U.S. patent application number 16/939777 was filed with the patent office on 2021-01-14 for detection and assay devices and methods of making and using the same.
The applicant listed for this patent is Sentilus Holdco, LLC. Invention is credited to Ashutosh CHILKOTI, Angus HUCKNALL.
Application Number | 20210011014 16/939777 |
Document ID | / |
Family ID | 1000005109544 |
Filed Date | 2021-01-14 |
United States Patent
Application |
20210011014 |
Kind Code |
A1 |
CHILKOTI; Ashutosh ; et
al. |
January 14, 2021 |
DETECTION AND ASSAY DEVICES AND METHODS OF MAKING AND USING THE
SAME
Abstract
An article such as a biomolecular detector or biosensor having a
nonfouling surface thereon includes:(a) a substrate having a
surface portion; (b) a linking layer on the surface portion; and
(c) a polymer layer formed on the linking layer; and (d) a first
member of a specific binding pair (e.g., a protein, peptide,
antibody, nucleic acid, etc.) bound to the polymer layer. Methods
of making and using the articles are also described.
Inventors: |
CHILKOTI; Ashutosh; (Durmam,
NC) ; HUCKNALL; Angus; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sentilus Holdco, LLC |
Norcross |
GA |
US |
|
|
Family ID: |
1000005109544 |
Appl. No.: |
16/939777 |
Filed: |
July 27, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16366054 |
Mar 27, 2019 |
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16939777 |
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14242355 |
Apr 1, 2014 |
10288607 |
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16366054 |
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12405300 |
Mar 17, 2009 |
8796184 |
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14242355 |
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61040223 |
Mar 28, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54366 20130101;
G01N 33/54353 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1-35. (canceled)
36. A method of detecting the presence of an analyte in a sample
suspected of containing the analyte, the method comprising (a)
contacting the sample suspected of containing the analyte to a
biomolecular detector, wherein the biomolecular detector comprises:
(i) a substrate having a surface portion, (ii) a linking layer on
the surface portion, (iii) a polymer layer formed on the linking
layer, wherein the polymer comprises monomeric units, with each of
the monomeric units comprising a monomer core group having at least
one protein-resistant head group coupled thereto, to thereby form a
brush molecule on the surface portion; wherein the brush molecule
comprises a stem formed from the polymerization of the monomer core
groups, and a plurality of branches formed from the head group
projecting from the stem, and (iv) a probe directly non-covalently
bound to the polymer layer; (b) incubating the sample on surface
portion of the biomolecular detector to permit specific binding
between the probe and the analyte, and (c) detecting the binding of
the analyte to the probe, wherein detection of the binding of the
analyte to the probe indicates the presence of the analyte in the
sample.
37. The method of claim 36, wherein the probe is a protein,
peptide, or nucleic acid.
38. The method of claim 36, wherein the probe is an antibody.
39. The method of claim 36, wherein the surface portion of the
biomolecular detector comprises a material selected from the group
consisting of metals, metal oxides, semiconductors, polymers,
silicon, silicon dioxide, and composites thereof.
40. The method of claim 36, wherein the surface portion of the
biomolecular detector comprises silicon dioxide or gold.
41. The method of claim 36, wherein the linking layer of the
biomolecular detector is continuous.
42. The method of claim 36, wherein the linking layer of the
biomolecular detector is patterned.
43. The method of claim 36, wherein the linking layer of the
biomolecular detector comprises a silane layer or a self-assembled
monolayer.
44. The method of claim 36, wherein the brush molecule of the
biomolecular detector is from 5 to 200 nanometers in length.
45. The method of claim 36, wherein the brush molecule of the
biomolecular detector occurs on the surface portion of the
biomolecular detector at a density from 10 to 500 milligrams per
meter.sup.2.
46. The method of claim 36, wherein the probe is bonded to the
polymer layer of the biomolecular detector at a density of from 1
milligram per meter.sup.2 to 50 grams per meter.sup.2.
47. The method of claim 36, wherein the analyte is present in the
sample at a concentration of from 0.1 to 100 femtomoles per
liter.
48. The method of claim 36, wherein the detecting comprises an
immunoassay.
49. The method of claim 36, wherein the sample comprises a
biological fluid.
50. The method of claim 49, wherein the biological fluid comprises
blood, blood plasma, serum, peritoneal fluid, cerebrospinal fluid,
tear, mucus, lymph fluid, semen, saliva, urine, and lavage fluid
from a wound or bodily orifice.
Description
RELATED APPLICATIONS
[0001] This application is related to Ashutosh Chilkoti, Non
fouling polymeric surface modification and signal amplification
method for biomolecular detection, US Patent Application Pub. No.
US 2007/0072220, published March 29, 2007 (Docket No. 5405-376)
(also published as PCT Application No. WO 2007/035527 on Mar. 29,
2007), the disclosure of which is incorporated by reference herein
in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to devices for biomolecular
detection.
BACKGROUND OF THE INVENTION
[0003] Microarrays are a powerful and established tool in
genomics..sup.1 In contrast, the development of antibody (Ab)
microarrays into an equivalent tool for proteomics has been limited
by: (1) the availability of high affinity and specificity
antibodies for capture and detection of protein biomarkers; (2) the
susceptibility of proteins to denaturation; and (3) the propensity
of Ab's and protein biomarkers to avidly adsorb to surfaces
(commonly referred to as the "non-specific adsorption" problem),
which can severely limit the ultimate sensitivity of protein
microarrays, especially from complex protein mixtures such as
plasma and serum..sup.2 One of the primary factors (others include
the intrinsic affinity of the capture antibody and the diffusion of
target to the microspot.sup.2,3) that controls the
limit-of-detection (LOD) of protein microarrays is the adventitious
adsorption of proteins (protein biomarkers and antibodies used for
detection).
