U.S. patent application number 15/055982 was filed with the patent office on 2017-06-29 for novel gold nanostructures and methods of use.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is Tammy Y. Olson, Adam Schwartzberg, Jin Z. Zhang. Invention is credited to Tammy Y. Olson, Adam Schwartzberg, Jin Z. Zhang.
Application Number | 20170184505 15/055982 |
Document ID | / |
Family ID | 41505471 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170184505 |
Kind Code |
A1 |
Zhang; Jin Z. ; et
al. |
June 29, 2017 |
Novel Gold Nanostructures and Methods of Use
Abstract
The invention is drawn to novel nanostructures comprising hollow
nanospheres and nanotubes for use as chemical sensors, conduits for
fluids, and electronic conductors. The nanostructures can be used
in microfluidic devices, for transporting fluids between devices
and structures in analytical devices, for conducting electrical
currents between devices and structure in analytical devices, and
for conducting electrical currents between biological molecules and
electronic devices, such as bio-microchips.
Inventors: |
Zhang; Jin Z.; (Santa Cruz,
CA) ; Schwartzberg; Adam; (Santa Cruz, CA) ;
Olson; Tammy Y.; (Santa Cruz, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zhang; Jin Z.
Schwartzberg; Adam
Olson; Tammy Y. |
Santa Cruz
Santa Cruz
Santa Cruz |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
41505471 |
Appl. No.: |
15/055982 |
Filed: |
February 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13284880 |
Oct 29, 2011 |
9276063 |
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15055982 |
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11784297 |
Apr 5, 2007 |
8137759 |
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13284880 |
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60790317 |
Apr 7, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54346 20130101;
H01L 29/0665 20130101; Y10S 977/896 20130101; H01L 29/0669
20130101; H01L 29/0673 20130101; Y10S 977/81 20130101; B22F 2998/10
20130101; B22F 9/24 20130101; G01N 21/658 20130101; G01N 33/54326
20130101; G01N 21/554 20130101; B82Y 10/00 20130101; B22F 2998/00
20130101; B82Y 35/00 20130101; H01L 29/127 20130101; Y10S 977/958
20130101; B22F 2304/05 20130101; B82Y 25/00 20130101; B22F 2301/255
20130101; Y10T 428/12431 20150115; B82Y 15/00 20130101; B22F
2998/00 20130101; B82Y 30/00 20130101; B22F 2009/245 20130101; Y10T
428/2982 20150115; G01N 2201/06113 20130101; H01F 1/0054 20130101;
G01N 33/5432 20130101; B22F 2001/0029 20130101; B22F 1/0025
20130101; Y10T 428/2975 20150115; Y10S 977/762 20130101; B22F 9/24
20130101 |
International
Class: |
G01N 21/65 20060101
G01N021/65; B22F 9/24 20060101 B22F009/24; B22F 1/00 20060101
B22F001/00; H01L 29/06 20060101 H01L029/06; G01N 33/543 20060101
G01N033/543 |
Goverment Interests
[0001] This invention was made partly using funds from the National
Science Foundation, the Petroleum Research Fund/American Chemical
Society, the University of California at Santa Cruz, the student
employee graduate research fellowship at Lawrence Livermore
National Laboratory. This work was performed under the auspices of
the U.S. Department of Energy by University of California Lawrence
Livermore National Laboratory under contract No. W-7405-Eng-48. The
US Federal Government has certain rights to this invention.
Claims
1-30. (canceled)
31. A method, comprising the step of: using a chemical sensor for
measuring at least one cellular process with a detecting molecule
bound to a surface of a synthetic nanotube to determine a cellular
process measurement, wherein the chemical sensor comprises the
synthetic nanotube, the synthetic nanotube comprising a
substantially homogenous polycrystalline uniform symmetrical metal
nanostructure, the synthetic nanotube being substantially hollow
and having dimensions of between about 20 nm and 100 nm in diameter
and at least between about 0.1 .mu.m and 4 .mu.m in length, the
synthetic nanotube further comprising the surface and the detecting
molecule, and wherein the detecting molecule is bound to the
surface.
