S. Pat.
Nos. 5,646,404 and 4,675,286 and throughout the prior art. For
example, procedures for non-covalent bonding are described in U.S.
Pat. No. 4,528,267. Procedures for covalently bonding antibodies to
solid supports are also described by Ichiro Chibata in IMMOBILIZED
ENZYMES; Halstead Press: New York (1978) and in A. Cuatrecasas, J.
Bio. Chem. 245:3059 (1970), the contents of both of which are
hereby incorporated by reference. Kawata et al., J. Exp. Med.,
160:653 (1984) discloses a method for isolating placental cell
populations by detecting target cells using cell-specific Abs, e.g.
monoclonal antibodies against human trophoblasts (anti-Trop-1 and
anti-Trop-2). U.S. Pat. No. 5,503,981 identifies three other
monoclonal Abs which can be used for this purpose.
[0045] The antibody is preferably bound to the solid post surfaces
indirectly, such as through the use of a surface layer or a coating
of long linkers to which the Abs are then attached. For example,
the surface can be first coated with a bifunctional or
polyfunctional agent, such as a protein; the agent is then coupled
with the antibody using a coupling agent, e.g., glutaraldehyde. The
antibody can also 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, or the antibody might be coupled to a hydroxylated
material by cyanogen bromide. Particularly preferred is the use of
a hydrophilic polyurethane-based hydrogel layer having free
isocyanate groups, which is described hereinafter in connection
with FIG. 9, or the use of a hydrophilic linker of substantial
length, such as one of PEG, polyglycine, as described hereinafter
in connection with FIG. 10.
[0046] The sequestering agents chosen are directed toward specific
capture of the biomolecule of interest, which 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. As mentioned above, such
microflow devices may also be used for negative enrichment by
targeting known contaminating cells.
[0047] When antibodies (Abs) 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.
[0048] With the antibodies or other sequestering agents in place
throughout the patterned post collection region, the microchannel
device is ready for use. A bodily fluid, such as a blood or urine
sample, or some other pretreated liquid containing the target cell
population, is caused to flow along a flow path through the
collection region 17, as by being discharged carefully from a
standard syringe pump into an inlet passageway 24a leading to the
inlet 15 for such a microchannel device or drawn by a vacuum pump
or the like therethrough from a sample reservoir provided by a
relatively large diameter inlet passageway 24a which serves as well
to hold the desired volume of sample for a test. The opening 24a
may contain a fitting (not shown) for mating with tubing connected
to such a syringe pump when such is used. The pump may be operated
to effect a flow of about 0.5-10 through the apparatus. 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.
[0049] To potentially increase the overall efficiency of a cell
separation method, it may be desirable to collect the sample
exiting the outlet 19 and cause it to flow through the microchannel
device more than once; such repeat treatment may be particularly
useful when the cells are particularly rare and thus are likely
very few in number in the sample. However, because of the high
efficiency of capture achieved by the apparatus, it is expected
that such repeat flow will seldom be needed. Alternatively, two
collection chambers linked in series might be used as earlier
mentioned. Moreover, if somewhat larger volumes of bodily fluid
samples are being processed, two or more microchannels could be
used in parallel on a substrate.
[0050] Sequestering agents (e.g. Abs) are attached to the base, the
facing surface, the posts and the sidewalls of the collection
regions in the microchannels; however, such sidewall surfaces are
not particularly effective in capturing cells as are the base,
facing surface and the posts which disrupt the flow. It has been
determined that flow of liquid containing cells or other
biomolecules through even a confined lumen results in the cells
being primarily present in the central flow stream region where
flow shear is the least; as a result, capture upon sidewalls that
carry sequestering agents is quite sparse in comparison to the
capture upon surfaces in the immediate regions where the transverse
posts have disrupted streamlined flow. In these regions,
sequestering agents that can assume their native 3-dimensional
configurations as a result of properly coupling are surprisingly
effective.