SUMMARY OF THE INVENTION
[0004] A first aspect of the present invention is an article
(preferably a biomolecular detector or biosensor such as a
microarray) having a nonfouling surface thereon, the article
comprising:
[0005] (a) a substrate having a surface portion;
[0006] (b) a linking layer on the surface portion; and
[0007] (c) a polymer layer formed on the linking layer (e.g., by
the process of surface-initiated polymerization (SIP) of monomeric
units thereon). Preferably, each of the monomeric units comprises a
monomer (for example, a vinyl monomer) core group having at least
one protein-resistant head group coupled thereto, to thereby form a
brush molecule on the surface portion. The brush molecule
preferably comprises a stem formed from the polymerization of the
monomer core groups, and a plurality of branches formed from the
head group projecting from the stem; and
[0008] (d) a first member of a specific binding pair (e.g., a
protein, peptide, antibody, nucleic acid, etc.) non-covalently
bound to the polymer layer.
[0009] A second aspect of the present invention is a method of
making an article (preferably a biomolecular detector such as a
microarray) having a nonfouling surface thereon, the method
comprising: (a) providing a substrate having a surface portion;
[0010] (b) depositing a linking layer on the surface portion; and
(c) forming a polymer layer on the linking layer by the process of
surface-initiated polymerization of monomeric units thereon, with
each of the monomeric units comprising a monomer (for example, a
vinyl monomer) core group having at least one protein-resistant
head group coupled thereto, to thereby form a brush molecule on the
surface portion; the brush molecule comprising a stem formed from
the polymerization of the monomer core groups, and a plurality of
branches formed from the hydrophilic head group projecting from the
stem; and then (d) non-covalently binding a member of a specific
binding pair to the polymer layer.
[0011] In some embodiments the polymer comprises a homopolymer of
hydroxy-terminated OEGMA. In another embodiment the polymer
comprises of a copolymer of methoxy-terminated OEGMA and
hydroxy-terminated OEGMA. In other embodiments the polymer
comprises of vinyl monomer bearing other head groups such as
hydroxyl (OH), glycerol, or groups known in the art as kosmotropes
(see, e.g., Kane et al., infra).
[0012] In some embodiments of the invention, the surface portion
comprises a material selected from the group consisting of metals,
metal oxides, semiconductors, polymers, silicon, silicon oxide, and
composites thereof.
[0013] In some embodiments of the invention the linking layer is
continuous; in some embodiments of the invention the linking layer
is patterned. In some embodiments of the invention the linking
layer is a self-assembled monolayer (SAM). In some embodiments of
the invention the linking layer comprises an initiator-terminated
silane or an initiator-terminated alkanethiol. In other embodiments
the linking layer comprises of the deposition of two layers in
separate steps. In the first step, an alkylsilane or alkanethiol is
deposited on a surface such as silicon dioxide or glass or gold,
and presents a terminal reactive functional group (e.g., amine). In
the next step, a bifunctional molecule, which comprises a first
functional group reactive towards the terminal group presented by
the first linking layer is reacted with the first linking layer
deposited in the first step. The second linker molecule contains a
second moiety group that acts as an ATRP or free radical
initiator.
[0014] In some embodiments of the invention the surface-initiated
polymerization is carried out by atom transfer radical
polymerization (ATRP); in some embodiments of the invention the
surface-initiated polymerization is carried out by free radical
polymerization.
[0015] In some embodiments, the article further comprises a
protein, peptide, oligonucleotide or peptide nucleic acid
non-covalently bound to the polymer layer. In some embodiments the
protein, peptide, oligonucleotide or peptide nucleic acid coupled
to the polymer layer or to the surface consists of or consist
essentially of a single preselected molecule (this is, one such
molecule is coupled to the surface portion via the brush molecule,
to the exclusion of other different molecules). The preselected
molecule may be a member of a specific binding pair, such as a
receptor.
[0016] A further aspect of the invention is a method of detecting a
second member of a specific binding pair in a sample, comprising
the steps of: (a) providing a detector as described herein; (b)
contacting a sample (e.g., an aqueous sample or biological fluid)
suspected of containing the second member(s) to the detector; and
then (c) determining the presence or absence of binding of the
second member to the first member, the presence of binding
indicating the presence of the second member in the sample. The
determining step can be carried out by any suitable technique, such
as by sandwich assay, as discussed further below.
[0017] Arrays. In some embodiments of the foregoing methods and
devices, useful for the detection of multiple different analytes,
the first member of said specific binding pair is non-covalently
bound to said polymer at a discrete probe location, and the
biomolecular detector further comprises: (e) a plurality of
additional first members of a specific binding pairs non-covalently
bound to said polymer layer at a plurality of additional discrete
probe locations to thereby form an array thereon. In some
embodiments the array has a density of 5 to 10,000 discrete probe
locations per cm.sup.2 thereon; in some embodiments the array has a
density of 10,000 to 1 million discrete probe locations per
cm.sup.2 thereon; and in some embodiments the array has a density
of 1 million to 1 billion discrete probe locations per cm.sup.2
thereon.
[0018] An advantage of the foregoing methods is the variety of
techniques by which the detecting step can be carried out. For
example, the detecting step may be carried out by: (a)
ellipsometry; (b) surface plasmon resonance (SPR); (c) localized
surface plasmon resonance using noble metal nanoparticles in
solution or on a transparent surface; (d) surface acoustic wave
(SAW) devices; (e) quartz-crystal microbalance with dissipation
(QCM-D) (e) atomic force microscopy, (f) fluorescence spectroscopy
or imaging; (g) autoradiography, (h) chemiluminescent imaging; and
(i) optical detection of metal nanoparticles either by extinction
or scattering. etc.
[0019] Still other aspects of the present invention are explained
in greater detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. A schematic diagram of an array of the present
invention.
[0021] FIG. 2. Synthesis of POEGMA brushes on glass via SI-ATRP.
Cleaned slides were functionalized with APTES in step 1, and
modified to present an ATRP initiator in step 2. Slides were then
immersed in a polymerization solution in step 3 to synthesize
surface tethered brushes of POEGMA.
