U.S. patent application number 14/775635 was filed with the patent office on 2016-01-28 for composite nanoparticle structures for chemical and biological sensing.
The applicant listed for this patent is THE TRUSTEES OF PRINCETON UNIVERSITY. Invention is credited to Stephen Y. Chou, Wei Ding.
Application Number | 20160025634 14/775635 |
Document ID | / |
Family ID | 51523990 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160025634 |
Kind Code |
A1 |
Chou; Stephen Y. ; et
al. |
January 28, 2016 |
Composite Nanoparticle Structures for Chemical and Biological
Sensing
Abstract
Described herein is a nanoparticle that enhances the interaction
of the nanoparticle and/or a molecule/material deposited on the
surface of the nanoparticle with light, comprising a pair of
stacked metallic disks separated by a non-metallic spacer, wherein:
(a) the dimensions of the disks and spacer are smaller than the
wavelength of the light; and (b) the nanoparticle enhance the light
interaction at least three times greater than that an individual
metallic disk. Methods for making the nanoparticle and methods for
using the nanoparticle in a variety of assays are also
provided.
Inventors: |
Chou; Stephen Y.;
(Princeton, NJ) ; Ding; Wei; (Princeton,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF PRINCETON UNIVERSITY |
Princeton |
NJ |
US |
|
|
Family ID: |
51523990 |
Appl. No.: |
14/775635 |
Filed: |
March 18, 2014 |
PCT Filed: |
March 18, 2014 |
PCT NO: |
PCT/US2014/031099 |
371 Date: |
September 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61801424 |
Mar 15, 2013 |
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61801096 |
Mar 15, 2013 |
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61793092 |
Mar 15, 2013 |
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61800915 |
Mar 15, 2013 |
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61801933 |
Mar 15, 2013 |
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61794317 |
Mar 15, 2013 |
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61802020 |
Mar 15, 2013 |
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61802223 |
Mar 15, 2013 |
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Current U.S.
Class: |
435/5 ; 427/551;
435/6.11; 435/7.1; 436/501 |
Current CPC
Class: |
B29C 59/026 20130101;
G01N 21/648 20130101; B29K 2995/0005 20130101; B29C 33/56 20130101;
G01N 21/658 20130101; B29C 59/02 20130101; B29C 33/424 20130101;
G01N 21/6486 20130101; B29C 2059/023 20130101; B29C 37/0003
20130101; G03F 7/0002 20130101 |
International
Class: |
G01N 21/65 20060101
G01N021/65; G01N 21/64 20060101 G01N021/64 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0004] This invention was made with government support under Grant
No. FA9550-08-1-0222 awarded by the Defense Advanced Research
Project Agency (DARPA). The government has certain rights in the
invention.
Foreign Application Data
Date |
Code |
Application Number |
Mar 17, 2014 |
US |
PCT2014 030624 |
Claims
1. A nanoparticle that enhances the interaction of the nanoparticle
and/or a molecule/material deposited on the surface of the
nanoparticle with light, comprising a pair of stacked metallic
disks separated by a non-metallic spacer, wherein: (a) the
dimensions of the disks and spacer are smaller than the wavelength
of the light; and (b) the nanoparticle enhance the light
interaction at least three times greater than that an individual
metallic disk.
2. The nanoparticle of any prior claim, wherein the light
interaction includes light absorption, light scattering, light
reflection, and light radiation.
3. The nanoparticle of any prior claim, wherein the light
interaction includes Raman scattering, color production, and
luminescence that includes fluorescence, electroluminescence,
chemiluminescence, and electrochemiluminescence.
4. The nanoparticle of any prior claim, wherein the light
interaction comprises interactions of light with a materials or
molecule that is deposited on the nanoparticle.
5. The nanoparticle of claim 4, wherein the molecules are analytes
that have been captured on the surface of the nanoparticle.
6. The nanoparticle of claim 4 or 5, wherein the analytes are
selected from proteins, peptides, DNA, RNA, nucleic acid, small
molecules, cells, and nanoparticles with different shape.
7. The nanoparticle of any prior claim, wherein the nanoparticle
further comprises two masking layers covering the exterior surfaces
of the metallic disks but a portion of the edges of the disks.
8. The nanoparticle of any prior claim, wherein the nanoparticle
further comprises a magnetic or magnezable disk that can be
attracted to a magnet.
9. The nanoparticle of claim 8, wherein the magnetic or magnezable
disk has a thickness in the range of 5 to 50 nm.
10. The nanoparticle of any prior claim, wherein the nanoparticle
further comprises at least one metallic nano-dot on the edge of the
spacer and/or the metallic disk.
11. The nanoparticle of claim 10, wherein the nano-dots have a
diameter in the range of 5 nm to 15 nm.
12. The nanoparticle of any prior claim, wherein the disks have the
shape selected from round, polygonal, pyramidal, elliptical,
elongated bar shaped, or any combination thereof.
13. The nanoparticle of any prior claim, wherein the metallic and
the spacer have the same and similar lateral dimension.
14. The nanoparticle of any prior claim, wherein, for light
enhancement in 800 nm wavelength and near by region, the disks have
a significantly round shape of diameter from 30 nm to 100 nm, the
top metallic disk thickness is from 5 nm to 30 nm, the spacer
thickness is from 2 to 30 nm, and the bottom metallic disk
thickness is from 5 nm to 30 nm.
15. The nanoparticle of any prior claim, wherein the nanoparticle
further comprises a magnetic or magnezible disk, that can be
attracted to a magnet.
16. The nanoparticle of any prior claim, wherein the stacked
metallic disks are made of the same or different materials.
17. The nanoparticle of any prior claim, wherein the material for
the metallic disks is selected from the group consisting of gold,
silver, copper, aluminum, alloys thereof, and combinations
thereof.
18. The nanodevice of any prior claim, wherein the distance between
the pair of the metallic disk is in the range of 0.1 nm to 20 nm,
for the light wavelength of 800 nm and around.
19. The nanodevice of any prior claim, wherein the lateral
dimension of said metallic disc is in the range from 5 nm to 150
nm.
20. The nanodevice of any prior claim, wherein said metallic disc
and the metallic back plane are spaced by a distance in the range
of 0.1 nm to 60 nm.
21. The nanodevice of any prior claim, wherein said at least one
metallic dot structure has dimensions in the range of 1 nm to 25
nm.
22. The nanodevice of any prior claim, wherein the distance between
said metallic dot structure and said metallic disc, and the
distance between said metallic dot structure and said metallic
backplane is in the range of 0.5 nm to 50 nm.
23. A method of making a free-standing nanoparticle, comprising:
(a) obtaining a template comprising a plurality of pillars on the
surface, wherein the height of the pillar is greater than the
thickness of the material to be deposited; (b) depositing one of
more materials on the top surface with the pillars using a beam of
the materials, in the direction substantially normal to the surface
of the template; (c) separating the deposited materials on top of
the pillars from the pillars, thereby producing said
nanoparticles.
24. The method of claim 23, wherein the pillars have a top area of
the shape selected from round, polygonal, pyramidal, elliptical,
elongated bar shaped, or any combination thereof.
25. The method of claim 23 or 24, wherein the method comprises a
step of depositing a magnetic or magnetizable layer on the stack
produced in step b).
26. An assay for analyte detection, comprising: (a) bringing an
analyte into proximity to or in contact with a free-standing
nanoparticle comprising a pair of stacked metallic disks separated
by a non-metallic spacer; (b) illuminating free-standing
nanoparticle with light that has a wavelength that is larger than
the dimensions of the disks and spacer; (c) detecting an outgoing
signal from said analyte and/or free-standing nanoparticle.
27. The assay of claim 26, wherein the signal is selected from the
group consisting of light absorption, light scattering, light
reflection, and light radiation.
28. The assay of claim 26 or 27, wherein the detecting is done by
detecting Raman scattering, color production, and/or luminescence
that includes fluorescence, electroluminescence, chemiluminescence,
and electrochemiluminescence.
29. The assay of any of claims 26-28, wherein the analyte is bound
by a capture agent that is on the surface of the nanoparticle.
30. The assay of any of claims 26-29, wherein the analyte is
selected from protein, peptides, DNA, RNA, nucleic acid, a small
molecule, cell, and nanoparticle with different shape.
Description
CROSS-REFERENCING
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 13/838,600, filed Mar. 15, 2013 (NSNR-003),
which application claims the benefit of U.S. provisional
application Ser. No. 61/622,226 filed on Apr. 10, 2012, and is a
continuation-in-part of U.S. patent application Ser. No.
13/699,270, filed on Jun. 13, 2013, which application is a
.sctn.371 filing of US2011/037455, filed on May 20, 2011, and
claims the benefit of U.S. provisional application Ser. No.
61/347,178, filed on May 21, 2010;
[0002] this application is also a continuation-in-part of U.S.
application Ser. No. 13/699,270, filed Jun. 13, 2013 (NSNR-001),
which application is a .sctn.371 filing of international
application serial no. US2011/037455, filed on May 20, 2011, which
application claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/347,178 filed on May 21, 2010; and
[0003] this application is also claims the benefit of: provisional
application Ser. No. 61/801,424, filed Mar. 15, 2013 (NSNR-004PRV),
provisional application Ser. No. 61/801,096, filed Mar. 15, 2013
(NSNR-005PRV), provisional application Ser. No. 61/800,915, filed
Mar. 15, 2013 (NSNR-006PRV), provisional application Ser. No.
61/793,092, filed Mar. 15, 2013 (NSNR-008PRV), provisional
Application Ser. No. 61/801,933, filed Mar. 15, 2013 (NSNR-009PRV),
provisional Application Ser. No. 61/794,317, filed Mar. 15, 2013
(NSNR-010PRV), provisional application Ser. No. 61/802,020, filed
Mar. 15, 2013 (NSNR-011PRV) and provisional application Ser. No.
61/802,223, filed Mar. 15, 2013 (NSNR-012PRV), all of which
applications are incorporated by reference herein for all
purposes.
BACKGROUND
[0005] There is a great need to develop new nanoparticle
structures, and new fabrication methods for applications in
chemical and biological sensing. The subject nanoparticle
structures can greatly enhance (e.g. amplify) optical signals
(particularly luminescence (e.g. fluorescence) and Surface Enhanced
Raman Scattering (SERS)) disposed on or near to the nanoparticle
surface; improve the detection sensitivity of the chemical and/or
biological properties of the molecules disposed on or near the
nanoparticles, improve nanoparticle performance in penetrating
biological cells or materials, and in reducing biological
toxicities. The subject fabrication methods allow the fabrication
of such new nanostructures which are otherwise either impossible or
hard to fabricate.
[0006] Conventional NPs, limited by their fabrication method
(chemical synthesis), have simple architecture (spheres, rods, and
shells), simple and smooth surfaces, and simple compositions
(either pure dielectric, pure metal, or dielectric enclosed
completely by metal (or vice versa)). All of these brought severe
drawback to in-vivo diagnosis. The four major drawbacks are: (a)
lower plasmonic enhancement than Au clusters or other plasmonic
structures made on substrate, (b) large particle size for in-vivo
diagnosis wavelength (e.g. 300 nm for Au NPs), (c)
bio-undegradeable (for metal particles or nanoshells), and (d)
similar surface property in entire surface rather surface location
selective. They also have large particle size variations
(.about.15%). These drawbacks lead (i) poor in-vivo performances of
low brightness (low enhancement in fluorescence or SERS), poor
in-vivo suitability (slow and low number NPs entering cells), poor
biocompability/safety (invasive and particle accumulation), and
poor selectivity
[0007] In bio-safe in-vivo plasmonic-based diagnosis and
therapeutics, one most significant roadblock is how to satisfy
simultaneously two completely conflicting requirements on
nanoparticle size: large (over 50 nm) for better therapeutic and
diagnostic efficacy (i.e. plasmonic effectiveness and decent blood
retention time), and ultra-small (sub10 nm) for lower toxicity
(i.e. rapid clearance from cell/body and hence zero
accumulation).
[0008] Another major roadblock is that conventional nanoparticle
fabrication methods prohibit current plasmonic nanoparticles from
having the complex structures needed for achieving an ultra-high
plasmonic enhancement that is several orders of magnitude higher
than the current ones.
[0009] For examples, to have decent plasmonic effects and decent
blood retention time, conventional approaches use gold spheres of
300 nm diameter, nanoshells of 60 nm diameter, and nanorods of 10
diameter and 60 nm long. These sizes are much larger than that for
quick clear-up in cells or bodies, which should be sub-10 nm.
Furthermore, for ultra-high plasmonic enhancements, it requires
complex particle structures, such as nanogaps and nano-sharp-edges,
which are missing in the current NPs, making them several orders of
magnitude less plasmonic effective than what we are able to achieve
(see section III). Clearly, the current NPs cannot simultaneously
satisfy the size requirements for plasmonic effectiveness and
bio-safety, because these nanoparticles are not biodegradable and
hence cannot change from large size for plasmonic effectiveness to
small size needed for bio clear-out.
[0010] In summary, all previous approaches cannot solve the major
roadblocks of efficacy-safety conflict caused by conflicting
particle-size requirements nor low plasmonic effects caused by
lacking complexity in nanostructure.
[0011] Therefore, to advance diagnosis (in vitro and in-vivo) and
single biological cell analysis, we need both new nanoparticle
platforms (different architectures and physical principles) and new
fabrication methods, that radically differ from conventional
approaches. This is subject of current invention.
SUMMARY
[0012] The following brief summary is not intended to include all
features and aspects of the present invention, nor does it imply
that the invention must include all features and aspects discussed
in this summary.