[0051] Following the completion of flow of the liquid sample
through the device, the targeted cells would, if present, have been
captured within the collection region, and purging is first carried
out with buffers so as to remove all of the extraneous biomaterial
that had been part of the sample and that has not been strongly
captured by the antibodies or other sequestering agents in the
collection region. Such purging with effective buffers is expected
to leave only the target cells attached in the collection region in
the microchannel device, having removed all nonspecifically bound
material.
[0052] Once purging with buffer has been completed, if the
objective of the treatment method is cell collection alone, the
captured cells are then suitably released. As mentioned
hereinafter, in some instances, it may be desired that some
analysis be carried out in situ. For example, the cells may be
counted while attached, or they may be lysed and then subjected to
PCR either in the collection chamber or downstream.
[0053] When release is to be effected, any method known in this art
may be used, such as mechanical (e.g. high fluid flow), chemical
(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
the sequestering agent or to cleave the bond between the agent and
the cells in order to release the target cells from the collection
region. For instance, trypsin or a specifically focused enzyme may
be used to degrade the Abs and/or the cell surface antigens.
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 rare cells, release may be effected by
treating with a solution containing trypsin or another suitable
protease, such as 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 outlet from the
microchannel is connected to a reservoir or other collector, and
the discharge stream carrying the released rare cells is collected
for further analysis. The microchannel device may be fabricated
with more than one exit passageway at the outlet and with valves
for regulating which exit is open; such allows one exit passageway
to be used for the waste discharge during the preliminary steps and
then a different exit passageway to direct the target cell stream
to a collection container.
[0054] It has been found that the placement and shape of the posts
23 in the patterned post collection region 17 can be engineered for
optimal fluid dynamics and enhancement of capture of target cells
through their specific surface characteristics. Very generally, in
most instances, the preferred shape of the horizontal cross-section
of the transverse fixed posts 23 avoids sharp angles which might
promote nonspecific binding to the transverse surfaces of the
posts. The posts 23 have rectilinear exterior surfaces and
preferably have either a generally circular cross sectional shape
or regular polygonal of 6 or more sides. Alternative shapes that
might be used are tear-drop shape where the tip is at the
downstream end and shallowly curved, or oval shape; however, should
more impact be desired, a square shape might be used. The pattern
of the posts should create a flow pattern in the liquid stream
which enhances the capture of target cells by the sequestering
agents attached to the surfaces of the posts, the base and the
facing surface. To achieve this end, it has been found that the
posts should be of different sizes and be arranged in a set random
pattern. Surprisingly, a random pattern of posts 23 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 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.
[0055] It is particularly preferred that the cross sectional area
of the posts, which all have sidewalls formed by parallel lines
which are perpendicular to the base, is such that they occupy
between and about 15 to 25% of the volume of the collection region.
Preferably the post pattern will be such that they occupy about 20%
of the volume of the collection region, leaving a void volume for
liquid flow of about 80%. The particular random pattern of post
locations shown in FIG. 2 appears to particularly enhance the
tendency of the cells to be captured by sequestering agents in
these regions where streamlined flow has been effectively
disrupted. The posts 23 are substantially spaced apart from one
another, e.g. by at least about 60 microns, and posts of different
sizes are preferably located upstream and downstream of one
another.
[0056] Smaller posts may create eddy regions downstream of larger
posts, and as a result of the flow pattern that is generated, the
surfaces in the vicinity, and particularly the bottom surface of
the collection region, will show particular effectiveness in
capturing target cells. As shown in FIG. 2, any straight line
extending longitudinally of the flowpath at a location more than
about 100 microns from a sidewall will intersect a plurality of
posts. As previously mentioned, the posts are integral with the
base 20 surface of the substrate and are preferably affixed at
their opposite or free ends to the facing surface, i.e. either a
flow-regulation plate 25 or a flat closure plate 27.
