U.S. patent application number 13/838600 was filed with the patent office on 2014-06-05 for structures for enhancement of local electric field, light absorption, light radiation, material detection and methods for making and using of the same..
This patent application is currently assigned to THE TRUSTEES OF PRINCETON UNIVERSITY. The applicant listed for this patent is THE TRUSTEES OF PRINCETON UNIVERSITY. Invention is credited to Stephen Y. Chou, Liang-Cheng Zhou.
Application Number | 20140154668 13/838600 |
Document ID | / |
Family ID | 50825785 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154668 |
Kind Code |
A1 |
Chou; Stephen Y. ; et
al. |
June 5, 2014 |
Structures for Enhancement of Local Electric Field, Light
Absorption, Light Radiation, Material Detection and Methods for
Making and Using of the Same.
Abstract
This disclosure provides, among other things, a nanosensor
comprising a substrate and one or a plurality of pillars extending
from a surface of the substrate, where the pillars comprise a
metallic dot structure, a metal disc, and a metallic back plane.
The nanosensor comprises a molecular adhesion layer that covers at
least a part of the metallic dot structure, the metal disc, and/or
the metallic back plane and a capture agent bound to the molecular
adhesion layer. The nanosensor amplifies a light signal from an
analyte, when the analyte is specifically bound to the capture
agent.
Inventors: |
Chou; Stephen Y.;
(Princeton, NJ) ; Zhou; Liang-Cheng; (Princeton,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF PRINCETON UNIVERSITY; |
|
|
US |
|
|
Assignee: |
THE TRUSTEES OF PRINCETON
UNIVERSITY
Princeton
NJ
|
Family ID: |
50825785 |
Appl. No.: |
13/838600 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13699270 |
Jun 13, 2013 |
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PCT/US2011/037455 |
May 20, 2011 |
|
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13838600 |
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61622226 |
Apr 10, 2012 |
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61347178 |
May 21, 2010 |
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Current U.S.
Class: |
435/5 ; 422/69;
427/162; 435/287.2; 435/6.1; 435/7.1; 436/501 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 21/6428 20130101; G01N 21/6486 20130101; G01N 2201/06113
20130101; G01N 21/648 20130101; G01N 2021/6439 20130101; G01N
21/658 20130101 |
Class at
Publication: |
435/5 ; 422/69;
436/501; 435/7.1; 435/6.1; 435/287.2; 427/162 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with United States government
support under Grant No. FA9550-08-1-0222 awarded by the Defense
Advanced Research Project Agency (DARPA) The United States
government has certain rights in this invention.
Claims
1. A nanodevice comprising: (a) a substrate; and (b) one or a
plurality of pillars extending from a surface of the substrate,
wherein at least one of said pillars comprises: i. a metallic disc
on top of the pillar; ii. a metallic back plane at the foot of the
pillar, said metallic back plane covering a substantial portion of
said substrate surface near the foot of the pillar; iii. a metallic
dot structure on sidewall of the pillar; and iv. a molecular
adhesion layer that covers at least a part of said metallic dot
structure, said metal disc, and/or said metallic back plane;
wherein said nanosensor amplifies a light signal that is proximal
to the exterior surface of said adhesion layer.
2. The nanodevice of claim 1, wherein the exterior surface of said
molecular adhesion layer comprises a capture agent-reactive group,
selected from an amine-reactive group, a thiol-reactive group, a
hydroxyl-reactive group, an imidazolyl-reactive group and a
guanidinyl-reactive group.
3. The nanodevice of claim 2, wherein said capture agent-reactive
group is a 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.
4. The nanodevice of claim 1, wherein said molecular adhesion layer
is attached to said metallic dot structure, said metal disc, and/or
said metallic back plane via a metal-sulfur bond.
5. The nanodevice of claim 1, wherein said molecular adhesion layer
has a thickness of 0.5 nm to 50 nm.
6. The nanodevice of claim 1, wherein the molecular adhesion layer
is a monolayer of alkanethiol or thio-poly(ethylene)glycol.
7. The nanodevice of claim 1, wherein the molecular adhesion layer
is attached to said metallic dot structure, said metal disc, and/or
said metallic back plane via a streptavidin/biotin interaction.
8. The nanodevice of claim 1, wherein the exterior surface of said
molecular adhesion layer comprises a biotin moiety or
streptavidin.
9. The nanodevice of claim 1, the exterior surface of said metallic
dot structure, said metal disc, and/or said metallic back plane
comprises a streptavidin group that can bind to a biotinylated
capture agent.
10. The nanodevice of claim 1, the exterior surface of said
metallic dot structure, said metal disc, and/or said metallic back
plane comprises a biotin moiety that can bind to a
streptavidin-linked capture agent.
11. The nanodevice of claim 1, wherein said nanosensor is disposed
within a container.
12. The nanodevice of claim 1, wherein said molecular adhesion
layer is a self-assembled monolayer (SAM), wherein each molecule of
the SAM comprises three parts: (i) a head group that has specific
affinity to the metal surfaces of the nanodevice, (ii) a terminal
group that specific affinity to the capture agent, and (iii) a
linker that links the head group and terminal group, wherein the
length of the linker determines the average spacing between the
metal surfaces and an attached capture agent can affects light
amplification of the nanodevice.
13. The nanodevice of claim 1, wherein the metal is selected from
the group consisting of gold, silver, copper, aluminum, alloys
thereof, and combinations thereof.
14. The nanodevice of claim 1, wherein the top of said pillar has a
shape selected from the group of shapes consisting of round,
polygonal, pyramidal, elliptical, elongated bar shaped, or any
combination thereof.
15. The nanodevice of claim 1, wherein the lateral dimension of
said metallic disc is in the range from 5 nm to 150 nm.
16. The nanodevice of claim 1, wherein said metallic disc and the
metallic back plane are spaced by a distance in the range of 0.1 nm
to 60 nm.
17. The nanodevice of claim 1, wherein said at least one metallic
dot structure has dimensions in the range of 1 nm to 25 nm.
18. The nanodevice of claim 1, 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.
19. The nanodevice of claim 1, wherein the spacing between the two
nearest pillars of said plurality of pillars is in the range from 2
nm to 200 nm.
20. The nanodevice of claim 1, wherein said pillar has a sidewall
surface that is columnar, sloped, or curved.
21. The nanodevice of claim 1, wherein the thickness of the said
metallic disc and metallic back plane is between 5 nm to 60 nm.
22. The nanodevice of claim 1, wherein said pillar has a lateral
dimension or a height less than the wavelength of said light.
23. The nanodevice of claim 1, wherein said metallic disc has
substantially the same lateral geometry as said pillar.
24. The nanodevice of claim 1, wherein said pillar comprises a
dielectric or semiconductor material selected from the group
consisting of polymers, silicon-dioxide, silicon-nitride, hafnium
oxide, aluminum oxide, silicon, gallium arsenide, and gallium
nitride.
25. The nanodevice of claim 1, wherein the lateral dimension of
said metallic disc is less than the wavelength of said light.
26. A method of making a nanosensor, comprising: attaching a
capture agent to the molecular adhesion layer of a nanodevice of
claim 1.
27. The method of claim 26, wherein said capture agent is a protein
or nucleic acid.
28. The method of claim 27, wherein said capture agent is an
antibody.
29. The method of claim 27, wherein said capture agent is an
oligonucleotide.
30. A nanosensor, comprising: (a) a nanodevice of claim 1; and (b)
a capture agent that specifically binds to an analyte, wherein said
capture agent is linked to the molecular adhesion layer of said
nanodevice; wherein said nanosensor amplifies a light signal from
an analyte, when said analyte is bound to said capture agent.
31. The nanosensor of claim 30, wherein said light signal is
luminescence or fluorescence.
32. The nanosensor of claim 30, wherein said capture agent is a
protein.
33. The nanosensor of claim 32, wherein said capture agent is an
antibody.
34. The nanosensor of claim 30, wherein said capture agent is a
nucleic acid.
35. The nanosensor of claim 30, wherein said capture agent is an
oligonucleotide.
36. The nanosensor of claim 30, further comprising a labeled
analyte that is specifically bound to said capture agent.
37. The nanosensor of claim 36, wherein said labeled analyte is
directly or indirectly labeled with a light-emitting label.
38. The nanosensor of claim 37, wherein said labeled analyte is
linked to said light-emitting label via a streptavidin/biotin
interaction.
39. The nanosensor of claim 30, wherein the thickness of said
molecular adhesion layer is selected to optimize the amplification
of said light signal.
40. The nanosensor of claim 30, wherein nanosensor is in a
multi-well format, wherein each well of a multi-well plate
comprises a nanosensor of claim 30, wherein the nanosensor in each
of the wells comprises a different capture agent.
41. A system comprising: (a) a nanosensor of claim 30; (b) a holder
for said nanosensor; (c) an excitation source that induces a light
signal from a label; and (d) a reader adapted to read said light
signal.
42. The system of claim 41, wherein said excitation source is a
light source.
43. The system of claim 41, wherein said excitation source is
electrical current.
44. The system of claim 41, wherein said reader is a photodetector,
a CCD camera, a CMOS camera, a spectrometer or an optical sensor
capable of producing a two dimensional spectral map of a surface of
said nanosensor.
45. The system of claim 41, wherein said nanosensor is in a
multi-well format, and said holder and/or said reader can be moved
so that reader can read a light signal from each of the wells
independently.
46. A method of detecting and/or quantifying an analyte,
comprising: (a) attaching a capture agent to the molecular adhesion
layer of a nanodevice of claim 1 to produce a nanosensor; (b)
contacting a sample containing a target analyte with said
nanosensor, wherein said target analyte specifically binds to said
capture agent and said contacting is done under conditions suitable
for said binding; and (c) reading a light signal from any target
analyte that is bound to said nanosensor; wherein said method
further comprises labeling said target analyte with a
light-emitting label, either prior to or after it is bound to said
capture agent.
47. The method of claim 46, wherein said reading applies an
exciting said light emitting label using light, electricity, a
chemical or combination of thereof, and measuring at least one
property of said light signal selected from intensity, wavelength,
and location.
48. The method of claim 46, wherein said analyte is labeled after
it is bound to the capture agent of said nanosensor.
49. The method of claim 46, wherein said labeling is done by
binding said target analyte to a detection agent that specifically
binds to said target analyte and that is linked to a light-emitting
label.
50. The method of claim 46, wherein said method comprise labeling
said light-emitting label after it is bound to the capture agent,
wherein said method further comprises removing any unbound
light-emitting label from the nanosensor prior to said reading.
51. The method of claim 49, wherein said detection agent is a
secondary antibody that comprises a light-emitting label.
52. The method of claim 49, wherein said detection agent is a
nucleic acid that comprises a light-emitting label.
53. The method of claim 46, wherein said light-emitting label is a
fluorescent, chemiluminescent or electroluminescent label.
54. The method of claim 53, wherein said light-emitting label is
labeled with IRDye800CW, Alexa 790 or Dylight 800.
55. The method of claim 46, wherein said label emits light at a
wavelength in the range of 300 nm to 1200 nm.
56. The method of claim 46, wherein said method comprises blocking
said nanosensor prior to said contacting step (b), thereby
preventing non-specific binding of said capture agents to
non-target analytes.
57. The method of claim 46, wherein said sample is a liquid
sample.
58. The method of claim 46, wherein said sample is a clinical
sample.
59. The method of claim 46, wherein said sample is derived from a
bodily fluid.
60. The method of claim 46, wherein said analyte is a protein.
61. The method of claim 46, wherein said capture agent and said
analyte are nucleic acids.
62. The method of claim 46, wherein said analyte is a cancer
biomarker.
63. The method of claim 46, wherein said analyte is a biomarker for
a neurological disease.
64. The method of claim 46, wherein said analyte is a biomarker for
cardiovascular diseases
65. The method of claim 46, wherein said analyte is a biomarker for
organic diseases
66. The method of claim 46, wherein said analyte is a biomarker for
an infectious or parasitic disease.
