U.S. patent application number 15/378938 was filed with the patent office on 2017-07-13 for capture, purification, and release of biological substances using a surface coating.
The applicant listed for this patent is Academia Sinica. Invention is credited to Ying-Chih CHANG, Po-Yuan TSENG, Han-Chung WU, Jen-Chia WU.
Application Number | 20170199184 15/378938 |
Document ID | / |
Family ID | 47424802 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170199184 |
Kind Code |
A1 |
CHANG; Ying-Chih ; et
al. |
July 13, 2017 |
CAPTURE, PURIFICATION, AND RELEASE OF BIOLOGICAL SUBSTANCES USING A
SURFACE COATING
Abstract
This invention relates to a surface coating for capture
circulating rare cells, comprising a nonfouling composition to
prevent the binding of non-specific cells and adsorption of serum
components; a bioactive composition for binding the biological
substance, such as circulating tumor cells; with or without a
linker composition that binds the nonfouling and bioactive
compositions. The invention also provide a surface coating for
capture and purification of a biological substance, comprising a
releasable composition to release the non-specific cells and other
serum components; a bioactive composition for binding the
biological substance, such as circulating tumor cells; with or
without a linker composition that binds the releasable and
bioactive compositions. The present invention also discloses a
novel microfluidic chip, with specific patterned microstructures to
create a flow disturbance and increase the capture rate of the
biological substance.
Inventors: |
CHANG; Ying-Chih; (Taipei,
TW) ; WU; Han-Chung; (Taipei, TW) ; TSENG;
Po-Yuan; (New Taipei, TW) ; WU; Jen-Chia;
(Magong City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academia Sinica |
Taipei |
|
TW |
|
|
Family ID: |
47424802 |
Appl. No.: |
15/378938 |
Filed: |
December 14, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14128354 |
May 20, 2014 |
9541480 |
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PCT/US2012/044701 |
Jun 28, 2012 |
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15378938 |
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61606220 |
Mar 2, 2012 |
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61502844 |
Jun 29, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/30 20130101;
G01N 1/405 20130101; G01N 33/54393 20130101; C07K 17/14 20130101;
G01N 33/54386 20130101; G01N 33/57492 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/574 20060101 G01N033/574 |
Claims
1.-63. (canceled)
64. A microfluidic chip for selectively enriching rare cells,
comprising: a first solid substrate and a second solid substrate,
wherein at least one of the first and second solid substrates
comprise a series of microstructures configured to interact with
cells, and wherein the first and second solid substrates are
configured to be bound parallel to one another; and a surface
coating for capturing the rare cells, wherein the surface coating
comprises a non-fouling composition and a bioactive composition
which selectively binds to the rare cells, wherein the non-fouling
composition of the surface coating is non-covalently associated
with the bioactive composition, wherein each of the first and
second solid substrates comprise the surface coating.
65. The microfluidic chip of claim 64, wherein the microstructures
are ordered such that progressing from one side of the microfluidic
chip to the other side of the microfluidic chip longitudinally, the
openings between the microstructures are staggered.
66. The microfluidic chip of claim 64, wherein the bioactive
composition comprises an antibody.
67. The microfluidic chip of claim 66, wherein the antibody is a
biotinylated EpCAM antibody.
68. The microfluidic chip of claim 64, wherein the non-fouling
composition comprises a lipid layer.
69. The microfluidic chip of claim 64, wherein the two solid
substrates comprises a glass substrate and a plastic substrate.
70. The microfluidic chip of claim 69, wherein the glass substrate
is located below the plastic substrate in a working
configuration.
71. The microfluidic chip of claim 64, wherein the first solid
substrate comprises the series of microstructures, and wherein the
first solid substrate is located above the second solid substrate
in a working configuration.
72. The microfluidic chip of claim 64, further comprising an
adhesive for bonding the first solid substrate to the second solid
substrate.
73. The microfluidic chip of claim 72, wherein the adhesive
comprises an inner hollow opening in a form of a channel.
74. The microfluidic chip of claim 73, wherein the channel is
configured to encompass the series of microstructures.
75. The microfluidic chip of claim 73, wherein the channel of the
adhesive determines a path for the rare cells to travel through for
the microfluidic chip.
76. The microfluidic chip of claim 72, wherein a thickness of the
adhesive determines a height of a channel of the microfluidic
chip.
77. The microfluidic chip of claim 64, wherein the binding moiety
comprises an antibody, and the antibody comprises a heavy chain and
a light chain that binds EpCAM, wherein (a) the heavy chain
comprises CDR1, CDR2, and CDR3 of SEQ ID No: 1, and (b) the light
chain comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 2.
78. The microfluidic chip of claim 64, further comprising a syringe
pump wherein the syringe pump is configured to apply buffer at a
flow rate configured to release non-specific cells from the
non-fouling layer without releasing cells selectively bound to the
bioactive composition.
79. The microfluidic chip of claim 64, further comprising a syringe
pump configured to aid rinsing the microfluidic chip with a buffer
at a shear force of about 2.5 to about 10 dyne/cm.sup.2.
80. The microfluidic chip of claim 64, wherein the non-fouling
composition is coupled to each of the first and second solid
substrates by a surface linker.
81. The microfluidic chip of claim 64, wherein the non-fouling
composition is from 2 nm to 300 um thick.
82. The microfluidic chip in accordance with claim 64, wherein the
surface coating is attached to the solid substrate by one of the
following non-covalent interactions: covalent bonding, hydrogen
bonding, electrostatic interaction, hydrophilic-hydrophilic
interaction, polar-polar interaction, magnetic force, or a
combination thereof.
83. The microfluidic chip of claim 64, wherein the non-fouling
composition is configured to completely coat each of the first and
second solid substrates.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a nonprovisional application of a U.S.
Patent Application Ser. No. 61/502,844, filed on 29 Jun. 2011, and
U.S. Patent Application Ser. No. 61/606,220, filed on 2 Mar. 2012,
which are incorporated by reference in their entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] NOT APPLICABLE
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] Table 1 is the amino acid sequence of EpAb4-1 antibody.
BACKGROUND OF THE INVENTION
[0004] The shedding of cells into the circulation is an intrinsic
property of the malignant tumor, and this feature provides
important information with regard to the diagnosis, staging,
treatment response and survival of cancer patients. For example,
Pantel et al found the number of circulating tumor cells (CTCs) in
the blood is correlated with the aggressiveness of the cancer as
well as the efficacy of the therapy. (Pantel, K. et. al.,
"Detection, clinical relevance and specific biological properties
of disseminating tumor cells", Nat Rev Cancer, 2008,
8(5):329-40).
[0005] However, CFCs, as few as one per 109 blood cells in patients
with metastatic cancer, are rare cells. This makes the detection
and isolation of CTCs technically challenging (sec Kahnet al.
Breast Cancer Res Treat 2004, 86:237-47). An enrichment process is
therefore necessary to effectively detect and isolate CTCs.