[0013] The invention is related to nanoparticle structures,
fabrication methods and applications in chemical and biological
sensing. The nanoparticles in the invention has very different
structures and materials from the conventional metallic
nanoparticles, which allows the new nanoparticle have many unique
properties desired in sensing and diagnostics, including much more
effective in enhancing a sensing light signal than the conventional
metallic nanoparticles while having a size much smaller. The small
sizes are important to in vivo testing and bio-safety. The unique
structures of the nanoparticles cannot be made by conventional
synthesis method, but are fabricated by template deposition and
exfoliations. The nanoparticles in the invention also can
biodegradable. In particular, the nanoparticle structures can
greatly enhance (e.g. amplify) light absorption, light scattering,
and light radiation, optical signals (particularly luminescence
(e.g. fluorescence) and Surface Enhanced Raman Scattering (SERS))
disposed on or near to the nanoparticle surface (the nanoparticle
can be inside biological cell and/or human body); improve the
detection sensitivity of the chemical and/or biological properties
of the molecules disposed on or near the nanoparticles, improve
nanoparticle performance in penetrating biological cells or
materials, and in reducing biological toxicities. The subject
fabrication methods allow the fabrication of such new
nanostructures which are otherwise either impossible or hard to
fabricate. The functionalized nanoparticles can be used as
biological and chemical assay for detection of biological and
chemical markers (also termed "analytes"), such as proteins, DNAs,
RNAs, and other organic and inorganic molecules, in single cells,
tissue, and in-vivo for human and animals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way. Some of the drawings are not in scale.
[0015] FIG. 1 Schematic of Stacked-nanoparticles (S-NPs): (a) a
pair of metal-disks separated by a single dielectric disk; (b) S-NP
with a magnetic disk on top; (c) S-NP with five disks; (d) 4 S-NPs
glued by another dielectric material into a single particle.
[0016] FIG. 2 Schematic of (a) DS-NPs: S-NP with metal nano-dots
self-assembled at the side wall, and (b) ES-NP (enhanced S-NP) with
two non-metallic disks 460, each of them covers the exterior
surface of the metallic disks of a S-NP.
[0017] FIG. 3 Biodegradation of stacked-nanoparticles (S-NPs).
[0018] FIG. 4 Schematics of coating a molecular adhesion layer and
then the capture agent.
[0019] FIG. 5 schematically illustrates an exemplary antibody
detection assay.
[0020] FIG. 6 schematically illustrates an exemplary nucleic acid
detection assay.
[0021] FIG. 7 schematically illustrates another embodiment nucleic
acid detection assay.
[0022] FIG. 8 shows a flow chart of Nano-PrinTED (nanoprint by
templated exfoliatable deposition) and Dip-print of biodegradable
dielectrics. Nano-PrinTED comprises three key steps: (i) have a
template with nanostructured protrusions or hollows (e.g. dense
nanopillar array on 4'' wafer), (ii) deposit a release layer
(optional) and then multiple-layer composite materials to form
nanoparticles on the pedestals of the template's protrusions or
inside the hollows, and (iii) exfoliate the nanoparticles from the
template by transfer printing or lift off. Dip-print is for
patterning biodegradable dielectrics, since such materials cannot
be thermally evaporated. Dip-print first puts a thin layer of
liquid polymer precursors with proper viscosity on a plate (termed
"material transfer plate"); and then presses the template used in
Nano-PrinTED against the material transfer plate, picking up the
polymer precursors only at the top of the pillars of the template.
Afterwards the polymer precursors will be cured to form a solid
polymer. The dip-printed polymers will be used for the dielectrics
between the stacked layers and for the dielectrics that glue
different columns into a single particle (Note different viscosity
liquid will be used depending upon the gap size between the
columns).
[0023] FIG. 9 Nano-PrinTED (nanoprint by templated exfoliateable
deposition)--a new nanoparticle fabrication technology. Schematics
of (a) Pillar template fabricated by lithography (e.g. NIL); (b)
multiple deposition and self-assembly to form D2-particles; (c)
transfer-print S-NPs to another substrate; and put in solution.
[0024] FIG. 10 Nano-PrinTED (nanoprint by templated exfoliateable
deposition)--method 2. Schematics of (a) hole template (polymer)
fabricated by lithography (e.g. NIL); (b) multiple deposition and
self-assembly to form D2-particles inside the holes; (c) lift off
the polymer (including the top stacked plane) around S-NPs; and put
in solution.
[0025] FIG. 11 Dip-print of biodegradable dielectrics. Schematics
of (a) pillar template fabricated by lithography (e.g. NIL); (b)
deposition thin metal disks on top of the pillars; (c) Puts a thin
layer of liquid polymer precursors with proper viscosity on a plate
(termed "material transfer plate"); and then presses the template
used in Nano-PrinTED against the material transfer plate, (d)
picking up the polymer precursors only at the top of the pillars of
the template. (e) add another deposition step to form top metal
disk if needed and (f) put S-NPs into solutions.
[0026] FIG. 12 Dip-print of biodegradable dielectrics that "glue"
columns. The same as dip printing the dielectric spacer, except
that the viscosity of polymer precursors may be changed to make the
polymer precursor fill the gaps between the columns. (a) 4 close
pillar template with 4 S-NPs on top. (b) pick up the polymer
precursors at the top of the S-NPs on the template, fill the gaps
in between and glue the 4 S-NPs. (c) put into solutions.
[0027] FIG. 13 Scanning electron microscopy (SEMs) of (a)
double-metal-disk and single dielectric (D-particle); (b)
triple-metal (or magnetic) dielectric-nanoparticle (TS-NP); (c)
D-particle after the "self-perfection by liquefaction" (SPEL) to
change the shape of 2 metal disks; (d) D-particles array on the
substrate after the template lift-off.
[0028] FIG. 14 Scanning electron microscopy (SEMs) of (a)
D-particles array on the substrate after the transfer printing. (b)
D-particles exfoliated into solution.
[0029] FIG. 15 Nano-PrinTED (nanoprint by templated exfoliateable
deposition)--a new nanoparticle fabrication technology. Top row:
Schematic. And bottom row: scanning electron microscope (SEM) of
experimental results. (a) Pillar template fabricated by lithography
(e.g. NIL); (b) Multiple deposition and self-assembly to form
D2-particles; (c) transfer-print DPs to another substrate; (d) put
in solution. (e-h), SEM images.
[0030] FIG. 16 Nano-PrinTED and Dip-print have far better precision
in controlling the NP structure dimensions (including the size and
shape of each individual components, their spacing, and final
particle). (a) SEM picture of D2-P before release and (b) Measured
size distribution. Measured size variation of D2-particle
fabricated by Nano-PrinTED (<5%) is 3 fold less than AuNP
manufactured by chemical synthesis (>15%).
[0031] FIG. 17 Measurements of extinction spectrum of D2-particles
with SiO.sub.2 layer thickness from 5 nm to 30 nm and constant Au
layer thicknesses of 20 nm. Plasmonic resonant peak wavelengths
redshift with increasing SiO.sub.2 layer thickness.
[0032] FIG. 18 Simulation of the size of nanoparticles with
different architectures required for the same resonant wavelength
at 800 nm. It clearly shows that S-NP has much smaller particle
sized than conventional metallic sphere and disks for a given
resonant wavelength.
[0033] FIG. 19 (a) Measured Surface Enhanced Raman Spectroscopy
(SERS) signal of BPE, and (b) fluorescence signal of IR-800 dye
with single D2-particle and gold nanoparticle. A single D2-particle
has a SERS/Fluorescence enhancement over 100/30 fold higher than a
single gold nanoparticle of similar diameter.
[0034] Corresponding reference numerals indicate corresponding
parts throughout the several figures of the drawings. It is to be
understood that the drawings are for illustrating the concepts set
forth in the present disclosure and are not to scale.
[0035] Before any embodiments of the invention are explained in
detail, it is to be understood that the invention is not limited in
its application to the details of construction and the arrangement
of components set forth in the following description or illustrated
in the drawings.
DEFINITIONS
[0036] Before describing exemplary embodiments in greater detail,
the following definitions are set forth to illustrate and define
the meaning and scope of the terms used in the description.
[0037] The term "molecular adhesion layer" refers to a layer or
multilayer of molecules of defined thickness that comprises an
inner surface that is attached to the S-NP and an outer (exterior)
surface can be bound to capture agents.
[0038] The term "capture agent-reactive group" refers to a moiety
of chemical function in a molecule that is reactive with capture
agents, i.e., can react with a moiety (e.g., a hydroxyl,
sulfhydryl, carboxy or amine group) in a capture agent to produce a
stable strong, e.g., covalent bond.
[0039] The term "capture agent" as used herein refers to an agent
that binds to a target analyte through an interaction that is
sufficient to permit the agent to bind and concentrate the target
molecule from a heterogeneous mixture of different molecules. The
binding interaction is typically mediated by an affinity region of
the capture agent. Typical capture agents include any moiety that
can specifically bind to a target analyte. Certain capture agents
specifically bind a target molecule with a dissociation constant
(K.sub.D) of less than about 10.sup.-6 M (e.g., less than about
10.sup.-7 M, less than about 10.sup.-8 M, less than about 10.sup.-9
M, less than about 10.sup.-19 M, less than about 10.sup.-11 M, less
than about 10.sup.-12 M, to as low as 10.sup.-16 M) without
significantly binding to other molecules. Exemplary capture agents
include proteins (e.g., antibodies), and nucleic acids (e.g.,
oligonucleotides, DNA, RNA including aptamers).
[0040] The term "nanosensor" refers to a nanoparticle that is
functionalize with a capture agent.
[0041] The terms "specific binding" and "selective binding" refer
to the ability of a capture agent to preferentially bind to a
particular target molecule that is present in a heterogeneous
mixture of different target molecule. A specific or selective
binding interaction will discriminate between desirable (e.g.,
active) and undesirable (e.g., inactive) target molecules in a
sample, typically more than about 10 to 100-fold or more (e.g.,
more than about 1000- or 10,000-fold).
[0042] The term "protein" refers to a polymeric form of amino acids
of any length, i.e. greater than 2 amino acids, greater than about
5 amino acids, greater than about 10 amino acids, greater than
about 20 amino acids, greater than about 50 amino acids, greater
than about 100 amino acids, greater than about 200 amino acids,
greater than about 500 amino acids, greater than about 1000 amino
acids, greater than about 2000 amino acids, usually not greater
than about 10,000 amino acids, which can include coded and
non-coded amino acids, chemically or biochemically modified or
derivatized amino acids, and polypeptides having modified peptide
backbones. The term includes fusion proteins, including, but not
limited to, fusion proteins with a heterologous amino acid
sequence, fusions with heterologous and homologous leader
sequences, with or without N-terminal methionine residues;
immunologically tagged proteins; fusion proteins with detectable
fusion partners, e.g., fusion proteins including as a fusion
partner a fluorescent protein, .beta.-galactosidase, luciferase,
etc.; and the like. Also included by these terms are polypeptides
that are post-translationally modified in a cell, e.g.,
glycosylated, cleaved, secreted, prenylated, carboxylated,
phosphorylated, etc., and polypeptides with secondary or tertiary
structure, and polypeptides that are strongly bound, e.g.,
covalently or non-covalently, to other moieties, e.g., other
polypeptides, atoms, cofactors, etc.
[0043] The term "antibody" is intended to refer to an
immunoglobulin or any fragment thereof, including single chain
antibodies that are capable of antigen binding and phage display
antibodies).
[0044] The term "nucleic acid" and "polynucleotide" are used
interchangeably herein to describe a polymer of any length composed
of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or
compounds produced synthetically (e.g., PNA as described in U.S.
Pat. No. 5,948,902 and the references cited therein) which can
hybridize with naturally occurring nucleic acids in a sequence
specific manner analogous to that of two naturally occurring
nucleic acids, e.g., can participate in Watson-Crick base pairing
interactions.
[0045] The term "complementary" as used herein refers to a
nucleotide sequence that base-pairs by hydrogen bonds to a target
nucleic acid of interest. In the canonical Watson-Crick base
pairing, adenine (A) forms a base pair with thymine (T), as does
guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced
by uracil (U). As such, A is complementary to T and G is
complementary to C. Typically, "complementary" refers to a
nucleotide sequence that is fully complementary to a target of
interest such that every nucleotide in the sequence is
complementary to every nucleotide in the target nucleic acid in the
corresponding positions. When a nucleotide sequence is not fully
complementary (100% complementary) to a non-target sequence but
still may base pair to the non-target sequence due to
complementarity of certain stretches of nucleotide sequence to the
non-target sequence, percent complementarily may be calculated to
assess the possibility of a non-specific (off-target) binding. In
general, a complementary of 50% or less does not lead to
non-specific binding. In addition, a complementary of 70% or less
may not lead to non-specific binding under stringent hybridization
conditions.
[0046] The terms "ribonucleic acid" and "RNA" as used herein mean a
polymer composed of ribonucleotides.
[0047] The terms "deoxyribonucleic acid" and "DNA" as used herein
mean a polymer composed of deoxyribonucleotides.
[0048] The term "oligonucleotide" as used herein denotes single
stranded nucleotide multimers of from about 10 to 200 nucleotides
and up to 300 nucleotides in length, or longer, e.g., up to 500 nt
in length or longer. Oligonucleotides may be synthetic and, in
certain embodiments, are less than 300 nucleotides in length.
[0049] The term "attaching" as used herein refers to the strong,
e.g, covalent or non-covalent, bond joining of one molecule to
another.
[0050] The term "surface attached" as used herein refers to a
molecule that is strongly attached to a surface.
[0051] The term "sample" as used herein relates to a material or
mixture of materials containing one or more analytes of interest.
In particular embodiments, the sample may be obtained from a
biological sample such as cells, tissues, bodily fluids, and stool.
Bodily fluids of interest include but are not limited to, amniotic
fluid, aqueous humour, vitreous humour, blood (e.g., whole blood,
fractionated blood, plasma, serum, etc.), breast milk,
cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime,
endolymph, perilymph, feces, gastric acid, gastric juice, lymph,
mucus (including nasal drainage and phlegm), pericardial fluid,
peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin
oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine and
exhaled condensate. In particular embodiments, a sample may be
obtained from a subject, e.g., a human, and it may be processed
prior to use in the subject assay. For example, prior to analysis,
the protein/nucleic acid may be extracted from a tissue sample
prior to use, methods for which are known. In particular
embodiments, the sample may be a clinical sample, e.g., a sample
collected from a patient.
[0052] The term "analyte" refers to a molecule (e.g., a protein,
nucleic acid, or other molecule) that can be bound by a capture
agent and detected.