[0057] As indicated before, the sequestering agents, such as
antibodies, may be attached throughout the collection region in a
manner such that they are able to perform more effectively by
coating the surfaces with (a) a thin layer of a particular
hydrophilic hydrogel substance or (b) hydrophilic linkers, such as
PEG, polyglycine or the like of a molecular weight of at least
about 1,000 daltons, preferably having a MW of about 2,000 to
100,000 daltons, and more preferably between about 3,000 and 50,000
daltons. Particularly preferred is the employment of (a), i.e. the
hydrophilic permeable hydrogel coating which is an
isocyanate-functional polymer containing PEG, PPG or a copolymer
thereof that is polymerized by urethane bonds and that contains
reactive isocyanate groups. A preferred hydrogel utilizes a
three-leg PEG molecule of approximately 6,000 molecular weight that
is created by the addition of ethylene oxide to glycerol. The
resultant polyol is reacted with isophorone diisocyanate and
trimethylol propane to prepare a prepolymer. A mixture of the
prepolymer with appropriate buffer, solvent and other components
for a particular application is crosslinked in situ upon surfaces
of the collection region in a microflow device. Schematically shown
in FIG. 8 is a representation of a collection region within a
microchannel wherein there are a plurality of posts 61 of varying
diameter that are randomly arranged to disrupt streamlined flow
through the chamber, wherein each of the posts 61 and the facing
flat surfaces carry an exterior coating 63. Sequestering agents 65
in the form of antibodies are depicted as being attached to the
hydrophilic permeable hydrogel coatings on the posts; as a result,
they retain their native three-dimensional confirmation, unaltered
by attachment to the hydrogel which is primarily water and thus
quite deformable.
[0058] FIG. 9 is provided as a schematic representation of
chemistry that may be employed when a hydrophilic permeable
hydrogel coating 49 of the preferred character is used, as in FIG.
8. Shown are representative sequences of attaching sequestering
agents, i.e., antibodies, to all the surfaces throughout the
collection region. Point 1 of FIG. 9 shows a surface following
amino-derivatization by treatment with an aminosilane or the like.
This step is followed by using non-fat milk to casein-coat the
surfaces, see Point 2. Point 3 represents the coated surface after
coating has been carried out. A prepolymer containing PEG of a
molecular weight of about 3400 that is end-capped with
toluenediisocyanate is dissolved in a water-miscible, organic
solvent, preferably an aprotic solvent such as a mixture of NMP and
CH.sub.3CN. The polymer preferably contains tri- or higher
functional polyols, e.g. PEGs and PPGs, and may contain
trifunctional isocyanate. An aqueous solution is then prepared
containing about 98.5 weight percent water, which solution is
pumped through the microchannel so that the surfaces of the posts
and the facing surfaces of the collection region become coated with
this hydrophilic hydrogel coating, as a result of reaction of some
of the end-capped isocyanate groups at the amine-derivatized
surfaces. The end result is represented at Point 3 in FIG. 9, which
is here a hydrogel which is created as a result of initial reaction
with water and later formation of urea bonds.
[0059] Point 4 represents the addition of antibodies which will
have surface amino groups. They can be attached directly to such
hydrophilic permeable hydrogel coatings of the posts, as shown in
Point 5, by covalent bonding of the Abs amines to either isocyanate
or thiocyanate groups carried by the hydrophilic coating.
Alternatively, the antibodies may first be thiolated as depicted at
Point 6 of FIG. 9, and these thiolated antibodies then supplied in
aqueous solution to the collection chambers, where they will in
turn readily covalently bond to the isocyanate groups of the coated
polymers, see Point 7.
[0060] As depicted at Points 8 and 9 in FIG. 9, when cells in a
liquid sample that is being caused to flow through the collection
chamber, as a result of the disrupted streamlined flow, come in
contact with the posts and/or the facing surfaces, antigens on the
cell surfaces specific to the antibodies become conjugated thereto,
effectively capturing the cells.