[0022] FIG. 3. (A) Example of signal and background intensities in
an array used for generation of IL-6 dose response curves (B) Dose
response curves of OPG in buffer and serum on POEGMA. (C) Dose
response curves of IL-6 in serum on POEGMA and nitrocellulose. In B
and C, the Y-axis shows the average background subtracted
fluorescence intensity in printed spots and the X-axis shows
analyte concentration in solution. Error bars represent one
standard deviation.
[0023] FIG. 4. Cy-5 labeled goat anti-rabbit IgG (Jackson) printed
on POEGMA substrates prepared for both covalent and non-covalent
attached to produce arrays.
[0024] FIG. 5. Incubation of the arrays of FIG. 4 with Cy5 labeled
goat anti-rabbit IgG yielded similar spot intensities for both
immobilization methods, however, background levels on the activated
slides (covalently coupled arrays) increased dramatically.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] The present invention will now be described more fully
hereinafter with reference to the accompanying drawings, in which
preferred embodiments of the invention are shown. This invention
may, however, be embodied in different forms and should not be
construed as limited to the embodiments set forth herein. Rather,
these embodiments are provided so that this disclosure will be
thorough and complete, and will fully convey the scope of the
invention to those skilled in the art.
[0026] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
disclosures of all United States patents cited herein are
incorporated by reference in their entirety.
1. Definitions.
[0027] "SI-ATRP" as used herein means surface initiated atom
transfer radical polymerization.
[0028] "OEGMA" as used herein refers to oligo(ethylene
glycol)methyl methacrylate. "Biological fluid" as used herein may
be any fluid of human or animal origin, including but not limited
to blood, blood plasma, serum, peritoneal fluid, cerebrospinal
fluid, tear, mucus, lymph fluid, semen, saliva, urine, lavage fluid
from a wound or bodily orifice, etc. Biological fluids generally
contain a mixture of a plurality of different proteins therein, and
typically contain other constituents such as other cells and
molecules. Biological fluids may be in their natural state or in a
modified state by the addition of ingredients such as reagents or
removal of one or more natural constituents (e.g., blood plasma),
but all typically comprise a mixture of a plurality of different
potential analytes, such as a plurality of different proteins.
[0029] "Kosmotrope", while originally used to denote a solute that
stabilized a protein or membrane, is also used by those skilled in
the art, and is used herein, to denote a substituent or "head
group" which, when deposited on a surface, renders that surface
protein-resistant. See, e.g., R. Kane, P. Deschatelets and G.
Whitesides, Kosmotropes Form the Basis of Protein-Resistant
Surfaces, Langmuir 19, 2388-2391 (2003). Numerous kosmotropes are
known and examples include but are not limited to OEGMA. "Polymer"
as used herein is intended to encompass any type of polymer,
including homopolymers, heteropolymers, co-polymers, ter-polymers,
etc., and blends, combinations and mixtures thereof.
[0030] "Specific binding pair" as used herein refers to two
compounds that specifically bind to one another, such as
(functionally): a receptor and a ligand (such as a drug), an
antibody and an antigen, etc.; or (structurally): protein or
peptide and protein or peptide; protein or peptide and nucleic
acid; and nucleotide and nucleotide etc. "Nucleic acids" may be any
natural or synthetic nucleic acids, including DNA and RNA, and are
typically from 10 to 1,000 nucleotides in length. Typically the
first member of the specific binding pair (or "probe") is a
protein, peptide or nucleic acid that specifically binds to the
second member (or "analyte") to be detected.
[0031] "Analyte" as used herein may be any second member of a
specific binding pair, as described above. Typically the analyte is
a constituent or found in a biological fluid as described herein.
Examples of such analytes include, but are not limited to: thyroid
stimulating hormone, glycosylated hemoglobin, parathormone,
prostate-specific antigen (psa), ferritin, natriuretic peptide,
folic acid, hepatitis b surface antigen, blood lipoproteins,
vitamin D, carcinoembryonic antigen, nuclear antigen antibody,
testosterone, homocystine, HIV-1 DNA, ck (cpk) gammaglobulin, etc.
The analyte can be a "marker" protein, peptide or other molecule
specifically found in patients infected or afflicted with a
microbial infection, examples of which include but are not limited
to Anthrax, Avian influenza, Botulism, Buffalopox, Chikungunya,
Cholera, Coccidioidomycosis, Creutzfeldt-Jakob disease,
Crimean-Congo haemorrhagic fever, Dengue fever, Dengue haemorrhagic
fever, Diphtheria, Ebola haemorrhagic fever, Ehec (E. Coli 0157),
Encephalitis, Saint-Louis, Enterohaemorrhagic escherischia coli
infection Enterovirus, Foodborne disease, Haemorrhagic fever with
renal syndrome, Hantavirus pulmonary syndrome, Hepatitis,
Influenza, Japanese encephalitis, Lassa fever, Legionellosis,
Leishmaniasis, Leptospirosis, Listeriosis, Lousebome typhus,
Malaria, Marburg haemorrhagic fever, Measles, Meningococcal
disease, Monkeypox, Myocarditis Nipah virus, O'Nyong-Nyong fever,
Pertussis, Plague, Poliomyelitis, Rabies, Relapsing fever, Rift
Valley fever, Severe acute respiratory syndrome (SARS),
Shigellosis, Smallpox vaccine--accidental exposure, Staphylococcal
food intoxication, Tularaemia, Typhoid fever, West Nile fever,
Yellow fever, etc. The analyte can be a "marker" protein, peptide
or other molecule specifically found in patients infected or
afflicted with a fungal infection, or viral infection. In all of
the preceding examples, the analyte may be derived from the
infectious agent itself, namely microbe, fungus or virus, or may be
proteins or other biomarkers (such as lipids, carbohydrates or DNA)
that are found in greater or lesser abundance in afflicted
individuals as compared to healthy individuals.
2. Substrates.
[0032] The present invention can be utilized to form surfaces on a
variety of different types of substrates.