67. A method for fabricating said nanodevice of claim 1,
comprising: (a) patterning at least one pillar on a top surface of
a substrate; (b) depositing a metallic material layer of said 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; (d) depositing a molecular adhesion layer on top of the
deposited metallic material, wherein the molecular adhesion layer
covers at least a part of said metallic dot structure, said metal
disc, and/or said metallic back plane, and wherein the exterior
surface of said molecular adhesion layer comprises a capture
agent-reactive group.
68. The method of claim 67, further comprising: attaching a capture
agent to said molecular adhesion layer.
69. The method of claim 67, wherein said patterning is an embossing
of a material.
70. A method of diagnosing a disease or condition, comprising: (a)
obtaining a liquid sample from a patient suspected of having said
disease or condition; (b) contacting said sample with a nanosensor
of claim 1, wherein the capture agent of said nanosensor
specifically binds to a biomarker for said disease and wherein said
contacting is done under conditions suitable for specific binding
of said biomarker with said capture agent; (c) removing any
biomarker that is not bound to said capture agent; and (d) reading
a light signal from biomarker that remain bound to said nanosensor,
wherein a light signal indicates that said patient has said disease
or condition; wherein said method further comprises labeling said
biomarker with a light-emitting label, either prior to or after it
is bound to said capture agent.
71. The method of claim 70, wherein said patient is suspected of
having cancer and said antibody binds to a cancer biomarker.
72. The method of claim 70, wherein said patient is suspected of
having a neurological disorder and said antibody binds to a
biomarker for said neurological disorder.
73. The method of claim 70, wherein said liquid sample comprises
amniotic fluid, aqueous humour, vitreous humour, whole blood,
fractionated blood, plasma, serum, 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 or exhaled condensate.
74. The method of claim 70, wherein said sensor is employed to
detect or quantify chemical compounds or biomolecules that
correlate with the stage of a diseases.
75. The method of claim 74, wherein said disease is cancer, a
cardiac disease, a pulmonary disease, a renal disease, or a mental
disorder,
76. The method of claim 70, wherein said sensor is employed to
detect or quantify a microorganism.
77. The method of claim 76, wherein said microorganism is a virus,
fungus or bacteria from the environment or a clinical sample.
78. The method of claim 70, wherein said sensor is employed to
detect or quantify chemical compounds or biological entities that
pose hazard to food safety or national security,
79. The method of claim 78, wherein said chemical compounds or
biological entities is toxic waste or anthrax.
80. The method of claim 70, wherein said sensor is employed to
quantify a vital parameter in a medical or physiological
monitor.
81. The method of claim 80, wherein said vital parameter is
glucose, blood oxygen level, or total blood count.
82. The method of claim 70, wherein said sensor is employed to
detector or quantify a specific DNA or RNA from a biosample.
83. The method of claim 70, wherein said sensor is employed to the
sequence and compare genetic sequences in DNA in the chromosomes or
mitochondria.
Description
CROSS-REFERENCING
[0001] This 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 Jan. 24, 2013, which application is a
.sctn.371 filing of US2011/037455, filed May 20, 2011 and claims
the benefit of U.S. provisional application Ser. No. 61/347,178,
filed on May 21, 2010, which applications are incorporated by
reference herein for all purposes.
BACKGROUND
[0003] There is a great need to enhance a luminescence signal (e.g.
a fluorescence signal) and detection sensitivity of biological and
chemical assays. The application is related to the
micro/nanostructures and molecular layers and methods for achieving
an enhancement (namely amplification of luminescence and
improvement of detection sensitivity), their fabrication and
applications.
SUMMARY
[0004] This disclosure provides, among other things, a nanosensor
comprising a substrate and one or a plurality of pillars extending
from a surface of the substrate, with a metallic dot structure on
pillar's sidewall, a metal disc on top of the pillar, and a
metallic back plane covering a significant area near the foot of
the pillar. The nanosensor further comprises a molecular adhesion
layer that covers at least a part of the metallic dot structure,
and/or the metal disc, and/or the metallic back plane and that
binds a capture agent. The nanosensor is coated with capture agent
that specifically captures targeted analytes (e.g. molecules, which
can be proteins or nucleic acids). The analytes can be optically
labeled directly or indirectly. In indirect labeling, a secondary
capture agent with an optical label (i.e. a labeled detection
agent) is used to bind and hence identify the presence of the
captured analyte. The nanosensor amplifies a light signal from a
the analyte, when the analyte is bound to the capture agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] 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.
[0006] FIG. 1 panels A and B schematically illustrate some features
of embodiment of a subject nanodevice. Panel C schematically
illustrates one way in which a subject nanodevice can be
manufactured.
[0007] FIG. 2 schematically illustrates an exemplary system.
[0008] FIG. 3 schematically illustrates an exemplary self-assembled
monolayer.
[0009] FIG. 4 schematically illustrates an exemplary antibody
detection assay.
[0010] FIG. 5 schematically illustrates an exemplary nucleic acid
detection assay.
[0011] FIG. 6 schematically illustrates another embodiment nucleic
acid detection assay.
[0012] FIG. 7 Disk-coupled dots-on-pillar antenna array (D2PA)
plate and immunoassay. (a) Schematic (overview and cross-section)
of D2PA plate without an immunoassay. D2PA has an array of dense
three-dimensional (3D) resonant cavity nanoantennas (formed by the
gold disks on top of periodic nonmetallic pillars and the gold
backplane on the pillar foot) with dense plasmonic nanodots inside,
and couples the metallic components through nanogaps. (b) Schematic
of the immunoassay on the D2PA, consisting of a self-assembled
monolayer (SAM) of adhesion layer, Protein-A (as capture layer) and
human-IgG pre-labeled with IRDye-800cw (as pre-labeled biomarker).
(c) Scanning electron micrograph (SEM) of D2PA with 200 nm period
(overview and cross-section). The gold nanodots rested on the
silica nano-pillar sidewalls are clearly observed.
[0013] FIG. 8 Measured absorbance spectrum of D2PA with (blue line)
and without (red line) the immunoassay being deposited. The peak
absorbance is 98% and 97%, and the resonance peak width is 165 nm
and 145 nm, respectively, with and without the immunoassay.
Deposition of the immunoassay slightly blue-shifted the absorption
peak from 795 nm to 788 nm and widened the absorption wavelength
range
[0014] FIG. 9 Measured area-average fluorescence intensity spectrum
of the human-IgG labeled with IRDye800CW captured by the assay on
the D2PA (red line) and the glass plate (blue line, which is
amplified 1000 times to be visible at given scales), respectively.
Compared with the assay on the glass plate, the average
fluorescence enhancement (dashed line) is 7,440 fold at the peak
wavelength of fluorescence (800 nm) and 7,220 fold when average
over the FWHM fluorescence. The plasmonic fluorescence enhancement
factor (EF) spectrum has much broader FWHM than the fluorescence
spectrum, which is consistent with the observed D2PA plasmonic
resonance spectrum (FIG. 5).
[0015] FIG. 10 Measured uniformity of fluorescence enhancement over
large area. (a) Measured immunoassay fluorescence enhancement
(factor) map over a total 5 mm.times.5 mm area of the D2PA. The map
has total 2,500 tiles (50.times.50), measured by using each tile
area (i.e. laser probe area) of 100 .mu.m.times.100 .mu.m and a
step-and-repeat distance of 100 .mu.m. (b) The corresponding
histogram of the measured enhancement factor gives a Gaussian
distribution variation of .+-.9%.
[0016] FIG. 11 A model direct assay of protein A and IgG. (A)
Fluorescence intensity vs. IgG concentration on D2PA (squares) and
glass plate reference (circles). The squares and circles are
measured data, and the curves were the fittings using
five-parameter logistic regression model to allow an extrapolation
of the data points between the measured ones. The limit of
detection (LoD) of D2PA and glass plate was found to be 0.3 fM and
0.9 nM, respectively, giving an enhancement of LoD of 3,000,000
fold. (B) Schematic of the immunoassay on the D2PA, consisting of a
self-assembled monolayer (SAM) of adhesion layer, Protein-A (as
capture layer) and human-IgG pre-labeled with IRDye-800cw (as
pre-labeled biomarker).
[0017] FIG. 12 Single molecule fluorescence of IRDye800CW labeled
IgG on D2PA plate. (a) 2D fluorescence image of 50 .mu.m.times.50
.mu.m area of a Protein A/IgG immunoassay on D2PA plate with an IgG
concentration of 10.sup.-10 M. Distinct "bright spots" are visible.
And (b) Fluorescence vs. time of a single bright spot. The binary
stepwise behavior indicates that the fluorescence is from a single
dye molecule placed at a hot spot (large electric field location)
of D2PA. Compared with the immunoassay on the glass reference, the
single molecule fluorescence at a hot spot is enhanced by
4.times.10.sup.6 fold.
[0018] FIG. 13. PSA immunoassay on D2PA plates. The experiment data
was fitted using 5-parameter logstic model (solid curve) in order
to calculate the LoD. An LoD.about.10 aM was achieved on D2PA.
Compared to glass plates, whose LoD was 0.9 pM, the sensitivity of
D2PA is 90,000 folds better. (Chou Group, to be published)
[0019] FIG. 14 CEA immunoassay on D2PA plates. Similar
configuration is used as the PSA immunoassay. For the tentative
trial so far, we managed to achieve an LoD.about.28 aM. Better
sensitivity (lower LoD) is expected once we manage to raise the
signal to noise ratio. (Chou Group, to be published)
[0020] FIG. 15 CA15.3 immunoassay on D2PA plates. A similar
configuration is used as the PSA immunoassay. For the tentative
trial so far, we managed to achieve an LoD.about.0.01 U/mL. Better
sensitivity (lower LoD) is expected once we manage to raise the
signal to noise ratio. (Chou Group, to be published)
[0021] FIG. 16 is two graphs showing the correlation between spiked
concentration and observed concentration.
[0022] FIG. 17 is two graphs showing the crossreactivity between
two antibodies.
[0023] FIG. 18 is two graphs showing reproducibility of
results.
[0024] FIG. 19 shows the results of a DNA hybridization assay, and
a schematic illustration of the same.
[0025] FIG. 20 shows a series of scanning electron micrographs.
[0026] FIG. 21 schematically illustrates an alternative
embodiment.
[0027] 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.
[0028] 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
[0029] 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.
[0030] 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 nanodevice and an outer
(exterior) surface can be bound to capture agents.
[0031] 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.
[0032] 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.-7M, less than about 10.sup.-8 M, less than about 10.sup.-9
M, less than about 10.sup.-10 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).
[0033] 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).
[0034] 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.
[0035] 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).
[0036] 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.
[0037] 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.
[0038] The terms "ribonucleic acid" and "RNA" as used herein mean a
polymer composed of ribonucleotides.
[0039] The terms "deoxyribonucleic acid" and "DNA" as used herein
mean a polymer composed of deoxyribonucleotides.
[0040] 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.
[0041] The term "attaching" as used herein refers to the strong,
e.g, covalent or non-covalent, bond joining of one molecule to
another.
[0042] The term "surface attached" as used herein refers to a
molecule that is strongly attached to a surface.
[0043] 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.
[0044] The term "analyte" refers to a molecule (e.g., a protein,
nucleic acid, or other molecule) that can bound by a capture agent
and detected.
[0045] The term "assaying" refers to testing a sample to detect the
presence and/or abundance of an analyte.
[0046] As used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations.
[0047] 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.
[0048] 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).
[0049] 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.
[0050] The terms "hybridizing" and "binding", with respect to
nucleic acids, are used interchangeably.
[0051] 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).
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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).
[0056] 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.
[0057] The term "streptavidin" refers to both streptavidin and
avidin, as well as any variants thereof that bind to biotin with
high affinity.
[0058] The term "marker" refers to an analyte whose presence or
abundance in a biological sample is correlated with a disease or
condition.
[0059] The term "bond" includes covalent and non-covalent bonds,
including hydrogen bonds, ionic bonds and bonds produced by van der
Waal forces.
[0060] 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.