[0006] An example of such enrichment process is the use of a highly
overexpressed cell surface biomarker with high specificity and
sensitivity for CTCs, such as the epithelial cell adhesion molecule
(EpCAM). The Cellsearch System.TM. (Veridex), the only FDA-approved
platform for CTC detection, utilizes anti-EpCAM antibody-coated
magnetic nanoparticles to capture and enrich CTCs, followed by
cytokeratin immunostaining. The AdnaTest (AdnaGen AG, Germany),
another commercially available system for CTC detection, adopts
similar immunomagnetic approach by using anti-EpCAM and Mucin 1
(MUC1) conjugated magnetic beads. More recently, "CTC chips" based
on anti-EpCAM antibody-coated microfluidics chip were developed for
CTC detection and enrichment (Nagrath et al, Nature 2007,
450:1235-9). However, the disadvantage of the above techniques is
the low detection rate of pure CTCs, due to the non-specific
binding of blood cells with anti-EpCAM antibody.
[0007] In order to maximize the detection and isolation of CTCs, it
is necessary to reduce the nonspecific binding of other circulating
blood cells. This can be achieved by surface modification with
bioinert materials. For example, Kaladhar et al. observed a
significant fewer circulating blood cells (e.g. platelets,
leukocytes, and erythrocytes) binding onto the solid substrate
modified with supported monolayer of various lipid compositions
containing phosphatidyl-choline, cholesterol, and glycolipid
(Kaladhar et al, Langmuir 2004, 20: 11115-22 and Kaladhar et al, J
Biomed Mater Res A 2006, 79A:23-35).
[0008] Despite the advance in the detection and isolation CTCs
technology, there is still a need for a more specific and effective
method for detecting, purification and releasing; CTCs and other
biological substances for further cultivation and
characterization.
BRIEF SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention is directed to a
surface coating to capture a circulating rare cell (CRC). This
surface coating increases the capture efficiency of a CRC, such as
CTC, circulating stem cells (e.g. tumor stem cell and bone marrow
stem cells), fetal cells, bacteria, virus, epithelial cells,
endothelial cells or the like and reduces the binding of
non-specific cells or protein adsorption.
[0010] The surface coating comprises 1) a nonfouling composition
that reduces the binding of nonspecific blood cells and adsorption
of other blood components, such as protein; and 2) a bioactive
composition that captures a CRC. The surface coating further
comprises a linker composition that attaches to the nonfouling
composition and the bioactive composition, as illustrated in FIG.
1A.
[0011] In another aspect, the present invention is directed to a
surface coating to capture and release a biological substance. This
surface coating increases the capture efficiency of a biological
substance, such as CTC, circulating stem cells (e.g. tumor stem
cell, liver stem cells and bone marrow stem cells), fetal cells,
bacteria, virus, epithelial cells, endothelial cells or the like
and enhances the removal or release of the non-specific cells or
protein from the surface coating.
[0012] The surface coating comprises 1) a releasable composition
for releasing or removing nonspecific blood cells and other blood
components, such as protein, from the surface coating; and 2) a
bioactive composition that captures a biological substance. The
surface coating further comprises a linker composition that
attaches to the releasable composition and the bioactive
composition.
[0013] The present invention is also directed to a microfluidic
device, with specific microstructure designs to create a disturbed
flow of blood, body fluid or biological samples to increase the
capture rate of the biological substance.
[0014] The present invention is also directed to a method of
manufacturing the surface coating, comprising a) forming the
nonfouling or the releasable composition; and b) attaching the the
linker composition with the nonfouling/releasable composition from
step a) and the bioactive composition, or c) attaching the
nonfouling/releasable composition from step a) with the bioactive
composition.
[0015] The present invention is also directed to methods to capture
and release the biological substance from the surface coating. The
biological substance on the surface coating can be purified by
removing the non-specific cells or protein. The captured biological
substance can be released by air bubbles, ultraviolet irradiation
and the like.
[0016] The present invention is also directed to uses of a
biotinylated anti-EpCam antibody, EpAb4-1 antibody, to capture a
CTC.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Embodiments of the present invention may be described with
reference to the accompanying drawings.
[0018] FIG. 1A illustrates schematically an embodiment of the
surface coating comprising a nonfouling composition, a linker
composition and a bioactive composition.
[0019] FIG. 1B illustrates schematically the binding of a
circulating tumor cell with the surface coating from FIG. 1A
[0020] FIG. 2A to FIG. 2F illustrate the chemical structures of
examples of nonfouling materials.
[0021] FIG. 3 illustrates the chemical reactions of conjugation
between the functional groups on the nonfouling composition and the
bioactive composition.
[0022] FIG. 4A illustrates schematically the attachment of the
surface coating and solid substrate without a surface linker.
[0023] FIG. 4B and FIG. 4C illustrate schematically a linker
composition with a cleavable functional group.
[0024] FIG. 4D illustrates schematically the attachment of the
surface coating and the solid substrate using a surface linker.
[0025] FIG. 5A and FIG. 5B illustrates schematically the formation
of the surface coating on a solid substrate.
[0026] FIGS. 6A and 6B illustrate schematically the components of a
microfluidic chip.
[0027] FIG. 6C illustrates schematically the microfluidic chip
assembly to capture CTCs from a biological sample.
[0028] FIG. 7A to FIG. 7H illustrate schematically the designs of
the microstructures the solid substrate.
[0029] FIGS. 7I and 7J illustrate the capture efficiency of various
microstructure designs in DMEM solution and blood respectively.
[0030] FIG. 8 illustrates the shear stresses of a buffer solution
to release the non-specific cells and purify the captured
biological substance.
[0031] FIG. 9. illustrates schematically the release of biological
substance by the air bubble method.
[0032] FIG. 10A illustrates schematically lie surface coating with
a cleavable linker composition on a solid substrate.
[0033] FIG. 10B illustrates schematics the release of the biologic
substance from the surface coating in FIG. 10A.
[0034] FIG. 11 illustrates QCM-D response of the surface coating
construction.
[0035] FIG. 12 illustrates the QCM-D response of the addition of
bovine serum albumin the surface coating.
[0036] FIG. 13 are the photographs of the non-specific cells (top
images) and the CTCs (bottom images) on the surface coating before
and after the buffer rinse.
[0037] FIG. 14A illustrates the capture efficiency and non-specific
blood cell binding of various surface coatings.
[0038] FIG. 14B are photo images which illustrate the non-specific
blood cell binding of various surface coatings before and after the
buffer rinse.
[0039] FIG. 15A to FIG. 15C are the photographs of the non-specific
cells and the biological substance on the surface coating before
and after the buffer rinse purification.
[0040] FIG. 16 illustrates the various shear stress and flushing
time for the removal of HCT116 and NTH-3T3 cell populations from
the surface coating.
[0041] FIG. 17 are the photographs of the CTCs released by the air
bubbles.
