Note: Descriptions are shown in the official language in which they were submitted.
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MOLECULAR NETS ON SOLID PHASES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application serial no.
61/783,189, filed March 14, 2013, and is a continuation-in-part of U.S. patent
application serial
nos. 13/511,364, filed May 22, 2012, and 13/938,055, filed July 9, 2013, which
are hereby
incorporated by reference in their entireties.
BACKGROUND
[0002] Current strategies for solid phase analyte capture, analyte
detection and analyte
measurement exist using a single layer of capture molecules absorbed or
covalently tethered to a
surface for direct real-time sensing or are used in conjunction with secondary
detection steps in
an indirect detection modality are well known in the art. Both direct and
indirect methods have
demonstrated limitations in sensitivity, specificity, signal-to-noise ratio
and/or cost.
[0003] There is need for analyte capture technology for solid phase
surfaces or devices
that can selectively capture analytes from a complex sample with little or no
sample preparation
and to position said selected analytes in a manner to maximize captured
analyte measurement
and/or detection in a manner that is compatible with most technologies.
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SUMMARY
[0004] Devices for capturing an analyte are described. In one embodiment,
a device may
comprise a solid phase and a molecular net coupled to at least a portion of a
surface of the solid
phase. The molecular net may include capture molecules of at least one type
coupled to each
other by linker molecules of a plurality of types to form a covalently-linked
multi-layered three-
dimensional matrix. The capture molecules may be configured to bind to the
analyte.
[0005] Methods of manufacturing a device for capturing an analyte are
also described. In
one embodiment, a method may comprise providing a solid phase, and placing a
molecular net
on at least a portion of a surface of the solid phase. The molecular net may
include capture
molecules of at least one type coupled to each other by linker molecules of a
plurality of types to
form a covalently-linked multi-layered three-dimensional matrix. The capture
molecules may be
configured to bind to the analyte.
[0006] Methods of measuring a quantity of an analyte in a sample are also
described. In
one embodiment, a method may comprise providing one or more devices each
comprising a solid
phase and a molecular net covering at least a portion of a surface of the
solid phase. The
molecular net may include capture molecules of at least one type coupled to
each other by linker
molecules of a plurality of types to form a covalently-linked multi-layered
three-dimensional
matrix. The capture molecules configured to bind to the analyte. The method
also comprises
exposing the devices to the sample and allowing at least a portion of the
analyte to bind to the
capture molecules of the molecular nets of the devices.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows a comparison of traditionally conjugated
microparticles and
Molecular Net microparticles in IgG purification.
[0008] FIG. 2 shows a traditional capture molecule conjugation to
microparticles and the
corresponding analyte measurement capability.
[0009] FIG. 3 shows an effectiveness of Molecular Net microparticles in
measuring
analyte.
[0010] FIG. 4 shows an effectiveness of a Molecular Net with topology in
measuring
analyte.
[0011] FIG. 5 shows a comparison of traditionally conjugated
microparticles and
Molecular Net microparticles in a Tau ELISA.
[0012] FIG. 6 shows a comparison of traditionally conjugated
microparticles and
Molecular Net microparticles in a TSH Luminex sandwich immunoassay.
[0013] FIG. 7 shows an exemplary Molecular Nets on particles.
[0014] FIG. 8 shows an exemplary Molecular Net topological features on
particles.
[0015] FIG. 9 shows an exemplary Molecular Nets for analyte delivery.
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DESCRIPTION
[0016] It has been shown in U.S. patent serial nos. 61/281,991,
61/337,257, 61/340,287,
61/343,467, 61/410,837, 61/489,646, and 61/489,648, each of which are hereby
incorporated by
reference, that the construction and use of a covalently-linked pseudorandom
multilayered three-
dimensional matrix enables the rapid and specific capture of protein, nucleic
acid, carbohydrate,
lipid, cell or other analytes from an unprocessed sample and that the use of
such Molecular Net
may be a significant improvement upon conventional analyte binding approaches.
Design and Fabrication of Molecular Nets
[0017] Properties of Molecular Nets may be imparted by: the capture
molecules selected
for use (examples of capture molecules may include antibodies, nucleic acid
probes, enzymes,
recombinant proteins, peptides and others); the resultant specificity said
capture molecules
impart; the size and number of selected capture molecules; the placement and
spacing of the
capture molecules in the molecular Net layer(s); the combination of capture
molecules; the order
in which the capture molecules may be used; and the ratio of capture molecules
to linker
molecules and spacer molecules used.
[0018] Properties of Molecular Nets may also be imparted by: the linker
molecules
selected for use (examples of linker molecules include homobifunctional,
heterobifunctional,
trifunctional and multifunctional types); the chemical specificity of the
linker molecules; the
Angstrom length of the linker molecules; the combination of linker molecules;
the order in which
the linker molecules may be used; and the ratio of capture molecules to linker
molecules and
spacer molecules used.
[0019] Properties of Molecular Nets may also be imparted by: the spacer
molecules
selected for use (examples of spacer molecules include PEG, polymer, nucleic
acid, albumin, Fc
region, peptide, and other); the chemical properties of the spacer molecules;
the size and number
of spacer molecules; the order in which the spacer molecules may be used; and
the ratio of
spacer molecules to linker molecules and capture molecules used.
[0020] Placement and spacing of the capture molecules, linker molecules
and spacer
molecules may: confer a characteristic topology on the Molecular Net surface;
confer a
characteristic density within each layer of a Molecular Net; confer a
characteristic porosity of a
Molecular Net; remove spatial constraints and thus stearic hindrance; improve
binding capacity;
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reduce non-specific binding; enable the binding of multiple forms of analyte
(for example,
simultaneous capture of degraded analyte, whole analyte and complexed
analyte), and other.
[0021] The porosity within a Molecular Net may be random, pseudorandom or
irregularly interspersed. Porosity of a Molecular Net may be used to filter a
sample; may be used
to discriminate binding potential molecules in a sample by size-exclusion; may
be used to enable
macromolecular or cellular binding due to the reduction in stearic hindrance,
or other. The pores
of a Molecular Net comprise capture molecules, linker molecules and may
comprise spacer
molecules. Traditional approaches to generate porosity on a solid phase
relates to the
mechanical modification of the surface of the solid phase and employ methods
such as laser
etching, laminating, lithography, laser printing or others to generate pores,
holes or other
structures in the solid surface. This solid surface is then prepared for
accepting conjugating
capture molecules. Use of a Molecular Net removes the need for mechanical
modification of a
surface and is thus more cost-effective. Additionally, traditional approaches
are still hampered
by the problem of high non-specific binding and require capture chemistry to
be bound to the
mechanically modified solid phase, which is not an improvement. Additionally,
flexibility may
be imparted into a Molecular Net as compared to the traditional capture format
due to size-
exclusion properties conferred by the porosity built into each layer of a
Molecular Net. In some
layers, pore diameter and depth may be similar or may vary depending on the
application. In
some layers, pore sizes may vary, the variance of which may depend on the
application.
[0022] Porosity that may be imparted on a Molecular Net may include but
are not limited
to picopores, nanopores, micropores, filtration pores, sieving pores, pockets
or other. Porosity
may be imparted into a Molecular Net by the selection of and method of
incorporation of specific
capture molecules, linker molecules and spacer molecules into each layer of a
Molecular Net.
Porosity may also be imparted into a Molecular Net by the selection of and
method of
incorporation of specific capture molecules, linker molecules and spacer
molecules used in the
fabrication of sequential layers.
[0023] Molecular Net porosity may range from about 6 Angstroms in
diameter to more
than about 1 um in diameter based on the identity of capture molecules, linker
and spacers used
in a layer. In some cases, the porosity of a Molecular Net may comprise a
range of pore
diameters. Exemplary diameter ranges may be from about 5 nm to about 50 nm,
from about 10
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nm to about 100 nm, from about 50 nm to about 200 nm, from about 250 nm to
about 500 nm,
from about 500 nm to about 1 um, and from about 800 nm to about 1.5 um.