[0061] Other specific binding conditions are known in the art and
may also be employed herein.
[0062] 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
[0063] The following detailed description illustrates some
embodiments of the invention by way of example and not by way of
limitation.
[0064] With reference to FIGS. 1A and 1B, disclosed herein is
nanodevice 100 comprising: (a) substrate 110; and (b) one or a
plurality of pillars 115 extending from a surface of the substrate,
wherein at least one of the pillars comprises a pillar body 120,
metallic disc 130 on top of the pillar, metallic back plane 150 at
the foot of the pillar, the metallic back plane covering a
substantial portion of the substrate surface near the foot of the
pillar; metallic dot structure 130 disposed on sidewall of the
pillar and molecular adhesion layer 160 that covers at least a part
of the metallic dot structure, and/or the metal disc, and/or the
metallic back plane. The underlying structure in this device has
been referred as "disk-coupled dots-on-pillar antenna array,
(D2PA)" and examples are them have been described (see, e.g., Li et
al Optics Express 2011 19, 3925-3936 and WO2012/024006, which are
incorporated by reference).
[0065] The exterior surface of molecular adhesion layer 160
comprises a capture-agent-reactive group, i.e., a reactive group
that can chemically react with capture agents, e.g., an
amine-reactive group, a thiol-reactive group, a hydroxyl-reactive
group, an imidazolyl-reactive group and a guanidinyl-reactive
group. For illustrative purposes, the molecular adhesion layer 160
covers all of the exposed surface of metallic dot structure 160,
metal disc 130, and metallic back plane 150. However, for practical
purposes, adhesion layer 160 need only part of the exposed surface
of metallic dot structure 160, metal disc 130, or metallic back
plane 150. As shown, in certain cases, substrate 110 may be made of
a dielectric (e.g., SiO.sub.2) although other materials may be
used, e.g., silicon, GaAs, polydimethylsiloxane (PDMS), poly(methyl
methacrylate) (PMMA). Likewise, the metal may be gold, silver,
platinum, palladium, lead, iron, titanium, nickel, copper,
aluminum, alloy thereof, or combinations thereof, although other
materials may be used, as long as the materials' plasma frequency
is higher than that of the light signal and the light that is used
to generate the light signal.
[0066] Nanodevice 100 is characterized in that it amplifies a light
signal that is proximal to the exterior surface of the adhesion
layer.
[0067] In some embodiments, the dimensions of one or more of the
parts of the pillars or a distance between two components may be
that is less than the wavelength of the amplified light. For
example, the lateral dimension of the pillar body 120, the height
of pillar body 120, the dimensions of metal disc 130, the distances
between any gaps between metallic dot structures 140, the distances
between metallic dot structure 140 and metallic disc 130 may be
smaller than the wavelength of the amplified light. As illustrated
in FIG. 1A, the pillars may be arranged on the substrate in the
form of an array. In particular cases, the nearest pillars of the
array may be spaced by a distance that is less than the wavelength
of the light. The pillar array can be periodic and aperiodic.
[0068] The nanodevice may be disposed within a container, e.g., a
well of a multi-well plate. The nanodevice also can be the bottom
or the wall of a well of a multi-well plate. The nanodevices may be
disposed inside a microfluidic channel (channel width of 1 to 1000
micrometers) or nanofluidic channel (channel width less 1
micrometer) or a part of inside wall of such channels.
[0069] As will be described in greater detail below (and as
illustrated in FIG. 10), a subject nanodevice 100 may be fabricated
by coating a so-called "disc-coupled dots-an-pillar antenna array"
200 (i.e., a "D2PA", which is essentially composed of substrate 110
and a plurality of pillars that comprise pillar body 120, metallic
disc 130, metallic back plane 150 and metallic dot structures 140
with a molecular adhesion layer 160. A detailed description an
exemplary D2PA that can be employed in a subject nanodevice are
provided in WO2012/024006, which is incorporated by reference
herein for disclosure for all purposes.
[0070] The first part of the description that follows below
describes certain features (i.e., the substrate, the pillar body,
the metallic disc, the metallic back plane and the metallic dot
structures) of the underlying D2PA structure. The second part of
the description that follows below describes the molecular adhesion
layer, the capture agents, and assays in which a subject
nanonsensor can be employed.
Disc-Coupled Dots-on-Pillar Antenna Arrays (D2PA)
[0071] A disc-coupled dots-on-pillar antenna array has a 3D plasmon
cavity antenna with a floating metallic disc or nanodisc that is
coupled to nanoscale metallic dots on a pillar body. Specifically,
in some embodiments the D2PA has a substrate, a pillar array on the
substrate, a metallic disc or nanodisc on top of each of the
pillars, nanoscale metallic dots on the pillar sidewall, with gaps
between the disc and some of the dots, gaps between the neighboring
dots, and a metallic back-plane which covers the most of the
substrate areas that are not occupied by the pillars.
[0072] In one embodiment, the pillar array is fabricated from
SiO.sub.2 with a 200 nm pitch, 130 nm height, and 70 nm diameter on
the substrate, formed from silicon. The metallic back-plane may be
formed from a 40 nm thick layer of gold, deposited on the pillar
array structures and substrate using e-beam evaporation along the
normal direction. The deposition process forms the metallic discs
in gold on top of each SiO.sub.2 pillar while simultaneously
forming the gold nanohole metallic back plane on the surface of the
silicon substrate. Each disc has a thickness of 40 nm and diameter
about 110 nm. During the evaporation process, with a deposition
rate of about 0.4 Ns, the gold atoms diffuse onto the sidewalls of
the SiO.sub.2 pillars and congregate into random particles with
granule sizes between 10 nm and 30 nm, forming the nanoscale
metallic dots.
[0073] A substrate with the gold nanodiscs, random gold
nanoparticle metallic dots, and bottom gold nanohole plate
(back-plane) is formed by the evaporation process. The gold
nanoparticles scattered on the sidewall of the SiO.sub.2 pillars,
forming the nanoscale metallic dots, have narrow gaps of about 0.5
nm-20 nm between them, which can induce highly enhanced electrical
fields. As used herein, the term "gap" is defined as the minimum
spacing between the two structures, such as the minimum spacing
between two discs or the spacing between a disc and an adjacent dot
structure. It also should be pointed out that the even a part of a
dot contacts with another dot, an enhancement effects achieved by
the present structures still exist, since there are other gaps
present between adjacent structures in other locations.
[0074] The D2PA structure can enhance light absorption through
plasma resonance and nanoantennas. The structure can enhance a
local electric field through the nanogaps between the discs and
nanodots and the nanogaps (140) between the nanodots themselves,
and assisted by the vertical cavity (for light) formed between the
discs and the back plane, and the lateral cavity formed by the disc
array.
[0075] More specifically, the structure can enhance the light
absorption through the array of nanopillars, and can enhance the
reflection of an optical signal from the surface through these
structures. It may have an enhanced vertical cavity light
absorption effect, formed by the discs through the dots and the
back plane to enhance the light absorption. It also can have a
lateral cavity light absorption effect through the back plane of
the metal to enhance the light absorption. It will be recognized by
those skilled in the art that any particular D2PA structure may
have one, several, or all of these functions, depending upon the
specific configuration of the structure, including the spacing in
the pillar array, size of the pillars, size of the discs, size of
the dots, and materials employed.
[0076] The enhancement of optical signals by the structure will be
a product of enhancement from the nanogaps between features of the
structure, from plasmon resonance, from antenna absorptions, from
antenna radiations, from vertical cavities, as well as lateral
cavities. The elements and functions of D2PA structure may be
viewed from a different angle. The discs and the spacing gap
between the disc and the adjacent metallic dots, as well as and
between the dots themselves, can affect the local electric field
enhancement provided by the structure. The dot position and number
of dots on each pillar body can also enhance the local electric
field. The diameter of each pillar and diameter of the capping disc
can affect the plasmon resonant frequency. The silicon dioxide
pillar height can affect the cavity length and number of nanogaps,
and also can affect the coupling of the disc and the gold back
planes. The number of pillars per unit cell can affect the active
areas, and the pitch (spacing) in the array of pillars can affect
coherent absorption and radiation of light. The gold back plane can
affect the antenna and cavity, and the pillar shape can determine
the light dependent absorption.
[0077] Within the structure, multiple variables may be "tuned" to
enhance signals. For example, the diameter of the discs and shape
of the pillars may be varied to alter the plasmon resonant
frequency, the metallic dots will effect local signal enhancement,
as well the disc-to-dot gap, dot position, and dot counts on each
pillar body; the height of the pillars will affect the resonant
cavity length and the number of nanogaps present, as well as the
coupling effect between the disc and the metallic back plane. The
total number of pillars per unit cell on the surface of the
structure defines the active areas, and the pillar spacing (pitch)
effects coherent absorption and radiation of optical energy.
Finally, the metallic back plane material and thickness is related
to antenna and cavity effects. Those of ordinary skill in this
field will recognize that each of these variable may be altered as
require from the exemplary embodiments shown herein to achieve a
structure 100 having desired characteristics or "tuning" to achieve
specific enhancements, without departing from the scope of the
present invention.
[0078] A variety of configurations for the structure are
envisioned. For example, the structure of the D2PA can have a layer
of SiO.sub.2 under the metal back plane and which contiguously
forms the pillars. Alternatively, the D2PA having a metallic back
plane without holes, such that the pillars are formed directly on
the back plane material, which in turn is deposited over a layer of
SiO.sub.2 on the underlying substrate.
[0079] When constructing the D2PA structure of the present
disclosure, the material for the underlying substrate can be an
insulator, a semiconductor, or a dielectric insulator. The
substrate need not be monolithic, but may be of a laminate
construction, comprising an insulator or semiconductor material top
layer (the layer next to the pillars) while the rest of the
substrate is made of any solid material.
[0080] The pillar bodies on the top layer of the substrate may be
formed from an insulating material, but may be semiconductors.
Exemplary materials for the formation of the pillars are
dielectrics: silicon-dioxide, silicon-nitride, hafnium oxide (HfO),
Aluminum oxide (AlO) or semiconductors: silicon, GaAs, and GaN.
Once formed, the pillars may have sidewalls which are columnar
(straight), sloped, curved, or any combination thereof. The height
of each pillar may be chosen from 5 nm to 7,000 nm, and a lateral
dimension of each pillar may be chosen from 5 nm to 8,000 nm. The
shape of the top surface of the pillar can be round, a point (of a
pyramid), polygon, elliptical, elongated bar, polygon, other
similar shapes or combinations thereof. The spacing between the
pillars in the array can be periodic or aperiodic. For some
applications, a periodic period is preferred and the period is
chosen to maximize the light absorption and radiation, which is
light wavelength dependent. The spacing (pitch) between adjacent
pillars in the array may be from 4 nm to 4000 nm.
[0081] Each pillar is topped with a metallic disc which may be
formed from either: (a) single element metal, such as gold, silver,
copper, aluminum, nickels; (b) a combination of the multiplayer
and/or multilayer of the single metals; (c) metallic alloys; (d)
semiconductors, (e) any other materials that generate plasmons, or
(f) any combination of (a), (b), (c), (d) and (e). The shape of
each disc can be a rounded, pointed (as in the form of a pyramid or
cone), polygonal, elliptical, elongated bar, polygon, other similar
shapes or combinations thereof. The shape of each disc can be the
same as, or different from, the shape of the top surface of the
associated pillar on which it is disposed. Preferably, a lateral
dimension of each disc is from 4 nm to 1500 nm, and a thickness of
the disc is from 1 nm to 500 nm. The diameter of the metal discs
can be either larger or smaller than the diameter of the supporting
pillar. The diameter difference can various from 0 to 200 nm
depending the working wavelength.
[0082] Disposed on the sidewalls of each pillar between the
metallic disc and the metallic back plane, the metallic dots have
shapes which are approximately spherical, discs-like, polygonal,
elongated, other shapes or combinations thereof. The metallic dots
on a pillar may all have approximately the same shape, or may be
individually varied. The dimensions of the metallic dots are
preferably between 3 nm to 600 nm, and may be different in three
dimensions. The exact dimension of the dots may be selected for a
specific light signal, as well regulated by fabrication convenience
and the fabrication of the associated gaps there between.