32. The method of claim 31, wherein the step of using the chemical
sensor further comprises the steps of (i) providing the chemical
sensor; (ii) providing a plurality of cells; (iii) incubating the
chemical sensor with the plurality of cells for a predetermined
period to bind the detecting molecule to the plurality of cells;
(iv) measuring the extent of binding between the detecting molecule
and the plurality of cells thereby measuring the cellular
process.
33. The method of claim 31, wherein the chemical sensor comprises
at least two detecting molecules bound to the surface of the
synthetic nanotube and the step using the chemical sensor further
comprises a step of using the chemical sensor for measuring at
least two cellular processes.
34. The method of claim 31, wherein the cellular process is
selected from the group consisting of intracellular lipid
metabolism, the cell cycle, actin skeleton regulation, cell
proliferation, and cellular motility.
35. The method of claim 31, where the plurality of cells comprises
human tissue, the human tissue selected from the group consisting
of tumor tissue, blood fluids, lymph fluids, hemolymph fluids,
pulmonary surfactant fluids, peritoneal fluids, gastric fluids,
xylem fluids, and phloem fluids.
36. A method, comprising the step of: producing a chemically stable
and electrically conducting nanotube, further comprising the steps
of (i) combining an aqueous solution of Co.sup.2+ salt with an
aqueous solution of citrate salt thereby forming a first mixture;
(ii) degassing the first mixture; (iii) purging at least once with
nitrogen gas; (iv) adding an aqueous solution of NaBH.sub.4 thereby
reducing the Co.sup.2+ to Co.sup.0, and thereby forming a second
mixture comprising Co.sup.0 particles, the step of adding being in
the presence of an induced magnetic field and wherein the presence
of the induced magnetic field aligns the Co.sup.0 particles; (v)
agitating the second mixture until hydrogen evolution is
substantially complete; (vi) adding the second mixture comprising
aligned Co.sup.0 particles to an aqueous solution of Au.sup.3+
salt; (vii) allowing the Au.sup.3+ to be reduced to crystalline
Au.sup.0 and the Co.sup.0 oxidized to Co.sup.2+, and wherein the
crystalline Au.sup.0 is deposited adjacent to the aligned Co.sup.0
thereby creating a nanotube comprising crystalline Au.sup.0; (viii)
Chemically controlling a length, a diameter, and a wall thickness
of the nanotube adjusting an initial concentration of each of the
Co.sup.2+ salt, of the citrate salt, and of the Au.sup.3+ salt; and
(ix) Magnetically controlling a position and a structural alignment
of the nanotube by adjusting a magnetic field; thereby producing a
chemically stable and electrically conducting nanotube.
37. The method of claim 36, wherein the step magnetically
controlling further comprises the step of magnetically controlling
the position and structural alignment of the nanotube by adjusting
a field strength of the magnetic field and/or adjusting a relative
position of the magnetic field.
38. The method of claim 36, wherein the initial concentration of
Co.sup.2+ salt is less than 0.4 M.
39. The method of claim 36, wherein the initial concentration of
Co.sup.2+ salt is 0.4 M.
40. The method of claim 36, wherein the initial concentration of
Co.sup.2+ salt is greater than 0.4 M.
41. The method of claim 36, wherein the initial concentration of
citrate salt is less than 0.1 M.
42. The method of claim 36, wherein the initial concentration of
citrate salt is 0.1 M.
43. The method of claim 36, wherein the initial concentration of
citrate salt is greater than 0.1 M.
44. The method of claim 36, wherein the initial concentration of
Au.sup.3+ salt is less than 0.1 M.
45. The method of claim 36, wherein the initial concentration of
Au.sup.3+ salt is 0.1 M.