[0042] FIG. 18 illustrates the cell cultures of the released CTCs
on day 1day 10 and day 14.
[0043] FIG. 19 illustrates schematically a CTC filtration
device.
[0044] FIG. 20 illustrates the CTC binding specificity of
biotinylated OC9801 antibody, biotinylated EpAb4-1 antibody,
biotinylated EpCam antibody and IgG antibody,
DETAILED DESCRIPTION OF THE INVENTION
[0045] The present invention is directed to a surface coating to
effectively capture a circulating rare cell (CRC), such as CTC,
circulating stem cells (e.g. tumor stem cell and bone marrow stem
cells), fetal cells, bacteria, virus, epithelial cells, endothelial
cells or the like.
[0046] In one embodiment, the surface coating for the capture of a
CRC comprises 1) a nonfouling composition that prevents the binding
of non-specific cells and adsorption of other blood components,
such as protein; and 2) a bioactive composition that captures the
circulating rare cells. The nonfouling composition and the
bioactive composition are joined by discrete functional groups or
moieties present in the nonfouling and bioactive compositions.
Generally, a linkage between the two compositions is formed by an
interaction comprising electrostatic interaction,
hydrophilic-hydrophilic interaction, polar-polar interaction,
complementary DNA binding, magnetic force, or combinations
thereof.
[0047] In one group of embodiments, complementary DNA fragments are
used for binding the nonfouling composition and the bioactive
composition. The fragments are attached to each of the compositions
and can be partially or completely complementary over their
lengths. A suitable length of DNA will generally be at least 15,
20, 25, 35, 50, 100 or more bases in length. An example of the DNA
used in the present invention is an DNA tweezer. (See, B Yurke et
al., A DNA-fuelled molecular machine made of DNA. Nature 2000,
406:605-608.)
[0048] In another group of embodiments, the surface coating
comprises 1) a nonfouling composition; 2) a bioactive composition;
and 3) a linker composition, which joins the nonfouling composition
to the bioactive composition. See FIG. 1A.
[0049] The present invention is also directed to a surface coating
to effectively capture a biological substance, such as CTC,
circulating stem cells (e.g. tumor stem cell, liver stem cells and
bone marrow stem cells), fetal cells, bacteria, virus, epithelial
cells, endothelial cells or the like, purify the biological
substance on the surface of the surface coating by releasing or
removing the non-specific cells and other serum components (e.g.
protein) through a buffer rinse, and release the captured
biological substance from the surface coating.
[0050] The surface coating for the capture and purification of a
biological substance comprises 1) a releasable composition for
releasing nonspecific blood cells and other blood components, such
as protein, through a buffer rinse; and 2) a bioactive composition
that captures a biological substance. The releasable composition
and the bioactive composition are joined by discrete functional
groups or moieties present in the releasable and bioactive
compositions. Generally, a linkage between the two compositions is
formed by an interaction comprising electrostatic interaction,
hydrophilic-hydrophilic interaction, polar-polar interaction,
complementary DNA binding, magnetic force, or combinations
thereof.
[0051] In one embodiment, the surface coating further comprises a
linker composition that attaches to the releasable composition and
the bioactive composition.
[0052] As will be explained in more detail below, the surface
coating can be incorporated into the following configurations: cell
cultural dishes, microfluidic channels, microfluidic chips,
filtration filter, capillaries, tubes, beads, nanoparticies, or the
like, with an inner diameter ranging from about 50 to about 1000
um.
Nonfouling and Releasable Composition
[0053] "nonfouling" composition (see FIG. 1A) reduces the binding
of non-specific cells and adsorption of the serum protein.
[0054] The "releasable" composition comprises a nonfouling
composition which also acts as a "lubricating" surface such that
only low flow shear stress is required to remove or release the
non-specific cells or blood components from the surface coating,
while the biological substance remains intact.
[0055] The nonfouling composition is selected from the group
consisting of: a supported lipid layer such as liposomes, supported
lipid bilayers (SLBs) or lipid multilayer, polypeptides,
polyelectrolyte multilayers (PEMs), polyvinyl alcohol, polyethylene
glycol (PEG) as illustrated in FIG. 2A, hydrogel polymers,
extracellular matrix proteins, carbohydrate, polymer brushes,
zwitterionic materials such as poly(carboxybetaine) (pCB)) as
illustrated in FIG. 2D, poly(sulfobetaine) (pSB) as illustrated in
FIG. 2E and pDMAEMA as illustrated in FIG. 2F, small organic
compounds, and the combination of above materials forming a single
or a multi-layer.
[0056] For those embodiments in which the nonfouling composition
comprises supported lipid bilayers (SLBs), the SLBs typically
comprise lipids such as, for example,
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)
(sodium salt) (b-PE) as illustrated in FIG. 2B and
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The
protein resistant property of a SLB can be explained by the
presence of neutral and zwitterionic phosphatidylcholine headgroups
in a wide pH range, as well as an aqueous thin film formed between
the hydrophilic lipid head groups and the bulk solution (see,
Johnson et al., Biophys J 1991, 59:289-94).
[0057] In another group of embodiments, the nonfouling composition
comprises PEG, preferably PEG with a molecular weight from about
100 to about 100,000 and exhibits a nonfouling property.
[0058] In yet another group of embodiments, the nonfouling
composition comprises polyelectrolyte multilayers (PEMS) or a
polymer brush. Examples of suitable PEMs useful in the present
invention include, but are not limited to,
poly-L-lysine/poly-L-glutamine acid (PLL/PLGA),
poly-L-lysine/poly-L-aspartic acid or similar counter ionic
polyelectrolytes. The polymer brush comprises
([2-(acryloyloxy)ethyl] trimethyl ammonium chloride,
TMA)/(2-carboxy ethyl acrylate, CAA) copolymer as illustrated in
FIG. 2C. Generally, the nonfouling layer has a thickness from a few
nanometers up to hundreds microns.
[0059] The nonfouling composition comprises functional groups
capable of covalent, non-covalent, or a combination of covalent and
non-covalent attachment, either directly to a functional group
present in the bioactive composition, or directly to a functional
group that is part of the linkage composition.
[0060] In some embodiments, the functional groups of the nonfouling
composition (prior to covalent attachment) are selected from:
hydroxy groups, amine groups, carboxylic acid or ester groups,
thioester groups, aldehyde groups, epoxy or oxirane groups,
hyrdrazine groups and thiol groups, which are selected to be
reactive with functional groups present in either the linker or
bioactive composition. In other embodiments, the functional groups
of the nonfouling composition (prior to non-covalent attachment)
which are first members of a binding pair, are selected from the
group using specific binding recognition consisting of avidin,
streptavidin DNA, RNA, ligand, receptor, antigen, antibody and
positive-negative charges, each of which is selected to bind to a
second member of the binding pair which is present in either the
linker or bioactive composition.