[0024] In some cases, capture molecules may be used to generate pores in
a Molecular
Net. In these instances, capture molecules may be pre-linked to one another
prior to being
incorporated into a Molecular Net layer. In some cases, linkers may be
selected based on
Angstrom length of the spacer arms. In some examples, extenders may be used to
connect a first
linker to a second linker to generate a long multi-functional linker. In some
cases, spacers may
be used to generate pores in a Molecular Net. Spacers may also be pre-linked
to one another
prior to being incorporated into a Molecular Net layer. In other examples,
inert physical plugs
may be used to build a pore, whereby each physical plug may be placed on a
previously built
layer while a new layer is being constructed. After curing, the physical plugs
may be removed,
thus leaving a pore of a specific diameter.
[0025] The flexible nature of the Molecular Net enables the use of
multiple types of
capture molecules. In some examples, a Molecular Net comprises a single type
of capture
molecule. In other examples, a Molecular Net comprises multiple types of
capture molecules. In
some examples, the use of more than one monoclonal antibody during the
fabrication of a
Molecular Net enables said Molecular Net to bind more than one epitope of an
analyte. Use of
more than one type of epitope-specific capture molecule enables improved
analyte capture by a
Molecular Net and relates to its performance. In some examples, the use of
more than one
nucleic acid sequence may be used during the fabrication process to generate a
Molecular Net
capable of binding to more than one epitope of an analyte. Examples of benefit
depend on the
use of the Molecular Net and may comprise improved performance in terms of
minimum levels
of detection, sensitivity, positive predictive value, negative predictive
value, ability to work with
degraded samples, ability to work with a diverse population, and others when
used in a test; may
comprise improved performance in binding capacity, purity, binding kinetics,
target analyte
depletion, and others when used as a purification tool; or other.
[0026] In some examples, the use of capture molecules directed against
mutually-
confirmatory analytes may be used in a Molecular Net and relates to its
performance. Use of
mutually-confirmatory capture molecules in a Molecular Net may be used in a
confirmatory
manner, whereby the capture of more than one analyte may provide a more
statistically
significant positive result; may provide a more robust test result; may
provide additional
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information regarding a sample; and other. Use of mutually-confirmatory
capture molecules in a
Molecular Net may also be used to qualify a sample or may be used as a control
in a test or may
be used to measure more than one related molecular variable linked to a
disease state or may be
used to measure more than one related molecular variable linked to the
treatment of a disease.
[0027] Examples of mutually-confirmatory analytes a Molecular Net may be
fabricated
to simultaneously capture from a sample may include: genetic sequence and
corresponding
protein product (for examples, cancer-related SNPs in BRCA1 and BRCA1
protein); the mRNA
and corresponding protein product (for example, human lactase mRNA and Lactase
protein); the
genetic sequence and the corresponding mRNA product (for example, disease-
related SNPs in
LMNA and pre-spliced or spliced Lamin A/C mRNA); miRNA and related mRNA or
protein
products (miR 9 and REST or CoREST mRNA, or miR 9 and REST protein); small
molecule
drugs and drug targets (tofacitinib and Janus kinase 3); epitope-specific
biologics and the
respective targets (for example, anti-TNF antibodies and circulating TNF
cytokine); epitope-
specific antibodies, epitope-specific T cells and/or epitope-specific B cells
or the like (for
example, anti-DNA autoantibodies, anti-DNA CD4 ' T cells and/or anti-DNA B
cells); or others.
Examples of benefit depend on use and may relate to improved performance in
test sensitivity,
positive predictive value, negative predictive value, specificity, diagnosis
of a disease, ability to
work with samples experiencing genetic drift, ability to measure response to a
therapeutic, ability
to measure effectiveness of a therapeutic, or other.
[0028] In one example, a Molecular Net may be fabricated in a manner to
capture and
position bound analytes in a manner that enhances the intensity of a
detectable signal or may
enhance detection of bound analytes, such as when used in a test with optical
detection.
Placement of captured analytes in a layered manner by pre-positioned layered
capture molecules
may enable the rapid detection of analyte by signal intensification. Examples
of signal
intensification by a Molecular Net may relate to fluorescence, fluorescence
resonance energy
transfer, absorbance, luminescence, light scatter, surface plasmon resonance,
optical heterodyne
detection, or other.
[0029] Said Molecular Net may be designed and fabricated to replace the
need for costly
and time-intensive methods for ultra-sensitive detection such as PCR, branched
DNA, or multi-
step detection methods required for signal amplification. Said Molecular Net
may also be
designed and fabricated to replace the need for costly and complicated
analytical devices.
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[0030] Generally, the number of capture molecules incorporated into a 3-
dimensional
Molecular Net matrix is less than or equivalent to the number of capture
molecules conjugated in
a 2-dimensional manner to a surface using conventional approaches. Two-
dimensional capture
molecule-surface conjugates may rely on the use of a single linker type or may
rely on the
sequential use of 2 linkers to conjugate capture molecules to a solid surface.
During the
fabrication of a layer of a Molecular Net, multiple linker types are used
simultaneously to link
capture molecule to capture molecule of a new layer and the linked capture
molecules of a new
layer to a spacer or capture molecule of a previous layer. Molecular Nets may
be fabricated in
solution prior to placement on a solid surface. Pre-fabricated Molecular Nets
may be absorbed
or covalently linked to a solid surface. Molecular Nets may also be fabricated
directly onto a
solid surface, layer by layer. Said Molecular Nets may be placed on a solid
surface using non-
covalent (electrostatic, van der Waals, or other) or covalent methods. In some
instances,
polystyrene, polyurethane, polyethylene or treated surfaces such as poly-L-
lysine coated
surfaces, modified surfaces comprising -COOH, NHS, amine or other may be
purchased from
commercial sources (examples of vendors may include Thermo, Millipore, Luminex
and other)
and used as solid phase surfaces for Molecular Net placement. In other
instances, solid phase
surfaces may be pre-treated by chemicals such as acid to activate the surface
moieties and thus to
generate attachment points between the solid phase surface and reactive
moieties of a Molecular
Net. In some examples, a solid phase may be pre-treated with linker to
covalently link a solid
phase surface to a Molecular Net.
[0031] Design and fabrication of a Molecular Net for use on a solid phase
surface may
result in a covalently-linked multilayered three-dimensional matrix of capture
molecules secured
by covalent connectors within each layer. Design and fabrication may occur in
a sequential
manner where a first layer is fabricated and subsequent layers are fabricated
in a sequential
manner whereby each layer may be interconnected in a covalent manner to
enhance structural
integrity, topology, porosity and/or stability. Selection of individual
capture molecules, linkers
and spacers may be made to contribute to one or more property of a Molecular
Net. Properties
may comprise analyte specificity, thermal stability, layer thickness, pore
diameter, absorbance
spectra, emission spectra, solid phase compatibility or other.
[0032] The use of capture molecules and linker molecules and spacers of
known lengths
and widths may be used to generate various topology on the surface of the
Molecular Net.
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Topological features that may be imparted on a Molecular Net may include but
are not limited to
dimples, pocks, stipples, pores, mounds, branches, filaments, fibers,
fissures, raised segments or
other and may be arranged in a Molecular Net in a random, pseudorandom or
irregular manner.
[0033] Topological features of a Molecular Net may be generated through
the use of
capture molecules and linkers; capture molecules, linkers and spacers; or
linkers and spacers. In
some cases, capture molecules may be used to generate topological features of
a Molecular Net.