[0053] The term "assaying" refers to testing a sample to detect the
presence and/or abundance of an analyte.
[0054] As used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations.
[0055] As used herein, the term "light-emitting label" refers to a
label that can emit light when under an external excitation. This
can be luminescence. Fluorescent labels (which include dye
molecules or quantum dots), and luminescent labels (e.g., electro-
or chemi-luminescent labels) are types of light-emitting label. The
external excitation is light (photons) for fluorescence, electrical
current for electroluminescence and chemical reaction for
chemi-luminscence. An external excitation can be a combination of
the above.
[0056] The phrase "labeled analyte" refers to an analyte that is
detectably labeled with a light emitting label such that the
analyte can be detected by assessing the presence of the label. A
labeled analyte may be labeled directly (i.e., the analyte itself
may be directly conjugated to a label, e.g., via a strong bond,
e.g., a covalent or non-covalent bond), or a labeled analyte may be
labeled indirectly (i.e., the analyte is bound by a secondary
capture agent that is directly labeled).
[0057] The term "hybridization" refers to the specific binding of a
nucleic acid to a complementary nucleic acid via Watson-Crick base
pairing. Accordingly, the term "in situ hybridization" refers to
specific binding of a nucleic acid to a metaphase or interphase
chromosome.
[0058] The terms "hybridizing" and "binding", with respect to
nucleic acids, are used interchangeably.
[0059] The term "capture agent/analyte complex" is a complex that
results from the specific binding of a capture agent with an
analyte. A capture agent and an analyte for the capture agent will
usually specifically bind to each other under "specific binding
conditions" or "conditions suitable for specific binding", where
such conditions are those conditions (in terms of salt
concentration, pH, detergent, protein concentration, temperature,
etc.) which allow for binding to occur between capture agents and
analytes to bind in solution. Such conditions, particularly with
respect to antibodies and their antigens and nucleic acid
hybridization are well known in the art (see, e.g., Harlow and Lane
(Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y. (1989) and Ausubel, et al, Short Protocols
in Molecular Biology, 5th ed., Wiley & Sons, 2002).
[0060] The term "specific binding conditions" as used herein refers
to conditions that produce nucleic acid duplexes or protein/protein
(e.g., antibody/antigen) complexes that contain pairs of molecules
that specifically bind to one another, while, at the same time,
disfavor to the formation of complexes between molecules that do
not specifically bind to one another. Specific binding conditions
are the summation or combination (totality) of both hybridization
and wash conditions, and may include a wash and blocking steps, if
necessary.
[0061] For nucleic acid hybridization, specific binding conditions
can be achieved by incubation at 42.degree. C. in a solution: 50%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0062] For binding of an antibody to an antigen, specific binding
conditions can be achieved by blocking a substrate containing
antibodies in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), followed by incubation with a sample containing analytes in
diluted blocking buffer. After this incubation, the substrate is
washed in washing solution (e.g. PBS+TWEEN 20) and incubated with a
secondary capture antibody (detection antibody, which recognizes a
second site in the antigen). The secondary capture antibody may
conjugated with an optical detectable label, e.g., a fluorophore
such as IRDye800CW, Alexa 790, Dylight 800. After another wash, the
presence of the bound secondary capture antibody may be detected.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise.
[0063] The term "a secondary capture agent" which can also be
referred to as a "detection agent" refers a group of biomolecules
or chemical compounds that have highly specific affinity to the
antigen. The secondary capture agent can be strongly linked to an
optical detectable label, e.g., enzyme, fluorescence label, or can
itself be detected by another detection agent that is linked to an
optical detectable label through bioconjugatio (Hermanson,
"Bioconjugate Techniques" Academic Press, 2nd Ed., 2008).
[0064] The term "biotin moiety" refers to an affinity agent that
includes biotin or a biotin analogue such as desthiobiotin,
oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide,
biocytin, etc. Biotin moieties bind to streptavidin with an
affinity of at least 10-8M. A biotin affinity agent may also
include a linker, e.g., --LC-biotin, --LC-LC-Biotin, --SLC-Biotin
or --PEGn-Biotin where n is 3-12.
[0065] The term "streptavidin" refers to both streptavidin and
avidin, as well as any variants thereof that bind to biotin with
high affinity.
[0066] The term "marker" refers to an analyte whose presence or
abundance in a biological sample is correlated with a disease or
condition.
[0067] The term "bond" includes covalent and non-covalent bonds,
including hydrogen bonds, ionic bonds and bonds produced by van der
Waal forces.
[0068] The term "amplify" refers to an increase in the magnitude of
a signal, e.g., at least a 10-fold increase, at least a 100-fold
increase at least a 1,000-fold increase, at least a 10,000-fold
increase, or at least a 100,000-fold increase in a signal.
[0069] The term "local" refers to "at a location",
[0070] Other specific binding conditions are known in the art and
may also be employed herein.
[0071] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise, e.g., when
the word "single" is used. For example, reference to "an analyte"
includes a single analyte and multiple analytes, reference to "a
capture agent" includes a single capture agent and multiple capture
agents, and reference to "a detection agent" includes a single
detection agent and multiple detection agents.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0072] The following detailed description illustrates some
embodiments of the invention by way of example and not by way of
limitation.
[0073] The invention is related to nanoparticle structures,
fabrication methods and applications in chemical and biological
sensing. The nanoparticles in the invention has very different
structures and materials from the conventional metallic
nanoparticles, which allow the new nanoparticle having many unique
properties desired in sensing and diagnostics, including much more
effective in enhancing a sensing light signal than the conventional
metallic nanoparticles while having a size much smaller. The small
sizes are important to in vivo testing and bio-safety. The unique
structures of the nanoparticles cannot be made by conventional
synthesis method, but are fabricated by template deposition and
exfoliations. The nanoparticles in the invention also can be
biodegradable. In particular, the nanoparticle structures can
greatly enhance (e.g. amplify) light absorption, light scattering,
and light radiation, optical signals (particularly luminescence
(e.g. fluorescence) and Surface Enhanced Raman Scattering (SERS))
disposed on or near to the nanoparticle surface (the nanoparticle
can be inside biological cell and/or human body); improve the
detection sensitivity of the chemical and/or biological properties
of the molecules disposed on or near the nanoparticles (e.g.
proteins, DNAs, RNAs, and other organic and inorganic molecules),
improve nanoparticle performance in penetrating biological cells or
materials, and in reducing biological toxicities. The subject
fabrication methods allow the fabrication of such new
nanostructures which are otherwise either impossible or hard to
fabricate.
[0074] The invention covers four areas: (1) nanoparticle
structures, (2) fabrication methods, (3) surface functioning, and
(4) applications in chemical and biological sensing.
[0075] Certain physical principles and certain materials,
dimensions gaps used in the invention are similar to "disk-coupled
dots-on-pillar antenna array" (D2PA) which is on a solid support
(as described in WO2012/024006 and WO2013154770 which are
incorporated by reference).
Nanoparticle Structures and Materials
Basic Structure
[0076] In one embodiment of the invention, as illustrated in FIG.
1, a nanoparticle 100, termed stacked-nanoparticle (S-NP), that
enhances the interaction of the nanoparticle and/or a
molecule/materials deposited on the surface of the nanoparticle
with light, comprises at least a pair of stacked metallic disks 110
and 120, separated by a non-metallic spacer 130, wherein (a) the
dimensions of the disks and spacer are smaller than the wavelength
of the light; and (b) the nanoparticle enhances the light
interaction at least three times greater than that of each said
metallic disk. The metallic disks 110 and 120 can be made of the
same or different materials and have the same or different
thickness. Furthermore, a thin adhesion layer may be between two
adjacent disks.
[0077] The non-metallic spacer 130 can be biodegradable, namely, it
can be dissolved in a bio-environment. As illustrated in FIG. 3,
when the spacer is degraded, a S-NP breaks into small pieces.
Breaking up into small pieces has advantages in in vivo
application; small pieces can come out biological cells and human
body much faster than large pieces, hence avoiding accumulation.
Since the space is not completely enclosed by the metallic disks
(non-biodegradable), fluid can access the spacer from the side of
the spacer. The biodegradation time can be controlled by the
biodegradable material and their dimensions.
[0078] The light interaction include light absorption, light
scattering, light radiation, Raman scattering, chromaticity, and
luminescence that includes fluorescence, electroluminescence,
chemiluminescence, and electrochemiluminescence.
[0079] A preferred wavelength range for the light that can be
enhanced by the S-NP is about from 20 nm to 10 micrometer. Another
preferred the wavelength range is from 300 nm to 4000 nm. For in
vivo applications, a preferred wavelength range (window) for a good
light penetration in biological tissue is about from 630 nm to 1316
nm.
[0080] The molecule/materials deposited on the S-NP include the
molecules/materials to be sensed, such as the analytes and/or their
labels including proteins, DNAs, RNAs, and other organic and
inorganic molecules in single cells, tissue, and in-vivo for human
and animals.
[0081] The "metallic" in this invention means that for a given
light wavelength the electrons in the materials can generate
plasmons. For example, the gold has a plasmon wavelength about 560
nm; for the wavelength longer than 560 nm, the gold behavior like
"metallic", for the wavelength significantly shorter than 560 nm,
the gold behavior like a non-metallic to the light.
[0082] The sensing property includes the sensing signal intensity,
sensing signal spectrum, limit of detection, detection dynamic
range, and signal variation reduction (smaller error bar) of the
sensing. The sensing includes the detection of the existence,
quantification of the concentration, and determination of the
states of the targeted analyte. The analyte includes proteins,
DNAs, RNAs, and other organic and inorganic molecules. The
invention can be used in the sensing in vitro, or in vivo.
[0083] The key reason for S-NP superior to the conventional NPs in
enhancement with smaller size is due to different physics.
Conventional NPs with metal all around follows the parabolic
function or the dispersion relation
k x 2 1 + k y 2 2 = .omega. 2 c 2 , ##EQU00001##
where .di-elect cons..sub.1 and .di-elect cons..sub.2 are
dielectric constants (permittivities) in two orthogonal directions
and for conventional NPs, they both have the same sign. Hence,
their isofrequency curve is elliptical, which leads to bounded
wavevectors, k, and relatively long wavelength. But for S-NP with
bipolar-permittivity (let .di-elect cons..sub.1=.di-elect
cons..sub.p and .di-elect cons..sub.2=-.di-elect cons..sub.v,
having opposite sign, while .di-elect cons..sub.p and .di-elect
cons..sub.v are positive values), the equation becomes
k v 2 p - k p 2 v = .omega. 2 c 2 , ##EQU00002##
a hyperbolic curve, which means that wavevector, k, in both
directions is unbounded, and can be very large. This allows a very
short wavelength inside the particle (outside the particle the
wavelength is still 800 nm), and consequently a very small particle
size for a large optical signal. Furthermore, a pair of disks forms
a resonant cavity for the light.
[0084] As shown in FIG. 4, when the surface of the S-NP 100 is
functionalized, it becomes a nanosensor 200 for sensing an analyte
in biological and chemical detection. The surface functionalization
can be many ways as discussed later, including attaching the
capture agents that selectively bond to a targeted analyte. The
analytes include proteins, peptides, DNA, RNA, nucleic acid, small
molecules, cells, nanoparticles with different shapes. The targeted
analyte can be either in a solution or in air or gas phase. The
sensing includes light absorption, light scattering, light
radiation, Raman scattering, chromaticity, luminescence that
includes fluorescence, electroluminescence, chemiluminescence, and
electrochemiluminescence. The sensing property includes the sensing
signal intensity, sensing signal spectrum, limit of detection,
detection dynamic range, and signal variation reduction (smaller
error bar) of the sensing. The sensing includes the detection of
the existence, quantification of the concentration, and
determination of the states of the targeted analyte. The invention
can be used in the sensing in vitro, or in vivo.
NP Structure Variations and Improvements
[0085] Dots-on-Sidewall S-NP (DS-SP).
[0086] One embodiment of S-NP (FIG. 1b), termed "dots-on-sidewall
S-NP" (DS-SP) 400 comprises metallic nanodots 440 on the sidewall
of the spacer and/or the metallic disks, which can have a higher
light signal enhancement than the S-NP without them. For 800 nm
wavelength, the diameter of the nanodots is about 3 to 15 nm.
[0087] Magnetic/Magnetizable S-NP (MS-NS).
[0088] S-NP can be made to be magnetic/magnetizable, so that they
can be attracted to a magnet, and is termed MS-NP. One embodiment
of MS-NP comprises a S-NP having at least one
magnetic/magnetizamble disk 140 stacked on a normal S-NP, as shown
in FIG. 1b.
[0089] Enhanced S-NP (ES-NP).
[0090] The S-NP light signal enhancement can be further increased.
For a S-NP, the light signal enhancement on its surface is not
uniform: the regions with sharp (i.e. small curvature) edges of
metallic materials and the small gaps between two metallic
materials are the high enhancement regions, while the other regions
are low enhancement regions (e.g. the flat exterior surface of the
metallic disk). The high enhancement region means that a molecule
or a material attached to that region will have its light signal
amplified more than that of attaching to a low enhancement region.
For example, a fluorophore (e.g. a fluorescence molecule) attached
to the high enhancement region will have a higher fluorescence
signal under a light excitation than that to attached a low
enhancement region.
[0091] One embodiment of the invention is to selectively mask the
low enhancement regions from a molecular binding and selectively
attach the molecules that its light signal will be amplified (e.g.
the capture agent that binds an analyte with a light label) to the
high enhancement regions. One way to achieve this is to add two
non-metallic disks as masking disks, one on each side of the S-NP,
to mask the exterior surface of the metallic disks of a S-NP from
the attachment of a molecule, as illustrated 150 and 160 in FIG.
1c, and 460 in FIG. 2. Such particle 400 is termed ES-NP (enhanced
S-NP), which has five disks (2 metallic disks and 3 non-metallic
disks). For example, a 3 nm thick of SiO.sub.2 disk can be used as
the masking disks and the molecules are DSU, which only attach to
the metal. Of course, one chose to mask one-side of disk or both
size. The masking disks may use different thickness and different
materials. For some applications, a part of disk edges are also
masked. Some other details have be disclosed in PCT/US14/29979,
filed on Mar. 15, 2014, and 61/801,424, filed on Mar. 15, 2013,
which are incorporated by reference herein.