The Linker Composition
[0061] The linker composition joins the nonfouling/releasable
composition and the bioactive composition and comprises functional
groups capable of covalent, non-covalent, or a combination of
covalent and non-covalent attachment directly to a functional group
present in the nonfouling/releasable composition and to a
functional group that is part of the bioactive composition.
[0062] In some embodiments, the linker composition comprises
functional groups (prior to covalent attachment) selected from:
hydroxy groups, amine groups, carboxylic acid or ester groups,
thioester groups, aldehyde groups, epoxy or oxirane groups,
hyrdrazine groups and thiol groups, which are selected to be
reactive with functional groups present in either the nonfouling or
bioactive composition.
[0063] In other embodiments, the linker composition comprises
functional groups (prior to non-covalent attachment) which are
first members of a binding pair, selected from the group using
specific binding recognition consisting of biotin, avidin,
streptavidin, DNA, RNA, ligand, receptor, antigen, antibody and
positive-negative charges, each of which is selected to bind to a
second member of the binding pair which is present on the
nonfouling/releasable composition or the bioactive composition.
[0064] The functional groups on the linker composition can also be
a cleavable functional group, selected from: a photosensitive
functional group cleavable by ultraviolet irradiation, an
electrosensitive functional group cleavable by electro pulse
mechanism, a magnetic material cleavable by the absence of the
magnetic force, a polyelectrolyte material cleavable by breaking
the electrostatic interaction, a DNA cleavable by hybridization,
and the like.
Bioactive Composition
[0065] The bioactive composition joins to either the linker
composition or the nonfouling composition, and comprises a binding
moiety selective for the detection of the biological substance or
CRC.
[0066] The bioactive composition comprises functional groups
capable of covalent, non-covalent, or a combination of covalent and
non-covalent attachment directly to a functional group present in
the nonfouling layer or to a functional group that is part of the
linker composition.
[0067] In some embodiments, the functional groups of the bioactive
composition (prior to covalent attachment) are selected from:
hydroxy groups, amine groups, carboxylic acid or ester groups,
thioester groups, aldehyde groups, epoxy or oxirane groups,
hyrdrazine groups and thiol groups which are selected to be
reactive with functional groups present in either the nonfouling or
linker composition. In other embodiments, the functional groups of
the bioactive composition (prior to non-covalent attachment) are
selected from the group using specific binding recognition
consisting of biotin, avidin, streptavidin, DNA, RNA, ligand,
receptor, antigen--antibody and positive-negative charges, each of
which is selected to bind to a second member of the binding pair
which is present on the nonfouling/releasable composition or the
linker composition.
[0068] The binding moiety of the bioactive composition has specific
affinity with the biological substance through molecular
recognition, chemical affinity, or geometrical/shape recognition.
Examples of the binding moiety for the detection of the biological
substance include, but are not limited to: synthetic polymers,
molecular imprinted polymers, extracellular matrix proteins,
binding receptors, antibodies, DNA, RNA, antigens or any other
surface markers which present high affinity to the biological
substance. A preferred antibody is the anti-EpCAM membrane protein
antibody (commercially available from many sources, including
R&D Systems, MN, USA), which provides high specificity for CTCs
because EpCAM is frequently overexpressed in the lung, colorectal,
breast, prostate, head and neck, and hepatic malignancies, but is
absent from haematologic cells. Another preferred antibody is
Anti-HER2, which has high specificity for CFCs but absent in
haematologic cells.
[0069] In one embodiment, the anti-EpCAM membrane protein antibody
is EpAb4-1 antibody, comprising a heavy chain sequence with SEQ ID
No:1 and a light chain sequence with SEQ ID NO: 2 shown in Table
1.
TABLE-US-00001 TABLE 1 Amino Acid Sequence of V.sub.H and V.sub.L
domains of EpAb4-I antibody FW1 CDR1 FW2 CDR2 SEQ ID
QIQLVQSGPELKKPGETV GYTFTNYG WVKQAPGKGLK INTYTGEP NO: 1 KISCKAS MN
WMGW (V.sub.H) SEQ ID DIVMTQAAFSNPVTLGTS RSSKSLLH WYLOKPGQSPQ
HMSNLAS NO: 2 ASISC SNGITYLY LLIY (V.sub.L) FW3 CDR3 FW4 Family SEQ
ID TYGDDFKGRFAFSLETSA FGRSVDF WGQGTSVTVSS V.sub.H9 NO: 1
STAYLQINNLKNEDTATY (V.sub.H) FCAR SEQ ID GVPDRFSSSGSGTDFILRI
AQNLENPR FGGGTKLEIK V.sub.K24/25 NO: 2 SRVEAEDVGIYYC T
(V.sub.L)
Complementary-determining regions 1-3 (CDR1-3), framework regions
1-4 (FW1-4) for both the V.sub.H and V.sub.L domains are shown. The
V domain families were aligned by VBASE2 database
www.vbase2.org).
[0070] The bioactive composition can have a variety of thicknesses,
selected so that it does not affect the function or the performance
of the surface coating.
[0071] In one embodiment, the conjugation linkers or catalysts for
the nonfouling composition and the bioactive compositions are
biotin/avidin or their derivatives. In another embodiment, the
conjugation linkers or catalysts for the nonfouling composition and
the bioactive composition are EDC/NHS. In yet another preferred
embodiment, the conjugation linkers or catalysts for the nonfouling
composition and the bioactive compositions are sulfo-SMCC. FIG. 3
schematically illustrates the chemical reactions of these
embodiments.
Solid Substrate
[0072] In some embodiments, the surface coating is attached to the
solid substrate without a surface linker, as illustrated in FIG.
4A. The nonfouling/releasable composition is attached to the solid
substrate via one of the following interactions: covalent bonding
(for PEG nonfouling composition), hydrogen bonding, electrostatic
interaction, hydrophilic-hydrophilic interaction (for SLB
nonfouling/releasable composition), polar-polar interaction,
complimentary DNA binding, magnetic force, or the like.
[0073] In other embodiments, the surface coating is attached to the
solid substrate with a surface linker, as illustrated in FIG. 4D.
Examples of the solid substrate used in the present invention
include, but are not limited to: metals, plastics, glass, silicon
wafers, hydroxylated poly(methyl methacrylate) (PMMA), and a
combination thereof. The shape of the solid substrate include, but
are not limited to: planar, circular and irregular shapes with
micro, or nano-structures such as nanoparticles, nanowires, and a
combination thereof.
[0074] The surface linker composition comprises functional groups
capable of covalent, non-covalent, or a combination of covalent and
non-covalent attachment directly to a functional group present in
the nonfouling/releasable composition and to a functional group
that is part of the solid substrate. Examples of the surface linker
for binding the surface coating to a glass substrate include, but
are not limited to, silane, aminopropyltriethoxy
aminopropyltrimethoxy silane, silane-PEG-NH.sub.2,
silane-PEG-N.sub.3 (PEG molecular weight is about 1,000 to about
30,000 daltons) and silane-PEG biotin.