[0083] In some embodiments, the gaps between the neighboring
metallic dots and the gap between the disc and adjacent metallic
dots is between 0.5 nm to 200 nm. For many applications, a small
gap is preferred to enhance the optical signals. The gaps may be
varied between each metallic dot on a pillar.
[0084] In the embodiment, the metallic back plane defines a
metallic layer on the substrate with a hole for each pillar. The
thickness of the metallic back plane is selected to be from 1 nm to
2000 nm, with a thickness in the range of 50 nm-200 nm preferred.
The material of the metallic back plane can be selected from the
same group as is used to form the metallic disc described above,
but for a given D2PA structure, the metallic back plane can be
formed from either the same or a different material as that used to
form the discs.
[0085] The above descriptions of the D2PA structure are
illustrative of the range of the materials, shapes, and dimensions
which may be employed, but are not considered to be exclusive.
Other materials, shapes, and dimensions may be used as required to
achieve a desired enhancement effect. The exact materials, shapes,
and dimensions for each D2PA structure will be determined by
particular requirements imposed by the light absorption to be
enhanced (wavelength, polarization), the light re-radiation to be
enhanced, and/or the local electric field to be enhanced.
[0086] A D2PA array may be fabricated using the following method.
The initial step is to provide the substrate with a layer of pillar
material, such as SiO.sub.2. The next step is to employ a
lithographic imprinting process to imprint a mold having a pattern
of pillars into a resist layer deposited over the layer of pillar
material. After imprinting the pattern into the resist layer to
create an etch mask, the residual material is removed via an
etching process to leave a pattern of pillar-like structures of the
resist layer. A layer of etch mask material, such as chromium (Cr)
or other material is then deposited over the pattern of pillar-like
structures, and the remaining resist material removed, resulting in
a pattern of Cr deposited directly on the layer of pillar material.
A final etching step which may be a dry etching such as
retro-etching, or a wet etching process, removes the unprotected
portions of pillar material, and leaves an array of pillars
disposed on the surface of the substrate. Any remaining etch mask
material (Cr) is optionally removed by either a dry or wet etching
process, and an evaporation process is employed to deposit the
metallic back plane material, disc material, and metallic dots onto
the structure in a substantially collimated deposition.
[0087] Those of ordinary skill will recognize that the various
lithography steps can use any variety of known lithography methods,
including electron-beam lithography, ion beam lithography,
photolithography, or nanoimprint lithography to form the pattern in
the resist material. Similarly, it will be recognized that the
etching mask material can be metal dielectric or insulators.
[0088] The etch mask material can be deposited on the resist layer
before or after the lithography step is performed. A liftoff
process will typically be used if the etch masking material is
deposited after the lithography step. Alternatively, if the step of
nanoimprint lithography is used to create a resist pattern first,
an etch mask material may be subsequently deposited into the
resulting trenches second, and then a liftoff process is performed.
Other methods for making a D2PA array are possible.
[0089] Through manipulation of the various parameters of the D2PA
structure, light of various wavelengths from about 100 nm to about
8000 nm may be manipulated.
[0090] The enhancement structure may be constructed with one or
more features specific to the wavelength of light to be detected.
These features include including the material selection, the
nanoscale pillar height, the nanoscale pillar sidewall shape, the
nanoscale metallic disc shape, the nanoscale metallic dot structure
spacing, the metallic materials, and the metallic backplane
configuration. The selection of the nanoscale metallic dot
structure spacing further includes selecting a gap distance between
adjacent nanoscale metallic dot structures and/or selecting a gap
spacing between the nanoscale metallic disc and adjacent nanoscale
metallic dot structures.
[0091] The substrate of the nanoscale structure may be an
electrical insulator, a dielectric insulator or a semiconductor.
Optionally the substrate may be a laminate structure, and wherein a
layer at the surface of the substrate is either an electrical
insulator or a semiconductor; and wherein a body of the substrate
below the surface layer consists of any solid material.
[0092] The pillar bodies may be formed from either an insulator or
a semiconductor, and has a top which has a shape selected from the
group of shapes consisting of round, pointed, polygonal, pyramidal,
elliptical, elongated bar shaped, or any combinations thereof. The
sidewall surface of the pillar may be columnar, sloped, or curved.
Preferably, the pillar has a height in the range from 5 nm to 7000
nm and a diameter in the range from 5 nm to 8000 nm. Optionally,
the pillar may be part of an array of pillars extending from the
surface of the substrate, with a spacing between adjacent pillars
in the range from 2 nm to 4,000 nm. The array of pillars may define
a periodic array with a spacing selected in relation to light of a
selected wavelength in order to maximize absorption or radiation of
the light using the nanoscale structure. Suitable materials for the
formation of the pillars on the nanoscale structure include
silicon-dioxide, silicon-nitride, hafnium oxide, aluminum oxide,
silicon, gallium arsenide, and gallium nitride.
[0093] The metallic discs of the nanoscale structure are formed on
top of the pillars from a metal such as gold, silver, copper,
aluminum, alloys thereof, or combinations thereof. The surface of
the metallic discs need not be uniform, and may have any
configuration such as round, pointed, polygonal, elliptical, bar or
combinations thereof. Preferably, a lateral dimension of the
metallic disc is in the range from 5 nm to 1500 nm and a vertical
thickness of the metallic disc is in the range from 1 nm to 500
nm.
[0094] The metallic dot structures disposed on the pillar sidewalls
of the nanoscale structure each have a shape selected from a group
of shapes consisting of approximately spherical, circular,
polygonal, elongated or combinations thereof, and have dimensions
in the range 3 nm to 600 nm. A gap between the metallic dot
structures and the metallic disc on a common pillar is in a range
from 0.5 nm to 600 nm, as is the gap between adjacent metallic dot
structures.
[0095] The metallic back plane of the nanoscale structure may be
configured either with holes through which the pillar bodies extend
from surface of the substrate, or may be substantially continuous,
with the pillar bodies disposed there on. Preferably, the metallic
back plane has a thickness ranging from 1 nm to 2000 nm, e.g., from
50 nm to 200 nm, and is composed of a metal selected from the group
of metals consisting of gold, silver, copper, aluminum, alloys
thereof, or combinations thereof. The metallic back plane may be
formed from either the same material as, or a different material
from, the metallic discs.
[0096] The nanoscale structure of the present disclosure may be
made by a variety of methods. An exemplary method for manufacture
of the nanoscale structure for enhancing local electric fields,
absorbing light or radiating light comprises the steps of:
providing a substrate comprising an outer surface of insulating or
semiconductive material; forming on the outer surface an array of
pillars having a height in the range 5 nm to 7000 nm and a lateral
dimension in the range 5 nm to 8000 nm; applying conductive
material to the tops of the pillars and to the underlying
substrate; and simultaneously (or subsequently) depositing
conductive dot structures on the pillar sidewalls. The array of
pillars is formed by a process comprising electron beam
lithography, ion-beam lithography, photolithography or nanoimprint
lithography.
[0097] In one embodiment that is configured for enhance light at a
wavelength of 800 nm, the D2PA nanostructure may be composed of a
periodic non-metallic (e.g. dielectric or semiconductor) pillar
array (200 nm pitch and .about.100 nm diameter), a metallic disk
(.about.135 nm diameter) on top of each pillar, a metallic
backplane on the foot of the pillars, metallic nanodots randomly
located on the pillar walls, and nanogaps between these metal
components. The disk array and the backplane (both are 55 nm thick)
form a 3D cavity antenna that can efficiently traps the excitation
light vertically and laterally. Each pillar has about 10 to 50
nanodots depending upon the pillar geometry; and the pillar density
is 2.5.times.10.sup.9 pillars/cm.sup.2.
[0098] The device may be configured to detect light having a
wavelength in the range of 400 to 1,000 nm range. In certain
embodiments, the average diameter for the nanodots is in the range
of 1 nm to 25 nm, and gaps between the nanodots, and the gaps
between the nanodots and the nanodisks may be in the range of 1 nm
to 10 nm. The metal is selected from the group consisting of gold,
silver, copper, aluminum, alloys thereof, and combinations thereof.
The top of the pillar has a shape selected from the group of shapes
consisting of round, polygonal, pyramidal, elliptical, elongated
bar shaped, or any combination thereof. The lateral dimension of
the metallic disc is in the range from 5 nm to 150 nm. The metallic
disc and the metallic back plane are spaced by a distance in the
range of 0.1 nm to 60 nm. At least one metallic dot structure has
dimensions in the range of 1 nm to 25 nm. The distance between the
metallic dot structure the metallic disc, and the distance between
the metallic dot structure and the metallic backplane are spaced by
a distance in the range of 0.5 nm to 50 nm.
[0099] In particular embodiments, the spacing between the two
nearest pillars of the plurality of pillars is in the range from 2
nm to 200 nm. The pillar has a sidewall surface that is columnar,
sloped, or curved. The thickness of the metallic disc and metallic
back plane is between 5 nm to 60 nm. The pillar has a lateral
dimension or a height less than the wavelength of the light. The
metallic disc has substantially the same lateral geometry as the
pillar. The pillar comprises a dielectric or semiconductor material
selected from the group consisting of polymers, silicon-dioxide,
silicon-nitride, hafnium oxide, aluminum oxide, silicon, gallium
arsenide, and gallium nitride. The lateral dimension of the
metallic disc is less than the wavelength of the light.
[0100] FIG. 21 shows two alternative embodiments, in which
embodiment (A) 400, there are no metallic dot structure on pillar
sidewall, but only the pillars 415 and the metallic disk 430 on top
of the pillars and the metallic back plane 450 on the foot of the
pillars, and the molecular adhesion layers 460 coated to the disks
and the back plane. It has a substrate 410, In embodiment (B) 500,
there are no pillar and metallic dot structure, but only a metallic
back plane 570 which now is a thin metal film 580 on a dielectric
or semiconductor substrate 510 with a thin film of an dielectric
(e.g. SiO2) or semiconductor 580 on top of the metal back plane,
and finally metallic disks and the molecular adhesion layers 560
coated to the disks only. It has a substrate 510,
[0101] The shape and size (laterial dimension and its thickness) of
the metallic disk are in similar to that in D2PA. For the
embodiment (A), the pillars and the backplane are similar to the in
D2PA. For the embodiment (B), the materials for the thin dielectric
and semiconductor layer are similar to the materials for the
pillars, but the film thickness is from 0.5 nm to 150 nm; and there
is no limitation on the thickness of the back plane (except no
thinner than 2 nm). In both embodiments, the materials for the
metals are similar to that in D2PA, except in the embodiment (B)
two dissimilar materials can be used for the disk and the back
plane.
Molecular Adhesion Layer and Attachment of Capture Agents
[0102] As shown in FIG. 1, nanodevice 100 comprises a molecular
adhesion layer 160 that covers at least a part of the metal
surfaces of the underlying D2PA. 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 nanodevice. 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 nanodevices on the other side.
[0103] 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.
[0104] 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 nanodevice'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 nanodevice. Such a SAM is illustrated in FIG. 3.
[0105] 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.
[0106] 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 nanodevice surface, or
synthesized together with the molecule chain before they are
assembled on the surface.
[0107] 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.
[0108] 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.
[0109] 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 nanodevice'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.
[0110] 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 sulfhydrl-reactive), although many others may
be used.
[0111] 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.
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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.
[0116] 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 nanodevice, in binding between molecular adhesion
layer and the capture agent, and in binding a light emitting table
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.
[0117] 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).
[0118] 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).
[0119] 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.
[0120] 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).
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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 D2PA nanodevice through physical
adsorption or strong binding. In one example, protein A can be
coated over the entire or partial areas of the surface of D2PA
nanodevice 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 D2PA nanodevice's surface. The
thickness of PEG molecule layer can be tuned by changing the PEG
polymer chain length. Another example is an amorphous SiO2 thin
film, which is attached to the surface of the D2PA nanodevice using
physical or chemical deposition methods, e.g., evaporation,
sputtering, sol-gel method. The thickness of the SiO2 thin film can
be precisely controlled during the deposition.