In these instances, capture molecules may be pre-linked to one another prior
to being
incorporated covalently into a Molecular Net layer. In some cases, linkers may
be selected based
on Angstrom length of the spacer arms. In some examples, spacers may be used
to connect a
first linker to a second linker to generate a long multi-functional linker. In
some cases, spacers
may be used to generate topological in a Molecular Net. Spacers may also be
pre-linked to one
another or to capture molecules prior to being incorporated into a Molecular
Net layer.
[0034] Molecular nets may be designed and fabricated to impart
characteristics such as
affinity, size exclusion, filtration, fluorescence, and other into each layer
of a Molecular Net.
Specific capture molecules, linker molecules and spacer molecules may be
selected based on
size, length, diameter, thickness, optical properties, chemical properties or
other for imparting
characteristics into a Molecular Net during the fabrication process.
[0035] Molecular Nets may be fabricated in a manner whereby one or more
capture
molecules may serve a structural role, may serve both a structural role and a
role in analyte
capture within the covalently-linked multilayered three-dimensional matrix.
Some examples of
capture molecules that may be used for structural and/or analyte capture roles
in a Molecular
Net.
[0036] The distance between capture molecules in each layer of a
Molecular Net may be
determined, in part, by the diameter, width and/or length of capture
molecules, linkers and
spacers used in the fabrication process for each layer, whereby the molar
relationship between
each linker-capture-spacer molecule may be similar or may be different and the
selection of said
molecules may be dependent on size and/or shape of the analyte to be captured,
the method used
to measure captured analyte and/or desired use.
[0037] Molecular Nets may be designed and fabricated in a manner whereby
each
capture molecule, linker and spacer component may have equivalent or non-
equivalent molar
ratios in a layer of said Molecular Net. Variance of molar ratios between said
components may
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be used from time to time to generate porosities or other topological features
within each layer.
Said porosities and topological features may have a range of diameters and may
have a range of
associated depths. Variance of molar ratios between Molecular Net components
may occur in a
single layer of Molecular Net or may occur in more than one layer of a
Molecular Net and is
dependent on the intended use of a Molecular Net.
TABLE 1. Examples of Molecular Net structural components with analyte capture
ability.
Examples of Molecular Net Approximate
Approximate
Structural/Capture Components Diameter (nm)
Length (nm)
IgG, IgE ¨9 16
IgM 37 37
IgA ¨9 ¨32
Streptavidin & recombinant variants ¨105 (tetrameric) N/A
Protein A & recombinant variants ¨3.2-5.3 N/A
Protein G & recombinant variants ¨3-5.4 N/A
MHC I 3.05 N/A
MHC II 2.99 N/A
TCR 3.34 N/A
CD28 2.75 N/A
TLR 4 2.62 N/A
B7x 4.52 N/A
Taq polymerase ¨6.49 N/A
poly(Arg9) peptide 1.43 N/A
HSP70 3.46 N/A
[0038] Some examples of analyte dimensions that may be considered during
the design
and fabrication process are provided in Table 2. Design and fabrication of
Molecular Net surface
chemistry, pore diameter, topology, layering or other may be based on analyte
shape; analyte
structure, analyte isoforms, analyte charge, analyte complex formation with
other molecules, and
other forms. Furthermore, Molecular Nets may be designed and fabricated to
bind and capture
said analyte or may be designed and fabricated to exclude said analyte.
Examples of analytes
and analyte sizes can be found in Table 2.
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TABLE 2. Examples of analytes and their dimensions.
Exemplary Analyte Approximate Diameter
Approximate Length
(um) (um)
E. coli 0.5 1-2
Klebsiella spp. 0.3-1 0.6-6
Pseudomonas spp. 0.6 3
Staphylococcus aureus 1 1
Staphylococcus aureus (cluster) >10 >10
Enterotoxin K ¨4.29 N/A
Peptidoglycan, gram negative ¨2-3, species dependent Highly species
dependent
bacteria
Outer membrane, gram negative ¨7, species dependent Species dependent
bacteria
IgG, IgE 0.009 0.016
IgA 0.009 0.032
IgM 0.037 0.037
B cell ¨ GO phase of cell cycle 4.5-5.5 N/A
B cell ¨ early G1 phase of cell 5.5-7 N/A
cycle
B cell ¨ late G1 and S phase of 7-10 N/A
cell cycle
B cell ¨ late S, G2 and M phases 10-12 N/A
of cell cycle
Monocyte ¨9-18 N/A
Macrophage 21, activation level N/A
dependent
Neutrophil 7.17-9.3, activation level N/A
dependent
IL6 monomer ¨4.11 N/A
IL6 multimer (variable) ¨6.16 N/A
IL 10 monomer ¨3.88 N/A
IL 10 multimer (variable) ¨7.7 N/A
microRNA-146 a ¨3-6 ¨7-9
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[0039] Molecular Nets comprising structural components and capture
components may
be arranged in the covalently linked 3-dimensional (3D) multilayered matrix
and may relate to
the capture of one or more analyte relating to one or more of the following
characteristics:
surface chemistry; analyte shape; analyte structure; analyte isoforms; analyte
charge; post-
translational modification; chemical modification; activity; or other.
[0040] Molecular Nets may comprise structural components that also act in
a manner
relating to the capture of analytes and may be arranged in the interconnected
3D multilayered
matrix of a Molecular Net by covalent linkers. A Molecular Net may also
comprise spacers to
interconnect said structure/capture molecules in a manner to maximize
structural reinforcement,
stability and/or specific analyte capture capability. Molecular Net examples
comprising capture
components/structural components, linkers and spacers are presented in Table
3.
[0041] Fabrication of the molecular Net is unique in that capture
molecules are secured
in a 3D matrix by covalent linker molecules. In numerous studies, Molecular
Nets have been
demonstrated to have improved thermal stability and extend shelf-life beyond
traditional capture
technologies.
TABLE 3. Examples of Molecular Nets and their Use.