[0075] In one group of embodiments, the surface linker comprises a
cleavable functional group selected from: a photosensitive
functional group cleavable by ultraviolet irradiation, an
electrosensitive functional group cleavable by electro-pulse
mechanism, an iron or magnetic material in which the absence of the
magnetic force will release the nonfouling composition a
polyelectrolyte material cleavable by breaking the electrostatic
interaction, an DNA cleavable by hybridization, and the like.
[0076] In one embodiment, the nonfouling composition comprises
silane-functionalized PEG and the solid substrate is preferably
selected from the group consisting of silicon, glass, hydroxylated
poly(methyl methacrylate) (PMMA) aluminum oxide, TiO.sub.2 and the
like. In another embodiment, the nonfouling composition comprises
thiol-functionalized compounds and the solid substrate is
preferably selected from the group consisting of Au, Ag, Pt, and
the like.
The Method of Manufacturing the Surface Coating
[0077] FIGS. 5A and 5B show the steps of forming the surface
coating: [0078] 1. Formation of the nonfouling/releasable
composition (e.g. SLB or PEG) with appropriate functional group
(biotin); [0079] 2. Attaching the functional group (streptavidin)
on the linker composition to the functional group (biotin) on the
nonfouling/releasable composition; [0080] 3. Formation of the
bioactive composition and attaching the functional group (biotin)
on the bioactive composition to the functional group (streptavidin)
on the linker composition.
[0081] he surface coating without a linker composition can be
formed by: [0082] 1. Formation of the nonfouling/releasable
composition with appropriate functional group (e carboxyl group of
N-glutaryl phosphatidylethanolamine or NGPE); [0083] 2. Formation
and attaching the functional group (primary amine) on the bioactive
composition to the functional group (carboxyl group of NGPE) on the
nonfouling/releasable composition in step 1.
[0084] The steps in paragraphs [0077] and [0078] can be
reversed.
Microfluidic Chip
[0085] As illustrated in FIG. 6A, the microfluidic chip comprises a
first solid substrate 1 (e.g. PMMA) and a second solid substrate 2
(e.g. glass), wherein the first and second solid substrates are
adhered together using an adhesive means 3 or other means.
[0086] Referring to FIG. 6B, the surface of one or both solid
substrates can be engraved with microstructures 4. In one group of
embodiments, the microstructures 4 are arranged in a linear
fashion. In another group of embodiments, the microstructures 4 are
arranged in herringbone fashion. The shaded region on the adhesive
3 in FIG. 6B is carved out to accommodate the microstructures 4 on
the surface of the solid substrate 1. A sealed channel 5 is created
by adhering the first solid substrate 1 and the second solid
substrate 2 together with an adhesive 3. The height of the channel
5 is determined by the thickness of the adhesive 3.
[0087] Once the microfluidic chip is formed, the surface coating
can be attached to one or both solid substrates. In one group of
embodiments, the surface coating is attached to the solid substrate
with a surface linker. In another group of embodiments, the surface
coating is attached to the solid substrate via one of the following
interactions: covalent bonding (for PEG nonfouling composition),
hydrogen bonding, electrostatic interaction,
hydrophilic-hydrophilic interaction (for SLB nonfouling/releasable
composition), polar-polar interaction, complimentary DNA binding,
magnetic force, or the like.
[0088] Referring to FIG. 6C, the microstructures 4 on the solid
substrate, arc perpendicular to the flow direction and create a
chaotic or disturbed flow of the blood, body fluid or biologic
sample as it passes through the sealed channel 5 of the
microfluidic chip. The disturbed flow enhances the biological
substance-surface coating contact.
[0089] Two factors govern the capture efficiency of the
microfluidic chip: [0090] (1) The linear speed of the blood, body
fluid or biological sample, which determines the contact time of
the biological substance and the surface coating. In a preferred
embodiment, the linear speed is about 0.1 mm/s to 1 mm/s. In a more
preferred embodiment, the linear speed is about 0.42 mm/s or 0.5
ml/h for Design E in FIG. 7F. [0091] (2) The flow disturbance of
the blood, body fluid or biological sample, created by the
microstructures 4 on the solid substrate(s). The flow disturbance
increases contact between the biological substance and the surface
coating.
[0092] FIG. 7A shows various designs of the microstructures 4 on
the solid substrate. The microstructures in Design F are arranged
in a herringbone pattern whereas the microstructures in Designs A-E
and H are arranged in a linear pattern. The dimensions of the
microstructures 4 are as follows: the length is about 50 mm fir O-D
and G and about 120 mm for E-F, the height is about 30 .mu.m, the
width is about 1.5 mm for O and A, about 3.0 mm for B, and about
5..5 mm for C-G. The height of the sealed channel 5 varies with the
thickness of the adhesive 3, preferably about 30-90 .mu.m, more
preferably about 60 .mu.m.
[0093] FIG. 7B-7H show the details of Designs A-C in FIG. 7A.
Design C in FIG. 7H is the preferred pattern, with the following
dimensions: the width of Microstructure (W) is about 150 .mu.m, the
length of microstructure (L) is about 1000 .mu.m, the distance
between two rows of microstructures (Sr) is about 250 .mu.m, the
distance between two adjacent microstructures (Sc) is about 350
.mu.m, the height of the microstructure (D) is about 30 .mu.m and
the height of the sealed channel 5 (H) is about 60 .mu.m.
[0094] The biological substance capture efficiency of the various
designs are shown in FIG. 7I and FIG. 7J. Capture rate is defined
as (captured biological substance/original biological substance in
the testing sample).times.100%. Channel O has no microstructure and
has the lowest biological substance capture rate, at 27% and 1% for
DMEM sample and blood sample, respectively. Design E has a 80%
capture rate for HCT116 cancer cells spiked in DMEM, and a 30%
capture rate for HCT116 cancer cells spiked in blood sample. Design
F has the best capture rate, on average over 70% of HCT116 cancer
cells spiked in blood sample were captured (see FIG. 7J).
Flow Purification
[0095] The biological substance on the surface coating can be
further purified by removing the non-specific cells and other blood
components on the surface of the nonfouling/releasable composition.
The nonfouling/releasable composition has low affinity for
non-specific cells and other blood components. Therefore, rinsing
the surface coating with a low flow buffer solution of about 0.8
dyne/cm.sup.2 to about 50 dyne/cm.sup.2 is sufficient to remove
non-specific cells and other blood components on the
nonfouling/releasable composition while the biological substance
remains on the surface coating.
[0096] In a preferred embodiment, the shear force of the buffer
rinse is about 2.5 to about 10 dyne/cm.sup.2. FIG. 8 shows that
when the shear stress of the buffer flow is about 3.3
dyne/cm.sup.2, 80% of the non-specific cells (i.e. white blood
cells) were removed while none of the biological substance (i.e.