46. The method of claim 36, wherein the initial concentration of
Au.sup.3+ salt is greater than 0.1 M.
47. An apparatus comprising the nanotube produced using the method
of claim 36.
48. The apparatus of claim 47, further comprising a miniature
electronic circuit adapted to interact with the nanotube.
49. The apparatus of claim 48, wherein the miniature electronic
circuit is selected from the group consisting of a memory and an
electrical circuit.
50. The apparatus of claim 48, further comprising the miniature
electronic circuit used with at least one biological medium,
wherein the biological medium is selected from the group consisting
of proteins, cell surface receptor proteins, and antibodies,
photosensitive compounds, chlorophyll, xanthocyanins, compounds
having oxidoreductase activity, cytochromes, haemoglobin,
myoglobin, and fluorescent compounds.
51. A method comprising the step of imaging at least one molecule
(molecular imaging) by use if a chemical sensor responding to a
sample binding a detecting molecule bound to a surface of a
synthetic nanotube contained in the chemical sensor.
52. The method of claim 51, wherein the step of molecular imaging
comprises the steps of (i) providing a chemical sensor, the
chemical sensor comprising a synthetic nanotube, the synthetic
nanotube comprising a substantially homogenous polycrystalline
uniform symmetrical metal nanostructure, the synthetic nanotube
being substantially hollow and having dimensions of between about
20 nm and 100 nm in diameter and at least between about 0.1 .mu.m
and 4 .mu.m in length, the synthetic nanotube further comprising a
surface and at least one detecting molecule, and wherein the
detecting molecule is bound to the surface; (ii) providing a
sample; (iii) mixing the chemical sensor with the sample, whereby
the detecting molecule of the chemical sensor binds to the sample;
(iv) measuring the SERS activity of the chemical sensor; (v)
analyzing the SERS activity, the method resulting in molecular
imaging.
53. The method of claim 51, wherein the sample is selected from the
group consisting of a biomedical sample, an electronic device, and
an industrial chemical.
54. The method of claim 53, wherein the biomedical sample is
selected from the group consisting of tumor tissue, blood fluids,
lymph fluids, hemolymph fluids, pulmonary surfactant fluids,
peritoneal fluids, gastric fluids, xylem fluids, and phloem
fluids.
55. The method of claim 53, wherein the electronic device includes
at least one member of the group consisting of a memory, a memory
chip, a microchip, and a chip.
56. The method of claim 53, wherein the industrial chemical
includes at least one member of the group consisting of a quantum
dot (QD) and a semiconductor quantum dot (SQD).
Description
FIELD OF THE INVENTION
[0002] The present invention relates to structures comprising a
metal with useful properties. The structures are hollow spheres and
tubes. The invention further relates to methods of using the
structures for detecting chemical and biological analytes and use
in electronics and in microfluidics.
BACKGROUND
[0003] During the 1980s Raman Scattering in fibers was demonstrated
by Lin, Stolen, and other co-workers of AT&T Bell Laboratories
in Holmdel, N.J., using lasers operating between 0.3 .mu.m to 2.0
.mu.m. In the early years of the Raman fiber before extensive work
had begun, no one perceived that a Raman fiber could be pumped by a
practical semiconductor laser-based source or that an efficient
CW-pumped Raman Fiber Laser was possible.
[0004] However, with the development of Cladding-pumped Fiber
Lasers and Fiber Bragg Gratings, diode-laser-based CW Raman Fiber
Lasers have been made efficient, emitting at various wavelengths
throughout the infrared spectrum a reality. (See van Gisbergen et
al., (1996) Chem. Phys. Lett. 259: 599-604.)
[0005] Raman spectroscopy is a powerful optical technique for
detecting and analyzing molecules. Its principle is based on
detecting light scattered off a molecule that is shifted in energy
with respect to the incident light. The shift, called Raman shift,
is characteristic of individual molecules, reflecting their
vibrational frequencies like molecular fingerprints. As a result,
the key advantage of Raman spectroscopy is its molecular
specificity while its main limitation is the small signal due to
low quantum yield of Raman scattering. One way to enhance the Raman
signal is to tune the excitation wavelength to be on resonance with
an electronic transition, so called resonance Raman scattering.