[0092] S-NP with More Stacked Metallic Disks.
[0093] In certain embodiments, S-NP has more than three disks; it
can have 4, 5, 6, or more disks as much as required in sensing. The
metallic disks also can be more a pair.
[0094] Bundled S-NPs (BS-NP) (S-NP with Multi-Columns).
[0095] One embodiment of S-NP is to pack several S-NPs together
into one larger particle (a bundle) using a biodegradable
dielectric material glue (FIG. 1.D). The reason is that such bundle
allows the bundle having the same or similar light signal
enhancement as a single S-NP with the same size, but after
biodegradable, the bundle has much smaller pieces and hence easy to
be cleared from biological cells and human body.
Disks Shape and Dimension
[0096] The lateral shapes of the disks of S-NP can be selected from
the shape of round, square, rectangle, polygon, elliptical,
elongated bar, polygon, other similar shapes or combinations
thereof. In general, each disk can have different lateral shapes
and dimensions from the others. In certain cases, as discussed
later, one way to fabricate the S-NP is by using a template,
deposition, exfoliation; such fabrication leads to similar lateral
shape and dimensions for all disks in a given S-NP. But by using
different templates each S-NP can have may different lateral
shapes.
[0097] The shape of the top and bottom surface of the disks can be
different, and can be flat, but also can be bulged, or a
half-sphere. An example of the fabricated S-NP for 800 nm light
wavelength are given in FIGS. 13, 14, and 15.
[0098] Dimensions for the metallic disks of S-NP and the spacer
should be less than the wavelength of the light that the S-NP
enhances. For a given wavelength, the light enhancement depends on
the S-NP size and a resonant peak (vs. the size). The spacer
between the pair of metallic disk has a thickness of 0.3 nm to 50
nm. This spacer's thickness has an important role in determine gap.
In general smaller the gap is better, but a small gap also changes
the resonant wavelength.
[0099] The disk diameters are often decided by balancing the light
signal enhancement and the other requirements in in vivo
application. For an example, to have an easy bio-cell and
bio-material penetration and exit and bio-safety, the particle size
should be as small as possible, but if the particle size is too
small, it reduces the light enhancement factor. One embody of the
invention is the S-NP that optimize both requirements.
[0100] As an example of DS-NP for 800 nm wavelength, the disks have
a round shape of diameter from 30 nm to 100 nm, the top metallic
disk thickness is from 5 nm to 30 nm, the spacer thickness is from
2 to 30 nm, and the bottom metallic disk thickness is from 5 nm to
30 nm, the self-assembled dots diameter is 5 nm to 15 nm, the
magnetic disk thickness is from 5 to 30 nm, and the adhesion layers
between the disks are titanium or Cr of a thickness from 0.5 nm-1
nm. Examples of the fabricated disks are shown in FIG. 13-16.
[0101] In a preferred DS-NP structure with light resonance
absorption around 800 nm wavelength, the disks have a round shape
of diameter of 70 nm, the top metallic (gold) disk thickness is 15
nm, the spacer (silicon dioxide) thickness is 20 nm, and the bottom
metallic (gold) disk thickness is 15 nm, the self-assembled gold
dots diameter is around 10 nm, and the adhesion layers between the
disks are titanium of a thickness of 0.5 nm.
Metallic Materials
[0102] The materials used for the metal components (e.g. disk and
dots) in S-NPs are chosen from materials that are metallic in the
working photon wavelength. For examples, the metallic materials can
be selected from gold, copper, silver, aluminum, their mixture,
alloys, and multilayers in visible light ranges and longer
wavelength, and certain metal oxides (as ITO, zinc oxide), for near
or mid infrared wavelength or longer wavelength, or semiconductor
(as silicon or gallium arsenide) for certain wavelength range. One
can use a single material or a combination of them.
Materials for Non-Metallic Spacer.
[0103] The materials for the non-metallic spacer and non-metallic
masking layers in S-NPs are chosen from dielectric materials and/or
semiconductors. The material can be bio-degradable or
non-bio-degradable. One important condition in selecting these
materials is their effects on the light enhancement of S-NP. In
many embodiments, such enhancement should be as high as
allowed.
[0104] The dielectric materials can be inorganic or organic either
in crystal, polycrystalline, amorphous, or hetero-mixture, and
combination of one or more thereof depends on the applications. For
examples, inorganic dielectric components can be selected from
silicon dioxide, diamond, graphite, titanium dioxide, other certain
metal oxides, and inorganic compounds in light wavelength range
smaller than their energy bandgap. Organic dielectric components
can be selected from polymers as biodegradable polymers (list
before) for certain applications, other polymer as biopolymer (e.g.
polynucleotides, cellulose), copolymer (e.g.
styrene-isoprene-styrene), conductive polymer (e.g.
poly(p-phenylene vinylene)), fluoropolymer, polyterpene, phenolic
resin, polyanhydrides, polyester, polyolefin, rubber,
superabsorbent polymer, vinyl polymer, etc.; or from small
molecules, e.g. fullerene derivative, benzene derivatives, etc. The
semiconductor materials can be inorganic or organic either in
crystal, polycrystalline, amorphous, or hetero-mixture, and
combination of one or more thereof depends on the applications. One
can use a single material or a combination of them.
Biodegradable Materials
[0105] The biodegradable polymers for S-NPs are a specific type of
polymers that is stable and durable enough for use in their
intended applications and easily breaks down (to form gases, salts,
or biomass) after its degradation.
[0106] These biodegradable polymers contains two major types:
agro-polymers (derived from biomass, e.g. poly(saccharide)s as the
starches in wood, cellulose, chitosan, proteins), and biopolyesters
(derived from micro-organisms or synthetically made from either
naturally or synthetic monomers, e.g. polyhydroxybutyrate and
polylactic acids). Most of biodegradable polymers consist of ester,
amide, or ether bonds. More examples of these biodegradable
polymers contains: poly(esters) based on polylactide (PLA),
polyglycolide (PGA), polycaprolactone (PCL), and their copolymers,
poly(hydroxyalkanoate)s of the PHB-PHV class, other hydrolytically
degradable polymers (as polyurethanes, poly(ester amide),
poly(ortho esters), polyanhydrides, poly(anhydride-co-imide),
pseudo poly(amino acide), poly(alkyl cyanoacrylates), etc.), other
enzymatically degradable polymers (proteins (as collagen,
elastion), poly(amino acids), polysaccharides, etc), and other
natural polymers. One can use a single material or a combination of
them.
Magnetic/Magnetizable Materials
[0107] The magnetic/magnetizable materials are those materials that
will experience a magnetic force in a magnetic field. The can be
selected from: ferromagnetic (e.g. iron, cobalt, nickel, some of
the rare earths (gadolinium, dysprosium), etc.), ferrimagnetic
(e.g. iron(II,III) oxide, yttrium iron garnet, cubic ferrites
composed of iron oxides and other elements such as aluminum,
cobalt, nickel, manganese and zinc, hexagonal ferrites, and
pyrrhotite, etc.), superparamagnetic, and other suitable magnetic
materials include oxides, e.g. ferrites, perovskites, chromites and
magnetoplumbites, a rare earth/cobalt alloy or any other
inorganic/organic compound with ferromagnetic/ferrimagnetic
properties. One can use a single material or a combination of
them.
[0108] Some key advantages are: the S-NPs have the right (large)
particle size and complex shape for high-performance in-vivo
plasmonic enhanced diagnostics and therapeutics, yet biodegradable,
afterwards (with controlled timing), into sub-10-nm particles with
a volume only 10% to 1% of original S-NP for quick cell/body
clearance. Furthermore, S-NPs offer a plasmonic enhancement several
orders of magnitudes higher than all current NPs.
[0109] To design different biocompatible and biodegradable
S-particles that are not only effective in in-vivo therapeutic and
diagnosis but also bio-safe, we need to control different S-NP's
architectures, materials, dimensions and shapes.
[0110] 1. Particle size requirements for efficacy of
plasmonic-based in-vivo therapeutic and diagnosis. Two factors that
determine the sizes: (a) effectiveness of plasmonic effects and (b)
the blood retention time--the time needed for sufficient
circulation in blood to deliver proper dosage of nanoparticles to
the specific sites.
[0111] To be plasmonic effective needs two things: first, the
nanoparticle size has to be in resonance with in coming wavelength;
and second, there should be small gaps and sharp edges. For the
first plasmonic requirement at the in vivo penetration light
wavelength of 800 nm (the entire window is NIR (670 to 890 nm)),
for example, conventional approaches use gold spheres of 300 nm
diameter, gold nanoshells of 60 nm diameter, and gold nanorods of
10 diameter and 60 nm long for decent plasmonic effects and decent
blood retention time. The size smaller than those above will make
these particles plasmonic extremely ineffective.
[0112] For the second plasmonic requirement, the conventional
particles do not have the complex structures (nanogaps and
nano-edges), and hence are much worse (orders of magnitude worse)
than those that have such structures, which has been proofed by our
experiment with S-particles.
[0113] To have sufficient blood retention time, the NP's size
should be -50 to 200 nm range, and cannot be too small either. Too
small particle size will make the particle cleared-out from cells
and body quickly (see below), hence failing to deliver proper
dosage, unless large nanoparticle dosage or repeated dosage of
nanoparticles are intravenously administered, which will become
unsafe and cause immunogenic response.
[0114] 2. Particle size requirements for bio-safety. Two
nanoparticle sizes are very important in bio-safety in in-vivo
diagnosis: (a) safe-particle-delivery size, which is the
nanoparticle size that can be safely delivered to the specific
sites without causing immunogenic response or any toxic effects,
assuming low NP dose and no NP accumulation, and (b) particle
clearance size, where NPs with such size can be easily cleared out
from cells/body. As shown in FIG. 11, the particle
safe-particle-delivery size has several bands, and the particle
clearance size should be <10 nm (<6 nm even better).
[0115] 3. Conflict in particle size requirements. Clearly, the size
requirements are conflicting. For conventional plasmonic particles,
which cannot change their size once are put in vivo, only way to
solve this size requirement conflict is to compromise both the
efficacy and the bio-safety to pick a particle size in the middle
to balance plasmonic enhancement and the circulation within the
body with the nanoparticles' ability to escape from the body.
[0116] The exact dimensions of these components depend on the light
wavelength and materials. In some cases particular for the light
wavelength of .about.800 nm and gold for the metallic materials and
silicon dioxide as the dielectrics, the dimensions of the
components can be in the range of 4 nm to 1500 nm and a thickness
of the disks may be from 1 nm to 500 nm, depending on the exact
wavelength of the light to be used in sensing. The gaps between
components (e.g., the gaps between the metallic disks) may be in
the range of 0.5 nm to 200 nm. For many applications, a small gap
(in the range of 5 nm to 50 nm) may be used to enhance the optical
signals.
[0117] This is one of the most challenging issues in
plasmonic-based particle in vivo. Previous approaches have very
limited success. For example, nanoparticle such as PEG-passivated
gold nanoparticles, Nanoshells and gold nanorods. These
nanoparticles employs surface coating or exotic shapes to achieve
optimum optical resonance while retain a proper size (<100 nm)
throughout the needed blood retention time. However, their ability
of tuning the optical responses is still limited, thus the optical
field enhancement is still weak. Moreover, these particles have a
size much larger than the clearance size and hence will be
accumulated in the body, becoming toxic.
S-NP Fabrication Methods
[0118] Nano-PrinTED-1 (Protrusion Template).
[0119] As shown in FIGS. 8 and 9, one embodiment of the fabrication
method, termed Nano-PrinTED using a protrusion template 1010,
comprising three key steps: (i) have a template with nanostructured
protrusions 1010 (e.g. dense nanopillar array on 4'' wafer (each
pillar of the same or similar diameter selected from 5 nm to 100
nm), (ii) deposit a release layer (optional) and then deposit
multiple-layers of materials 1020 needed to form nanoparticles on
both the pedestals of the template's protrusions (each nanoparticle
size is determined by the diameter of the template nanopillar) and
inside the trenches, and (iii) exfoliate the nanoparticles 100 from
the template.
Solvent Dissolve/Ultra-Sonic Exfoliation.
[0120] The template with S-NPs is put inside the container with
particular solvent to dissolve the release layer under the S-NPs.
The container can be put in an ultrasonicator to speed up the
exfoliation process. S-NPs will be exfoliated in the solvent.
[0121] The exfoliation can be done in several ways: (1) Transfer
printing exfoliation: The template with S-NPs is printed onto
another substrate with a thin adhesion layer 1030 (e.g. certain
polymer thin film). The larger adhesion force between the particles
and the adhesion layer exfoliate all the S-NPs onto the new
substrate. The S-NPs can be further released into solution by
dissolving the adhesion layer on the new substrate similar to
previous method. (2) Spin-on peel-off exfoliation: A kind of
adhesion thin film (e.g. certain polymer) is spinned onto the
template with S-NPs. After the curing of the film, the S-NPs are
adhered in the adhesion layer, following a peel-off process to
exfoliate all the S-NPs. The S-NPs can be further released into
solution by dissolving the adhesion film as previous method. And
(3) Wash exfoliation: The template with S-NPs are tilted above a
beaker (or other container), and washed with certain solution (with
spray gun). With the force of turbulent solution, the S-NPs will be
exfoliated from the template and into the solution.
[0122] In certain embodiments, the same metal deposition for 1020
also form the nanodots on the disk sidewall to form S-NP, due to
the fact that a thin metal on the sidewall self-assembled into
nanodots, as shown from the experimental results in FIGS. 13, 14
and 15.