[0033] In some embodiments, the article is a label-free optical or
mass detector (e.g., a surface plasmon resonance energy detector,
an optical wave guide, an ellipsometry detector, etc.) and the
surface is a sensing surface (e.g., a surface portion that would be
in contact with a biological fluid). Examples of such articles
include but are not limited to those described in U.S. Pat. Nos.
6,579,721; 6,573,107; 6,570,657; 6,423,055; 5,991,048; 5,822,073;
5,815,278; 5,625,455; 5,485,277; 5,415,842; 4,844,613; and
4,822,135.
[0034] In other embodiments, the article is a biosensor, an assay
plate, or the like. For example, the present invention may be
utilized with optical biosensors such as described in U.S. Pat. No.
5,313,264 to Ulf et al., U.S. Pat. No. 5,846,842 to Herron et al.,
U.S. Pat. No. 5,496,701 to Pollard-Knight et al., etc. The present
invention may be utilized with potentiometric or electrochemical
biosensors, such as described in U.S. Pat. No. 5,413,690 to Kost,
or PCT Application WO98/35232 to Fowlkes and Thorp. The present
invention may be utilized with a diamond film biosensor, such as
described in U.S. Pat. No. 5,777,372. Thus, the solid support may
be organic or inorganic; may be metal (e.g., copper or silver) or
non-metal; may be a polymer or nonpolymer; may be conducting,
semiconducting or nonconducting (insulating); may be reflecting or
nonreflecting; may be porous or nonporous; etc. For example, the
solid support may be comprised of polyethylene,
polytetrafluoroethylene, polystyrene, polyethylene terephthalate,
polycarbonate, gold, silicon, silicon oxide, silicon oxynitride,
indium, tantalum oxide, niobium oxide, titanium, titanium oxide,
platinum, iridium, indium tin oxide, diamond or diamond-like film,
etc.
[0035] The present invention may be utilized with substrates for
"chip-based" and "pin-based" combinatorial chemistry techniques.
All can be prepared in accordance with known techniques. See. e.g.,
U.S. Pat. No. 5,445,934 to Fodor et al., U.S. Pat. No. 5,288,514 to
Ellman, and U.S. Pat. No. 5,624,711 to Sundberg et al., the
disclosures of which are incorporated by reference herein in their
entirety.
[0036] Substrates as described above can be formed of any suitable
material, including but not limited to a material selected from the
group consisting of metals, metal oxides, semiconductors, polymers
(particularly organic polymers in any suitable form including
woven, nonwoven, molded, extruded, cast, etc.), silicon, silicon
oxide, and composites thereof.
[0037] Polymers used to faun substrates as described herein may be
any suitable polymer, including but not limited to: poly(ethylene)
(PE), poly(propylene) (PP), cis and trans isomers of
poly(butadiene) (PB), cis and trans isomers of poly(ispoprene),
poly(ethylene terephthalate) (PET), polystyrene (PS), polycarbonate
(PC), poly(epsilon-caprolactone) (PECL or PCL), poly(methyl
methacrylate) (PMMA) and its homologs, poly(methyl acrylate) and
its homologs, poly(lactic acid) (PLA), poly(glycolic acid),
polyorthoesters, poly(anhydrides), nylon, polyimides,
polydimethylsiloxane (PDMS), polybutadiene (PB), polyvinylalcohol
(PVA), polyacrylamide and its homologs such as poly(N-isopropyl
acrylamide), fluorinated polyacrylate (PFOA),
poly(ethylene-butylene) (PEB), poly(styrene-acrylonitrile) (SAN),
polytetrafluoroethylene (PTFE) and its derivatives, polyolefin
plastomers, and combinations and copolymers thereof, etc.
[0038] If desired or necessary, the substrate may have an
additional layer such as a gold or an oxide layer formed on the
relevant surface portion to facilitate the deposition of the
linking layer, as discussed further below.
3. Linking (or "Anchor") layers.
[0039] Anchor layers used to carry out the present invention are
generally formed from a compound comprising an anchor group coupled
(e.g., covalently coupled) to an initiator (e.g., directly coupled
or coupled through an intermediate linking group).
[0040] The choice of anchor group will depend upon the surface
portion on which the linking layer is formed, and the choice of
initiator will depend upon the particular reaction used to form the
brush polymer as discussed in greater detail below.
[0041] The anchoring group may be selected to covalently or
non-covalently couple the compound or linking layer to the surface
portion. Non-covalent coupling may be by any suitable secondary
interaction, including but not limited to hydrophobic bonding,
hydrogen bonding, Van der Waals interactions, ionic bonding,
etc.
[0042] Examples of substrate materials and corresponding anchoring
groups include, for example, gold, silver, copper, cadmium, zinc,
palladium, platinum, mercury, lead, iron, chromium, manganese,
tungsten, and any alloys thereof with sulfur-containing functional
groups such as thiols, sulfides, disulfides (e.g., --SR or --SSR
where R is H or alkyl, typically lower alkyl, or aryl), and the
like; doped or undoped silicon with silanes and chlorosilanes
(e.g., --SiR.sub.2Cl wherein R is H or alkyl, typically lower
alkyl, or aryl); metal oxides such as silica, alumina, quartz,
glass, and the like with carboxylic acids as anchoring groups;
platinum and palladium with nitrites and isonitriles; and copper
with hydroxamic acids. Additional suitable functional groups
suitable as the anchoring group include benzophenones, acid
chlorides, anhydrides, epoxides, sulfonyl groups, phosphoryl
groups, hydroxyl groups, phosphonates, phosphonic acids, amino acid
groups, amides, and the like. See, e.g., U.S. Pat. No.
6,413,587.
[0043] Any suitable initiator may be incorporated into the
anchoring group by introduction of a covalent bond at a location
non-critical for the activity of the initiator. Examples of such
initiators include, but are not limited to, bromoisobutyrate,
polymethyl methacrylate-Cl, polystyrene-Cl, AIBN,
2-bromoisobutyrate, chlorobenzene, hexabromomethyl benzene,
hexachloromethyl benzene, dibromoxylene, methyl bromoproprionate.