[0061] FIG. 10 is provided as a schematic representation of
chemistry that may be employed when an elongated PEG or PPG linear
polymer, instead of a hydrogel, is used to tether a sequestering
agent, particularly an antibody, to the surfaces in a collection
region. The linear polymer is selected so as to be of such length
that the antibody will be able to assume its native three
dimensional configuration in an aqueous environment where capture
is being carried out. Point 1 of FIG. 10 shows the surface
following amino-derivatization by treatment with an aminosilene or
the like. This step is again followed by using non-fat milk solids
to casein-coat the surfaces as described above. Following washing,
all the surfaces are treated with a linear PEG or PPG having a
molecular weight of at least about 2000, and preferably at least
about 3000, which has a NHS moiety at one end and maleimidyl moiety
at the opposite end. The N-hydroxy-succinimidyl ester moiety reacts
readily with the amino groups on the surfaces to provide a coating
at least about 1 micron thick. After suitable incubation, the
microchannel is drained and washed with a suitable buffer, leaving
the maleimido-PEG-coated surfaces as represented by point 3 of FIG.
10. Point 4 represents antibodies which are specific for
trophoblasts and which inherently have surface amino groups. The
antibodies are preferably thiolated using a suitable reagent, such
as Traut's reagent, to reach the point depicted as point 5 in FIG.
10. The thiolated antibody is then conjugated with the
maleimido-PEG-coated posts by introduction of the purified
thiolated antibody into the microchannel in a buffered solution and
allowing it to appropriately incubate. The microchannel is then
washed with a suitable buffer, and the conjugated arrangement
depicted as point 6 is obtained.
[0062] Point 7 of the schematic representation of FIG. 10 shows the
capture of a trophoblast by an antibody that is tethered to a
surface by the linear PEG coupling agent.
[0063] A more preferred, alternative construction of a microflow
apparatus is illustrated in FIGS. 12-15. A microflow device 71 is
shown which resembles that shown in FIGS. 1-3, but which is
constructed to take advantage of gravity to create uniform flow, to
improve contact between a liquid being treated and the surfaces
interior of the device in the collection region, and to minimize
attachment of the targeted cells in regions outside of the
collection chamber, such as the inlet and outlet. In this respect,
the apparatus is designed for use inclined at an angle of between
about 30.degree. and 60.degree. to the horizontal, preferably at
about 45.degree.. Accordingly, the device includes a body or
substrate 73 that includes a microchannel 75 that extends
therethrough from an inlet passageway 77 to an outlet passageway
79, with a collection region 81 being located therebetween which
includes an entrance region 83 and an exit region 85 as before. The
collection region 81 is formed as a recess or a cavity in a flat
surface 87 of the body, and the collection region itself has a flat
base surface 89 that is substantially parallel to the surface 87 of
the body. A plurality of posts 91, as described previously, extend
from the base surface 89, perpendicular thereto, to the level of
the body flat surface 87, together with flow dividers in the
entrance and exit regions.
[0064] Except for the orientation of the entrance and exit
passageways 83, 85, the construction of the device 71 is
essentially the same as described hereinbefore with regard to those
devices depicted in FIGS. 1-5. As shown in FIGS. 12 and 14, the
cavity which constitutes the microchannel 75 is closed with a flat
solid plate 93 which is preferably of greater dimensions than the
body 73 to facilitate handling of the completed apparatus during
treatment and analysis of a liquid sample. The flat closure plate
93 may be made of glass or a suitable impermeable polymeric
material as described hereinbefore. The plate 93 may be coated with
a layer of the same polymeric material as that from which the body
is cast, molded or otherwise suitably constructed. More preferably,
a PDMS polymer is used, and if a standard glass slide about 25 mm
by 75 mm is used, it may be coated with a layer or a thin film 95
of PDMS as indicated in FIG. 12. Attachment of the closure plate 93
could affix its flat surface to the end surfaces of the posts 91,
as described hereinbefore, or they might simply be left to
substantially abut.