[0123] In the deposition (ii), the deposition uses a beamed
materials (i.e. the material is deposited in one direction not in
the other directions) and in the angle substantially normal to the
template surface. Due the height of the pillars, the materials
deposited in on the foot of the pillars are not connected to the
materials deposited on the top of the pillar (i.e. pillars'
pedestals), making the materials deposited on the pedestals forming
S-NPs. The exfoliation free these S-NP from the template. The
template can be used repeatedly until the trenches between the
pillars are filled with material. When that happens, a cleaning
step can be used to remove the deposited materials, and then the
template can be reused again.
[0124] Nano-PrinTED-2 (Concave Template).
[0125] As shown in FIG. 10, one embodiment of the fabrication
method, termed Nano-PrinTED using a concave template, comprising
three key steps: (i) have a template with nanostructured wells
1100, (ii) deposit a release layer (optional) and then deposit
multiple-layers of materials 1120 needed to form nanoparticles in
the wells of the template's protrusions (each nanoparticle size is
determined by the diameter of the well), and (iii) exfoliate the
nanoparticles 100 from the template. Due the depth of the wells,
the materials deposited on the bottom of the wells are not
connected to the materials deposited on the top surface of the
template, making the materials deposited inside well forming
S-NPs.
[0126] All the templates can be in the form of a plate, a roller or
roll or sheet, and can be in regard material or flexible. The
template can be fabricated by nanoprint. The template materials can
be any materials that mechanical strong enough for templating and
chemical inner enough.
[0127] Methods of Deposition.
[0128] The methods to shadow deposit materials can be any method,
as long as it is more or less directional, and can evaporate the
intended materials. The deposition methods include evaporation,
sputtering and chemical or molecular beams. The evaporation further
includes the evaporation by chemical vapors, molecular beams,
electron beam heating thermal heating, laser heating, and other
heating methods. The sputtering includes the sputtering by ion,
electron, plasmon, photon (i.e. laser), and other energetic
particles.
[0129] Dip-Print.
[0130] The fabrication method of Dip-print is for patterning
biodegradable dielectrics, since such materials cannot be thermally
evaporated. The fabrication method of Dip-print, as shown in FIG.
12, first puts a thin layer of liquid polymer precursors with
proper viscosity on a plate (termed "material transfer plate"); and
then presses the template used in Nano-PrinTED against the material
transfer plate, picking up the polymer precursors only at the top
of the pillars of the template. Afterwards the polymer precursors
will be cured to form a solid polymer. The dip-printed polymers
will be used for the dielectrics between the stacked layers and for
the dielectrics that glue different columns into a single particle
(Note different viscosity liquid will be used depending upon the
gap size between the columns).
[0131] The key advantages of this new fabrication technique are (a)
form the complex structures that are needed for enhance plasmonic
effects but cannot be formed in conventional fabrication methods,
(b) have far better precision in controlling the NP structure
dimensions (including the size and shape of each individual
components, their spacing, and final particle), (c) a new way to
solve the problem in patterning biodegradable materials, and (d)
scalable to large volume and low-cost (e.g. As having demonstrated,
the fabrication rate is 2.times.10 11 particles per 4'' wafer per
run, and it can be scaled up by over three orders of magnitude in
throughput by roll-to-roll technology.) (ii) Fabrication: to
advance new nanoparticle fabrication methods, nano-PrinTED and
Dip-print, and use them together with polymer chemistry to
fabricate the S-particles with desired architectures, shapes,
dimensions, and materials with high precision. One major goal is to
achieve such precision fabrication for pillars of diameter of 6 nm
or smaller, which means to sub-6 nm size particles after
biodegradation.
Surface Functioning for Sensing
[0132] The S-NP 100 becomes a nanosensor 200 after the surface
functionalization. Surface functioning of S-NP is to modify the
properties of the S-NP's surface to control the five key surface
properties: surface shape, chemical bonding, surface charge,
hydrophobic and hydrophilic properties, and active targeting (FIG.
12).
[0133] Surface Shape.
[0134] The shape of a nanoparticle has effects of the NP's ability
to enter and exit of a biological materials, such as cell and cell
nuclei. One embodiment is to change S-NP shape after the
fabrication as needed to a desired shape. The methods of changing
the shape including coating a polymer or multilayer polymer, and
biodegradable materials.
[0135] Chemical Bonding.
[0136] One of the most important surface chemistry modifications is
to have the linkers--the materials that link different kinds of
biochemical reagent (e.g. targeting agent) onto nanoparticles.
Bio-compatible block co-polymer coatings, such as polyethylene
glycol (PEG), will act as a cross-linker in this proposed research.
We will investigate several assembling methods, particularly two
kinds. One kind is to function one end of the polymer ligands with
thiol-group to form strong bonding to the nanoparticles' gold
surface, and the other end of the linker with NHS-group to form
strong covalent bond to the primary amine-site on the antibodies or
proteins. We will also try to replace the thiol-end with silane so
that the cross-linker can be efficiently linked to dielectric
surface such as silica. The other method is using bio-affinity
reactions such as avidin-biotin bonding. Other linkers are
discussed in the section of molecular adhesion layer.
[0137] Surface Charges and Wetting Properties.
[0138] The physicochemical characteristics of a polymeric
nanoparticle such as surface charge and functional groups can
affect its uptake by the cells. For phagocytic system, it is well
accepted that positively charged nanoparticles have a higher rate
of cell uptake compared to neutral or negatively charged
formulations (due to the negatively charged character of the cell
plasma membrane). The coating on nanoparticles for positive charge
is generally based on (or coated with) cationic polymers (e.g. the
most widely used being the polysaccharide chitosan). In addition,
we need to give further consideration to the surface wetting
properties of the coating material, because for biocompatible
materials that facilitate biodegradation, it is preferred that the
coating is hydrophilic and water soluble. We will choose PEG and
PGA as the positive and negative surface, respectively. Both
materials are water soluble, non-toxic, biodegradable and have long
been used to passivate colloid gold nanoparticle to facilitate both
permeation and retention in body [13, 14]. We will test the effect
of charge on the S-nanoparticle's biodistribution and clearance on
both cellular level and organism level.
[0139] Effect of Active Targeting.
[0140] We will use active targeting method, where we further
enhance the delivery specificity by conjugation of targeting
ligands to the surface of nanoparticles. These ligands can include
antibodies, engineered antibody fragments, proteins, peptides,
small molecules, and DNA or RNA aptamers. We will study the effect
of active targeting on both cellular level and organism level.
[0141] Molecular Adhesion Layer
[0142] The capture agents for the target analytes are immobilized
either directly on S-NPs 100 or through a molecular adhesion/spacer
layer (MAL) 160. As shown in FIG. 6, S-NP 100 comprises a molecular
adhesion layer 160 that covers at least a part of the metal
surfaces of the underlying S-NP. The molecular adhesion layer has
two purposes. First, the molecular adhesion layer acts a spacer.
For optimal fluorescence, the light-emitting labels (e.g.,
fluorophores) cannot be too close to the metal surface because
non-radiation processes would quench fluorescence. Nor can the
light-emitting labels be too far from the metal surface because it
would reduce amplification. Ideally, the light-emitting labels
should be at an optimum distance from the metal surface. Second,
the molecular adhesion layer provides a good adhesion to attach
capture agent onto the S-NP. The good adhesion is achieved by
having reactive groups in the molecules of the molecular adhesion
layer, which have a high affinity to the capture agent on one side
and to the S-NPs on the other side.
[0143] The molecular adhesion layer (MAL) 160 can have many
different configurations, including (a) a self-assembled monolayer
(SAM) of cross-link molecules, (b) a multi-molecular layers thin
film, (c) a combination of (a) and (b), and (d) a capture agent
itself.
[0144] In the embodiment of MAL (a), where the molecular adhesion
layer 160 is a self-assembled monolayer (SAM) of cross-link
molecules or ligands, each molecule for the SAM comprises of three
parts: (i) head group, which has a specific chemical affinity to
the S-NP's surface, (ii) terminal group, which has a specific
affinity to the capture agent, and (iii) molecule chain, which is a
long series of molecules that link the head group and terminal
group, and its length (which determines the average spacing between
the metal to the capture agent) can affect the light amplification
of the S-NP. Such a SAM is illustrated in FIG. 3.
[0145] In many embodiments, the head group attached to the metal
surface belongs to the thiol group, e.t., --SH. Other alternatives
for head groups that attach to metal surface are, carboxylic acid
(--COOH), amine (C.dbd.N), selenol (--SeH), or phosphane (--P).
Other head groups, e.g. silane (--SiO), can be used if a monolayer
is to be coated on dielectric materials or semiconductors, e.g.,
silicon.
[0146] In many embodiments, the terminal groups can comprise a
variety of capture agent-reactive groups, including, but not
limited to, N-hydroxysuccinimidyl ester,
sulfo-N-hydroxysuccinimidyl ester, a halo-substituted phenol ester,
pentafluorophenol ester, a nitro-substituted phenol ester, an
anhydride, isocyanate, isothiocyanate, an imidoester, maleimide,
iodoacetyl, hydrazide, an aldehyde, or an epoxide. Other suitable
groups are known in the art and may be described in, e.g.,
Hermanson, "Bioconjugate Techniques" Academic Press, 2nd Ed., 2008.
The terminal groups can be chemically attached to the molecule
chain after they are assembled to the S-NP surface, or synthesized
together with the molecule chain before they are assembled on the
surface.
[0147] Other terminal groups are Carboxyl --COOH groups (activated
with EDC/NHS to form covalent binding with --NH2 on the ligand);
Amine, --NH2, group (forming covalent binding with --COOH on the
ligand via amide bond activated by EDC/NHS); Epoxy, Reacted with
the --NH2 (the ligand without the need of a cross-linker);
Aldehyde, (Reacted with the --NH2 on the ligand without the need of
a cross-linker); Thiol, --SH, (link to --NH2 on the ligand through
SMCC-like bioconjugation approach); and Glutathione, (GHS) (Ideal
for capture of the GST-tagged proteins.
[0148] The molecular chain can be carbon chains, their lengths can
be adjusted to change the distance between the light emitting label
to the metal for optimizing the optical signal. In one embodiment,
as will be described in greater detail in example section, the SAM
layer is dithiobis(succinimidyl undecanoate), whose head group is
--SH that binds to gold surface through sulfer-gold bond, and
terminal group is NHS-ester that bind to the primary amine sites of
the capture agent, and the molecule alkane chain with length of 1.7
nm.
[0149] In many embodiments, the molecule chains that link head
groups and terminal groups are alkane chain, which is composed of
only hydrogen and carbon atoms, with all bonds are single bonds,
and the carbon atoms are not joined in cyclic structures but
instead form a simple linear chain. Other alternatives for molecule
chain can be ligands that are from polymers such as poly(ethylene
glycol) (PEG), Poly(lactic acid) (PLA), etc. The molecule chains
are chemically non-reactive to neither the metal surface that the
head groups attach to, nor the capture agent that the terminal
groups attach to. The chain length, which determines the distance
of analyte to the S-NP's surface, can be optimized in order to
achieve the maximum signal amplification. As will be described in
greater detail below, the molecule chains may have a thickness of,
e.g., 0.5 nm to 50 nm.
[0150] The molecular adhesion layer used in the subject nanosensor
may be composed of a self-assembled monolayer (SAM) that is
strongly attached to the metal at one side (via, e.g., a sulfur
atom) and that terminates a capture-agent-reactive group, e.g., an
amine-reactive group, a thiol-reactive group, a hydroxyl-reactive
group, an imidazolyl-reactive group and a guanidinyl-reactive
group, at the other (exterior) side. The monolayer may have a
hydrophobic or hydrophilic surface. The most commonly used
capture-agent reactive groups are NHS (which is amine-reactive) and
maleimide (which is sulfhydryl-reactive), although many others may
be used.
[0151] In some embodiments, the molecular adhesion layer may be a
self-assembled monolayer of an alkanethiol (see, e.g., Kato Journal
of Physical Chemistry 2002 106: 9655-9658), poly(ethylene)glycol
thiol (see, e.g., Shenoy et al Int. J. Nanomedicine. 2006 1:
51-57), an aromatic thiol or some other chain that terminates in
the thiol.
[0152] Thiol groups may be used because (a) the thiol sulfur
interacts with gold and other metals to form a bond that is both
strong and stable bond (see, e.g., Nuzzo et al J. Am. Chem. Soc.
1987 109:2358-2368) and (b) van der Waals forces cause the alkane
and other chains chains to stack, which causes a SAM to organize
spontaneously (see, e.g., Love et al. Chem. Rev. 2006
105:1103-1169). Further, the terminal group is available for either
direct attachment to the capture molecule or for further chemical
modifications.
[0153] Alkanethiol may be used in some embodiments. It has been
estimated that there are 4.times.10.sup.14 alkanethiol
molecules/cm.sup.2 in a packed monolayer of alkanethiol (Nuzzo et
al, J. Am. Chem. Soc. 1987 109:733-740), which approximately
corresponds to an alkanethiol bond to every gold atom on the
underlying surface. Self-assembled monolayers composed of
alkanethiol can be generated by soaking the gold substrate in an
alkanethiol solution (see, e.g., Lee et al Anal. Chem. 2006 78:
6504-6510). Gold is capable of reacting with both reduced
alkanethiols (--SH groups) and alkyldisulfides(--S--S--) (see,
e.g., Love et al Chem. Rev. 2005 105:1103-1169).
[0154] Once a self-assembled monolayer of poly(ethylene)glycol
thiol or alkanethiol has been produced, a large number of
strategies can be employed to link a capture to the self-assembled
monolayer. In one embodiment, a capture agent such as streptavidin
(SA) can be attached to the SAM to immobilize biotinylated capture
agents.
[0155] In one embodiment, streptavidin (SA) itself can be use as a
functional group (e.g. terminal group) the SAM to crosslink capture
agent molecules that have high binding affinity to SA, such as
biotinylated molecules, including peptides, oligonucleotides,
proteins and sugars.
[0156] The functional group of avidin, streptavidin have a high
affinity to the biotin group to form avidin-biotin. Such high
affinity makes avidin/streptavidin serve well as a functional group
and the biotin group as complementary functional group binding.