Additional examples of initiators include those initators described
in U.S. Pat. No. 6,413,587 to Hawker (particularly at columns 10-11
thereof) and those initiators described in U.S. Pat. No. 6,541,580
to Matyjaszewski et al.
[0044] As noted above, a linking group or "spacer" may be inserted
between the anchoring group and initiator. The linker may be polar,
nonpolar, positively charged, negatively charged or uncharged, and
may be, for example, saturated or unsaturated, linear or branched
alkylene, aralkylene, alkarylene, or other hydrocarbylene, such as
halogenated hydrocarbylene, particularly fluorinated
hydrocarbylene. Preferred linkers are simply saturated alkylene of
3 to 20 carbon atoms, i.e., --(CH.sub.2).sub.4-- where n is an
integer of 3 to 20 inclusive. See, e.g., U.S. Pat. No. 6,413,587.
Another preferred embodiment of the linker is an
oligoethyleneglycol of 3 to 20 units, i.e.,
(CH.sub.2CH.sub.2O).sub.n where n ranges from 3 to 20.
[0045] The anchoring layer may be deposited by any suitable
technique. It may be deposited as a self-assembled monolayer. It
may be created by modification of the substrate by chemical
reaction (see, e.g., U.S. Pat. No. 6,444,254 to Chilkoti et al.) or
by reactive plasma etching or corona discharge treatment. It may be
deposited by a plasma deposition process. It may be deposited by
spin coating or dip coating. It may be deposited by spray painting.
It may also be deposited by deposition, printing, stamping, etc. It
may be deposited as a continuous layer or as a discontinuous (e.g.,
patterned) layer.
[0046] In some preferred embodiments, the substrate is glass,
silicon oxide or other inorganic or semiconductor material (e.g.,
silicon oxide, silicon nitride) and compound semiconductors (e.g.,
gallium arsenide, and indium gallium arsenide) used for microarray
production.
[0047] In some preferred embodiments, the anchoring group is a
silane or chlorosilane (e.g., -SiR.sub.2Cl wherein R is H or alkyl,
typically lower alkyl, or aryl).
[0048] In some preferred embodiments, the linking layer comprises
of the deposition of two layers in separate steps. In the first
step, an anchoring layer of alkylsilane or alkanethiol is deposited
on a surface such as silicon dioxide or glass or gold, and presents
a terminal reactive functional group (e.g., amine). In the next
step, a bifunctional molecule, which comprises a first functional
group reactive towards the terminal group presented by the first
linking layer is reacted with the first linking layer deposited in
the first step. The second functional group of the bifunctional
molecule contains a moiety group that acts as an ATRP or free
radical initator.
4. Brush polymer formation.
[0049] The brush polymers are, in general, formed by the
polymerization of monomeric core groups having a protein-resistant
head group coupled thereto. Any suitable core vinyl monomer
polymerizable by the processes discussed below can be used,
including but not limited to styrenes, acrylonitriles, acetates,
acrylates, methacrylates, acrylamides, methacrylamides, vinyl
alcohols, vinyl acids, and combinations thereof.
[0050] Protein resistant groups may be hydrophilic head groups or
kosmotropes. Examples include but arc not limited to
oligosaccharides, tri(propyl sulfoxide), hydroxyl, glycerol,
phosphorylcholine, tri(sarcosine) (Sarc), N-acetylpiperazine,
betaine, carboxybetaine, sulfobetaine, perrnethylated sorbitol,
hexamethylphosphoramide, an intramolecular zwitterion (for example,
--CH.sub.2N.sup.+(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3.sup.-)
(ZW), and mannitol. Additional examples of kosmotrope protein
resistant head groups include, but are not limited to:
[0051] -(EG).sub.6OH;
[0052] --O(Mannitol);
[0053]
--C(O)N(CH.sub.3)CH.sub.2(CH(OCH.sub.3)).sub.4CH.sub.2OCH.sub.3;
[0054]
--N(CH.sub.3).sub.3.sup.+Cl.sup.-/--SO.sub.3.sup.-Na.sup.+;
[0055] --N(CH.sub.3).sub.2.sup.+CH.sub.2CH.sub.2SO.sub.3 ,
[0056] --C(O)Pip(NAc);
[0057] --N(CH.sub.3).sub.2.sup.+CH.sub.2CO.sub.2 ;
[0058] --O([Blc-.alpha.(1,4)-Gle-.beta.(1)-]);
[0059] --C(O)(N(CH.sub.3)CH.sub.2C(O)).sub.3N(CH.sub.3).sub.2;
[0060]
--N(CH.sub.3).sub.2.sup.+CH.sub.2CH.sub.2CH.sub.2SO.sub.3.sup.-;
[0061]
--C(O)N(CH.sub.3)CH.sub.2CH.sub.2N(CH.sub.3)P(O)(N(CH.sub.3).sub.2)-
.sub.2; and
[0062] --(S(O)CH.sub.2CH.sub.2CH.sub.2).sub.3S(O)CH.sub.3.
See, e.g., R. Kane et al., Langmuir 19, 2388-91 (2003)(Table
1).
[0063] A particularly preferred protein resistant head group is
poly(ethylene glycol), or "PEG", for example PEG consisting of from
3 to 20 monomeric units.
[0064] Free radical polymerization of monomers to form brush
polymers can be carried out in accordance with known techniques,
such as described in U.S. Pat. No. 6,423,465 to Hawker et al.; U.S.
Pat. No. 6,413,587 to Hawker et al.; U.S. Pat. No. 6,649,138 to
Adams et al.; US Patent Application 2003/0108879 to Klaerner et
al.; or variations thereof which will be apparent to skilled
persons based on the disclosure provided herein.
[0065] Atom or transfer radical polymerization of monomers to thin'
brush polymers can be carried out in accordance with known
techniques, such as described in U.S. Pat. No. 6,541,580 to
Matyjaszewski et al.; U.S. Pat. No. 6,512,060 to Matyjaszewski et
al.; or US Patent Application 2003/0185741 to Matyjaszewski et al.,
or variations thereof which will be apparent to skilled persons
based on the disclosure provided herein.