Analyte to Capture Molecules to Methods of
Anticipated Use
Capture Use for Affinity Generating Size
Capture Exclusion
E. coli Antibodies against Covalently linked Diagnostics:
Food
surface antigens (e.g., antibodies ¨ IgG, safety, infectious
LPS, 0-antigen, pili, IgM, covalent disease, water
safety;
other); PNA probes linkers, spacers Molecular tools:
against chromosomal polymicrobial
and/or plasmid DNA sampling, microbiome
sampling, molecular
biology
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Klebsiella spp. Antibodies against Covalently linked Diagnostics:
Food
surface antigens (e.g., antibodies ¨ IgG, safety, infectious
LPS, other); PNA IgM, covalent disease, water
safety;
probes against linkers, spacers Molecular tools:
chromosomal and/or polymicrobial
plasmid DNA sampling, microbiome
sampling, molecular
biology
Pseudomonas spp. Antibodies against Covalently linked Diagnostics:
Food
surface antigens (e.g., antibodies ¨ IgG, safety, infectious
LPS, V antigen, other), IgM, covalent disease, water
safety;
excreted materials linkers, spacers Molecular tools:
(e.g., heat shock polymicrobial
proteins, alginate, sampling, microbiome
other); PNA probes sampling, molecular
against chromosomal biology
and/or plasmid DNA
Staphylococcus Antibodies against Covalently linked Diagnostics: Food
aureus surface antigens (e.g., antibodies ¨ IgG,
safety, infectious
protein A, IgM, covalent disease, water
safety;
peptidoglycan, other), linkers, spacers Molecular tools:
excreted materials polymicrobial
(e.g., heat shock sampling, microbiome
proteins, exotoxins, sampling, molecular
other); PNA probes biology
against chromosomal
and/or plasmid DNA
Staphylococcus Antibodies against Covalently linked Diagnostics: Food
aureus (cluster) surface antigens (e.g., antibodies ¨ IgG,
safety, infectious
protein A, IgM, covalent disease, water
safety;
peptidoglycan, other), linkers, longer Molecular tools:
excreted materials spacers polymicrobial
(e.g., heat shock sampling, microbiome
proteins, exotoxins, sampling, molecular
other); PNA probes biology
against chromosomal
and/or plasmid DNA
IgG, IgE Antibodies against Fc Covalently linked Diagnostics:
Immune
IgG or IgE; Antibodies antibodies, response profiling,
against Fab; antigens antigens, covalent vaccination,
antibody
linkers, spacers titering; Molecular
tools: immunologic
studies, pre-clinical
studies
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IgA Antibodies against Fe Covalently linked Diagnostics:
Immune
IgA; Antibodies antibodies, response profiling,
against Fab IgA; antigens, covalent vaccination,
antibody
antigens linkers, spacers titering; Molecular
tools: immunologic
studies, pre-clinical
studies
IgM Antibodies against Covalently linked Diagnostics:
Immune
IgM; Antibodies antibodies, response profiling,
against 5 IgM; antigens, covalent vaccination,
antibody
antigens linkers, spacers titering; Molecular
tools: immunologic
studies, pre-clinical
studies
B cell ¨ GO phase Antibodies against Covalently linked Diagnostics:
Immune
of cell cycle PAX5, CD19, CD20, antibodies ¨ IgG, response
profiling,
CD79a, others; IgM, covalent disease monitoring;
antigens; TCR:antigen; linkers, spacers, Molecular tools:
MHC I:antigen; MCH MHC:antigen immunologic studies,
II:antigen; cytokines complexes, pre-clinical studies
(e.g., IL10, IL6, TGFb, cytokines
other)
B cell ¨ early G1 Antibodies against Covalently linked Diagnostics:
Immune
phase of cell cycle PAX5, CD19, CD20, antibodies ¨ IgG,
response profiling,
CD79a, others; IgM, covalent disease monitoring;
antigens; TCR:antigen; linkers, spacers, Molecular tools:
MHC I:antigen; MCH MHC:antigen immunologic studies,
II:antigen; cytokines complexes, pre-clinical studies
(e.g., IL10, IL6, TGFb, cytokines
other)
B cell ¨ late G1 and Antibodies against Covalently linked Diagnostics:
Immune
S phase of cell PAX5, CD19, CD20, antibodies ¨ IgG, response
profiling,
cycle CD79a, others; IgM, covalent disease monitoring;
antigens; TCR:antigen; linkers, spacers, Molecular tools:
MHC I:antigen; MCH MHC:antigen immunologic studies,
II:antigen; cytokines complexes, pre-clinical studies
(e.g., IL10, IL6, TGFb, cytokines
other)
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B cell ¨ late S, G2 Antibodies against Covalently linked
Diagnostics: Immune
and M phases of PAX5, CD19, CD20, antibodies ¨ IgG, response
profiling,
cell cycle CD79a, others; IgM, covalent disease monitoring;
antigens; TCR:antigen; linkers, spacers, Molecular tools:
MHC I:antigen; MCH MHC:antigen immunologic studies,
II:antigen; cytokines complexes, pre-clinical studies
(e.g., IL10, IL6, TGFb, cytokines
other)
Macrophage Complement, Covalently linked Diagnostics:
Immune
antibodies against antibodies ¨ IgG, response
profiling,
mannose receptor Ab, IgM, covalent infectious disease
anti-Ly6C, or other; linkers, spacers, monitoring;
antibodies against Ml, MHC:antigen vaccination
M2a, M2b, M2c complexes, monitoring; chronic
markers; TLR agonists; cytokines, TLR inflammatory disease
cytokines; DAMPs; agonists, DAMPs, monitoring;
Molecular
PAMPs; alarmins PAMPs, alarmins tools: immunologic
studies, pre-clinical
studies
Neutrophil Complement, Covalently linked Diagnostics:
Immune
antibodies against antibodies ¨ IgG, response
profiling,
CD15, Ly6G or other; IgM, covalent infectious disease
antibodies against linkers, spacers, monitoring;
neutrophil markers; MHC:antigen vaccination
TLR agonists; complexes, monitoring; chronic
cytokines; DAMPs; cytokines, TLR inflammatory disease
PAMPs; alarmins agonists, DAMPs, monitoring;
Molecular
PAMPs, alarmins tools: immunologic
studies, pre-clinical
studies
Cytokines Antibodies against one Covalently linked Diagnostics:
Immune
or more epitope of antibodies ¨ IgG, response
profiling,
cytokine; cytokine IgM, covalent infectious disease
binding domain of linkers, spacers, monitoring;
cytokine receptor; PNA vaccination
probes against cytokine monitoring; chronic
gene and/or cytokine inflammatory disease
mRNA; or other monitoring; Molecular
tools: immunologic
studies, pre-clinical
studies
[0042] In some examples, the solid phase may be particles ranging from
about 2 nm in
diameter to about 200 mm in diameter and Molecular Nets may be attached to the
surface of said
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particle. Particles may comprise polystyrene, polyethylene, silica, composite,
nylon, PVDF,
nitrocellulose, cellulosic, carbon, or other may be magnetic, paramagnetic,
fluorescent, barcoded
or other.
[0043] Molecular Nets may be absorbed or covalently linked to the surface
of a particle
in manner to generate pseudorandom or ordered porosities in a single layer of
said Molecular Net
or throughout. In its most basic form, a particle may be initially coated with
a layer of Molecular
Net, which may be connected to a second layer, which may be connected to a
third layer.
Molecular Net layers may comprise the same capture molecules at the same or at
different
concentrations in each layer. Molecular Net particles may also comprise
different capture
molecules in each layer and may be fabricated in a manner to incorporate the
same or different
concentrations of capture molecules compared to previous layers.
[0044] In some examples, Molecular Nets may be attached to a particle
surface in a
manner to generate an asymmetric particle having a pre-determined polarity.
Such a particle
may be designed and fabricated with an initial layer comprising structural
molecules with a large
diameter, width and/or length and may be linked to a particle in an asymmetric
manner to
generate a polarity. A second layer may be linked to a first layer and a third
layer may be
connected to a second layer and so on. The number of layers in a Molecular Net
particle may
vary depending on use.
[0045] In some examples, Molecular Nets may be attached to a segment of a
particle to
generate an asymmetric particle having a pre-determined polarity. Such a
particle is constructed
whereby the initial layer is coated onto a segment of a particle and whereby a
second layer is
connected to the initial layer onto the same segment of said particle, and
whereby a third layer is
connected to the second layer onto the same segment of said particle, and
whereby a fourth layer
is connected to the third layer onto the same segment of said particle.
[0046] In some examples, Molecular Nets may be passively absorbed to a
nonfunctionalized particle surface. In other examples, particle surfaces may
be functionalized
and may require activation prior to attachment. In other examples, particle
surfaces may be
activated prior to functionalization, at which time a Molecular Net may be
attached. Yet in other
examples, Molecular Nets may be constructed directly on the particle surface.
Attachment of
Molecular Nets to a particle may change the physical and/or chemical features
of said particle. In
some examples, Molecular Nets may comprise pseudorandom topological features
placed on the
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surface of a particle. In some examples, Molecular Net particles may comprise
topological
features, the topological features comprising capture molecules and linkers
and may also
comprise spacers. Examples of various topological features may include
appendages, spikes,
plateaus, planes, mounds, fissures, pellicles, stipples, channels, pores and
other and may be
comprised of capture components directly linked within and/or linked to one or
more layer of a
Molecular Net.
[0047] Other examples of topological features may comprise be pockets,
pillars, bumps,
branches, projections, ridges, clefts, trellis-like structures, flakes,
pellets, spheres, or others.
Topological features may be pre-formed in solution and linked to the Molecular
Net or may be
formed at the time each layer is constructed.
[0048] Molecular Nets on particles may comprise heterogeneous capture
molecules
within one or more layer of a Molecular Net. Benefits of a heterogeneous
design may relate to
the capture of a plurality of analytes having a plurality of surface
chemistries on a single particle.