This can usually produce an enhancement on the order of
10.sup.2-10.sup.3.
[0006] Another technique to enhance Raman scattering is surface
enhancement by roughened metal surfaces, notably silver and gold,
that provides an enhancement factor on the order of
10.sup.6-10.sup.8. This is termed surface enhanced Raman
spectroscopy (SERS). Similar or somewhat larger enhancement factors
(.about.10.sup.8-10.sup.10) have been observed for metal, mostly
silver or gold, nanoparticles.
[0007] In the last few years, it has been shown that an even larger
enhancement (.about.10.sup.10-10.sup.15) is possible for aggregates
of metal nanoparticles (MNPs), silver and gold. The largest
enhancement factor of 10.sup.14-10.sup.15 has been reported for
rhodamine 6G (R6G) on single silver nanoparticle aggregates. This
huge enhancement is thought to be mainly due to significant
enhancement of the local electromagnetic field of the nanoparticle
aggregate that strongly absorbs the incident excitation light for
the Raman scattering process. With such large enhancement, many
important molecules that are difficult to detect with Raman
normally can now be easily detected. This opens many interesting
and new opportunities for detecting and analyzing molecules using
SERS with extremely high sensitivity and molecular specificity.
[0008] SERS can also be developed into a molecular imaging
technique for biomedical and other applications. Existing Raman
imaging equipment should be usable for SERS imaging. SERS will
provide a much-enhanced signal and thereby significantly shortened
data acquisition time, making the technique practically useful for
medical or other commercial and industrial applications including
chip inspection or chemical monitoring. SERS is also useful for
detecting other cancer biomarkers that can interact or bind to the
MNP surface. For example, Sutphen et al. have recently shown that
lysophospholipids (LPL) are potential biomarkers of ovarian cancer
(Sutphen et al., (2004) Cancer Epidemiol. Biomarker Prey. 13:
1185-1191).
[0009] For many practical applications, for example SERS and
optical filters, it is highly desirable to narrow the distribution
of size/shape of nanoparticle aggregates. For SERS in particular,
the incident light has to be on resonance with the substrate
absorption. Only those nanoparticle aggregates that have resonance
absorption of the incident light are expected to be SERS active. It
is thus extremely beneficial to have a narrow size/shape
distribution and thereby narrow optical absorption.
[0010] Fluorescent nanoparticles (quantum dots (QDs) such as
semiconductor quantum dots, SQDs) have been used recently as
fluorescent biological markers and have been found to be extremely
effective. They offer advantages including higher stability,
stronger fluorescence, tunability of color, and possibility of
optical encoding based on different sized or colored SQDs.
[0011] Metal nanoparticles have been recognized for their unique
optical properties that could be exploited in optoelectronic
devices. Nanoparticle systems composed of gold, for example, have
distinct optical properties that make them amenable to study by
Raman scattering. The Raman spectrum of the adsorbed species is
significantly enhanced by 10 to 15 orders of magnitude when the
metal nanoparticles have aggregated, leading to enhanced
electromagnetic field effects near the surface that increases the
Raman scattering intensity. The greater sensitivity found in the
SERS of metal nanoparticle aggregates facilitates the detection and
analysis of a whole host of molecules that were previously
difficult to study. Wang et al. disclose a method of using SQDs
(dye-conjugated CdTe nanoparticles, CT-NPs) to detect interactive
binding between Ag-CT-NPs and Ab-CT-NPs (Wang et al., (2002)
NanoLett. 2: 817-822). The interactions were determined by
differential quenching or enhancement fluorescence activity of two
different sized SQDs (red or green) measured during the
analysis.