[0065] As best seen in FIG. 14, the inlet passageway 77 is aligned
at an acute angle of 30.degree. and 60.degree. to the flat face
surface of the microchannel which is parallel to the flow path
therethrough; more preferably at an angle of between 40.degree. and
50.degree. and most preferably at about 45.degree. thereto. This
arrangement allows the feed sample to be fed vertically downward
into the microflow apparatus 71, as schematically shown in FIG. 15,
with the apparatus clamped along its upper edge to the oblique
surface of a base 96 by a plate 98 so that the flow path
therethrough is inclined to the vertical, e.g. at about 45.degree..
Thus, the feed sample will fill the wide entrance region 83 to the
collection region 81, and gravity assists in keeping the collection
region, where the posts are located, full and promoting a desired
slow and uniform flow therethrough. More importantly, because the
liquid flow will be linearly through the chamber in a direction
parallel to the upper and lower surfaces, and because the cells are
heavier than the liquid aqueous buffer in which they are being
transported, gravity will create a force vector on these cells
different from the flow vector of the transporting liquid. As a
result, in addition to the disruption of streamline flow that is
created by the random pattern of posts of different sizes in the
collection region, there is a further vector which causes the cells
to escape from the flowing stream and adhere to a surface by
attachment to a sequestering agent. This effect has been found to
substantially improve cell collection even when there is a
relatively slow flow through the collection region.
[0066] The inlet and outlet passageways 77, 79 are preferably
located in the same vertical plane, and the orientation of the
outlet passageway is preferably at about 90.degree. thereto. Thus,
when the apparatus 71 is clamped to the base 96 as shown in FIG.
15, with the inlet passageway 77 vertical, the outlet passageway 79
is horizontal. This allows the sample containing cells and other
biomaterials that have not been bound by the Abs to exit
horizontally, thus facilitating their removal and minimizing
non-specific attachment within the flow channel and sedimentation
in the outlet region.
[0067] FIG. 15 also depicts the presently preferred method of
running a treatment wherein a sample is fed vertically into the
inlet passageway to the microflow apparatus, and the outlet
passageway 79 is connected via suitable tubing to the suction side
of a syringe pump or the like which then facilitates the flow of
sample through the apparatus at the desired flow rate. The flow
rate is carefully controlled so that the liquid sample is withdrawn
at a rate so that the average velocity of liquid flow in the
collection region is between about 0.1 and about 1 mm/sec,
preferably between about 0.2 and about 0.8 mm/sec and more
preferably between about 0.25 and about 0.5 mm/sec, which has been
found to maximize capture of the targeted cells as a result of the
flow disruption of the posts and the force vectors resultant from
gravity. Higher velocities are found to be less effective and may
injure delicate cells that might be sought. Although the apparatus
is illustrated in its simplest form, it should be understood that
various valves and ancillary components, as known in these MEMS
devices and as generally described hereinbefore, can be
incorporated together with the microflow device 71.
[0068] The following examples illustrate effective use of prototype
microchannel devices of this type to sequester trophoblast cells
from an extract of cervical mucus. The overall method is outlined
in the flow sheet attached as FIG. 11. The examples 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 which are
appended at the end of this description.
Example 1
[0069] A microflow device for separating biomolecules is
constructed using a prototype substrate as generally illustrated in
FIG. 1. The substrate is formed from PDMS and is bonded to a flat
glass plate to close the flow channel. The interior surfaces
throughout the collection region are derivatized by incubating for
30 minutes at room temperature with a 10 volume % solution of Dow
Corning Z-6020. 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 the interior with a permeable
hydrogel formed from a prepolymer of isocyanate-capped PEG triols,
average MW of 6000, using 1 part by weight prepolymer to 6 parts of
organic solvent, i.e. acetonitrile and DMF, and mixing it with
water and flowing it through the channel as described with respect
to FIG. 9.