Heterogeneous capture molecules incorporated into a Molecular Net during
fabrication may be
randomly distributed throughout each layer; may be stratified throughout each
layer; or other,
depending on use.
[0049] Molecular Nets may be attached to particles to increase surface
area of said
particle. Molecular Nets may also be used to increase particle diameter.
Topological features of
a Molecular Net on a particle may relate to an increased particle size in
addition to analyte
capture capacity.
[0050] In some examples, a first layer of Molecular Net may be attached
to a particle
surface to modify the physical and/or chemical properties of particle. In many
commercial
particles, "bead effects" or "surface effects" can hamper results and are
still not well understood.
Traditional conjugation techniques that result in 2D conjugates and 2D
conjugated surfaces often
suffer from surface effects. Molecular Nets may be used to minimize or
neutralize bead effects
to minimize non-specific binding to a bead surface, bead autofluorescence,
bead interference
with in an assay or other. In some examples, Molecular Net particles may
impart increased
analyte binding capacity and may also impart blockade of non-specific binding
of undesired
analytes to increase the signal-to-noise ratio in an assay, yield and purity
of purified analyte or
other.
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[0051] In some examples In yet another aspect, the invention features
molecular Net on
particle containing more than one layer, wherein each layer contains capture
molecules directed
against analyte, wherein each layer contains distinct capture molecules
directed against distinct
analyte, wherein different layers can be directed against different analytes
to enable the capture
of analyte or a plurality of analytes.
[0052] In yet another aspect, a Molecular Net placed on a solid phase
surface may be
used to increase the purity of one or more analyte recovered from a sample.
Molecular Net-
coated surfaces may reduce non-specific binding of undesired analyte compared
to commercial
2D functionalized surfaces.
[0053] In some examples, Molecular Nets placed on particles may
significantly increase
analyte capture capacity of a particle. Additional layering of a Molecular Net
may further
increase the number of bound analyte per particle and may be used to enhance
recovery or yield
of analyte from a sample and may be used to deplete one or more analyte from a
sample.
Advantages of using Molecular Nets
[0054] Molecular and cellular testing strategies employ the use of single-
plex or multi-
plex immunoassays, PCR assays, next-generation sequencing techniques or other
to identify the
presence of or to measure the amount of one or more analyte in a sample.
[0055] In multiplexed assays, reactions may be separated spatially or may
be combined
into a single testing reaction and may employ solid phases comprising unique
identifiers to
provide information. Some examples of unique identifiers may comprise the use
of different
barcodes, different fluorescence emissions, different chemistries, different
ordered nucleotide
tags, or other.
[0056] Solid phases may be used in single-plex and multi-plex assays may
rely on the
specific binding of target analyte to produce a measurable signal or a
measurable change in
signal and may be used in a direct assay or may be used in an indirect assay.
Measurable signals
may be generated from a positive test and may comprise electric, thermal,
magnetic, optical,
vibrational, isotopic, or other measurable characteristic.
[0057] Many of the difficulties in achieving sensitive and reproducible
measurements
using current strategies result in high non-specific binding, lower
sensitivity, low signal-to-noise
and thereby require upstream sample processing steps to remove as many non-
specific
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components from a sample, coupled with the use of highly sensitive reader
technologies and
complex algorithms which may be required in order to determine real signal
from the noise,
which make them difficult to translate to truly real-time, easy-to-use
molecular diagnostics and
analyte measurement tools.
[0058] Molecular Nets may be used in place of current commercial
approaches and may
generate specific and sensitive analyte capture, detection and measurement
from a sample.
Examples of results obtained from the use of Molecular Nets in place of
current approaches for
analyte capture are presented in Figures 1-6. Improvements in assay
sensitivity, minimum levels
of analyte detection, and other features may be obtained through the use of a
Molecular Net in
place of current 2D approaches for analyte capture and measurement. Reduction
in background
noise may be obtained through the use of a Molecular Net in place of current
2D approaches and
may be used to improve analyte purification, analyte purity, and assay
sensitivity.
[0059] Advantages of a Molecular Net are presented in Table 4 and may
include: the
rapid capture of one, several or a plurality of molecular and cellular
analytes in a raw sample;
ability to generate sensitive and specific signals when used in a test
involving indirect and direct
detection methods; ability to generate a signal having enhanced fluorescent
intensity; ability to
concentrate bound analyte; ability to spatially separate bound analyte in a
manner that reduces
stearic hindrance between analytes and/or between detection molecules;
enhanced stability;
reduced background and others.
TABLE 4. Demonstrated Advantages of Molecular Nets
Demonstrated Advantages Anticipated Impact
Finger stick by lancet (-50 uL) Displaces the need for venipuncture
No sample prep ¨ compatible with raw, Displaces the need for sample
processing ¨ both
unprocessed sample centrifugation & serum isolation (saves
time)
Point-of-use testing Displaces the need and cost of sample
transport,
with ultimate potential for testing at home or in
field settings
Portability, no complex capital equipment Displaces the need and cost for off-
site CLIA
labs
Provides immediate answers (<30 mins) Enables point-of-care treatment and
patient
monitoring
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Multiplexing (multi-analyte analysis) Delivers more robust answer and
eliminates the
cost of having to run separate tests per sample
Simple test procedure Displaces the need for high-complexity
testing
(Western blot, Luminex, bead arrays, and PCR)
Capable of producing simple actionable Enables health care provider (and/or
ultimately
readouts (Binary-No/Yes; Semi- the patient) to make decision sooner;
flexible data
quantitative-Low, Mid, High; Fully output enables numerous applications
quantitative)
Stability of test (enzyme-free) = longer Increases shelf life, & reduces
costs associated
shelf life with storage issues
Cost effective Disposable cassette with the potential for
multiple-tests-in-1
High signal, very low noise (demonstrated Significantly more sensitive than
current
femtogram range in multiple test types) immunoassays
Molecular Nets and their Use
[0060] Molecular Nets may be used in applications where analyte binding
efficiency,
analyte binding kinetics, analyte binding capacity, analyte detection, analyte
measurement,
analyte enrichment, analyte purification and analyte delivery may be
important. Molecular Nets
may be used in fluid phase or may be attached to a solid phase.
[0061] Molecular Nets may be attached through absorption or covalent
processes on a
receptive surface. Examples of solid phases include but are not limited to
nanotubes, metals,
particles, microtiter plates, slides, cassettes, probes, lateral flow tests,
stents, catheters, valves,
blood tubes, needles, solid phase devices or other. Examples of chemistries of
various solid
phases that may be compatible for Molecular Net attachment include but are not
limited to
plastics, other polymer, thin film, colloidal metals, silica, carbon nanotube,
protein,
carbohydrate, lipid, nucleic acid, cell, tissue or other.
[0062] Molecular Nets may be attached to a solid phase device surface to
capture, purify
or deplete one or more analyte from a sample. An example of using a Molecular
Net for analyte
capture and/or purification from a sample is presented in Figure 1. Some other
examples of
Molecular Nets that may be used to capture and purify analyte from a sample
are: Protein A,
Protein G or Protein L Net-coated microspheres for immunoglobulin capture;
Streptavidin Net-
coated microspheres for biotin capture; TNF Net-coated microspheres for anti-
TNF biologic
capture; IL6 Net-coated microspheres for anti-1L6 biologic capture; IgM Net-
coated
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microspheres for RNA virus capture; Ig Fe Net-coated microspheres for
complement capture;
antigen Net-coated microspheres for antigen-specific immunoglobulin; antigen-
specific immune
cell capture; and others. Molecular Nets may be used in chromatography methods
for the
capture and purification of one or more analyte from a sample.