[0125] 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.
[0126] 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.
[0127] 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 nanodevice'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.
[0128] 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 nanodevice (i.e. D2PA). 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.
6. In this embodiment, the nucleic acid comprises a headgroup that
binds directly the nanodevice. The remainder of the steps are
performed as described in FIG. 5.
[0129] 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.
[0130] 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.
[0131] 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 sulfhydrl-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.
[0132] 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.
[0133] In certain instances, a subject nanodevice 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.
[0134] 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.
Nanosensors
[0135] A nanosensor made by the method set forth above is provided.
In certain embodiments, the nanosensor comprises (a) a substrate;
and (b) one or a plurality of pillars extending from a surface of
the substrate, wherein at least one of the pillars comprises: i. a
metallic disc on top of the pillar; ii. a metallic back plane at
the foot of the pillar, the metallic back plane covering a
substantial portion of the substrate surface near the foot of the
pillar; iii. a metallic dot structure disposed on sidewall of the
pillar; iv. a molecular adhesion layer that covers at least a part
of the metallic dot structure, the metal disc, and/or the metallic
back plane; and v. a capture agent that specifically binds to an
analyte, wherein the capture agent is linked to the molecular
adhesion layer. The nanosensor is characterized in that it
amplifies a light signal from an analyte, when the analyte is bound
to the capture agent.
[0136] The light amplication comes from one or several following
factors: the nanosensor can (a) absorb light excitation effectively
(e.g. the light at a wavelength that excites fluorescent moieties),
(b) focus the absorbed light into certain locations, (c) place the
analytes into the regions where most of light are focused, and (d)
radiate efficiently the light generated by analytes from the
locations where the analytes immobilized.
[0137] Depending on how the analyte is labeled, light signal that
is amplified may be luminescence (e.g., chemiluminescent or
electroluminescent, or fluorescence). FIG. 3 illustrates a
biosensor in which the capture agent is a protein, e.g., an
antibody; FIG. 4 illustrates a biosensor in which the capture agent
is a nucleic acid, e.g., an oligonucleotide. In some embodiments,
the thickness of the molecular adhesion layer is selected to
optimize the amplification of the light signal.
[0138] In some embodiments, different capture agents are attached
to the nanosensor surface with each capture agent coated on a
different location of the surface, e.g., in the form of an array,
hence providing multiplexing in detections of different analysts,
since each location is specific for capturing a specific kind of
analyte.
[0139] In some embodiments, the nanosensor may be implemented in a
multi-well format, e.g., a 24-well, a 96-well or 384 well format,
where each well of a multi-well plate comprises a nanosensor (e.g.
the nanosensor is in each of the wells or is the bottom or a part
sidewall of each well). The capture agent in each well can be the
same or different. In some embodiments, multiple different capture
agents, each coated on different location can be placed in a well,
which provide multiplexing of detections for different analyst. In
these embodiments, several analytes in a sample may be analyzed in
parallel. In some embodiments, the nanosensor can be a part of
micro or nanofluidic channel.
[0140] In particular embodiments, a subject nanosensor may further
comprise labeled analyte that is specifically bound to the capture
agent. As noted above, the labeled analyte may be directly or
indirectly labeled with a light-emitting label. In embodiments in
which an analyte is indirectly labeled with a light-emitting label,
the analyte may be bound to a second capture agent, also termed:
detection agent (e.g., a secondary antibody or another nucleic
acid) that is itself optically labeled. The second capture agent
may be referred to as a "detection agent" in some cases.
[0141] In other embodiments, a subject nanosensor may be disposed
inside a microfluidic channel (channel width of 1 to 1000
micrometers) or nanofluidic channel (channel width less 1
micrometer) or a part of inside wall of such channels. The
nanosensors may be disposes at multiple locations inside each
channel and be used in multiple channels. The nanosensors in
different locations or different fluidic channels may later coated
with different capture agents for multiplexing of detections.
Systems
[0142] 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.
[0143] 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). An exemplary system is illustrated in FIG. 2. With
reference to FIG. 2, the excitation system may comprise a laser,
laser optics (including a beam expander, lens, mirror and a laser
line-pass filter), a reader (e.g., a spectrometer with a CCD
sensor), further optics (e.g., a long wavelength pass filter, a
beam splitter, and a lens), and a holder for the nanosensor. In
certain cases, the holder may be on a motorized stage that has an
X-Y and Z movement.
[0144] 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 absorption 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.
[0145] 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.
Assay Methods
[0146] 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 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).
[0147] 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.
[0148] In certain embodiments, the method comprises attaching a
capture agent to the molecular adhesion layer of a subject
nanodevice 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 the nanodevice 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] Some of the steps of an assay are shown in FIGS. 4 and 5.
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.
[0153] Some of the steps of an exemplary antibody binding assay are
shown in FIG. 4. In this assay, nanodevice 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 nanodevice. 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.
[0154] Some of the steps of an exemplary nucleic acid binding assay
are shown in FIG. 5. In this assay, nanodevice 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 200 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.
[0155] 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 D2PA nanodevice's metal surface through
sulfur-gold reaction. Then targeted DNA is added to the nanodevice
to be captured by the capture DNA. Finally the fluorescence labeled
detection DNA is added to the nanodevice to detect the immobilized
targeted DNA. After washing off the unbound detection DNA, the
fluorescence signal emanate from the nanodevices' surface is
measured for the detection and quantification of targeted DNA
molecules.
[0156] In the embodiments shown in FIGS. 4 and 5, 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.
[0157] 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.
[0158] 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 6 E10 and detection G210,
in which case 6 E10 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.
[0159] 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
[0160] 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.
[0161] 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.
[0162] 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 immobilized on the
nanodevices' 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 nanodevice. Therefore by detecting the
fluorescence emission emanate from the nanodevices' 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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
[0170] 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).
[0171] 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.
[0172] 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.
[0173] 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
[0174] Aspects of the present teachings can be further understood
in light of the following examples, which should not be construed
as limiting the scope of the present teachings in any way.
[0175] The following example provides a description of how
fluorescence and detection sensitivity of an immunoassay with
Protein A and the human-immunoglobulin G (IgG) tagged with near IR
dye (IRDye800CW) was measured using a new plasmonic structure,
D2PA, with a self-assembled monolayer (SAM) of molecular spacer.
The average fluorescence of the immunoassay on D2PA is enhanced by
7,400 fold, and the detection sensitivity by 3,000,000 fold (the
limit of detection is reduced from 0.9 nM to 0.3 fM), compared to
identical assays performed on glass plates. Furthermore, the
average fluorescence enhancement has a dynamic range of 8 orders of
magnitude (from 100 nM to 1 fM), and uniform over the entire large
sample area with a spatial variation .+-.9%. When a single molecule
fluorophore is placed at a "hot spot" of D2PA, its fluorescence is
enhanced by 4.times.10.sup.6 fold, which indicates the potential to
further increase the average enhancements and the detection
sensitivity significantly. The observed enhancements are orders of
magnitude higher than previously reported. The large enhancements
are attributed to the unique 3D architecture of the D2PA that can
overcome some key shortcomings in current plasmonic structure
design, as well as the use of the thin SAM molecular spacer. It is
suspected that D2PA may concentrate biomarkers in a solution into
the hot spots of the plasmonic structure, which can further improve
the enhancement. The fabrication method of D2PA plates is simple,
inexpensive and scalable. Together with good spatial uniformity,
wide dynamic range, and ease to manufacture, the giant enhancements
in immunoassay's fluorescence and detection sensitivity should have
broad applications in biology study, medical diagnosis, and many
others.
Materials and Methods I
[0176] Plasmonic Structure.
[0177] The plasmonic architecture described herein will be referred
to as "disk-coupled dots-on-pillar antenna array, (D2PA)", has an
array of dense three-dimensional (3D) resonant cavity nanoantennas
with dense plasmonic nanodots inside and the nanogaps that couple
the metallic components (illustrated in FIG. 7; See also Li et al
Optics Express 2011 19, 3925-3936). The 3D antennas greatly
increase the efficiency in receiving and radiating light, the
metallic nanodots and the nanogaps further "focus" the light to
small regions to increase local electric fields, and the high
densities increase the average enhancement and uniformity.
Particularly, the D2PA consists of a periodic non-metallic (e.g.
dielectric or semiconductor) pillar array (200 nm pitch, .about.100
nm diameter, and .about.65 nm height), a metallic disk (.about.135
nm diameter) on top of each pillar, a metallic backplane on the
foot of the pillars, metallic nanodots randomly located on the
pillar walls, and nanogaps between these metal components (FIG. 7).
The disk array and the backplane (both are 55 nm thick) form a 3D
cavity antenna that can efficiently traps the excitation light
vertically and laterally. The nanodots have diameters of
.about.5-20 nm, and the nanogaps between them and the nanodisks are
1-10 nm. Each pillar has about 10 to 50 nanodots depending upon the
pillar geometry; and the pillar density is 2.5.times.10.sup.9
pillars/cm.sup.2. The exact diameter and height of the pillar and
metal disks, which were optimized to match the wavelengths of
excitation laser and fluorescence, as well as the roles of each
element of D2PA in plasmonic enhancement have been discussed
elsewhere (Li et al Optics Express 2011 19, 3925-3936).
[0178] The D2PA structures were fabricated on 4'' fused silica
wafers by a nanofabrication approach that combines nanoimprint
(top-down) with self-aligned self-assembly (bottom-up). The pillars
were patterned first in the silica wafer by nanoimprint and
reactive ion etching. Then a thin gold layer was evaporated onto
the wafer in a direction normal to the wafer surface, which
simultaneously deposited the gold nanodisk on the pillar top, the
gold backplane, and gold nanodots on the pillar sidewall. The gold
deposited on the pillar sidewall is much thinner than that on the
top of the nanodisks and the backplane. Such thin gold is unstable
and diffuses at the elevated evaporation temperature; and together
with the non-wetting property of gold on SiO2 surface, the gold
self-assembles into nanodots with a small gap in between and
self-aligned precisely next to the gold nanodisk. Other details of
D2PA structure and fabrication are described elsewhere (Li et al,
supra; Chou et al Science 1996 272: 85-87 and Chou et al Applied
Physics Letters 1995 67: 3114-3116, which are all incorporated by
reference for those teachings).
[0179] FIG. 20 shows scanning electron micrographs of
nanodevices--Disk-Coupled Dots-on-Pillar Antenna-Array (D2PA). (A)
Top view and (B) side view of the same D2PA with 70 nm diameter
metallic disk and metallic dot structure on sidewall of size
ranging from 5 nm to 30 nm. And (C) top view and (D) sideview of
another D2PA with metallic disk diameter of 100 nm and metallic dot
structure on sidewall of size ranging from 1 nm to 15 nm. In (C)
two of four disks are missing. Both D2PAs have a period of 200
nm.
[0180] Immunoassay, Fluorophores, Adhesion Layer and Reference.
[0181] The immunoassay was used to test the enhancement in
fluorescence and detection sensitivity was a direct assay of
Protein A and human immunoglobulin G (IgG), which has been widely
used as the simplest model assay for such testing. Protein-A on a
solid plate surface served as the capture agent to capture the IgG
(the targeted analyte) in a solution which are already labeled with
infrared fluorescent dye (IRDye800CW (Li-COR)), hence there was no
need to use additional detection agent. Protein A catch human IgG
through the strong Fc (Fragment, crystallizable) binding. The
concentration of IgG in solution was then quantified by measuring
its fluorescent labels.