Such functional group can be in binding the molecular adhesion
layer to the S-NP, in binding between molecular adhesion layer and
the capature agent, and in binding a light emitting lable to the
secondary capture agent. In one embodiment, a molecular adhesion
layer containing thiol-reactive groups may be made by linking a
gold surface to an amine-terminated SAM, and further modifying the
amine groups using
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC) to yield a maleimide-activated surface.
Maleimide-activated surfaces are reactive thiol groups and can be
used to link to capture agents that contain thiol- (e.g., cysteine)
groups.
[0157] In another embodiment, a molecular adhesion layer containing
an amine-reactive group (N-hydroxl succinimide (NHS)) can be
produced by, e.g., by soaking the gold substrate in a 1-10 mM
solution of succinimidyl alkanedisulfides such as
dithiobis-sulfosuccinimidylpropionate (DSP) or
dithiobis(succinimidyl undecanoate) (see, e.g., Peelen et al J.
Proteome Res. 2006 5:1580-1585 and Storri et al Biosens.
Bioelectron. 1998 13: 347-357).
[0158] In another embodiment, a molecular adhesion layer containing
an amine-reactive group (NHS) may be produced using
carboxyl-terminated SAM such as 12-carboxy-1-undecanethiol. In this
case, the surface of the SAM may be linked to the NHS in the
presence of 1-ethyl-3(3dimethylaminopropyl)carbodiimide HCl (EDC)
to yield an inter-mediate which forms stable amide bonds with
primary amines (see, e.g., Johnsson et al Anal. Biochem. 1001 198:
268-277).
[0159] In another embodiment, a molecular adhesion layer may
contain Protein A which binds with high affinity to Fc region of
IgGs, other immunoglobulin form, e.g., IgE.
[0160] In another embodiment, an imidazole group (which is also
reactive with amines) may be added by reacting a
carboxyl-terminated SAM with 1,1'-carbonyldiimidazole (CDI).
[0161] In further embodiments, aldehyde-terminated alkanethiol
monolayers can be used to immobilize both proteins and
amine-terminated DNA oligonucleotides, and his-tagged fusion
proteins can be immobilized on nitrilotriacetic (NTA)-modified gold
surfaces.
[0162] Thiol-reactive groups can link to synthetic DNA and RNA
oligonucleotides, including aptamers, which can be readily
synthesized commercially with a thiol terminus. Thiol-reactive
groups can also link to proteins that contain a cysteine groups,
e.g., antibodies. Thiolated molecules can be attached to
maleimide-modified surfaces (see, e.g., Smith et al Langmuir 2002
19: 1486-1492). For in certain cases, one may use an amino acid
spacer (e.g., Ser-Gly-Ser-Gly) inserted after a terminal Cys, which
improves the amount of binding relative peptides that lacking
spacers. For oligonucleotides, an alkane spacer can be used.
Carbohydrates synthesized to contain with terminal thiols can be
been tethered to gold in the same way.
[0163] Amine-reactive groups can form bonds with primary amines,
such as the free amine on lysine residues. In addition to proteins,
amine-reactive surfaces can be used to immobilize other
biomolecules, including peptides containing lysine residues and
oligonucleotides synthesized with an amine terminus.
[0164] In the embodiment of MAL (b), in which the molecular
adhesion layer 160 is a multi-molecular layer thin film, the
molecules may be coated on the S-NP through physical adsorption or
strong binding. In one example, protein A can be coated over the
entire or partial areas of the surface of S-NP surface, in which
case the protein A can be deposited through physical adsorption
process and has a thickness of 4 nm to 5 nm. In another example,
the layer may be a thin film of a polymer such as polyethylene
glycol (PEG), which has a functional head group on one end, e.g.,
thiol (--SH). The functioned PEG molecule layer forms a strong bond
to S-NP's surface. The thickness of PEG molecule layer can be tuned
by changing the PEG polymer chain length. Another example is an
amorphous SiO.sub.2 thin film, which is attached to the surface of
the S-NP using physical or chemical deposition methods, e.g.,
evaporation, sputtering, sol-gel method. The thickness of the
SiO.sub.2 thin film can be precisely controlled during the
deposition.
[0165] In the embodiment of MAL (c), where the molecular adhesion
layer 160 is a combination of a multi-molecular layer thin film and
a SAM, the SAM layer may be deposited first, followed by a
multi-molecular layer.
[0166] In one example, the molecular adhesion layer may contain a
monolayer of streptavidin first, followed by other layers of
molecules that have high binding affinity to streptavidin, such as
biotin, biotinylated molecules, including peptides,
oligonucleotides, proteins, and surgars.
[0167] In one example, the molecular adhesion layer, may contain a
SAM layer dithiobis(succinimidyl undecanoate) (DSU) and a Protein A
layer. The DSU SAM layer binds to S-NP's metal surface through
sulfer-gold bond, and has a terminal group of NHS-ester that binds
to the primary amine sites on Protein A. In a particular case,
capture antibodies bond to such bilayer of protein A on top of DSU
through their Fc region. The protein A can ensure the orientation
of antibodies for better capture efficiency.
[0168] In the embodiment of MAL (d), where the molecular adhesion
layer 160 is a capture agent itself, the capture agent has a
headgroup that have a high affinity to the metal or pillar sidewall
of the subject S-NP. One of the common headgroup is thiol-reactive
group. Thiol-reactive groups can link to synthetic DNA and RNA
oligonucleotides, including aptamers, which can be readily
synthesized commercially with a thiol terminus. Thiol-reactive
groups can also link to proteins that contain a cysteine groups,
e.g., antibodies. Another example where the MAL itself is used as
the capture agent is a layer of antibody fragments, e.g., half-IgG,
Fab, F(ab')2, Fc. The antibody fragments bond to metal surface
directly through the thiol-endopeptidase located in the hinge
region. This embodiment is illustrated in FIG. 8. In this
embodiment, the nucleic acid comprises a headgroup that binds
directly the S-NP. The remainder of the steps are performed as
described in FIG. 7.
[0169] The thickness of molecular adhesion layer should be in the
range of 0.5 nm to 50 nm, e.g., 1 nm to 20 nm. The thickness of the
molecular adhesion layer can be optimized to the particular
application by, e.g., increasing or decreasing the length of the
linker (the alkane or poly(ethylene glycol) chain) of the SAM used.
Assuming each bond in the linker is 0.1 nM to 0.15 nM, then an
optimal SAM may contain a polymeric linker of 5 to 50 carbon atoms,
e.g., 10 to 20 carbon atoms in certain cases.
[0170] A nanosensor may be made by attaching capture agents to the
molecular adhesion layer via a reaction between the capture agent
and a capture-agent reactive group on the surface of the molecular
adhesion layer.
[0171] Capture agents can be attached to the molecular adhesion
layer via any convenient method such as those discussed above. In
many cases, a capture agent may be attached to the molecular
adhesion layer via a high-affinity strong interactions such as
those between biotin and streptavidin. Because streptavidin is a
protein, streptavidin can be linked to the surface of the molecular
adhesion layer using any of the amine-reactive methods described
above. Biotinylated capture agents can be immobilized by spotting
them onto the streptavidin. In other embodiments, a capture agent
can be attached to the molecular adhesion layer via a reaction that
forms a strong bond, e.g., a reaction between an amine group in a
lysine residue of a protein or an aminated oligonucleotide with an
NHS ester to produce an amide bond between the capture agent and
the molecular adhesion layer. In other embodiment, a capture agent
can be strongly attached to the molecular adhesion layer via a
reaction between a sulfhydryl group in a cysteine residue of a
protein or a sulfhydryl-oligonucleotide with a sulfhydryl-reactive
maleimide on the surface of the molecular adhesion layer. Protocols
for linking capture agents to various reactive groups are well
known in the art.
[0172] In one embodiment, capture agent can be nucleic acid to
capture proteins, or capture agent can be proteins that capture
nucleic acid, e.g., DNA, RNA. Nucleic acid can bind to proteins
through sequence-specific (tight) or non-sequence specific (loose)
bond.
[0173] In certain instances, a subject S-NP may be fabricated using
the method: (a) patterning at least one pillar on a top surface of
a substrate; (b) depositing a metallic material layer of the top
surface; (c) allowing the metallic material deposited on the pillar
tops to form a disc, the metallic material deposited on the pillar
feet to form a metallic back plane, and the metallic material
deposited on the sidewall to form at least one metallic dot
structure; and, as described above, (d) depositing a molecular
adhesion layer on top of the deposited metallic material, wherein
the molecular adhesion layer covers at least a part of the metallic
dot structure, the metal disc, and/or the metallic back plane, and
wherein the exterior surface of the molecular adhesion layer
comprises a capture agent-reactive group.
[0174] Furthermore, the patterning in (a) include a direct
imprinting (embossing) of a material, which can be dielectric or
semiconductor in electric property, and can be polymers or polymers
formed by curing of monomers or oligomers, or amorphous inorganic
materials. The material can be a thin film with a thickness from 10
nanometer to 10 millimeter, or multilayer materials with a
substrate. The imprinting (i.e. embossing) means to have mold with
a structure on its surface, and press the mold into the material to
be imprinted to for an inverse of the structure in the material.
The substrates or the top imprinted layers can be a plastic (i.e.
polymers), e.g. polystyring (PS), Poly(methyl methacrylate) (PMMA),
Polyethylene terephthalate (PET), other acrylics, and alike. The
imprinting may be done by roll to roll technology using a roller
imprinter. Such process has a great economic advantage and hence
lowering the cost.
Sensing Systems
[0175] Also provided is a system comprising a subject nanosensor, a
holder for the nanosensor, an excitation source that induces a
light signal from a label (i.e. light emitting label); and a reader
(e.g., a photodetector, a CCD camera, a CMOS camera, a spectrometer
or an imaging device capable of producing a two dimensional
spectral map of a surface of the nanosensor) adapted to read the
light signal. As would be apparent, the system may also has
electronics, computer system, software, and other hardware that
amplify, filter, regulate, control and store the electrical signals
from the reader, and control the reader and sample holder
positions. The sample holder position can be move in one or all
three orthogonal directions to allow the reader to scan the light
signal from different locations of the sample.
[0176] The excitation source may be (a) a light source, e.g., a
laser of a wavelength suitable for exciting a particular
fluorophore, and a lamp or a light emitting diode with a light
filter for wavelength selection; or (b) a power source for
providing an electrical current to excite light out of the
nanosensor (which may be employed when an electrochemiluminescent
label is used).
[0177] In particular cases, laser-line pass filter filters out
light whose wavelength is different from the laser, and the long
wavelength pass filter will only allow the light emanate from the
optically detectable label to pass through. Since different
fluorescence labels absorb light in different spectral range, the
fluorescence label should be chosen to match its peak absorption
wavelength to the laser excitation wavelength in order to achieve
optimum quantum efficiency. In many embodiments, the light signal
emanating from the fluorescence label on the nanosensors are at a
wavelength of at least 20 nm higher than the laser wavelength. Thus
the nanosensor's plasmonic resonance should be tuned to cover the
fluorescence label's abosprtion peak, emission peak and laser
excitation wavelength. In some embodiments, the excitation and
fluorescence wavelength range can be from 100 nm to 20,000 nm. The
preferred range is from 300 nm to 1200 nm. The 600-850 nm range is
preferable due to low background noise.
[0178] It is apparent there are other ways to achieve the functions
of light excitation and reading.
[0179] As would be apparent from the above, certain nanosensors may
be implemented in a multi-well format. In these embodiments, the
stage can move moved so that reader can read a light signal from
each of the wells of the multi-well plate, independently.
Applications in Chemical and Biological Sensing and Assay
Methods
[0180] The functionalized S-particles can be used as biological and
chemical sensing, including detection of biological and chemical
markers, such proteins, DNAs, RNAs, and other organic and inorganic
molecules, in single cells, tissue, and in-vivo for human and
animals, and diagnosis.
[0181] Here diagnosis means to assess the condition of certain
disease or condition by quantitative detection of certain
biomarkers or biomolecules. Such diagnosis by S-NPs include
detection of proteins, nucleic acids, micro-organisms (virus,
bacteria, etc.) and mall molecules (hormones, etc.).
[0182] The methods of diagnosis by S-NPs include In vitro
detection, fluorescence based detection, homogeneous fluorescence
immunoassay (Alpha-LISA), flow-cytometery based detection,
colorimetric detection, bio-bar-code Assay, SERS-based detection,
SERS label homogeneous immunoassay, multiplex SERS label, in vivo
detection, fluorescence Imaging (e.g. tumor cells), optical
tomography and MRI.
[0183] The subject nanosensor may be used to detect analytes in a
sample. This method may comprise: (a) contacting a sample
comprising an analyte with a nanosensor under conditions suitable
for specific binding of an analyte in the sample with the capture
agent; and (b) reading an optically detectable signal from the
nanosensor, wherein the optically detectable signal indicates that
the analyte is bound to the capture agent. In the above step (a),
before the bonding to the capture agent, the analyte may be labeled
with a light-emitting label or not labeled (also referred as
labeled directly or indirectly). In embodiments in which an analyte
is no labeled with a light-emitting label before the bonding, the
analyte, after the bonding to the capture agent, may be bound to a
second capture agent (i.e. detection agent) (e.g., a secondary
antibody or another nucleic acid) that is itself optically labeled,
labeled secondary capture agent or labeled detection agent, (such
process is also referred as indirectly labeling of an analyte). In
a sensing using indirectly labeling, the labeled secondary capture
agents unbounded to analytes are removed before the above reading
step (b). In a sensing using directly labeling, the optical labels
unbounded to analytes are removed before the above reading step
(b).
[0184] In reading the light emitting labels on the assay, an
excitation (photo, electro, chemical or combination of them) are
applied to light emitting label, and the properties of light
including intensity, wavelength, and location are detected.