[0066] In general, the brush molecules formed by the processes
described herein (or other processes either known in the art or
which will be apparent to those skilled in the art based upon the
present disclosure), will be from 2 or 5 up to 100 or 200
nanometers in length, or more, and will be deposited on the surface
portion at a density of from 10, 20 or 40 up to 100, 200 or 500
milligrams per meter.sup.2, or more.
[0067] In some preferred embodiments, the polymer layer is formed
by SI-ATRP of OEGMA to form a poly(OEGMA) film. In particularly
preferred embodiments, the polymer layer is a functionalized
poly(OEGMA) film prepared (preferably in a single step) by
copolymerization of a methacrylate and methoxy terminated
OEGMA.
[0068] Preparation of substrate and polymer layer far deposition.
Prior to deposition of the first member of the specific binding
pair, the substrate and polymer layer are macroscopically dry or at
least macroscopically dry (that is, dry to the touch or dry to
visual inspection, but retaining bound water or water of hydration
in the polymer layer). To enhance immobilization, it is preferable
that the polymer layer retain bound water or water of hydration (or
stated otherwise, that the article includes water consisting of or
consisting essentially of waters of hydration, but not bulk surface
water). When the substrate with polymer layer has been stored in
desiccated form, this can be achieved by quickly hydrating,
dipping, or contacting the polymer layer to water and then blow
drying the surface (e.g., with a nitrogen or argon jet), or by
simply exposing the polymer layer to ambient air for a time
sufficient for water of hydration to be bound from the atmosphere
by the polymer layer.
[0069] Deposition and post-deposition drying. The first member of
the specific binding pair (as described above) can be deposited on
the polymer layer by any suitable technique such as microprinting
or microstamping, including piezoelectric or other forms of
non-contact printing and direct contact quill printing.
[0070] When an array is being fomied by the deposition of multiple
first binding pairs, or "probes", at discrete probe locations on
the polymer layer, probe densities of 1, 3, 5 or 10, up to 100 or
1000 probe locations per cm.sup.2 can be made. Modern non-contact
arrayers can be used in the deposition step to produce arrays
having up to 1,000,000 probe locations per cm.sup.2. With dip-pen
nanolithography, arrays with up to 1 billion discrete probe
locations per cm.sup.2 can be made. It will be appreciated that the
specific molecular species at each probe location can be different
from the others, or that some can be the same (e.g., to provide
some redundancy or control), depending upon the particular purpose
of the array.
[0071] After deposition of the first member of the specific binding
pair, the device is optionally but preferably dried, e.g., by mild
desiccation, blow drying, lyophilization, or exposure to ambient
air at ambient temperature, for a time sufficient for the article
to be macroscopically dry or at least macroscopically dry as
described above. Again, water of hydration may remain bound by the
polymer layer even though the device is macroscopically dry. Once
the device is macroscopically dry or at least macroscopically dry,
it may be sealed in a container (e.g., such as an impermeable or
semipermeable polymeric container) in which it can be stored and
shipped to the user. Once sealed in the container, the device
preferably has, in some embodiments, a shelf life of at least 2 to
4 months, and preferably up to 6 months or more, when stored at a
temperature of 25 .degree. C. (e.g., without loss of more than 20,
30 or 50 percent of binding activity).
5. Uses and applications of articles.
[0072] In some embodiments the present invention is utilized by (a)
providing an article as described herein; and then (b) contacting
the article to a biological fluid or other composition.sub.;
containing a second member of the specific binding pair, wherein
the second member of the specific binding pair binds to the surface
portions. Such uses are particularly appropriate where the article
is a sensor or biosensor as described in greater detail above.
[0073] Any suitable "second member" or "analyte" as described above
can be detected. For example, the second member may be a compound
found in or marker for: [0074] HEP AJB/C/E, Influenza A/B [0075]
Common and Antibiotic-Resistant Cocci, TB, Syphillis [0076] HIV,
HCV, HTLV, HPV, Herpes Simplex, Chlamydia, Ghanaian, West Nile,
Chlarnydiazyme [0077] CMV, Rubella, TOXO, TPHA, Lyme disease [0078]
ehrlichiosis, anaplasmosis, bartonellosis, typhus, Q fever,
tickborne spotted fevers, actinomycete
[0079] Fungal infection markers: [0080] aspergillosis,
blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis,
histoplasmosis, Pneumocystis carinii
[0081] The second member may be one or more of, for example: [0082]
A human cytokine, such as IL-1.alpha., IL-1.beta., IL-2, IL-4,
1L-5, IL-6, IL-8, IL-10,IL-12, IL-13, IFN-.gamma.and TNF.alpha.;
[0083] A human IR chemokine, such as ENA-78, Eotaxin, GRO.alpha.,
IP-10, MCP-1, MDC, MIG, MIP-1.alpha., MIP-1.beta., MPIF-1, RANTES
and TARC [0084] A human angiogenic factor, such as ANG-2, FGF
Basic, HB-EGF, HGF, KGF, PDGF-BB, THVIP-1, Tpo, VEGF, FGF basic,
HGF, PDGF-BB, VEGF; or
[0085] A cardiac marker, such as: Apo A-1, Apo B-100, Fibrinogen,
Fibronectin and CRP; Acrp-30, A-SAA, MPO, MMP-2 and MMP-9; AI-1
Active, NT-proBNP, P-Selectin, IL-8, IL-6, OPG, PAPP-A and RANKL;
and [0086] a diabetes and/or obesity marker, such as IGFBP-1,
IGFBP-3, Prolactin, Resistin, CRP, ICAM-1, Acrp-30 and MMP-2;
MMP-9, TNF-RII, VCAM-1 and E-Selectin; Leptin, IL-6, C-peptide and
HG.