[0182] Because Protein A does not bind to the metal surface well,
for the D2PA plate, an additional adhesion layer was used between
the metal and Protein A. This adhesion layer will add an additional
material to the spacer, and could, potentially, weaken the
plasmonic enhancement. To limit the total spacer thickness while
providing good binding of Protein A to the metal (gold in our
case), we used a self-assemble-monolayer (SAM) of dithiobis
succinimidyl undecanoate (DSU) as the adhesion layer. The DSU
molecule has one end of sulfide which strongly binds to a gold
surface and the other end of N-hydroxysuccinimide (NHS) ester group
which binds well to Protein A's amine group.sup.26. The SAM was
.about.1.7 nm thick with refractive index n=1.50. Together with the
Protein A layer which is estimated to be 4.8 nm, the total spacer
thickness is 6.5 nm.
[0183] For comparison with the D2PA plate's immunoassay
fluorescence enhancement measurements, we used plain flat glass
plates as the reference. The immunoassay on the D2PA plates and the
reference was prepared in the same manner and the same batch.
[0184] Preparation of Fluorophore Label and Immunoassay.
[0185] The human IgG (Rockland Immunochemicals) was labeled with
the infrared fluorescent dye, IRDye800CW, in house. NHS ester group
on the dye molecule was coupled to the amine group on IgG by mixing
the reactive dye with IgG solution and letting them react for 2
hours at 20.degree. C. in dark environment. Free dye was separated
through buffer exchange by using desalting spin columns (Pierce
Zeba). Each IgG has an average of 1.3 IRdye800CW molecules.
[0186] For coating DSU SAM on the D2PA, the plates were immersed in
a solution of 0.5 mM DSU (Dojindo, Japan) in 1,4-dioxane
(Sigma-Aldrich), and incubated overnight at room temperature in a
sealed container to form the SAM spacer. After incubation, they
were rinsed extensively in 1,4-dioxane and dried with argon gas and
ready for Protein A immobilization.
[0187] Protein A (Rockland Immunochemicals) in phosphate buffered
saline (PBS) buffer solution (pH=7.4, Sigma-aldrich) was dropped on
the D2PA and reference plates, and each plate was incubated in a
sealed container for 120 min at room temperature. Then the plates
were washed 3 times in wash solution (R&D systems) for 15
minutes each to remove the unbounded molecules. After coating
Protein A, we diced the plates into 5 mm.times.5 mm square pieces
for testing. Then fluorescence-labeled IgG in PBS solution were
then dropped on the Protein A layer and incubated in a sealed
container for 60 min at room temperature. After another washing in
the same manner, the plates were gently rinsed in streams of
deionized water to remove any salt content. After drying with argon
gas, the plates were optically measured immediately.
[0188] To precisely control the concentrations of IgG on the
plates, we first prepare different concentrations of IgG in PBS
solution from 1 .mu.M to 10 aM through serial dilution from stock
solution (using dispense pipette with .+-.0.6% inaccuracy). Then we
precisely dropped 3 .mu.L solution of each concentration on
individual square pieces (each 5 mm.times.5 mm). The 3 .mu.L volume
is chosen to make sure that the solution layer, after completely
wetting the plate top surface, will not spill to the plate's back
surface.
[0189] Optical Measurements.
[0190] The average fluorescence of the immunoassays on the D2PA
plates and the reference plates were measured using a commercial
laser scanning confocal spectrometer (ARAMIS, Horiba Jobin Yvon)
with a 785 nm laser excitation. The system uses a microscope lens
to focus the excitation laser beam normally on the sample surface
and uses the same lens to collect the generated fluorescence. The
laser beam was in a rapid raster-scanning (by a scanning galvo
mirror system) to homogenize the excitation over an area, termed
"laser scan area", which can be varied from a laser spot size
(diffraction limited focal point) up to 100 .mu.m.times.100 .mu.m.
By a step-and-repeat of the laser scan area using an x-y stage, up
to 20 mm.times.20 mm of the sample area can be measured
automatically. The optical signals from a sample were sent to a
spectrometer which consists of gratings and a CCD for spectrum
measurement. Typically, we used a 10.times. objective (Numerical
Aperture (N.A.)=0.25), and 100 .mu.m.times.100 .mu.m laser scan
area. For measuring single molecule fluorescence, another optical
set-up that gives 2D maps of optical signal over the excitation
area was used.
Results and Discussion I
[0191] Plasmonic Resonance of D2PA Plate. The D2PA nanostructure
was optimized to make its plasmonic resonance close to the
wavelengths of excitation laser (785 nm) as well as the absorption
and emission peak of the fluorescent dye (IRDye-800CW) used in the
immunoassay (780 nm and 800 nm respectively). Absorbance of the
D2PA was obtained by measuring the transmission (T) and reflection
(R) spectrum using a white light source, and calibrated to same
measurement performed on a glass plate (T=94%) and silver mirror
standard (R=98%) respectively. The absorbance (1-T-R) was found to
have a resonance peak of 97% at 795 nm and a resonant full width at
half maximum (FWHM) of 145 nm for the optimized D2PA plates without
any molecular coating. After applying the immunoassay, the peak
absorbance becomes 98%, and the resonance peak is blue-shifted
slightly to 788 nm, and the FWHM is 165 nm--slightly wider (FIG.
8). A blue-shift, rather than a common red-shift, which was also
observed in another plasmonic system, has been attributed to
negative molecular polarizability that destructively interferes
with the oscillating polarization from the surface plasmon.
[0192] Large-Area Average Fluorescence Enhancement over 7,400 Fold
with Good Uniformity (.+-.9% Variation). The typical fluorescence
spectrum of the Protein A/IgG immunoassay on the D2PA plate and the
reference (the flat glass plate) are given in FIG. 9. Both spectra
were measured on the assays with 10 nM fluorescent-labeled IgG with
a laser scan area of 100 .mu.m.times.100 .mu.m). The laser power
and the detector integration time were 3 .mu.W and 1 second for the
D2PA, and 212 .mu.W and 8 second for the reference glass plate; but
were normalized in plotting FIG. 9. Compared to the glass reference
sample, the immunoassay's fluorescence intensity on the D2PA is
significantly enhanced. On the other hand, the fluorescence peak
wavelength is the same (800 nm) and the full-width at the
half-maximum (FWHM) is nearly the same (.about.30 nm) as the
reference, which are due to the fact that D2PA's plasmonic
resonance has its peak optimized at 788 nm and a FWHM of .about.165
nm--5 times wider than the dye fluorescence peak, hence making the
plasmonic enhancement factor nearly constant over the entire
wavelength range of the IRDye800CW fluorescence.
[0193] The average fluorescence enhancement of the immunoassay on
the D2PA plate over a reference (the glass plate) was obtained
by:
EF ( .lamda. ) = I fluo . D 2 PA ( .lamda. ) I fluo . REF ( .lamda.
) I Exc . REF I Exc . D 2 PA , ( 1 ) ##EQU00001##
[0194] where I.sub.Exc.REF and I.sub.Exc.D2PA is the intensity of
laser excitation, and I.sub.Fluo.REF and I.sub.Fluor.D2PA is the
measured average fluorescence intensity for the reference and D2PA
plate respectively. Note that we measured the average fluorescence
enhancements using the same IgG concentration for both the
reference and the D2PA. This is to avoid the errors caused by the
effects of different IgG concentrations. Moreover, to ensure the
average accuracy, at least, a total of 5 different laser scan areas
(each 100 .mu.m.times.100 .mu.m) over a sample were measured.
[0195] Using the above approach, the measured average fluorescence
enhancement of the D2PA plate at 10 nM fluorescent-labeled IgG was
7,440 fold over the reference when the fluorescence peaks are
compared, and 7,220 fold when the fluorescence intensities are
integrated over the FWHM of the fluorescence spectrum. FIG. 9 shows
that the average fluorescence enhancement spectrum has much broader
FWHM than the fluorescence spectrum, which is consistent with the
observed D2PA plasmonic resonance spectrum (FIG. 8). For 100 nM
fluorescent-labeled IgG concentration, the average peak enhancement
is 8,460 fold. The immunoassay fluorescence enhancements observed
here are two orders of magnitude higher than previous plasmonic
enhanced fluorescence in immunoassays (Zhang et al Optics Express
2007 15 2598-2606; Tabakman et al Nature Communications 2011 2:
466).
[0196] Another important feature of D2PA is the uniformity of the
giant average fluorescence enhancement over a large area. The
uniformity of D2PA plates was measured by mapping the fluorescence
intensities of 10 nM fluorescence-labeled IgG concentration over
the entire 5 mm.times.5 mm area of the D2PA plate. A laser scan
area (100 .mu.m.times.100 .mu.m) was used, termed a "tile" in
mapping; hence there are a total 2,500 tiles (50.times.50) (FIG.
10a). The laser power was 3 .mu.W and the integration time per tile
was one second. Statistics performed on the mapping measurements
showed that the average fluorescence enhancement over such large
sample surface is 7,000 fold with a variation (defined as the
variation of a Gaussian distribution) of 18% or .+-.9% from the
mean--very uniform everywhere (FIG. 10b).
[0197] For the laser excitation power density and excitation time
used in above measurements, we have not observed either saturation
or noticeable bleaching, which are essential to ensure accurate
enhancement measurements. In fact, the fluorescence signals from
both D2PA and the reference samples are found to be linear over a
wide range of laser power density and dye concentration, which
indicate no saturation. Moreover, the fluorescence vs. time
measurement showed that under the laser intensities used, even for
a time period much longer than the typical measurement time we
used, there is still no noticeable bleaching.
[0198] Sub-Femto-Molar Detection Sensitivity and Wide Dynamic
Range. In clinical diagnostic applications, the detection
sensitivity (i.e. the limit of detection (LoD)) and dynamic range
have more practical meaning than the fluorescence enhancement
factor. To test these, we measured the immunoassay's fluorescence
signals from the D2PA and the reference prepared with different IgG
concentrations: from 1 .mu.M to 10 aM (with a series dilution
factor of 10). The LoD, determined using the well-accepted
standard, is the IgG concentration corresponding to the
fluorescence signal that is equal to the background optical noise
plus three times of its standard deviation (i.e. the
root-mean-squared deviation). In our experiments, the background
optical noise were obtained by performing the exactly same optical
measurements on a blank sample as the sample with IgG (i.e. the
same optical setup, sample area, laser power and integration time).
The blank sample was prepared on identical substrates using the
same preparation protocol except that the normal step of dropping
fluorescent-labeled IgG is replaced by dropping of pure buffer
solution (i.e. no IgG).
[0199] FIG. 11 shows the logarithm plot of the fluorescence signals
versus the fluorescent-labeled IgG concentration deposited on the
D2PA and the reference (the response curve). Error bars are the
standard deviation, calculated from the measurements at five
different sample areas for each concentration. To determine the
LoD, we first used five-parameter logistic regression model to
create fitting curves which allow an extrapolation of the data
points between the measured ones. The LoD of the immunoassay on
D2PA plates was found to be 0.3 fM (3.times.10.sup.-16 M), and the
dynamic range (where the fluorescence is linear with IgG
concentration) is over eight (8) orders of magnitude (from 1 .mu.M
to 1 fM) (FIG. 11). On the other hand, the LoD of identical
immunoassays performed on the reference plates (planar glass
plates) was found to be 0.9 nM (0.9.times.10.sup.-9 M). Therefore
the detection sensitivity is enhanced by 3,000,000 fold (over 6
orders of magnitude) on the D2PA plate compared to the glass plate.
This detection sensitivity enhancement is over two orders of
magnitude higher than the previous work using plasmonic structures
(Zhang et al Optics Express 2007 15 2598-2606; Tabakman et al
Nature Communications 2011 2: 466).
[0200] Fluorescence Enhancement of Single Molecule Fluorophore at
Hot Spot up to 4.times.10.sup.6 Fold. To explore the potential in
further enhancing fluorescence and detection sensitivity, the
fluorescence enhancement of the immunoassay was measured from a
single labeled IgG molecule which was placed at a "hot spot" of
D2PA (namely the region where the local electric field is the
strongest). Such single molecule fluorescence can be visible when
the IgG molecules are far apart from each other (i.e. a very low
IgG concentration) and a sensitive CCD camera is used.