[0185] In certain embodiments, the method comprises attaching a
capture agent to the molecular adhesion layer of a subject S-NP to
produce a nanosensor, wherein the attaching is done via a chemical
reaction of the capture agent with the capture agent-reactive group
in the molecules on the molecular adhesion layer, as described
above. Next, the method comprises contacting a sample containing a
target-analyte with the nanosensor and the contacting is done under
conditions suitable for specific binding and the target-analyte
specifically binds to the capture agent. After this step, the
method comprises removing any target-analytes that are not bound to
the capture agent (e.g., by washing the surface of the nanosensor
in binding buffer); Then detection agent conjugated with optical
detectable label is added to detect the target-analyte. After
removing the detection agent that are not bound to the
target-analyte, The S-NP can then be used, with a reading system,
to read a light signal (e.g., light at a wavelength that is in the
range of 300 nm to 1200 nm) from detection agent that remain bound
to the nanosensor. As would be apparent, the method further
comprises labeling the target analytes with a light-emitting label.
This can be done either prior to or after the contacting step,
i.e., after the analytes are bound to the capture agent. In certain
embodiments, analytes are labeled before they are contacted with
the nanosensor. In other embodiment, the analytes are labeled after
they are bound to the capture agents of the nanosensor. Further, as
mentioned above, the analyte may be labeled directly (in which case
the analyte may be strongly linked to a light-emitting label at the
beginning of the method), or labeled indirectly (i.e., by binding
the target analytes to a second capture agent, e.g., a secondary
antibody that is labeled or a labeled nucleic acid, that
specifically binds to the target analyte and that is linked to a
light-emitting label). In some embodiments, the method may comprise
blocking the nanosensor prior to the contacting step (b), thereby
preventing non-specific binding of the capture agents to non-target
analytes.
[0186] The suitable conditions for the specific binding and the
target-analyte specifically binds to the capture agent, include
proper temperature, time, solution pH level, ambient light level,
humidity, chemical reagent concentration, antigen-antibody ratio,
etc.
[0187] In certain embodiments, a nucleic acid capture agent can be
used to capture a protein analyte (e.g., a DNA or RNA binding
protein). In alternative embodiments, the protein capture agent
(e.g., a DNA or RNA binding protein) can be used to capture a
nucleic acid analyte.
[0188] The sample may be a liquid sample and, in certain
embodiments, the sample may be a clinical sample derived from
cells, tissues, or bodily fluids. Bodily fluids of interest include
but are not limited to, amniotic fluid, aqueous humour, vitreous
humour, blood (e.g., whole blood, fractionated blood, plasma,
serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen
(earwax), chyle, chime, endolymph, perilymph, feces, gastric acid,
gastric juice, lymph, mucus (including nasal drainage and phlegm),
pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum,
saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid,
tears, vomit, urine and exhaled condensate.
[0189] Some of the steps of an assay are shown in FIGS. 7 and 8.
General methods for methods for molecular interactions between
capture agents and their binding partners (including analytes) are
well known in the art (see, e.g., Harlow et al., Antibodies: A
Laboratory Manual, First Edition (1988) Cold spring Harbor, N.Y.;
Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed.,
Wiley & Sons, 1995). The methods shown in FIGS. 4 and 5 are
exemplary; the methods described in those figures are not the only
ways of performing an assay.
[0190] Some of the steps of an exemplary antibody binding assay are
shown in FIG. 5. In this assay, S-NP 100 is linked to an antibody
in accordance with the methods described above to produce a
nanosensor 200 that comprises antibodies 202 that are linked to the
molecular adhesion layer of the S-NP. After nanosensor 200 has been
produced, the nanosensor is contacted with a sample containing a
target analyte (e.g., a target protein) under conditions suitable
for specific binding. The antibodies 202 specifically bind to
target analyte 204 in the sample. After unbound analytes have been
washed from the nanosensor, the nanosensor is contacted with a
secondary antibody 206 that is labeled with a light-emitting label
208 under conditions suitable for specific binding. After unbound
secondary antibodies have been removed from the nanosensor, the
nanosensor may be read to identify and/or quantify the amount of
analyte 204 in the initial sample.
[0191] Some of the steps of an exemplary nucleic acid binding assay
are shown in FIGS. 6 and 7. In this assay, S-NP 100 is linked to a
nucleic acid, e.g., an oligonucleotide in accordance with the
methods described above to produce a nanosensor 300 that comprises
nucleic acid molecules 302 that are linked to the molecular
adhesion layer. After nanosensor 300 has been produced, the
nanosensor is contacted with a sample containing target nucleic
acid 304 under conditions suitable for specific hybridization of
target nucleic acid 304 to the nucleic acid capture agents 302.
Nucleic acid capture agents 304 specifically binds to target
nucleic acid 304 in the sample. After unbound nucleic acids have
been washed from the nanosensor, the nanosensor is contacted with a
secondary nucleic acid 306 that is labeled with a light-emitting
label 308 under conditions for specific hybridization. After
unbound secondary nucleic acids have been removed from the
nanosensor, the nanosensor may be read to identify and/or quantify
the amount of nucleic acid 304 in the initial sample.
[0192] One example of an enhanced DNA hybridization assay that can
be performed using a subject device is a sandwich hybridization
assay. The capture DNA is a single strand DNA functioned with thiol
at its 3'-end The detection DNA is a single strand DNA functioned
with a fluorescence label e.g., IRDye800CW at its 3'-end. Both the
capture and detection DNA has a length of 20 bp. They are
synthesized with different sequences to form complementary binding
to a targeted DNA at different region. First the capture DNA is
immobilized on the S-NP's metal surface through sulfur-gold
reaction. Then targeted DNA is added to the S-NP to be captured by
the capture DNA. Finally the fluorescence labeled detection DNA is
added to the S-NP to detect the immobilized targeted DNA. After
washing off the unbound detection DNA, the fluorescence signal
emanate from the S-NPs' surface is measured for the detection and
quantification of targeted DNA molecules.
[0193] In the embodiments shown in FIGS. 5 and 6, bound analyte can
be detected using a secondary capture agent (i.e. the "detection
agent") may be conjugated to a fluorophore or an enzyme that
catalyzes the synthesis of a chromogenic compound that can be
detected visually or using an imaging system. In one embodiment,
horseradish peroxidase (HRP) may be used, which can convert
chromogenic substrates (e.g., TMB, DAB, or ABTS) into colored
products, or, alternatively, produce a luminescent product when
chemiluminescent substrates are used. In particular embodiments,
the light signal produced by the label has a wavelength that is in
the range of 300 nm to 900 nm). In certain embodiments, the label
may be electrochemiluminescent and, as such, a light signal can be
produced by supplying a current to the sensor.
[0194] In some embodiments, the secondary capture agent (i.e. the
detection agent), e.g., the secondary antibody or secondary nucleic
acid, may be linked to a fluorophore, e.g., xanthene dyes, e.g.
fluorescein and rhodamine dyes, such as fluorescein isothiocyanate
(FITC), 6-carboxyfluorescein (commonly known by the abbreviations
FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX),
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J),
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T),
6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G.sup.5
or G.sup.5), 6-carboxyrhodamine-6G (R6G.sup.6 or G.sup.6), and
rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins,
e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258;
phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes;
carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes,
e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline
dyes. Specific fluorophores of interest that are commonly used in
subject applications include: Pyrene, Coumarin,
Diethylaminocoumarin, FAM, Fluorescein Chlorotriazinyl,
Fluorescein, R110, Eosin, JOE, R6G, Tetramethylrhodamine, TAMRA,
Lissamine, ROX, Napthofluorescein, Texas Red, Napthofluorescein,
Cy3, and Cy5, IRDye800, IRDye800CW, Alexa 790, Dylight 800,
etc.
[0195] The primary and secondary capture agents should bind to the
target analyte with highly-specific affinity. However, the primary
and secondary capture agents cannot be the molecule because they
need to bind to different sites in the antigen. One example is the
anti-human beta amyloid capture antibody 6E10 and detection G210,
in which case 6E10 binds only to the 10.sup.th amine site on human
beta amyloids peptide while G210 binds only to the 40.sup.th amine
site. Capture agent and secondary capture agent do not react to
each other. Another example uses rabbit anti-human IgG as capture
antibody and donkey anti-human IgG as detection antibody. Since the
capture and detection agents are derived from different host
species, they do not react with each other.
[0196] Methods for labeling proteins, e.g., secondary antibodies,
and nucleic acids with fluorophores are well known in the art.
Chemiluminescent labels include acridinium esters and sulfonamides,
luminol and isoluminol; electrochemiluminescent labels include
ruthenium (II) chelates, and others are known.
Applications
[0197] The subject methods and compositions find use in a variety
applications, where such applications are generally analyte
detection applications in which the presence of a particular
analyte in a given sample is detected at least qualitatively, if
not quantitatively. Protocols for carrying out analyte detection
assays are well known to those of skill in the art and need not be
described in great detail here. Generally, the sample suspected of
comprising an analyte of interest is contacted with the surface of
a subject nanosensor under conditions sufficient for the analyte to
bind to its respective capture agent that is tethered to the
sensor. The capture agent has highly specific affinity for the
targeted molecules of interest. This affinity can be
antigen-antibody reaction where antibodies bind to specific epitope
on the antigen, or a DNA/RNA or DNA/RNA hybridization reaction that
is sequence-specific between two or more complementary strands of
nucleic acids. Thus, if the analyte of interest is present in the
sample, it likely binds to the sensor at the site of the capture
agent and a complex is formed on the sensor surface. Namely, the
captured analytes are immobilized at the sensor surface. After
removing the unbounded analytes, the presence of this binding
complex on the surface of the sensor (i.e. the immobilized analytes
of interest) is then detected, e.g., using a labeled secondary
capture agent.
[0198] Specific analyte detection applications of interest include
hybridization assays in which the nucleic acid capture agents are
employed and protein binding assays in which polypeptides, e.g.,
antibodies, are employed. In these assays, a sample is first
prepared and following sample preparation, the sample is contacted
with a subject nanosensor under specific binding conditions,
whereby complexes are formed between target nucleic acids or
polypeptides (or other molecules) that are complementary to capture
agents attached to the sensor surface.
[0199] In one embodiment, the capture oligonucleotide is
synthesized single strand DNA of 20-100 bases length, that is
thiolated at one end. These molecules are are immobilized on the
S-NPs' surface to capture the targeted single-strand DNA (which may
be at least 50 bp length) that has a sequence that is complementary
to the immobilized capture DNA. After the hybridization reaction, a
detection single strand DNA (which can be of 20-100 bp in length)
whose sequence are complementary to the targeted DNA's unoccupied
nucleic acid is added to hybridize with the target. The detection
DNA has its one end conjugated to a fluorescence label, whose
emission wavelength are within the plasmonic resonance of the S-NP.
Therefore by detecting the fluorescence emission emanate from the
S-NPs' surface, the targeted single strand DNA can be accurately
detected and quantified. The length for capture and detection DNA
determine the melting temperature (nucleotide strands will separate
above melting temperature), the extent of misparing (the longer the
strand, the lower the misparing). One of the concerns of choosing
the length for complementary binding depends on the needs to
minimize misparing while keeping the melting temperature as high as
possible. In addition, the total length of the hybridization length
is determined in order to achieve optimum signal amplification.
[0200] A subject sensor may be employed in a method of diagnosing a
disease or condition, comprising: (a) obtaining a liquid sample
from a patient suspected of having the disease or condition, (b)
contacting the sample with a subject nanosensor, wherein the
capture agent of the nanosensor specifically binds to a biomarker
for the disease and wherein the contacting is done under conditions
suitable for specific binding of the biomarker with the capture
agent; (c) removing any biomarker that is not bound to the capture
agent; and (d) reading a light signal from biomarker that remain
bound to the nanosensor, wherein a light signal indicates that the
patient has the disease or condition, wherein the method further
comprises labeling the biomarker with a light-emitting label,
either prior to or after it is bound to the capture agent. As will
be described in greater detail below, the patient may suspected of
having cancer and the antibody binds to a cancer biomarker. In
other embodiments, the patient is suspected of having a
neurological disorder and the antibody binds to a biomarker for the
neurological disorder.
[0201] The applications of the subject sensor include, but not
limited to, (a) the detection, purification and quantification of
chemical compounds or biomolecules that correlates with the stage
of certain diseases, e.g., infectious and parasitic disease,
injuries, cardiovascular disease, cancer, mental disorders,
neuropsychiatric disorders and organic diseases, e.g., pulmonary
diseases, renal diseases, (b) the detection, purification and
quantification of microorganism, e.g., virus, fungus and bacteria
from environment, e.g., water, soil, or biological samples, e.g.,
tissues, bodily fluids, (c) the detection, quantification of
chemical compounds or biological samples that pose hazard to food
safety or national security, e.g. toxic waste, anthrax, (d)
quantification of vital parameters in medical or physiological
monitor, e.g., glucose, blood oxygen level, total blood count, (e)
the detection and quantification of specific DNA or RNA from
biosamples, e.g., cells, viruses, bodily fluids, (f) the sequencing
and comparing of genetic sequences in DNA in the chromosomes and
mitochondria for genome analysis or (g) to detect reaction
products, e.g., during synthesis or purification of
pharmaceuticals.
[0202] The detection can be carried out in various sample matrix,
such as cells, tissues, bodily fluids, and stool. Bodily fluids of
interest include but are not limited to, amniotic fluid, aqueous
humour, vitreous humour, blood (e.g., whole blood, fractionated
blood, plasma, serum, etc.), breast milk, cerebrospinal fluid
(CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces,
gastric acid, gastric juice, lymph, mucus (including nasal drainage
and phlegm), pericardial fluid, peritoneal fluid, pleural fluid,
pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat,
synovial fluid, tears, vomit, urine and exhaled condensate.
[0203] In some embodiments, a subject biosensor can be used
diagnose a pathogen infection by detecting a target nucleic acid
from a pathogen in a sample. The target nucleic acid may be, for
example, from a virus that is selected from the group comprising
human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2), human
T-cell leukaemia virus and 2 (HTLV-1 and HTLV-2), respiratory
syncytial virus (RSV), adenovirus, hepatitis B virus (HBV),
hepatitis C virus (HCV), Epstein-Barr virus (EBV), human
papillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus
(CMV), herpes-simplex virus 1 and 2 (HSV-1 and HSV-2), human
herpesvirus 8 (HHV-8, also known as Kaposi sarcoma herpesvirus) and
flaviviruses, including yellow fever virus, dengue virus, Japanese
encephalitis virus and West Nile virus. The present invention is
not, however, limited to the detection of DNA sequences from the
aforementioned viruses, but can be applied without any problem to
other pathogens important in veterinary and/or human medicine.