[0063] Molecular Nets may be used to capture analyte from a sample for
downstream
analyte measurement by an independent method, referred to herein as sample
prep. Examples of
independent methods may include mass spectrometry, immunoassay, PCR, next-
generation
sequencing, qRT-PCR, digital PCR, microscopy, fluorescence, flow cytometry,
bead cytometry,
or other.
[0064] In another aspect, the invention improves signal-to-noise ratios
when used in an
assay.
[0065] Molecular Nets may be attached to a solid phase device surface to
measure the
presence, absence, modification or concentration of one or more analyte.
Examples of using
Molecular Nets for analyte detection and/or measurement are presented in
Figures 3 and 4. In
some other examples, Molecular Nets may be used to simultaneous detect and
measure 2 or more
specific analytes in a direct or indirect manner. Indirect capture by a
Molecular Net may relate
to the capture of a primary analyte by a specific capture molecule of a
Molecular Net that may
enable the detection of one or more related secondary associated with the
captured primary
analyte. Molecular Nets may be used as discovery tools capture primary
analytes from a sample
and enables the identification, detection or measurement of secondary analytes
that are captured-
by-association. Molecular Nets may be used in this manner for drug discovery,
pathway
mapping, and in proteomics, transcriptomics, glycomics, lipidomics,
metabolomics, functional
genomics, foodomics, nutrition, pharmacology, toxicology and others.
[0066] In some examples, Molecular Nets may be used to detect drug
resistance in a cell.
Cells may be tumor cells, immune cells, microbial cells or other cells.
Molecular Nets for these
applications may comprise capture molecules directed against one or more
unique features of a
cell type. Molecular Nets may additionally be fabricated in a manner to impart
surface topology
relating to the capture of in tact cells.
[0067] In other examples, Molecular Nets may be fabricated and used in:
immune cell
reactivity measurement; immune response monitoring; immune response
classification;
immunoglobulin titering; biotinylated molecule capture; multiplex
immunoassays; singleplex
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immunoassays; next-generation sequencing reactions; PCR; microbiome capture;
microbiome
discovery; mRNA and encoded protein measurement; SNP (single nucleotide
polymorphisms)
mapping; SNP detection; disease marker sample preparation; miRNA capture
and/or
measurement; post-translation modification discovery and/or capture and/or
measurement;
kinase activity measurement; or other.
[0068] Molecular Nets may have measurable characteristics imparted during
the
fabrication process and may be used in a direct or indirect manner as a
sensor. A measurable
change in one or more characteristic of a Molecular Net sensor may be detected
using
commercial approaches employing the use of optical sensing, electrochemical
sensing,
electromagnetic sensing, electrical impedence, or other. In one example, a
Molecular Net sensor
may be used to capture and bind an analyte. Analyte binding may result in a
measurable change
in a characteristic of a Molecular Net sensor. A binding event or modifying
event pertaining to
the Molecular Net sensor may be monitored over a period of time, and the
changes in Molecular
Net sensor characteristics may be detected, relayed and collected by a device.
Other examples of
using a Molecular Net as a sensor may include an analyte binding event,
enzymatic reaction,
analyte modification event, cell differentiation, cell-cell interaction, or
other.
[0069] Examples of measurable characteristics include but are not limited
to: physical
shape, height, density, fluorescence intensity, wavelength shift (FRET or
FRAP), vibrational
frequency, absorbance, flexibility, refractiveness, conductance, impedence,
resistance, melting
temperature, denaturation temperature, freezing temperature, and other.
[0070] Measuring devices that may be compatible for use with a Molecular
Net sensor
may comprise: photonic multichannel analyzers, spectrometers, magnetic
resonance imagers,
magnetic field detectors, optical fibers, glass pipettes, circuits,
fluorometers, spectroscopic
analyzers, flow cytometers, CCD cameras, microscopes, acoustic chambers,
microphones,
luminometers, and other. The measuring devices may be used to measure changes
in: thickness,
topology, charge, insulation, capacitance, voltage, color, acoustics,
vibration, magnetism,
enzymatic activity or other characteristics of a Molecular Net used as a
sensor.
[0071] Additionally, Molecular Nets may be used in flexible circuits,
whereby the
capture molecules and/or structural molecules may be connected to conductive
molecules.
Molecular Net circuits may be used in single-sided flexible circuits, double
access (back bared
flex circuits), sculptured flex circuits, double-sided flex circuits,
multilayered flex circuits, ridge
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flex circuits, ridge-flex boards, polymer thick film flex circuits or other.
Most flexible circuits
are passive wiring structures that are used to interconnect electronic
components such as
integrated circuits, resistor, capacitors and the like, however some are used
only for making
interconnections between other electronic assemblies either directly or by
means of connectors.
Molecular Nets for use in circuits or for use as a component of a circuit may
be comprised of
synthetic components or may be comprised of biochemical capture molecules
and/or cells and
may be fabricated in a manner to be used in a flexible circuit. Molecular Net
circuits may also
be used as a sensor.
[0072] In some examples, Molecular Net circuits may have specific
electrochemical
properties and may be used to monitor various parameters such as pH, current,
voltage,
impedence, or other in an electrochemical/electrolyte cell. Binding events and
modifying events
that may occur to Molecular Net may be measurable and may be reflected by a
change in the
conductance, current, or voltage. More specifically, the introduction of a
sample containing an
analyte that has specific binding affinity for, or is reactive towards, a
component in a Molecular
Net circuit may be monitored by a change in the electrochemical properties of
the Molecular Net
and/or the surrounding environment.
[0073] Examples of binding events on a Molecular Net used in a circuit
may include:
antibody-antigen interaction, nucleic acid-nucleic acid interaction, enzyme-
substrate interaction,
drug-target interaction, enzyme-co-factor interaction, ligand-cell
interaction, or any other
specific surface-chemistry-driven non-covalent interaction. Analyte capture by
a Molecular Net
may be determined by a change in pH, current or voltage an
electrochemical/electrolyte cell.
Measurement of a change in Molecular Net characteristics may also result from
one or more, or
an accumulation of modifying events to one or more component in a Molecular
Net or to a
capture analyte. Examples of modifying events may include: enzyme cleavage;
post-
translational modification (such as phosphorylation, sulfonation,
glycosylation, methylation, or
other); removal of a post-translational modification (such as de-
phosphorylation); or other
similar modification. Modifying events may be determined by a change in pH,
current or
voltage in the electrochemical/electrolyte cell resulting from a change in
Molecular Net
characteristics or in the surrounding buffer system.
[0074] Methods to determine changes in electrochemical properties of a
Molecular Net
used in a circuit may include the use of scanning ion current microscopy,
nanofluidic diodes,
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nanopores or nanochannels that display voltage-gated ion current, ion
nanogating, nanopore-
based sensing platforms and other methods for measuring the flow, or changes
in flow of
electrical charge through a medium. More specifically, the inherent
sensitivity of many solid-
state nanopore sensors is the selective permeability of electrolytes, or ion
current, when a bias is
applied across the nanopore. Molecular Nets may be coated onto the surface of
a nanopore and
the change in current, voltage, and impedance can be monitored.
[0075] Molecular Net can also be coated onto the surface of a carbon
nanotube and
whereby the molecular Net can be constructed in a manner to generate size
exclusion and affinity
requirements for analyte sensing.
[0076] Bio-Layer Interferometry (BLI) is a label-free technology for
measuring
biomolecular interactions. It is an optical analytical technique that analyzes
the interference
pattern of white light reflected from two surfaces: a layer of immobilized
molecular Net on the
biosensor tip, and an internal reference layer. Any change in the number of
molecules bound to
the biosensor tip causes a shift in the interference pattern that can be
measured in real-time. The
binding between a ligand immobilized on the molecular Net-coated biosensor tip
and an analyte
in solution produces an increase in optical thickness at the biosensor tip,
which results in a
wavelength shift, AX, which is a direct measure of the change in thickness of
the biological layer.