[0201] An IgG concentration of 100 pM was used to study single
molecule fluorescence, which gives an average distance between two
immobilized IgG about 420 nm. We mapped the two-dimensional
fluorescence of the immunoassay using an inverted microscope
(Nikon, USA) with 40.times. objective lens (N.A.=0.6). A 785 nm
laser beam was expanded uniformly to illuminate a 50 .mu.m.times.50
.mu.m area on D2PA plates. Images were continuously collected by an
electron multiplying charge-coupled device (EM-CCD, Andor) of
512.times.512 pixel resolution (hence .about.390 nm per pixel for
the given laser scanning area). The CCD pixel size oversamples the
fluorescence intensity distribution imaged at optical
diffraction-limit (0.8 .mu.m determined by Rayleigh criterion).
[0202] From the fluorescence imaging of 100 pM fluorescent-labeled
IgG on D2PA plate (FIG. 12a), distinct fluorescence "bright spots"
that were randomly distributed in a uniform background were
observed. The fluorescence intensity of individual bright spot as a
function of time was shown to have a binary stepwise behavior (FIG.
12b), which indicates that a single molecule at or near a D2PA's
hot spot first emits fluorescence and then gets bleached.
[0203] To estimate the fluorescence enhancement factor for single
molecule at a hot spot, g.sub.Hotspot, we used two methods. For the
first method, g.sub.Hotspot is the ratio of the single molecule
fluorescence signal at a "hot spot" of D2PA, S.sub.Hot.Spot, to the
average fluorescence signal per molecule on reference sample (which
equals to the area-average fluorescence intensity on reference
sample, I.sub.Ref.Avg, divided by the average IgG molecules per
unit area on reference sample, n.sub.Ref.Avg):
g Hot . Spot = S Hot . Spot I Exc . Ref ( I Ref . Avg / n Ref . Avg
) I Exc . D 2 PA ( 2 ) ##EQU00002##
[0204] where I.sub.Exc.D2PA and I.sub.Exc.Ref is the excitation
intensity for the D2PA and reference plates, respectively.
According to FIG. 12(a), S.sub.Hotspot=1,200 counts,
I.sub.Ref.Avg=3,088 counts/.mu.m.sup.2, and
n.sub.Ref.Avg=7.22.times.10.sup.5 molecule/.mu.m.sup.2,
I.sub.Ref.Exc=1.74 mW and I.sub.Exc.D2PA=110 .mu.W. We found the
fluorescence enhancement is g.sub.Hotspot=4.4.times.10.sup.6, which
is 3 orders of magnitude larger than most of the reported
fluorescence enhancement for a single molecule in the "hot
spot".sup.31.
[0205] For the second method, the average fluorescence intensity
per molecule for the reference was deducted from the average
fluorescence intensity per molecule for the D2PA plate
(I.sub.D2PA.Avg/n.sub.D2PA.Avg) divided by the fluorescence
enhancement factor (EF). The division of EF scales the signal from
D2PA plate to a regular glass plate:
g Hot . Spot = S Hot . spot ( I D 2 PA . Avg / n D 2 PA . Avg ) /
EF , ( 3 ) ##EQU00003##
[0206] For I.sub.D2PA.Avg=19 counts, EF=7,220 and
n.sub.Avg.about.7.22 molecule/.mu.m.sup.2, we found
g.sub.Hotspot=3.28.times.10.sup.6. Both methods gave consistent
results for calculating the single molecule fluorescence
enhancements. The average of the two methods gives
g.sub.Hotspot.about.4.times.10.sup.6.
[0207] Origin of Giant Assay Fluorescence and Detection Sensitivity
Enhancement and Uniformity. Without wishing to be bound to any
particular theory, it is believed that that there are three primary
reasons for the observed enhancement in fluorescence and detection
sensitivity. The first and the most important is the unique D2PA
plasmonic structure. The second is a proper ultra-thin spacer
layer. And the third is the possibility that the D2PA structure
might concentrate the biomarkers into the hot spots of the
D2PA.
[0208] Again without wishing to be bound to any particular theory,
it is believed that the plasmonic structure D2PA enhances the
fluorescence significantly through four key factors. (1) The 3D
antennas array is extremely efficient in receiving excitation light
and radiating fluorescent light. As already shown in FIG. 8, the
measured absorbance of the optimized D2PA is .about.97% at the
excitation laser wavelength of 785 nm. A good light absorber is
also a good radiator. (2) The small metallic dots and the small
gaps in the D2PA can strongly focus light to small regions to
significantly enhance local electric fields, as already
demonstrated in SERS study (Li et al, supra). The smaller the dots
and the gaps, the stronger the focusing (and local electric field
enhancements) will be (Schuller et al Nature Materials 2010 9:
193-204 and Nie et al Science 1997 275: 1102-1106). However, it is
also well-known that small metallic structures of subwavelength
size are extremely poor light absorber and radiator (Fromm et al
Nano Letters 2004 4 957-961 and Farahani et al Physical Review
Letters 2005 95, article no. 017402). Hence only the small dots and
gap alone without antenna will not make a good fluorescence
enhancer, since it only can concentrate a small portion of the
incoming photons while most of the photons are thrown away, and it
cannot efficiently radiate the fluorescence generated in the near
field into the far field. (3) The effective coupling between the
D2PA's antennas and the nanostructures through the nanogaps between
them, making the D2PA effective both in receiving and radiating
light and in locally focusing the light to small spots. And (4) the
high densities of antennas, dots, and gaps allow larger percentage
of targeted molecules to be near the hot spots, hence increasing
the fluorescence's average enhancement and uniformity and reducing
the performance sensitivity to the device geometry variations.
Since the final plasmonic enhancement is a product of all four
factors, any plasmonic structures that lacking one of the four
factors could end up a poor plasmonic enhancer, which is exactly
the problem suffered by the most previous plasmonic structures, and
the exact reason why the D2PA, which improves all four factors
together, is superior.
[0209] To maximize the overall MEF fluorescence, the proper
ultra-thin spacer layer plays a key role in balancing the
fluorescence excitation and quenching by the same metal. In a
separate experiment where the only spacer between a D2PA's metal
and a fluorophore is a SiO2 layer (i.e. without any assay), we
found that a 5 nm SiO2 thickness offers the best balance between
MEF and quenching.sup.30. Considering the total spacer layer
thickness for our D2PA's assay is 6.5 nm (does not include IgG) and
the effective permittivity is 3, this spacer has a total effective
dielectric distance very close to the 5 nm SiO2 spacer. Therefore,
the choice of self-assembled adhesion layer, DSU, offers a proper
spacing for MEF.
[0210] That theory the D2PA structure may concentrate the
biomarkers (targeted analytes) in a solution into the hot spots
(i.e. large local electric field region) is just a speculation.
Three possible reasons may account for such concentration. (1) The
drying of liquid on D2PA surface will occur first outside pillar
sidewall, but the last inside the gaps. Hence the liquid movement
during the drying may bring biomarkers from other locations into
the gaps. (2) The local built-in electric field in D2PA could moves
biomarkers (if they are polar molecules) to hot spots. And (3) the
SAM adhesion layer, DSU, adheres only the gold not the SiO2, which
could led to more Protein A and hence more fluorescent-labeled IgG
on gold nanodots than the SiO2 sidewalls that are not covered by
the gold.
[0211] There are two more important experimental facts that need
discussions. (1) The measured immunoassay fluorescence signal
intensity does not drop as fast as the biomakers' concentration
(i.e. not in 1:1 ratio). This, known to the fluorescence
immunoassays, is believed to be caused by the fact that the bonding
of IgG to Protein A during the incubation and the loss of the IgG
bonded on Protein A during the washing may be different for
different IgG concentrations. And (2) the detection sensitivity
(LoD) enhancement by D2PA (3,000,000) is over 400 times higher than
the fluorescence enhancement (7,400) at 10 nM fluorescent-labeled
IgG. We suspect this might be due to biomarkers in a solution being
concentrated into the hot spots of D2PA.
[0212] It should also be pointed out that the fluorescence
enhancement by plasmonic structures is known to depend on the
intrinsic quantum efficient (QE) of a dye: stronger enhancement for
a lower QE. At a low QE, the fluorescence enhancement is inversely
proportional to the QE. The IRDye800cw dye has a quantum efficiency
of 7%, similar to the QE of the dyes used in other assay
experiments. Even when the difference of the different dyes's QEs
is scaled, the observed average fluorescence enhancements are still
two orders of magnitude higher than previous experiments.
[0213] Finally, the large fluorescence enhancement factor of single
fluorophore at a hot spot may indicate that one might be able to
further increase the assay detection sensitivity significantly if
biomarkers are placed into the hot spots.
Materials and Methods II
Ultrasensitive Detection of Prostate Specific Antigen Preparation
of PSA Immunoassay on Nanosensor (Also Termed: D2PA Plate)
[0214] The D2PA immunoassay plate is made up of two components: (1)
the aforementioned D2PA plasmonic nanostructure and (2) a mixed
self-assembled layers of Protein A layer on top of ithiobis
succinimidyl undecanoate (DSU). The DSU molecules provide strong
cross-link of protein A to gold surface by providing one end of
sulfide that strongly binds to gold and the other end of
N-hydroxysuccinimide (NHS) ester group that binds well to Protein
A's amine group. These molecular layers (Protein A and DSU) have
two functions: (1) with a combined thickness of 6.5 nm they will
act as a spacer layer that can suppresses metal's fluorescence
quenching effect and (2) Since antibodies will bind to protein A
through their Fc region, the molecule layers on D2PA can increase
the quality of antibody orientation and immobilization, which will
further improve the capture efficiency of the antibodies.
[0215] For coating DSU SAM and Protein A on the D2PA, freshly
fabricated D2PA substrate was first diced into 5 mm.times.5 mm
pieces and immersed in a solution of 0.5 mM DSU (Dojindo, Japan) in
1,4-dioxane (Sigma-Aldrich), and incubated overnight at room
temperature in a sealed container. After incubation, the D2PA
substrates were rinsed extensively in 1,4-dioxane and dried with
argon gas. We immediately place these DSU coated D2PA substrates in
separated wells of a standard 96-well plates (Pierce, USA). They
were then immersed in 100 uL of 10 ug/mL Protein A (Rockland
Immunochemicals) in phosphate buffered saline (PBS) solution
(pH=7.2, Sigma-Aldrich) and incubated in a sealed condition
overnight in the fridge at 4 C. The solution is then aspirated and
each individual D2PA plates is washed 3 times in washing solution
(R&D systems) for 15 minutes each to remove the unbonded
protein A. The plates were then gently rinsed in streams of
deionized water to remove any salt content. After drying with argon
gas, the D2PA immunoassay plate was ready for immediate immunoassay
testing or stored at -20 C degree for later use.
[0216] A fluorescence-based sandwich assay of PSA, which is widely
used in both research and clinical diagnosis, was performed on the
D2PA immunoassay plates. Capture antibodies (mouse anti-human
Kallikrein 3) were first immobilized by immersing the D2PA
immunoassay plate in 100 uL of capture antibody solution with
concentration of 180 ug/mL and incubate for 2 hours at room
temperature. We then aspirate the solution and wash the plates with
wash buffer, followed with blocking of each individual plate by
immersing in 100 uL of blocking solution (R&D systems) and
incubate at room temperature for 1 hour. After the same
aspiration/wash process, the D2PA plates in each well were then
immersed in 100 uL of PSA (R&D systems) in PBS solution at
concentrations from 10 aM to 10 pM with a dilution factor of 10.
They were then incubated at room temperature for 2 hours. After
another washing, 100 uL of detection antibody (goat anti-human
Kallikrein 3) at concentration of 200 ng/mL was added to each
individual plate and incubated at room temperature for 1 hour. We
then repeated the aspiration/wash process again and added 50 uL of
diluted IRDye800CW labeled streptavidin at 50 ng/mL concentration
(Rockland Immunochemicals) to each D2PA plate and incubate at room
temperature for 1 hour. After the final washing, the D2PA plates
were rinsed gently in deionized water and dried with argon gas. The
plates were optically measured immediately after the immunoassay
was developed.