[0087] In one embodiment of the invention, a substrate of the
invention contains a plurality (e.g., one, two or three, up to 20,
30, or 40 or more) of different first members that each bind to a
different one of the foregoing second member/analytes at separate
and discrete locations on the substrate polymer layer to form an
array or microarray that can be used to test for a plurality of
different analytes in the same biological fluid sample. The
plurality of different first members (selected for the
corresponding second members/analytes to which they bind) can be
selected and deposited on the array to provide a "panel" test for a
particular purpose, such as a human cytokine array; a human IR
chemokine array; a human angiogenic factor array, a cardiac marker
array, a diabetes and/or obesity marker array, a cancer array
etc.
[0088] Binding of the second member of the specific binding pair
(analyte) can be detected by any suitable technique. In some
embodiments the analyte is detected by immunometric assay such as a
sandwich assay. In some embodiments of a "sandwich" assay, a third
binder, that also specifically binds to the second member of the
binding pair (the "analyte"), is bound to the analyte, and the
binding of that third binder is detected (e.g., by labelling of the
third binder with a detectable group such as an enzyme, fluorescent
group, or radioactive group). Such sandwich assays are well known.
Numerous assay formats are known which can be used or adapted to
carry out the present invention. See, e.g., U.S. Pat. Nos.
7,312,041; 7,270,970; 7,267,951; 7,247,500; 7,229,775; 7,202,028;
7,195,883; 7,166,469; 7,148,016, etc.
[0089] The present invention is explained in greater detail in the
following non-limiting Examples.
EXPERIMENTAL
[0090] Herein, we demonstrate that eliminating background
adsorption in protein microarrays can decrease the LOD by 100-fold
in buffer and serum over the same protein microarrays printed on a
conventional substrate (that displays high binding capacity but
significant adventitious adsorption) without need for any other
changes to the assay protocol. Notably, the LODs are equivalent for
assays performed in either buffer or serum typically, the LODs for
most immunoassays obtained in buffer are severely compromised when
complex protein mixtures such as serum are probed..sup.4
[0091] We chose to use a poly(oligo(ethylene glycol) methacrylate)
(POEGMA) polymer brush as the microarray substrate because it can
be conveniently grown on glass as a high-density brush that limits
protein adsorption..sup.5 The procedure (SI) used to grow the
POEGMA brushes on glass is summarized in FIG. 2. Ellipsometry in
air of POEGMA brushes grown on oxidized silicon wafers under
identical conditions indicated a POEGMA thickness of 105.+-.2
nm.
[0092] A non-contact PerkinElmer Piezorray was used to print Ab
microarrays onto POEGMA substrates at room temperature and humidity
using coated slides that had been stored on the benchtop in a
closed container for up to two months (substrate storage time had
no observed effect on assay performance). Antibodies for IL-6 and
Osteoprotegerin (OPG) (R&D Systems) were printed from 50 gg/mL
solutions and allowed to non-covalently absorb into the 100 nm
thick polymer brush. After printing, drying of the spots was
facilitated by placing the printed slides under vacuum. This
printing and drying process provides stable immobilization of
antibody, as arrayed spots of Cy-5 labeled goat anti-rabbit IgG
(Jackson) were still visible after high power sonication in a 1%
Tween-20 solution (SI). An advantage of this approach over chemical
activation of the POEGMA brushes and subsequent covalent
attachment.sup.6 is the extreme simplicity of the process, as no
slide activation/deactivation steps are required. Non-covalent
immobilization via dehydration resulted in equivalent levels of
immobilized capture antibody when compared to covalent
immobilization via disuccinimidyl carbonate (DSC) and
carbonyldiimidazole (CDI) activation procedures (SI). A large
increase in background was observed by printing on CDI-activated
POEGMA, presumably because of incomplete deactivation of the
surface after printing (SI), which highlights another important
advantage of printing directly on the polymer brush as opposed to
covalent coupling. Furthermore, we found that drying the arrays
after printing does not prevent the recognition of analytes by the
capture antibodies, nor does it result in bleeding of the spots
upon subsequent exposure to liquids during the interrogation of the
arrays (FIG. 3a). We hypothesize that the 100 nm thick POEGMA brush
functions as a quasi-3D hydrogel that retains sufficient
interfacial water, even during macroscopic drying of the printed
arrays, to allow retention of antibody structure and hence function
future experiments will test this hypothesis. After printing the
capture antibodies, the arrays were stored in vacuum for eventual
use in protein assays. Storage time of the Ab arrays from 24 h to
two weeks had no obvious effect on array activity. An example of
this observation can be seen in FIG. 3b arrayed slides used to
produce the two dose response curves were printed simultaneously,
however the assays in buffer preceded the assays in serum by two
weeks.
[0093] IL-6 and OPG Ab arrays were used to directly probe a
dilution series of analyte-spiked PBS and serum (assay details
below). To compare the performance of these arrays against a
commonly used array material, we also printed the same Ab arrays on
commercially available nitrocellulose membranes (Whatman), which
are used because of their ability to provide high print densities
of the capture antibody and hence high signal. Assays on
nitrocellulose substrates were performed according to the
manufacturer's suggested protocol.
[0094] The fluorescence intensity after scanning and background
subtraction for different concentrations of IL-6 as a function of
analyte concentration in serum are shown in FIG. 2C for
nitrocellulose and POEGMA. The data clearly show that the
fluorescence signal from the printed capture Ab spots on
nitrocellulose are only visible to a concentration of 10 pg/ml,
while the signal on POEGMA is clearly visible down to a
concentration of 100 fg/ml. Furthermore, despite the incubation and
rinse steps, there was no bleeding of the spots (FIG. 3A), which
confirmed the stable immobilization of the capture antibody. The
image in FIG. 3A also shows that the POEGMA matrix retains its
ability to resist non-specific protein adsorption throughout the
entire array fabrication and assay process fluorescence intensities
of the background areas surrounding printed spots measured prior to
the assay show no increase in intensity upon completion of the
procedure (the only background fluorescence detected on the POEGMA
substrates is due to the autofluoresence of the glass slide). This
elimination of background signal allows the POEGMA substrates to
achieve LODs (signal was considered significant if greater than
three standard deviations above the average of the same Ab spots
exposed to non-spiked serum) that are up to two orders of magnitude
more sensitive when compared to traditional nitrocellulose
substrates, as shown by the dose-response curves in FIG. 3C.