[0070] For this test, it is desired to isolate trophoblasts from a
sample of cervical mucus, and antibodies to Trop-1 and Trop-2 are
selected which are specific to ligands carried by the exterior
surfaces of trophoblasts which are of fetal origin. Antibody (0.1
mg) was dissolved in 100 .mu.l of 0.2M sodium borate/0.15 M NaCl
containing 5 mM EDTA (pH 8.3) and reacted with 5 .mu.l of 40 mM
Traut's reagent at RT for one hour to effect thiolation. Excess
Traut's reagent is reacted with 10 .mu.l of 100 mM glycine followed
by purification of the thiolated antibody on the Centricon-30.TM.
membrane. Thiolation was confirmed by standard laboratory
procedures.
[0071] About 5 micrograms total of the thiolated anti-Trop-1 and 2
in aqueous solution, at a concentration of about 0.5 mg/ml, are
supplied to the pretreated microflow device, and the solution is
left to incubate for 2 hours at 25.degree. C. Following this
incubation period, the flow channel is flushed with a 1% PBS/BSA to
give antibody-coated surfaces which were then used to try to
isolate fetal trophoblast cells.
[0072] Cervical mucus from expectant mothers (8-12 weeks gestation)
was 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 were spun at 1500 RPM
for 30 minutes. The cell pellet was resuspended in HAM's media (100
.mu.l) and passed through the Trop-1 and Trop-2 coated microchannel
by hooking the microflow separation device up to outlet tubing from
a Harvard Apparatus syringe pump which is filled with about 50
microliters of this cell suspension of cervical mucus extract. The
syringe pump is operated to produce a slow continuous flow of the
sample liquid through the microflow device at room temperature and
a rate of about 10 .mu.l/min. During this period, the Trop-1 and
Trop-2 Abs, that have been attached to the surfaces in the
collection region where the random pattern of transverse posts is
located, capture trophoblasts that are present in the sample. After
the entire sample has been delivered by the syringe pump, a slow
flushing is carried out with a 1% PBS/BSA aqueous buffer. About 100
.mu.l of this aqueous buffer is fed through the device over a
period of about 10 minutes, which effectively removes all
non-specifically bound bio material from the flow channel in the
device. Two additional washings are then carried out, each with
about 100 .mu.l of 1% PBS plus 1% BSA over a period of about 10
minutes.
[0073] At this time, inasmuch as the device is made of optically
clear material, microscopic examination can be made of the effects
of the capture, as by using photomicroscopy. The bound cells were
stained with cytokeratin 7 and cytokeratin 17 that are specific to
captured cells which are of trophoblast origin. By counting Cells
in such photomicrographs, it is estimated that substantially 97% of
the trophoblasts estimated to be present in the sample have been
captured in the patterned post collection region, which is
considered to be a very excellent result.
[0074] In a repeat of this procedure through the capture and
washing steps, instead of staining in situ, the captured
trophoblasts are released by causing a solution of 100 .mu.l of a
0.25% solution of trypsin to slowly flow through the flow channel
at 27.degree. C. over a period of 20 minutes. This reagent causes
digestion of the Abs, releasing the trophoblasts into the aqueous
flow where they pass through the outlet and are collected. Analysis
of the collected cells by PCR. and FISH based technologies shows
that they are indeed the trophoblasts that were targeted by the Abs
that were employed.
Example 2
[0075] Another microflow device for separating biomolecules is
constructed using a prototype substrate as described in Example 1.
The interior surfaces of the substrate are derivatized, washed with
ethanol, and treated with nonfat milk as in Example 1. Following
washing with 10% ethanol in water, a treatment is effected using
the prepolymer, BSA, and antibodies Trop-1 and Trop-2 in borate
buffer. 1 mg/ml antibody aqueous solution in 100 mM sodium borate
pH 8.0 containing BSA is used. The specific formulation comprises
100 mg of the same prepolymer in Acn/DMF; 350 .mu.L of 0.25 mg/ml
antibody mix in borate buffer; and 350 .mu.L of 1 mg/ml BSA in
borate buffer, and it contains about 2% polymer by weight.