[0217] For comparison with the D2PA immunoassay plates'
fluorescence enhancement and sensitivity improvement, we used plain
flat glass plates coated with Protein A as the reference. Identical
Sandwich PSA immunoassay was prepared on the reference using the
reagent from the same batch and treated in the same manner.
[0218] Optical Measurements.
[0219] Fluorescence-based PSA immunoassays on the D2PA plates and
the reference plates were both measured using a commercial laser
scanning confocal spectrometer (ARAMIS, Horiba Jobin Yvon) with a
785 nm laser excitation. Excitation light and fluorescence signals
were measured above the sample surface at normal angle. The system
uses the same microscope lens to focus the excitation laser beam as
well as to collect the generated fluorescence light. The laser beam
was in a rapid raster-scanning (by a scanning galvo mirror system)
to homogenize the excitation over an area, termed "laser scan
area", which can be varied from a laser spot size (diffraction
limited focal point) up to 100 .mu.m.times.100 .mu.m. By a
step-and-repeat of the laser scan area using an x-y stage, up to 20
mm.times.20 mm of the sample area can be measured automatically.
Fluorescence signals from the sample were coupled to a spectrometer
that consists of gratings and a CCD for spectrum measurement. In
this report, we used a 10.times. objective (Numerical Aperture
(N.A.)=0.25), and 100 .mu.m.times.100 .mu.m laser scan area and
measured over the entire 5 mm.times.5 mm D2PA immunoassay plates
surface.
Results II
[0220] Giant Fluorescence Enhancement Over 1,700 Fold Over Large
Area with Good Uniformity (.+-.10% Variation).
[0221] FIG. 13 shows the typical fluorescence spectrum of a PSA
immunoassay measured on D2PA plates and the glass plate references.
Both spectra were obtained on the assay with 10 pM PSA
concentration. The laser power and detector integration time were 3
uW and 10 second for the D2PA, and 212 uW and 8 second for the
reference glass plate. We had to use larger laser excitation power
for the measurements on reference glass plate in order to get
spectrum with good Signal-to-noise ratio (SNR). For the laser
excitation power density and excitation time used in above
measurements, we have observed neither saturation nor noticeable
bleaching, which is essential for the accurate enhancement factor
measurements. In fact, the fluorescence signal intensity was
constant over a long period of time due to the raster scanning of
laser focal spot--bleaching effect is thus minimized in our
experiments.
[0222] The enhancement factor estimated here still hold for higher
concentrations. Using the above approach, the measured average
fluorescence enhancement of the D2PA plate at 10 pM PSA was 1,700
fold over the glass reference plate when the fluorescence peak
intensities are compared.
[0223] Ten Atto-Molar Detection Sensitivity and Wide Dynamic
Range.
[0224] In order to demonstrate how the giant fluorescence
enhancement will improve the immunoassay sensitivity, i.e. LoD, and
dynamic range, we measured the PSA immunoassay's fluorescence
signals response of PSA concentration from 10 aM to 10 pM (with
series dilution factor of 10) on the D2PA and the reference glass
plate. The LoD, determined using the well-accepted standard, is the
PSA concentration corresponding to the fluorescence signal that is
equal to the background optical noise plus three times of its
standard deviation (i.e. the root-mean-squared deviation). In PSA
immunoassay, the background optical noises were obtained by
performing the exactly same optical measurement on an assay during
which the PSA solution is replaced by pure PBS buffer solution
(i.e. no PSA), while the other steps of preparation protocol remain
the same.
[0225] FIG. 13 shows the D2PA and reference glass plate's
fluorescence immunoassay signals as a function of PSA concentration
in logarithm scale (i.e. the response curve). Error bars are the
standard deviation calculated from the measurements on five
replicates (different sample plates with identical immunoassay and
same PSA concentration). We used a five-parameter logistic
regression function to create fitting curves, which allow an
extrapolation of the data points between the measured PSA
concentrations. We then determined the LoD of the immunoassay on
D2PA plates to be 10 aM (0.3 fg/mL), and the dynamic range over 6
decades (from 1 fM to 1 uM). We also found the LoD of identical
immunoassay performed on the reference glass plates to be 0.9 pM
(27 pg/mL). Therefore, the D2PA immunoassay plate's detection
sensitivity was enhanced by 90,000 fold compared to the glass
plate. In addition, the detection sensitivity we reported here is
at least 30 times better than previous reports using competitive
techniques.
Ultra-Sensitive Detection of Breast Cancer Biomarkers CEA and
CA15.3
[0226] The nanosensor described above has demonstrated (a) LoD of
Carcinoembryonic Antigen (CEA) (see FIG. 14) and CA15.3 (breast
cancer biomarkers) (see FIG. 15) of 28 aM (.about.0.8 fg/mL) and
0.001 U/mL respectively, which is 3-5 orders of magnitude better
than commercial ELISA kits (CEA: R&D systems and CA15.3:
Abcam), and 5-6 orders of magnitude more sensitive than the
clinical cut-off level (4 ng/mL for CEA and 25 U/mL for CA15.3 in
blood plasma). (b) both new assays have a linearity of 8 dynamic
orders.
[0227] For the modified three-layer-sandwich CEA assays on the D2PA
plate, we have achieved an LoD of 28 aM (.about.0.8 fg/mL) in
buffer with 8 order dynamic range, respectively, when using a
conventional plate reader (area-averaged fluorescence intensity)
(FIG. 14). The new assay's LoDs are 170,000-fold better than an
identical assay performed on a standard glass plate; 20-fold more
sensitive than the current best CEA immunoassays (e.g. random gold
island); and 5-6 orders of magnitude more sensitive than the
typical CEA level in blood plasma (4 ng/mL).
[0228] For CA15.3 Immunoassay, we have achieved an LoD of 0.001
U/mL (unit/mL, 1 unit is an arbitatry value related to a maintained
reference antigen preparation. A conversion between U/mL to
molarity is not available) and 4-5 orders of magnitude more
sensitive than typical CA15.3 level in blood plasma (25 U/mL).
Ultra-Sensitive Detection of Alzheimer's Disease Biomarkers Beta
Amyloid 1-40 and 1-42
[0229] The new assay has demonstrated (a) LoD of .beta.-amyloid
(A.beta.) in buffer of 2.3 fM (10.3 fg/mL) for A.beta. 42 and 0.2
fM (0.9 fg/mL) for A.beta. 40; (b) a linearity of over 8 dynamic
order, (c) a recovery rate of 88% and 106.8% for serum and saliva
spike-and-recovery assessment respectively, (d) 0.06% cross
reactivity of A.beta.40 over 42 (for 1 pg/ML A.beta.42 conc.), and
(e) consistent and repeatable detection of A.beta. 42 in both blood
and saliva. See FIGS. 16 and 17.
[0230] In the detecting A.beta.-42, the bottom of an ordinary 96
well-plate is replaced with the new assay plate; and commercial
"A.beta.-42 and 40 ELISA kits" (Covance USA) were modified, where
we only attach commercial streptavidin-conjugated fluorescence
(IRDye800CW) labels (Rockland USA) to Covance's biotinylated
detection antibody (agent) through biotin-avidin reaction (no
enzyme was used in our assay). We then used the rest of the kit as
is, and followed the conventional protocol. The capture and
detection agent in the Covance kit is, respectively, 6 E10 and
anti-A.beta.42 for A.beta.-42 and 6 E10 and anti-A.beta.40 for
A.beta.-40.
[0231] The one that we used for A.beta. assay is a bilayer of
dithiobis (succinimidyl undecanoate) (DSU) and Protein A. DSU has
one end of sulfide which strongly binds to the gold surface on D2PA
and the other end of N-hydroxysuccinimide (NHS) ester group which
binds well to the amine-group on Protein A, which, in turn, binds
to the capture antibody through the Fc region.
[0232] (a) Sub-100 fM limit of detection (LoD) with 8 order dynamic
range. In the first test on the D2PA plate, we used a two layer
model immunoassay of Protein A and fluorescence labeled IgG, and
achieved LoD enhancement over a glass plate by more than one
million times (.times.10 6). See FIG. 11. For the modified
three-layer-sandwich A.beta. assays on the D2PA plate, we have
achieved an LoD of 2.3 fM (10.3 fg/mL) and 0.2 fM (0.9 fg/mL) for
detecting A.beta. 42 and 40 in buffer with 8 order dynamic range,
respectively, when using a conventional plate reader (area-averaged
fluorescence intensity). The new assay's LoDs are 5,000-fold better
than an identical assay performed on a standard glass plate; 500
times more sensitive than the current best commercial A.beta.
assays (e.g. Meso Scale Discovery); and .about.4 and 5 orders of
magnitude more sensitive than the typical level A.beta.-42 and 40
in blood plasma (C.sub.A.beta.42.about.50 pg/mL,
C.sub.A.beta.40.about.200 pg/mL), and .about.3 and 4 orders of
magnitude more sensitive saliva (C.sub.A.beta.42.about.5 pg/mL and
C.sub.A.beta.40.about.25 pg/mL) respectively.
[0233] (b) Excellent Linearity of 8 dynamic orders has been
achieved in all tests (See, e.g., FIG. 11).
[0234] (c) Recovery rate of 88% for blood serum and 102% for human
saliva are achieved in the performed spike-and-recovery test for
the A.beta.-42 immunoassay on D2PA plate. (See, e.g., FIG. 16)
[0235] (d) Cross-Reactivity of 0.06% of A.beta. 42 immunoassay over
A.beta. 40 at 1 pg/mL A.beta. concentration and much better
cross-reactivity at a higher A.beta. concentration have been
achieved (FIG. 17)
[0236] (e) Excellent Parallelisms for A.beta. immunoassays on D2PA
plate (FIG. 18) and other assays on D2PA have been demonstrated. In
fact, we have made 2 independent production runs and achieved
consistent results.
Ultra-Sensitive DNA Hybridization Assay
[0237] One of the example for enhanced DNA hybridization assay
using D2PA nanodevice is a sandwich hybridization assay. The
capture DNA is a single strand DNA functioned with thiol at its
5'-end. (3'-GAAGAAGATAGACTTACATG-5'-SH) The detection DNA is a
single strand DNA functioned with a fluorescence label e.g.,
IRDye800CW at its 3'-end (IR800-3'-TTTGGCTTGTGGTAGTTAGA-5'). 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. The targeted DNA is
5'-ACCGAACACCATCAATCTCTTCTATCTGAATGTACTTTTT-3')
[0238] First the capture DNA is immobilized on the D2PA
nanodevice's metal surface by incubating the D2PA nanodevice with 5
.mu.M DNA solution diluted with 1.times.TE buffer with 1M NaCl
concentration. Then targeted DNA is diluted in DNA hybridization
buffer (H7140, Sigma-Aldrich) and added to the nanodevice to
hybridize with the capture DNA. The temperature for hybridization
is controlled below the melting temperature at 35.degree. C.
Finally the fluorescence labeled detection DNA with concentration
of 100 .mu.M is added to the nanodevice to hybridize with the
immobilized targeted DNA. The hybridization buffer and temperature
remain the same as the last step. After washing off the unbound
detection DNA, the fluorescence signal emanate from the
nanodevices' surface is measured for the detection and
quantification of targeted DNA molecules.
[0239] We measured the fluorescence signal intensity from the DNA
hybridization assay with different concentrations of targeted DNA.
FIG. 19 shows the fluorescence response curve (standard curve) for
the hybridization assay performed on D2PA nanodevices. The limit of
detection, calculated as the background signal plus three times of
the standard deviation of background signal, is 71 fM.
Sequence CWU 1
1
3120DNAartificial sequenceSynthetic oligonucleotide 1gaagaagata
gacttacatg 20220DNAartificial sequencesynthetic oligonucleotide
2tttggcttgt ggtagttaga 20340DNAartificial sequencesynthetic
oligonucleotide 3accgaacacc atcaatctct tctatctgaa tgtacttttt 40
* * * * *