HCT 116 cancer cells) were removed from the surface coating. When
the shear stress of the buffer flow was increased to 8
dyne/cm.sup.2, almost all of the non-specific cells were removed
while none of the biological substance was removed from the surface
coating.
Release of the Biological Substance
[0097] After removing the majority of the non-specific cells and
blood components by flow purification, the, biological substance
can be released from the surface coating.
[0098] If the nonfouling/releasable composition comprises a lipid
or a mixture of lipid, the captured biological substance can be
released by introducing an air bubble solution or oil phase. As
shown in FIG. 9, the surface coating comprises a nonfouling
composition A (lipid bilayer) and a bioactive composition B
(antibody) and is bound to a solid substrate S. The biological
substance, CTC, is bound to the bioactive composition B, whereas
other cells were repelled by the nonfouling composition A. As the
air bubble approaches the lipid bilayer, the hydrophobic tails of
the lipid bilayer are turned upside down due to its high affinity
with the air inside the air bubble, which is also hydrophobic. This
breaks up the hydrophilic-hydrophilic interaction at the surface of
the lipid Hayes and allows the air bubble to "lift off" the top
layer of the lipid bilayer, together with the CTC bound on the
bioactive composition.
[0099] If the nonfouling compost ion comprises a composition other
than a lipid or a mixture of lipid, the captured biological
substance can be released by breaking the cleavable functional
group on the linker composition or on the surface linker. This
release mechanism is illustrated in FIGS. 10A and 10B. FIG. 10A
shows a surface coating on a solid substrate, wherein the surface
coating comprises a bioactive composition B, a linker composition
with a cleavable functional group C, and a nonfouling composition
A. The surface coating is attached to a solid substrate S (e.g.
glass) by a surface linker 1. FIG. 10B shows the release of the
biologic substance (e.g. CTC) from the surface coating in FIG. 10A.
The biologic substance is bound to the bioactive composition B,
whereas other cells were repelled by the nonfouling composition A.
The surface coating is irradiated with 365 nm ultraviolet light,
which breaks the cleavable functional group on the linker
composition C and the biologic substance is released for subsequent
analysis but maintaining the viability.
[0100] The biological substance can also be released by other
mechanisms. In one group of embodiments, the linker composition or
the surface linker comprises an electrosensitive cleavable
functional group, and the biological substance is released by
electro pulse mechanism. In another group of embodiments, the
linker composition or the surface linker comprises a magnetic
material as the cleavable functional group, and the absence of the
magnetic field or force releases the biological substance. In yet
another group of embodiments, the linker composition or the surface
linker comprises a PEM as the cleavable functional group, and the
biological substance is released by changing the electrostatic
interaction between the layers. In yet another group of
embodiments, the linker composition or the surface linker comprises
an DNA piece as the cleavable functional group, and the biological
substance is released by DNA hybridization.
EXAMPLES
[0101] The following examples further illustrate the present
invention. These examples are intended merely to be illustrative of
the present invention and are not to be construed as being
limiting.
Example 1
Preparation of the Two-Layer Surface Coating
Preparation of the Nonfouling Composition:
[0102] Supported lipid bilayer (SLB) was prepared by the following
steps: [0103] (1) POPC and b-PE (commercially available from Avanti
Polar Lipids, USA) were dissolved in chloroform and the final lipid
concentration was 5 mg/mL. The POPC/b-PE solution was vortex dried
under a slow stream of nitrogen to form a thin, uniform POPC/b-PE
film. The POPC/b-PE film was further dried in a vacuum chamber
overnight to remove residual chloroform. [0104] (2) The
POPC/biotin-PE film in step (1) was dispersed in and mixed with a
phosphate buffer containing 10 mM of phosphate buffered saline, 150
mM of sodium chloride aqueous solution, and 0.02% (w/v) of sodium
azide (NaN.sub.3, commercially available from Sigma-Aldrich, USA),
with the pH adjusted to 7.2, The mixed solution was filtered
through the 100-nm, followed by the 50-nm Nuclepore.RTM.
track-etched polycarbonate membranes (Whatman Schleicher &
Schnell, Germany) at least 10 times under 150 psi at room temp.
[0105] (3) The filtered solution in step (2) was passed through the
LIPEX.TM. Extruder (Northern Lipids, Inc. Canada) to generate a
homogenous population of unilamillar vesicles. The size of the
POPC/biotin-PE vesicles was about 65.+-.3 nm, determined by the
dynamic laser light scattering detector (Zetasizer Nano ZS, Malvern
Instruments, Germany).
Preparation of the Bioactive Composition
[0106] Biotinylated EpCAM Antibody was prepared by the following
steps: [0107] (1) The anti-EpCAM monoclonal antibody (OC98-1 or
EpAb4-1) was generated by method described by Chen et al (Clip
Vaccine Immunol 2007; 14:404-11). [0108] (2) The antibody in step
(1) was dissolved in a buffer solution containing 10 mM of PBS and
150 mM of NaCl, with a pH about 7.2, The concentration of the
antibody buffer solution was about 0.65 mg/mL, determined by
Nanodrop 1000 spectrophotometer (Thermo Scientific, USA). [0109]
(3) The antibody solution in step (2) was mixed with 10 mM of Sulfo
NHS-LC-Biotin (with a molar ratio of 1 to 10) and dissolved in
Milli-Q water (Milli-Q RO system, USA) at room temperature for 30
min. Excess biotin was removed by dialysis in phosphate buffered
saline at 4.degree. C. for 24 h, with a buffer change every 12 h.
[0110] (4) The ratio of biotin and antibody in the biotinylated
anti-EpCAM antibody (bOC98-1 or bEpAb4-1) was 1.5 to 1, determined
by the HABA assay using a biotin quantitation kit (Pierce,
USA).
[0111] Alternatively, commercially available biotinylated goat
anti-human anti-EpCAM antibody from R and D Systems (Minneapolis,
Minn.) could be used.
Preparation of Solid Substrates of the Present Invention
[0112] Glass substrate (such as microscope coverslips from
Deckglaser, Germany) were cleaned with 10% DECON 90 (Devon
Laboratories Limited, England), rinsed with Milli-Q water, dried
under nitrogen gas, and exposed to oxygen plasma in a plasma
cleaner (Harrick Plasma, Ithaca, N.Y., U.S.A.) at 100 mtorr for 10
min. Prior to each use, the glass substrate was rinsed with ethanol
and dried under nitrogen gas.
[0113] Silicon oxide based solid substrates (e.g. silicon wafer or
glass coverslips) were cleaned with piranha solution (70% sulfuric
acid and 30% hydrogen peroxide (v/v)) at 120.degree. C. tor 40 min,
subsequenctly washed with distilled water and rinsed with acetone.
The solid substrates were dried under a stream of nitrogen and
treated with a plasma cleaner.