Interactions may be measured in real time, providing the ability to monitor
binding specificity,
rates of association and dissociation, or concentration, with precision and
accuracy. Only
molecules binding to or dissociating from the Molecular Net biosensor may
shift the interference
pattern and generate a response profile. Unbound molecules, may change the
refractive index of
the surrounding medium, or may change flow rate but will not affect the
interference pattern.
This is a unique characteristic of BLI and extends its capability to perform
in crude samples used
in applications for analyte - capture molecule binding, quantitation,
affinity, and kinetics.
[0077] Molecular Net particles may also be used to deliver an active
agent. Active
agents may be pre-loaded onto capture molecules located in one or more layer
of a Molecular
Net. Active agents may comprise: drugs, therapeutics, toxins, viruses,
allergens, vaccine
components, antigens, immune modulators, surfactants, microbes,
oligonucleotides, nutrients, or
other. Molecular Net particles may be used in drug or therapeutic delivery,
vaccine delivery, in
biofermentation or other. Molecular Nets may comprise one or more targeting
agent on a
surface-exposed layer to facilitate specificity in targeting said Molecular
Net particle to a
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specific cell type, tissue type, organ type or other. Targeting agents may be
capture molecules of
a Molecular Net. Targeting agents may comprise: antibodies, receptors,
ligands, anti-ligands, or
other. Targeting agents in a Molecular Net may be covalently linked to capture
molecules,
linkers, and spacers in a surface-exposed layer. Targeting agents may also
contribute to the
topological features of a Molecular Net.
EXAMPLES
Example 1. Comparison of Conventional 2D and 3D Molecular Net
microparticles for analyte purification.
[0078] Molecular Nets comprised of monomeric protein G and linked protein
G and
crosslinkers BS3, EMCS, EGS, BMPH and others were used in fabrication.
Molecular Net
fabrication occurred in real-time on 0.8-10 um magnetic polystyrene
microparticle and 45 um
nitrocellulose microparticle surfaces. In some examples, the capture molecule,
protein G was
used as the only source of structural support. In some examples, pre-linked
protein G and
monomeric protein G were mixed to serve as additional structural support for
fabrication of some
layers of the Molecular Net. Yet in some other examples, a first layer of
Molecular Net
comprised protein G and Ig Fc region to serve as structural support for
fabrication of additional
layers of the Molecular Net. In some examples, a protein G Molecular Net
comprised 2 layers
and in other examples, a protein G Molecular Net comprised 3 layers. The last
layer of the
Molecular Net comprised topological features to enhance analyte (in this case
IgG) binding and
recovery from a sample. Figure 1 is an example of data obtained using protein
G Molecular Net
microparticles in comparison to commercial protein G microparticles. Briefly,
IgG-Alexa 647
was spiked into human serum (1 ug/tube). Uncoated microparticles, commercial
protein G
microparticles and protein G Molecular Net microparticles were incubated with
spiked sample
for 15-60 min at RT (at 100,000 particles (uncoated control), 100,000
particles (commercial) and
25,000 particles (Molecular Net). Particles were isolated from samples using
magnetic
separation and were washed 3x in PBST. Particles were resuspended in 2xLSB,
boiled and
loaded onto an SDS-PAGE. Recovered IgG was measured by Coomassie-stained band
densitometry compared to input control. Depicted in Figure 1 is the percent
recovery of input for
each purification type. Use of an optimized Molecular Net can reduce
background noise in an
assay and increase a visible signal.
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Example 2. Effectiveness of Conventional 2D Conjugates for analyte detection.
[0079] Traditional approaches to covalently link capture antibody to a
surface is a 2-
dimensional approach (X and Y planes). as there are no additional layers added
onto the surface
of the linked antibodies on a surface. Traditional methods used to covalently
link capture
antibody to a surface involve a single type of linker, for example EDC, NHS,
sulfo-NHS or
other. Occasionally, a second linker is used to secure the capture antibody to
a surface, but
involves removal of the first linker and does not add additional height or
layering to the
antibody-conjugated surface. In an example, per manufacturer instructions,
anti-human
neuroserpin antibody was coupled to Luminex particles (bead region #54) by
linker. Particles
were then quenched, blocked, and washed prior to use. Particles were incubated
for 15 min in
pre-cleared serum + neuroserpin at a concentration range of (0-1 ng/mL). Bound
particles were
washed and incubated with biotin-anti-Neuroserpin (10 ng/mL) for 15 min.
Neuroserpin
detection was visualized by avidin-PE (30 ng/mL) for 15 min. Washed particles
were then
analyzed on a Luminex 100, collecting 100 particles per sample. Presented in
Figure 2 is the
median fluorescence intensity (Fl) at each dilution above background
fluorescence intensity.
FIG. 3. Effectiveness of Molecular Net microparticles in measuring analyte.
[0080] Molecular Net comprised of identical anti-human neuroserpin
antibody (as Figure
2) and linkers Sulfo-NHS, EMCS, EGS, BMPH and others was fabricated to provide
a 3-
dimensional multi-layered (X, Y, and Z planes) matrix. The Molecular Nets were
then
covalently linked to Luminex microparticles (bead region #54). Assay
performance with the 4-
layered Molecular Nets are presented in Figure 3. Improved assay MFI was
observed using a 3-
dimensional multi-layered Molecular Net.
FIG. 4. Effectiveness of a Molecular Net with topology in measuring analyte.
[0081] Molecular Net comprised of identical anti-human neuroserpin
antibody (as
Figures 2 and 3) and linkers Sulfo-NHS, EMCS, EGS, BMPH and others was
fabricated to
provide a 3-dimensional multi-layered (X, Y, and Z planes) matrix. The
Molecular Nets were
then covalently linked to Luminex microparticles (bead region #54). Assay
performance with
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the 5-layered Molecular Nets are presented in Figure 4. Improved assay MFI was
observed
using a 3-dimensional multi-layered Molecular Net with enhanced topology in
the outer layers.
FIG. 5. Comparison of traditionally conjugated microparticles
and Molecular Net microparticles in an ELISA.
[0082] Molecular Nets comprised of monoclonal antibody directed against
human-TauF
and crosslinkers Sulfo-NHS, EMCS, EGS, BMPH and others were used in
fabrication.
Molecular Net fabrication occurred in real-time on 0.5, 6.3 and 10 um magnetic
microparticle
surfaces. In some examples, the capture molecule, anti-Tau mAb, was used as
the only source of
structural support. In some examples, the spacer, albumin, was mixed with the
anti-Tau mAb in a
first layer at a 1.5:1.0 Molar ratio (albumin:anti-Tau Ab) to serve as
additional structural support
for fabrication of the first layer. In some examples, a second capture
molecule, human tubulin
was used and provided both structural support and capture roles within a
Molecular Net. Figure 5
is an example of data obtained using an anti-Tau Molecular Net (LV.P6 Cap-
TECH) in
comparison to a commercial Tau microparticle (Partner LV) ELISA (identical
assay conditions,
identical antibody pair, etc.). Figure 5 is an example of using a Molecular
Net to reduce
background noise in an assay and increase a visible signal in an ELISA.
FIG. 6. Comparison of traditionally conjugated microparticles
and Molecular Net microparticles using Luminex.
[0083] Molecular Net comprised of monoclonal antibody directed against
human-thyroid
stimulating hormone and crosslinkers EDC, BS(PEG)9, EMCS, EGS, BMPH and others
were
used in fabrication. Molecular Net fabrication occurred in real-time on
Luminex magnetic
microparticle surfaces. In some examples, the capture molecule, anti-TSH mAb,
was used as the
only source of structural support. In some examples, the spacers, PEG, heat-
denatured lysozyme
and others were mixed with the anti-TSH mAb in a first layer at a 1.0:2.0
Molar ratio
(spacer:anti-TSH Ab) to serve as additional structural support for fabrication
of the first layer. In
some examples, an anti-TSH Molecular Net comprised 4 layers and in other
examples, an anti-
TSH Molecular Net comprised 6 layers with the last layer comprising
topological features to
enhance analyte binding and performance in a Luminex. Figure 6A is an example
of using a
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Molecular Net to increase the overall MFI in a Luminex assay. Figure 6B is
exemplary data
obtained in a Luminex assay to increase the minimum levels of detection in a
Luminex assay.