[0076] The antibodies are not thiolated, and about 5 microliters
total of the Trop-1 and 2 aqueous hydrogel solution are supplied to
the pretreated microflow device. The solution is left to incubate
for about 30 minutes at 25.degree. C., and following this
incubation period, the flow channel is flushed with mineral oil
which is slowly pushed into the flow channel to displace and push
out excess hydrogel. This results with an oil-filled flow channel
which has a thin layer of hydrogel coating separating the oil from
the PDMS material. After 3 hours, the hydrogel has fully cured, and
oil is flushed out with a 1.times.PBS/0.1% Tween solution. The
device is then filled with 1.times.PBS solution to preserve the
Abs.
[0077] Cervical mucus from expectant mothers is diluted, treated,
filtered, centrifuged and resuspended in 100 .mu.l of HAM's media.
The liquid cell suspension sample is passed through the Trop-1 and
Trop-2 coated microchannel using a Harvard Apparatus syringe pump,
as in Example 1. After the entire sample is delivered by the
syringe pump, a slow flushing is carried out with a 1% PBS/BSA
aqueous buffer. About 100 .mu.l of this aqueous buffer is fed
through the device over a period of about 10 minutes to effectively
remove all non-specifically bound biomaterial from the flow channel
in the device. Two additional washings are then carried out, each
with about 100 .mu.l of 1% PBS plus 1% BSA over a period of about
10 minutes.
[0078] Microscopic examination is again made of the effects of the
capture by using photomicroscopy, after staining the bound cells
with cytokeratin 7 and cytokeratin 17. By counting cells in such
photomicrographs, it is determined that excellent capture of the
trophoblasts estimated to be present in the sample is achieved.
Example 3
[0079] Another microflow device for separating biomolecules is
constructed using a prototype substrate as described in Example 1.
The interior surfaces of the substrate are derivatized, washed with
ethanol, and treated with nonfat milk as in Example 1.
[0080] Following washing with 10% ethanol in water, a treatment is
effected using 10 .mu.l of 2.5 mM NHS-polyglycine (ave. MW about
4500) in 0.2 MOPS/0.5 M NaCl, pH 7.0, by incubating at RT for 2
hours with gentle pumping of the solution back and forth in the
channel to provide agitation. The microchannel is washed three
times with 500 .mu.l of pH 7.0 MOPS buffer to obtain
maleimido-polyGly-coated channels.
[0081] Antibodies Trop-1 and Trop-2, which are specific to ligands
carried by the exterior surfaces of trophoblasts are treated as in
Example 1 to thiolate them.
[0082] About 5 micrograms total of thiolated anti-Trop-1 and 2 in
aqueous solution, at a concentration of about 0.25 mg/ml, are
supplied to the pretreated microflow device, and the solution is
left to incubate for 2 hours at 25.degree. C. Following this
incubation period, the flow channel is flushed (3 times) with a 1%
PBS/BSA to provide the antibody-coated surfaces which are then used
to try to isolate fetal trophoblast cells.
[0083] Cervical mucus from expectant mothers is diluted, treated,
filtered, centrifuged and resuspended in 100 .mu.l of HAM's media.
The liquid cell suspension sample is passed through the Trop-1 and
Trop-2 coated microchannel using a Harvard Apparatus syringe pump.
After the entire sample is delivered by the syringe pump, a slow
flushing is carried out with a 1% PBS/BSA aqueous buffer. About 100
.mu.l of this aqueous buffer is fed through the device over a
period of about 10 minutes to effectively remove all
non-specifically bound biomaterial from the flow channel in the
device. Two additional washings are then carried out, each with
about 100 .mu.l of 1% PBS plus 1% BSA over a period of about 10
minutes.
[0084] Microscopic examination is again made of the effects of the
capture by using photomicroscopy after staining the bound cells
with cytokeratin 7 and cytokeratin 17. By counting cells in such
photomicrographs, it is determined that good capture of the
trophoblasts estimated to be present in the sample is achieved.