[0114] For the vapor phase silanization reaction, clean silicon
oxide substrates and a Petri-dish containing 150 .mu.L of
3-(aminopropyl)-triethoxysilane (Sigma, USA) were placed in a
desiccator (Wheaton dry-seal desiccator, 100 nm) under reduced
pressure at .about.0.3 Torr for 16 h. The substrates were cleaned
by acetone and dried under nitrogen stream.
Construction of the SLB Surface Coating on a Solid Substrate
[0115] 0.25 mg/ml of POPC/b-PE vesicle solution from paragraph
[0084] was added to the cleaned solid substrate to form a SLB
coated solid substrate. This was followed by an extensive rinse
with a phosphate buffer containing 10 mM PBS and 150 mM NaCl
(pH=7.2) to remove excess POPC/b-PE vesicles. Biotin was the
functional group in the SLB which binds with the functional group
(streptavidin) in the linker composition
[0116] 0.1 mg/mL of streptavidin (SA) solution (commercially
available from Pierce Biotechnology, Rockford, Ill., USA) was added
to the SLB coated solid substrate and incubated for 1 hour,
followed with a PBS buffer rinse to remove excess SA.
[0117] About 0.05 mg/mL of b-Anti-EpCAM solution was added to the
SA-SLB coated solid substrate to form the surface coating of the
present invention.
Construction of the PEG Surface Coating on a Solid Substrate
[0118] The biotinylated PEG si lane solution (Si-bPEGs) was added
to the clean glass substrate and incubated for 1 hour to form a
Si-bPEG nonfouling composition on the glass substrate, followed by
an ethanol rinse to remove excess Si-bPEGs. Silane was the surface
linker and the biotin was the functional group that bind with the
functional group (SA) in the linker composition.
[0119] 0.1 mg/mL of SA solution was added to the Si-bPEGs coated
solid substrate and incubated for 1 hour, followed by a PBS buffer
rinse to remove excess SA.
[0120] 0.05 mg/mL of b-Anti-EpCAM solution was added and bound with
SA-Si-bPEGs surface coating, followed by PBS buffer rinse to remove
excess b-Anti-EpCAM.
Construction of the PENT Surface Coating on a Solid Substrate
[0121] Physical deposition of PEM films was performed by batch and
static conditions as follows: initially, all polypeptides were
dissolved in 10 mM Tris-HCl buffer with 0.15 M NaCl, pH 7.4. Solid
substrates were then immersed in PLL (MW 15000-30000; Sigma, St
Louis, Mo.) solution (1 mg/mL) for 10 min at room temperature,
followed by rinsing with 1 mL of Tris-HCl buffer for 1 min. To
couple PLGA, the PLL-coated slide was subsequently immersed in the
PLGA solution (MW 3000-15000, Sigma, St Louis, Mo., 1 mg/mL) for 10
min, followed by rinsing with 1 mL of Tris-HCl buffer for 1 min.
Lastly, substrates were cleaned with fresh PBS to remove uncoupled
polypeptides. The resulting c-(PLL/PLGA)i, where i was denoted as
the number of polyelectrolyte pairs generated by repeating the
above steps: i) 0.5 was referred to c-PLL only, i) I was referred
to c-(PLL/PLGA)1, and the like.
QCM-D Characterization of the SLB Surface Coating
[0122] The construction of the surface coating was monitored by
quartz crystal microbalance with dissipation (QCM-D). The QCM-D
response in FIG. 11 shows the construction of the surface coating
on a SiO.sub.2-pretreated quartz crystal. First, 0.25 mg/mL of
POPC/b-PE vesicle mixture (in phosphate buffer) was dispensed into
the QCM chamber at point (I). The normalized frequency change F and
dissipation shift D were 26.0.+-.0.7 Hz and 0.19.+-.0.03.times.10-6
respectively, which are the characteristics of a highly uniformed
lipid bilayer. After two buffer washes (denoted as *), 0.1 mg/mL,
of SA solution was dispensed at point II. .cndot. SA binding was
saturated at F=52.8.+-.5.4 Hz and D=3.84.+-.0.54.times.10-6. At
point (III), 0.025 mg/mL of OC98-1 antibody solution was dispensed
into the QCM chamber and there was no frequency or dissipation
change. This shows there was no interaction between the OC98-1
antibody and the SA-lipid bilayer surface. In contrast, adding
biotinylated antibody solution (OC98-1 or bEpAb4-1) at point (IV)
resulted in frequency and dissipation change, with equilibrated
shifts of F=39.4.+-.6.8 Hz and D=1.63.+-.0.28.times.10-6. This
demonstrates the binding of biotinylated antibody to SA-lipid
bilayer surface.
[0123] The characteristics of the SLB nonfouling composition on the
surface coating were examined using QCM-D (FIG. 12). Bovine serum
albumin (BSA, commercially available from Sigma-Aldrich, USA) was
added to the surface coating and there was a sudden change in
frequency and dissipation, with equilibrated shifts of F=6.9 Hz and
D=3.35.times.10-6. This indicates an immediate BSA adsorption.
Three buffer rinses (*) caused an increase in frequency and a
decrease in disspation, with saturated shifts of F=6.1 Hz and
D=3.16.times.10-6. This indicates the adsorbed BSA can be easily
removed from the surface coating and thus, a very weak interaction
between BSA and SLB.
Example 2
Preparation of the Microfluidic Chip
[0124] The microfluidic p can be prepared by the following steps:
[0125] 1. A commercial CO.sub.2 laser scriber (Helix 24, Epilog,
USA) was used to engrave the microtrenches to form microstructures
on the PMMA substrate. [0126] 2. The PMMA substrate, glass
substrate and nuts were cleaned with MeOH, detergent and water,
followed by 10 min sonication. The nuts and the solid substrates
were dried by nitrogen gas and baked for 10 min at 60.degree. C.
[0127] 3. The PMMA substrate gas bon led with nuts by chloroform
treatment. [0128] 4. PMMA substrate and the glass slide were joined
together using an adhesive (e.g. 3M doubled sided tape from 3M,
USA).
Example 3
CTCs Binding to the Anti-EpCAM Functionalized SLB Surface
Coating
[0129] Eight blood samples were used to determine the CTC capture
rate of the Anti-EpCAM functionalized SLB surface coating in a
microfluidic chip in Example 2. Each blood sample contained 2 ml of
blood from a stage IV colon cancer patient and the sample was
introduced to the sealed channel of the microfluidic chip at 0.5
ml/hr, controlled by a syringe pump. Subsequently, the sealed
channel in the microfluidic chip was rinsed with 0.5 ml of PBS
buffer at the flow rate of 1 ml/h, followed by in situ
immunostaining.
[0130] The number of CTCs captured per ml of blood for these 8
samples were 26, 34, 36, 39, 47, 67 79, and 99. 25% of the blood
samples had 79 or higher CTC count per ml of testing sample and the
median CTC count was 43 per ml of testing sample. There was minimal
binding of the non-specific cells and proteins after the buffer
rinse.
[0131] As a comparison, the CTC count for the FDA approved Veridex
CellSearch is as follows: 25% of the samples had 3 or more CTCs per
7.5 ml of testing sample and the median CTC counts was 0.