[0095] The POEGMA substrates also provide an improved dynamic range
and can quantify protein concentration across six orders of
magnitude, as seen in the dose response curves in FIG. 2 and
summarized in Table 1. OPG dose response curves in buffer and serum
are shown in FIG. 2B to illustrate the important point that the Ab
arrays on POEGMA have virtually identical LODs in buffer and serum.
This is in contrast to most other fluorescence immunoassays, where
the LOD is typically orders of magnitude greater in complex
physiological solutions containing high concentrations of
extraneous proteins when compared to LODs determined in buffer.
TABLE-US-00001 TABLE 1 Limits of Detection (LOD) and Dynamic Ranges
of Serum-Based Microarray Assays on POEGMA Analyte LOD Dynamic
Range IL-6 100 fg/mL 100 fg/mL-10 ng/mL IL-.beta. 100 fg/mL 100
fg/mL-10 ng/mL TNF-.alpha. 100 fg/mL 100 fg/mL-10 ng/mL IL-8 100
fg/mL 100 fg/mL-10 ng/mL OPG 1 pg/mL .sup. 1 pg/mL-10 ng/mL
[0096] In conclusion, we have demonstrated antibody arrays on
POEGMA brushes with several significant features: first, the direct
physical printing of the capture Abs provides a simple and robust
procedure for the stable immobilization of the capture Abs that
avoids the need for chemical activation and deactivation of the
surface.
[0097] Second, the printed microarrays have a practical shelf-life
of at least several weeks with no loss in performance. Third,
antibody arrays printed on the POEGMA brushes require during
interrogation of the array, which simplifies the assay. Finally,
the resistance of the POEGMA brushes to protein adsorption from
solution eliminates background noise in the microarrays stemming
from adventitious protein adsorption and leads to LODs as low as
100 fg/mL in serum (which corresponds to 4 fM for IL-6). The
femtomolar LODs in serum and the wide dynamic range suggest that
these microarrays will he useful for the quantification of low
abundance protein biomarkers directly from complex mixtures with
minimal sample pre-processing.
Methods:
[0098] Synthesis of POEGMA surfaces: The POEGMA brushes were
fabricated on glass as follows (FIG. 2, all chemicals purchased
from Sigma): first, glass slides (VWR) were cleaned in a solution
of 3:1 H-)SO.sub.4:H.sub.2O.sub.2 for 30 minutes. After rinsing
with deionized H.sub.2O and drying, the cleaned slides were
immersed in 10% aminopropyltriethoxysilane (APTES) in ethanol for
30 min and were then rinsed with ethanol and dried at 120.degree.
C. for 3 h (step 1). Slides were then immersed in a solution of 1%
bromoisobutyryl bromide and 1% triethylamine in dichloromethane for
30 min, rinsed with dichloromethane and ethanol, and blown dry with
N.sub.2 (step 2). Slides were then immersed for 12 h in a degassed
polymerization solution of 5 mg/mL Cu(I)Br, 12mg/mL bipyridine and
300mg/mL oligo(ethylene glycol) methacrylate under argon (step 3).
Finally, slides were rinsed with deionized H.sub.2O and blown dry
with N.sub.2.
[0099] Antibody Immobilization: Cy-5 labeled goat anti-rabbit IgG
(Jackson) was printed on POEGMA substrates to produce arrays seen
in FIG. 4. The arrays were dehydrated for 24 hours to promote
immobilization and then subjected to high power sonication for 10
minutes in a 1% Tween-20 PBS solution.
[0100] Covalent vs. non-covalent attachment: Identical arrays of
mouse anti-goat IgG were printed onto unmodified POEGMA substrates
as well as substrates that had been activated with either
disuccinimidyl carbamate (DSC) or carbonyldiimidazole (CDI).
Subsequent incubation with Cy5 labeled goat anti-rabbit IgG yielded
similar spot intensities for both immobilization methods, however,
background levels on the activated slides increased dramatically,
as shown in FIG. 5. We suggest that the use of ethanolamine to
deactivate unused conjugation sites, and the subsequent
incorporation of large numbers of amide bonds into the POEGMA
layer, as well as residual reactive groups, led to the increase in
non-specific protein adsorption and coupling in subsequent
steps.
[0101] CDI activation protocol: Slides were immersed in a 0.5M
solution of CDI in dry dioxane for two hours at 37.degree. C. with
stirring and then rinsed with dry dioxane, dried, and used
immediately for printing.
[0102] DSC activation protocol: Slides were immersed in a solution
of 0.6M DSC and 0.6M 4-(dimethylamino)pyridine in dry acetone for 6
hours with stirring and then rinsed with dry acetone, dried, and
used immediately for printing.
[0103] Deactivation protocol: Printed slides were immersed in a
0.1M Na Borate buffer at pH 8.5 for 1 hour, and were then
transferred to a 0.1M Na Borate buffer at pH 8.5 with 1M
ethanolamine for 1 hour.
[0104] Multiplexed sandwich immunoassay details: Arrays were first
incubated with a dilution series of 100 ml of analyte-spiked PBS or
serum for 2 h with stirring, followed by 100 ml 1 mg/ml
biotinylated secondary antibody in PBS with 1% (w/v) BSA for 1 h.
Finally, the arrays were developed by incubation in 100 ml of 1
ug.sup.-/m1 streptavidin-Cy5 for 30 min, and then scanned with an
Axon Genepix 4200 fluorescence microarray scanner. After each
incubation step, arrays were washed twice for 30 s with 1% BSA
(w/v) and 0.1% (w/v) Tween-20 in PBS.
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[0111] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
* * * * *