Example 4
[0085] A plurality of microflow devices, similar to that employed
in Example 1 and as shown in FIG. 3, are formed to test the
improvement that results from operation at 45.degree. as opposed to
horizontal. The improved effectiveness of such an angularly
disposed microflow device 71 is tested by employing a feed liquid
that employs a mixture of BeWo and Jurkat cells. 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, The interior surface of the microflow device 71 is
pretreated and then coated with a permeable hydrogel as described
in Example 2 using Anti-Trop-1 and Anti-Trop-2 in aqueous solution
in the coating formulation. The interior of the microflow device is
filled with the Ab coating solution and allowed to incubate for
about 30 minutes at 25.degree. C. Flushing is carried out using
mineral oil and then PBS buffer as in Example 2.
[0086] Sufficient test feed solution is prepared for six test runs;
it contains about 3,000 BeWo cells and about 3,000 Jurkat cells in
a 1% BSA/PBS buffer. The feed solution is split into six aliquots,
with each of the aliquots containing about 500 BeWo cells and about
500 Jurkat cells. Three of the identical microflow devices 71 arc
arranged horizontally, 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. Three different rates of flow are used
for the three test devices: flow rates of 1 .mu.l/min, of 5
.mu.l/min and of 10 .mu.l/min.
[0087] Following flow through these three test devices, washing was
carried out with a PBS buffer, and each individual device was then
examined by microscopy. Each of the two groups of captured cells
was separately counted manually by microscopy. With respect to the
targeted BeWo cells, it was found that, at the lowest flow rate,
about 47% of the BeWo cells were captured in the inlet region and
only about 32% in the collection channel, with the remainder
residing in the outlet region. At the flow rate of 5 .mu.l/min, the
percentage of BeWo cells captured in the collection channel region
dropped slightly to about 27%, and although more cells were still
captured in the inlet region, the largest percentage of cells
collected in the outlet region. At the highest flow rate through
the device in the horizontal orientation, only 10% of the BeWo
cells are captured in the collection channel region, whereas about
65% of the cells collect in the outlet region. With respect to the
Jurkat cells, at the lowest flow rate, about 20-25% of the cells
were captured in each of the inlet and channel regions, and at the
middle flow rate, only about 10% of the Jurkat cells were captured
in the collection channel region. As expected, the amount of these
cells which collect in the outlet region increases with each
increase in flow rate, where all the cells that are not retained in
the device collect as a mixture.
[0088] The experiment is then run using three more, identical,
microflow devices, with each oriented at 45.degree. from the
vertical. This time, flow rates of 1, 3 and 5 .mu.l/min are used.
The improvement in the desired cell capture of BeWo cells in the
collection channel region is striking. At the lowest flow rate,
about 75% of the BeWo cells are now 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, although the
nonspecific binding of the Jurkat cells in the collection region is
relatively high at the lowest flow rate, i.e. about 45%, it drops
to only about 15% at 3 .mu.l/min, and less than 5% at the highest
flow rate. Accordingly, the improvement in performance at the
45.degree. orientation versus horizontal is very substantial when
operating at flow rates of about 3-5 .mu.l/min, collecting over 80%
of the cells rather than only about 27%. 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.
[0089] 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 separating 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 the sample contains specific
subpopulations of cells, the target cells to be captured may be a
group of unwanted cells to be separated from rare cells or the
like. Moreover, once targeted cells have been collected, they may
also be lysed in situ to provide the cell DNA, which may be
collected for analysis downstream or alternatively subjected to PCR
within the collection chamber. U.S. Published Application
2003/0153028 teaches lysing such bound cells to obtain the nucleic
acid that is released. If there are two different subpopulations of
target cells in a sample, different sequestering agents may be
attached to the posts in a pair of upstream and downstream
collection chambers. In another situation, one genus of cells may
be first collected in an upstream collection chamber, released, and
then screened again in a downstream chamber to isolate a subgenus
of cells.
* * * * *