[0132] The anti-EpCAM functionalized SLB surface was incubated with
150 uL of HCT116 cancer cell spiked human blood (with HCT116 cancer
cell density of approximately 10 to 100 per 100 .mu.L of blood),
followed by a buffer rinse to remove non-specific cells. FIG. 13
shows the surface coating before and after the buffer rinse. Prior
to the buffer rinse, the surface coating was covered with
non-specific cells (upper left) and four HCT116 cancer cells (lower
left). After the buffer rinse, almost all of the non-specific cells
were removed (upper right) but the four HCT116 cancer cell (lower
right) remained on the surface coating.
[0133] The results show the surface coating of the present
invention effective in capturing CTCs and releasing the
non-specific cells.
Example 4
Comparison of Capture Efficiency and Nonfouling Property of Various
Surface Conditions
[0134] The capture rate of HCT116 cancer cells (biological
substance) and the nonfouling property of six different surface
conditions are illustrated in FIG. 14A,
[0135] The results show that the surface coatings of the present
invention (lipid/SA/b-anti-EpCAM and PEG(15 mM)/SA/b-anti-EpCAM)
are more effective in capturing the biological substance. There is
less binding of the non-specific cells (white blood cells or WBC)
on the surface coatings of the present invention compare to a
surface coating without a nonfouling composition (glass only).
[0136] FIG. 14B shows the non-specific, blood cell binding of the
following surfaces: (A) Glass only; (B) biotinylated SLB (b-SLB),
(C) Streptavidin conjugated-bSLB, and (D) OC98-1-conjugated bSLB.
These surfaces were incubated with diluted human blood from healthy
donor (1 uL, of blood in 100 uL PBS buffer) for 4 hours, followed
by a PBS buffer rinse. Images (E) to (H) are the after rinse images
which correspond to the surface coatings in (A) to (D). The results
show that after a buffer rinse, there is less non-specific blood
cell on the surface coatings with a releasable composition (i.e.
SLB) compare to the surface coating without a releasable
composition (i.e. glass only).
Example 5
Purification by Flow
[0137] The differentiated flow shear could selectively "flush" out
the non-specific cells based on the affinity of these cells to the
nonfouling composition, while the biological substance remains on
the surface coating.
[0138] In this study, the surface coating comprised a SLB, a linker
composition and fibronectin as the bioactive composition. FIG. 15A
shows fibroblast 3T3 (green) and colon cancer cell line HCT116
(red) were incubated on the surface coating for 4 h. The surface
coating was rinsed with a buffer solution, which has a shear stress
of 3 dyne/cm.sup.2.
[0139] The HCT 116 cells (red) were flushed away from the surface
coating within 5 min of the buffer rinse, as shown in FIG. 15B. The
fibroblast 3T3 cells (green) remained on the surface coating after
30min of buffer rinse, as shown in FIG. 15C, due to its high
affinity to fibronectin.
[0140] The result shows a shear stress about 3 dyne/cm.sup.2 is
sufficient to remove the non-specific cells from the releasable
composition.
[0141] FIG. 16 summarizes the respective shear stress and flushing
time for the HCT116 and NIH-3T3 cell populations (non-specific
cells). To remove HCT116 cells from the releasable composition of
the surface coating, the shear stress is about 3 to about 4.5
dyne/cm.sup.2. To remove NIH-3T3 cells from the releasable
composition of the surface coating, the shear stress is about 8.5
to about 12 dyne/cm.sup.2 (N/N0 is the percentage of the cells
remains attached to the surface coating using various shear
stresses, N is the final cell number and N0 is the initial cell
number.)
Example 6
Release of CTCs from the Surface Coating
[0142] The captured HCT116 cancer cells on the surface coating in
Example 3 were released by introducing air bubbles. FIG. 17 shows
HCT116 cells in the red circle were removed from the surface
coating within 3 seconds of introducing air bubbles.
Example 7
Culture of Released CTCs From the Surface Coating
[0143] The captured CTCs were incubated with 5 mM of EDTA at
37.degree. C. for 5 to 10 min and released by flowing a culture
medium into the sealed channel of the microfluidic chip. A total of
18 colo205 cells were released from this procedure. The released
colo205 cells, together with a serum-containing culture medium and
antibiotics (penicillin+streptomycin+gentamicin), were placed into
a 48-well tissue cultured polystyrene plate for cultivation.
[0144] FIGS. 18A-18C show a portion of 18 colo205 cells on day 1,
on day 10 and day 14 respectively. This study demonstrates the
released colo205 cells retained their viability for subsequent cell
culture.
Example 8
Capture CTCs Through a CTC Filtration Device
[0145] Any membranes, tubes, capillaries, beads, nanoparticles or
channels can be coated with the surface coating of the present
invention. FIG. 19 illustrates schematically a filtration device,
wherein the filtration filter is coated with the surface coating of
the present invention. The filter could accommodate high volume
blood flow and capture a biological substance for a diagnostic or
therapeutic purpose. To access the patient's blood or body fluid, a
catheter can be inserted into the patient's vein or fistula and the
patient's blood flows through the CTC filtration device, wherein
the surface coating on the filters captures the CTCs. The filtered
blood flows back to the patient.
Example 9
Capture CTCs Through a Biotinylated EpAb4-1 Antibody
[0146] The binding specificity of biotinylated OC9801 antibody,
biotinylated EpAb4-1 antibody and biotinylated EpCam antibody
(commercially available from R&D system, USA) were examined
using the HCT116 (colorectal) CTCs and SAS (tongue) CTCs.
[0147] The CTCs were spiked in a buffer solution (about 10.sup.5
CTCs/ ml). The CTC-spiked buffer solution was introduced to the
surface coatings with the following bioactive composition:
biotinylated OC9801 antibody, biotinylated EpAb4-1 antibody,
biotinylated EpCam antibody and IgC1 antibody.
[0148] The CTC binding specificy of the antibodies was determined
by colorimetric method, by measuring the absorption optical density
at 490 nm. FIG. 20 shows biotinylated EpAb 4-1 is effective in
capturing HCT116 CTCs and SAS CTCs.
Sequence CWU 1
1
21116PRTArtificial Sequencesynthetic anti-epithelial cell adhesion
molecule (EpCAM) membrane protein antibody EpAb4-1 heavy chain V-H9
domain 1Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly
Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys
Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu
Pro Thr Tyr Gly Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser
Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn
Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Phe
Gly Arg Ser Val Asp Phe Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr
Val Ser Ser 115 2112PRTArtificial Sequencesynthetic anti-epithelial
cell adhesion molecule (EpCAM) membrane protein antibody EpAb4-1
light chain V-kappa24/25 domain 2Asp Ile Val Met Thr Gln Ala Ala
Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser
Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr
Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr His Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Ala
Gln Asn 85 90 95 Leu Glu Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110
* * * * *
References