[0204] Human papillomaviruses (HPV) are further subdivided on the
basis of their DNA sequence homology into more than 70 different
types. These types cause different diseases. HPV types 1, 2, 3, 4,
7, 10 and 26-29 cause benign warts. HPV types 5, 8, 9, 12, 14, 15,
17 and 19-25 and 46-50 cause lesions in patients with a weakened
immune system. Types 6, 11, 34, 39, 41-44 and 51-55 cause benign
acuminate warts on the mucosae of the genital region and of the
respiratory tract. HPV types 16 and 18 are of special medical
interest, as they cause epithelial dysplasias of the genital mucosa
and are associated with a high proportion of the invasive
carcinomas of the cervix, vagina, vulva and anal canal. Integration
of the DNA of the human papillomavirus is considered to be decisive
in the carcinogenesis of cervical cancer. Human papillomaviruses
can be detected for example from the DNA sequence of their capsid
proteins L1 and L2. Accordingly, the method of the present
invention is especially suitable for the detection of DNA sequences
of HPV types 16 and/or 18 in tissue samples, for assessing the risk
of development of carcinoma.
[0205] In some cases, the nanosensor may be employed to detect a
biomarker that is present at a low concentration. For example, the
nanosensor may be used to detect cancer antigens in a readily
accessible bodily fluids (e.g., blood, saliva, urine, tears, etc.),
to detect biomarkers for tissue-specific diseases in a readily
accessible bodily fluid (e.g., a biomarkers for a neurological
disorder (e.g., Alzheimer's antigens)), to detect infections
(particularly detection of low titer latent viruses, e.g., HIV), to
detect fetal antigens in maternal blood, and for detection of
exogenous compounds (e.g., drugs or pollutants) in a subject's
bloodstream, for example.
[0206] The following table provides a list of protein biomarkers
that can be detected using the subject nanosensor (when used in
conjunction with an appropriate monoclonal antibody), and their
associated diseases. One potential source of the biomarker (e.g.,
"CSF"; cerebrospinal fluid) is also indicated in the table. In many
cases, the subject biosensor can detect those biomarkers in a
different bodily fluid to that indicated. For example, biomarkers
that are found in CSF can be identified in urine, blood or saliva,
for example.
TABLE-US-00001 Marker disease A.beta.42, amyloid beta-protein (CSF)
Alzheimer's disease. fetuin-A (CSF) multiple sclerosis. tau (CSF)
niemann-pick type C. secretogranin II (CSF) bipolar disorder. prion
protein (CSF) Alzheimer disease, prion disease Cytokines (CSF)
HIV-associated neurocognitive disorders Alpha-synuclein (CSF)
parkinsonian disorders (neuordegenerative disorders) tau protein
(CSF) parkinsonian disorders neurofilament light chain (CSF) axonal
degeneration parkin (CSF) neuordegenerative disorders PTEN induced
putative kinase 1 (CSF) neuordegenerative disorders DJ-1 (CSF)
neuordegenerative disorders leucine-rich repeat kinase 2 (CSF)
neuordegenerative disorders mutated ATP13A2 (CSF) Kufor-Rakeb
disease Apo H (CSF) parkinson disease (PD) ceruloplasmin (CSF) PD
Peroxisome proliferator-activated receptor PD gamma coactivator-1
alpha (PGC-1.alpha.)(CSF) transthyretin (CSF) CSF rhinorrhea (nasal
surgery samples) Vitamin D-binding Protein (CSF) Multiple Sclerosis
Progression proapoptotic kinase R (PKR) and its AD phosphorylated
PKR (pPKR) (CSF) CXCL13 (CSF) multiple sclerosis IL-12p40, CXCL13
and IL-8 (CSF) intrathecal inflammation Dkk-3 (semen) prostate
cancer p14 endocan fragment (blood) Sepsis: Endocan, specifically
secreted by activated-pulmonary vascular endothelial cells, is
thought to play a key role in the control of the lung inflammatory
reaction. Serum (blood) neuromyelitis optica ACE2 (blood)
cardiovascular disease autoantibody to CD25 (blood) early diagnosis
of esophageal squamous cell carcinoma hTERT (blood) lung cancer
CAI25 (MUC 16) (blood) lung cancer VEGF (blood) lung cancer sIL-2
(blood) lung cancer Osteopontin (blood) lung cancer Human
epididymis protein 4 (HE4) (blood) ovarian cancer Alpha-Fetal
Protein (blood) pregnancy Albumin (urine) diabetics albumin (urine)
uria albuminuria microalbuminuria kidney leaks AFP (urine) mirror
fetal AFP levels neutrophil gelatinase-associated lipocalin (NGAL)
Acute kidney injury (urine) interleukin 18 (IL-18) (urine) Acute
kidney injury Kidney Injury Molecule -1 (KIM-1) (urine) Acute
kidney injury Liver Fatty Acid Binding Protein (L-FABP) (urine)
Acute kidney injury LMP1 (saliva) Epstein-Barr virus oncoprotein
(nasopharyngeal carcinomas) BARF1 (saliva) Epstein-Barr virus
oncoprotein (nasopharyngeal carcinomas) IL-8 (saliva) oral cancer
biomarker carcinoembryonic antigen (CEA) (saliva) oral or salivary
malignant tumors BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and Lung
cancer LZTS1 (saliva) alpha-amylase (saliva) cardiovascular disease
carcinoembryonic antigen (saliva) Malignant tumors of the oral
cavity CA 125 (saliva) Ovarian cancer IL8 (saliva) spinalcellular
carcinoma. thioredoxin (saliva) spinalcellular carcinoma. beta-2
microglobulin levels - monitor activity of HIV the virus (saliva)
tumor necrosis factor-alpha receptors - monitor HIV activity of the
virus (saliva) CA15-3 (saliva) breast cancer
[0207] As noted above, a subject nanosensor can be used to detect
nucleic acid in a sample. A subject nanosensor may be employed in a
variety of drug discovery and research applications in addition to
the diagnostic applications described above. For example, a subject
nanosensor may be employed in a variety of applications that
include, but are not limited to, diagnosis or monitoring of a
disease or condition (where the presence of an nucleic acid
provides a biomarker for the disease or condition), discovery of
drug targets (where, e.g., an nucleic acid is differentially
expressed in a disease or condition and may be targeted for drug
therapy), drug screening (where the effects of a drug are monitored
by assessing the level of an nucleic acid), determining drug
susceptibility (where drug susceptibility is associated with a
particular profile of nucleic acids) and basic research (where is
it desirable to identify the presence a nucleic acid in a sample,
or, in certain embodiments, the relative levels of a particular
nucleic acids in two or more samples).
[0208] In certain embodiments, relative levels of nucleic acids in
two or more different nucleic acid samples may be obtained using
the above methods, and compared. In these embodiments, the results
obtained from the above-described methods are usually normalized to
the total amount of nucleic acids in the sample (e.g., constitutive
RNAs), and compared. This may be done by comparing ratios, or by
any other means. In particular embodiments, the nucleic acid
profiles of two or more different samples may be compared to
identify nucleic acids that are associated with a particular
disease or condition.
[0209] In some examples, the different samples may consist of an
"experimental" sample, i.e., a sample of interest, and a "control"
sample to which the experimental sample may be compared. In many
embodiments, the different samples are pairs of cell types or
fractions thereof, one cell type being a cell type of interest,
e.g., an abnormal cell, and the other a control, e.g., normal,
cell. If two fractions of cells are compared, the fractions are
usually the same fraction from each of the two cells. In certain
embodiments, however, two fractions of the same cell may be
compared. Exemplary cell type pairs include, for example, cells
isolated from a tissue biopsy (e.g., from a tissue having a disease
such as colon, breast, prostate, lung, skin cancer, or infected
with a pathogen etc.) and normal cells from the same tissue,
usually from the same patient; cells grown in tissue culture that
are immortal (e.g., cells with a proliferative mutation or an
immortalizing transgene), infected with a pathogen, or treated
(e.g., with environmental or chemical agents such as peptides,
hormones, altered temperature, growth condition, physical stress,
cellular transformation, etc.), and a normal cell (e.g., a cell
that is otherwise identical to the experimental cell except that it
is not immortal, infected, or treated, etc.); a cell isolated from
a mammal with a cancer, a disease, a geriatric mammal, or a mammal
exposed to a condition, and a cell from a mammal of the same
species, preferably from the same family, that is healthy or young;
and differentiated cells and non-differentiated cells from the same
mammal (e.g., one cell being the progenitor of the other in a
mammal, for example). In one embodiment, cells of different types,
e.g., neuronal and non-neuronal cells, or cells of different status
(e.g., before and after a stimulus on the cells) may be employed.
In another embodiment of the invention, the experimental material
is cells susceptible to infection by a pathogen such as a virus,
e.g., human immunodeficiency virus (HIV), etc., and the control
material is cells resistant to infection by the pathogen. In
another embodiment of the invention, the sample pair is represented
by undifferentiated cells, e.g., stem cells, and differentiated
cells.
[0210] Although the foregoing embodiments have been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it is readily apparent to those of
ordinary skill in the art in light of the above teachings that
certain changes and modifications can be made thereto without
departing from the spirit or scope of the appended claims.
EXAMPLES
[0211] Various S-NP particles have been fabricated by Nano-PrinTED
and Dip-print and tested for enhancements of both SERS and
fluorescence (tested with light wavelength of .about.800 nm).
Exemplary results are given below.
[0212] For the S-NP structure with light resonance absorption
around 800 nm wavelength, the disks have a round shape of diameter
of 70 nm, the top metallic (gold) disk thickness is 15 nm, the
spacer (silicon dioxide) thickness is 20 nm, and the bottom
metallic (gold) disk thickness is 15 nm, the self-assembled gold
dots diameter is around 10 nm, and the adhesion layers between the
disks are titanium of a thickness of 0.5 nm.
[0213] In fabrication, first, the dense nanostructured protrusions
template with was patterned on 4 inch substrate by nanoimprint
lithography and reactive ion etching (RIE). The pillar height or
etching depth was precisely controlled by RIE (FIG. 15a). The mold
can be a daughter mold duplicated by nanoimprint from a master mold
fabricated with interference lithography, e-beam lithography,
sphere lithography and others. Second, the multiple depositions: a
release layer, metal layers (e.g. gold), adhesion layers (e.g.
titanium), dielectric layers (e.g. silicon dioxide) were deposited
in a sequence onto the protrusions template in a normal direction
to the surface by evaporation as shown in FIG. 15b. Guided by the
protrusions, the materials deposited on the top formed DS-NP
arrays, and at the pillar foot, a multi-layer nano-hole backplane
was also formed, the two are not connected. Third, transfer-print
S-NPs to another substrate (FIG. 15c), presents the schematic of
the S-NPs array that has been transferred onto another substrate.
Before transferring, a thin layer of buffer layer around 50-100 nm
was spinned onto the substrate served as an adhesion layer. The
transferring process was taken under low pressure (e.g. 50 PSI) and
room temperature, thus not damaged the substrate nor D2-Particle
arrays. Then the template peeled off the S-NPs from the templates.
And fourth, solvent was used to dissolve the buffer layer and
released the S-NPs into the solutions to make S-NPs (FIG. 15d).
[0214] FIG. 13 shows the scanning electron microscopy (SEMs) of (a)
double-metal-disk and single dielectric (D-particle); (b)
triple-metal (or magnetic) dielectric-nanoparticle (TS-NP); (c)
D-particle after the self-perfection by liquefaction (SPEL) to
change the shape of 2 metal disks; (d) D-particles array on the
substrate after the template lift-off.
[0215] FIG. 14 shows the scanning electron microscopy (SEMs) of (a)
D-particles array on the substrate after the transfer printing. (b)
D-particles exfoliated into solution.
[0216] FIG. 15 shows the Nano-PrinTED (nanoprint by templated
exfoliateable deposition) fabrication of S-NP at each step. Top
row: Schematic. And bottom row: scanning electron microscope (SEM)
of experimental results. (a) Pillar template fabricated by
lithography (e.g. NIL); (b) Multiple deposition and self-assembly
to form D2-particles; (c) transfer-print DPs to another substrate;
(d) put in solution. (e-h), SEM images.
[0217] As shown in FIG. 16, Nano-PrinTED and Dip-print have far
better precision in controlling the NP structure dimensions
(including the size and shape of each individual components, their
spacing, and final particle). (a) SEM picture of D2-P before
release and (b) Measured size distribution. Measured size variation
of D2-particle fabricated by Nano-PrinTED (<5%) is 3 fold less
than AuNP manufactured by chemical synthesis (>15%).
[0218] FIG. 17 shows the measurements of extinction spectrum of
D2-particles with SiO.sub.2 layer thickness from 5 nm to 30 nm and
constant Au layer thicknesses of 20 nm. Plasmonic resonant peak
wavelengths redshift with increasing SiO.sub.2 layer thickness.
[0219] FIG. 18 shows the simulation of the size of nanoparticles
with different architectures required for the same resonant
wavelength at 800 nm. It clearly shows that S-NP has much smaller
particle sized than conventional metallic sphere and disks for a
given resonant wavelength.
[0220] As shown in FIG. 19, (a) Measured Surface Enhanced Raman
Spectroscopy (SERS) signal of BPE, and (b) fluorescence signal of
IR-800 dye with single D2-particle and gold nanoparticle. A single
D2-particle has a SERS/Fluorescence enhancement over 100/30 fold
higher than a single gold nanoparticle of similar diameter. The
sophisticated architectures of PDS-NPs allow simultaneously
improving of all three key factors for plasmonic enhancement and
hence a large final enhancement. In PDS-NPs, the metallic disks
(25-60 nm diameter) create antenna for good absorption of
excitation light and radiation of the generated optical signal,
while the smaller gaps (between the disks or additional nanodots)
and sharp edges offer large local field enhancements.
* * * * *