FIG. 7. Exemplary Molecular Nets on particles.
[0084] Figure 7 depicts some examples in which Molecular Nets may be
placed onto a
particle surface. In some examples, a Molecular Net is placed on a particle
surface (Fig. 7A,
1001) in a circumferential manner whereby a Molecular Net having X, Y and Z
spatial
orientation may be fairly symmetrical and where each layer (examples of 3
layers, 1002, 1003
and 1004) adds to the Z plane of the particle. In some examples, Molecular
Nets may be placed
onto a particle surface (Fig. 7B, 1005) in an asymmetrical manner whereby a
Molecular Net
having X, Y and Z spatial orientation may be placed onto the surface of a
particle in a manner to
generate a polarity of the particle and where each layer (examples of 3
layers, 1006, 1007 and
1008) adds to the Z plane of the particle in a layer-dependent manner (for
example, some layers
may have less height and other layers may have more height). In other
examples, Molecular
Nets may be placed onto a portion of particle surface (Fig. 7C, 1009) in an
asymmetrical manner
whereby a Molecular Net having X, Y and Z spatial orientation may be placed
onto the surface
of a particle in a manner to generate a polarity of the particle and where
each layer (examples of
3 layers, 1010, 1011 and 1012) adds to the Z plane of the particle in a layer-
dependent manner
(for example, some layers may have specificity for an analyte and other layers
may have
specificity for other analytes).
FIG. 8. Exemplary Molecular Net topological features on particles.
[0085] Figure 8 depicts some examples in which Molecular Nets may be
placed onto a
particle surface. In some examples, a Molecular Net is placed on a particle
surface (Fig. 8A,
2001) in a circumferential manner whereby a Molecular Net having X, Y and Z
spatial
orientation may be fairly symmetrical in one layer (2002) and where each layer
(examples of 3
layers, 2002, 2003 and 2004) adds to the Z plane of the particle in differing
and asymmetric
ways (for example, topology is generated). In some examples, Molecular Nets
may be placed
onto a particle surface (Fig. 8B, 2005) in an asymmetrical manner whereby a
Molecular Net
having X, Y and Z spatial orientation may be placed onto the surface of a
particle in a manner to
generate structural features (2008) throughout layers 1 (2006), 2 (2007) and 3
(2008) of the
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particle and where each layer adds to the Z plane of the particle in a layer-
dependent manner (for
example, some layers may have less height and other layers may have more
height).
Additionally, the structural elements in each layer may also serve an analyte
capture role in a
Molecular Net. In other examples, Molecular Nets may be placed onto a portion
of particle
surface (Fig. 8C, 1009) in an asymmetrical manner whereby a Molecular Net
having X, Y and Z
spatial orientation may be placed onto the surface of a particle in a manner
to generate a polarity
of the particle and where each layer (examples of 4 layers, 2010, 2011, 2012
and 2013) adds to
the Z plane of the particle in a layer-dependent manner and whereby each layer
may serve both
structural and analyte capture roles. For example, some layers may have
specificity for an
analyte based on size (e.g., Fig. 8C, 2010) and outer layers (e.g., Fig. 8C,
2013) may have
specificity for analyte of a larger size.
FIG. 9. Exemplary Molecular Nets for analyte delivery.
[0086] Figure 9 depicts some examples in which Molecular Nets may be
placed onto a
particle surface for use in analyte capture or targeted analyte delivery. In
some examples, a
Molecular Net is placed on a particle surface (Fig. 9A, 3001) in a
circumferential manner
whereby a Molecular Net having X, Y and Z spatial orientation may be fairly
symmetrical in one
layer (3002) and where each layer (examples of 3 layers, 3002, 3003 and 3004)
adds to the Z
plane of the particle in differing and asymmetric ways (for example, topology
is generated). In
some examples, analyte cargo (3003) may be pre-loaded onto capture molecules
in one or more
layer of a Molecular Net. In outer layers, different capture molecules may be
linked into a
Molecular Net to generate a topology and/or an affinity for a different target
analyte. In some
examples, a pre-loaded analyte may comprise a drug, a therapeutic, siRNA,
miRNA, dsRNA,
virus, toxin, immunogen or other. Pre-loaded cargo may be non-covalently
associated with one
or more type of capture molecule in a layer of a Molecular Net. In some
examples, different
capture molecules (3004) of a Molecular Net may be arranged in the outer
layers of a Molecular
Net and may serve topological and analyte capture roles. The different capture
molecules may
have specificity for one or more different analyte, the analyte of which may
comprise an
antibody, an anti-ligand, a ligand, a receptor, an antigen or other and may
serve one or more
structural and/or affinity and/or targeting role.
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[0087] In some examples, Molecular Nets may be placed onto a particle
surface (Fig. 9B,
3005) in a manner whereby analyte cargo (3006) may be pre-loaded in all layers
of a Molecular
Net. In some layers, capture molecules may be used to generate topological
features (3008) that
serve particle-targeting roles. In some examples, outer layers of a Molecular
Net particle may
target said particle to a specific cell, tissue, organ or other.
FIG. 10. Molecular Nets for Analyte Purification from a Sample
[0088] Molecular Nets may be used in sample purification processes
(example is
provided in Figure 10). In some examples, Molecular Net designed and
fabricated to deplete one
or more analyte may be used to treat a sample for analyte depletion. Exemplary
methods may
include incubation of Molecular Net with sample for about 15 minutes to about
24 hours in a
batch slurry or in a chromatography column. Sample supernatant or flowthrough
may be
collected depending on preferred method. Molecular Nets may be collected and
analyzed using
various methods for the presence and amount of captured analyte. Molecular Net-
treated sample
may be collected and analyzed using various methods to determine the residual
presence of
analyte in the sample or may be analyzed for other analytes in the sample.
FIG. 11. Molecular Nets for Analyte Detection & Measurement from a Sample
[0089] Molecular Nets may be used in an analyte measurement tool or a
diagnostic tool
(example is provided in Figure 11). In some examples, Molecular Net designed
and fabricated to
capture one or more analyte may be used to treat a sample for analyte
detection and
measurement. Exemplary methods may include incubation of Molecular Net with
sample for
about 15 minutes to about 2 hours in a batch slurry, cassette, slide,
microtiter plate or other.
Sample supernatant or flowthrough may be collected depending on preferred
method. Molecular
Nets may be collected and analyzed using various methods for the presence and
amount of
captured analyte(s). Molecular Net-treated sample may also be collected and
analyzed using
various methods to measure other analytes. Methods for measuring changes in
Molecular Net
characteristics may include optical, electrophoretic, electrical, magnetic,
chemical, thermal or
other.
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[0090] While the present invention has been described with reference to
the specific
embodiments thereof, it should be understood by those skilled in the art that
various changes can
be made and equivalents can be substituted without departing from the scope of
the invention. In
addition, many modifications can be made to adapt a particular situation,
material, composition
of matter, process, process step or steps, to achieve the benefits provided by
the present invention
without departing from the scope of the present invention. All such
modification are intended to
be within the scope of the claims appended hereto.
[0091] All publications and patent documents cited herein are
incorporated herein by
reference as if each such publication or document was specifically and
individually indicated to
be incorporated herein by reference. Citation of publications and patent
documents is not
intended as an indication that any such document is pertinent prior art, nor
does it constitute any
admission as to the contents or date of the same.
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