[0012] The use of SERS for analyte detection of biomolecules has
been previously studied. U.S. Pat. No. 6,699,724 to West et al.
describes a chemical sensing device and method (nanoshell-modified
ELISA technique) based on the enzyme-linked immunoadsorbant assay
(ELISA). The chemical sensing device can comprise a core comprising
gold sulfide and a surface capable of inducing surface enhanced
Raman scattering (SERS). In much of the patent disclosure, the
nanoparticle is disclosed as having a silica core and a gold shell.
The patent discloses that an enhancement of 600,000-fold
(6.times.10.sup.5) in the Raman signal using conjugated
mercaptoaniline was observed.
[0013] In the nanoshell-modified ELISA technique, antibodies are
directly bound to the metal nanoshells. Raman spectra are taken of
the antibody-nanoshell conjugates before and after the addition of
a sample containing a possible antigen, and binding of antigen to
antibody is expected to cause a detectable shift in the
spectra.
[0014] The conjugation of quantum dots to antibodies used for
ultrasensitive nonisotopic detection for use in biological assays
has also been studied. U.S. Pat. No. 6,468,808 B1 to Nie et al.
disclosed an antibody is conjugated to a water-soluble quantum dot.
The binding of the quantum dot-antibody conjugate to a targeted
protein will result in agglutination, which can be detected using
an epi-fluorescence microscope. In addition, Nie et al. described a
system in which a quantum dot is attached to one end of an
oligonucleotide and a quenching moiety is attached to the other.
The preferred quenching moiety in the Nie patent is a
nonfluorescent organic chromophore such as
4-[4'-dimethylaminophenylazo]benzoic acid (DABCYL).
[0015] Raman amplifiers are also expected to be used globally as a
key device in next-generation optical communications, for example,
in wavelength-division-multiplexing (WDM) transmission systems.
Raman scattering occurs when an atom absorbs a photon and another
photon of a different energy is released. The energy difference
excites the atom and causes it to release a photon with low energy;
therefore, more light energy is transferred to the photons in the
light path.
[0016] Improving the consistency of SERS probes requires the use of
single, SERS active nano-sized structures. Nano-crescents, and
core-shell systems are examples of cleverly engineered
nanostructures capable of providing sufficient SERS intensity from
individual particles due to their ability to strongly localize
surface electromagnetic fields. (See in particular, Lu, Y., Liu, G.
L., Kim, J., Mejia, Y. X., and Lee, L. P., Nano Lett. 2005, 5,
119-124; Talley, C. E., Jackson, J. B., Oubre, C., Grady, N. K.,
Hollars, C. W., Lane, S. M., Huser, T. R., Nordlander, P., and
Halas, N. J., Nano Lett. 2005, 5, 1569-1574.) However, the
relatively large size of these nanostructures will ultimately limit
their accessibility to some sub-cellular organelles. To push the
size boundary of sensing, as required by systems biology, even
smaller probes will be required. Of interest is a subset of
core-shell structures, hollow metal structures, a unique class of
nanomaterials explored, most notably, by Sun et al. (Sun, Y. G.,
Mayers, B., and Xia, Y. N., Advanced Materials 2003, 15, 641-646).
Utilizing the galvanic replacement of silver with gold and other
metals, they have produced a variety of different sized and shaped
hollow structures and have recently demonstrated the SERS activity
of these structures (Chen, J. Y., Wiley, B., Li, Z. Y., Campbell,
D., Saeki, F., Cang, H., Au, L., Lee, J., Li, X. D., and Xia, Y.
N., Advanced Materials 2005, 17, 2255-2261).
[0017] In solid spherical particles there is a single resonance at
approximately 520 nm for gold and 400 nm for silver, varying
slightly depending on size and embedding media. However, when one
axis is extended, for example, a nanorod, the resonance will break
into two absorption bands, one corresponding to the short axis, or
transverse mode, and another to the long axis, or longitudinal mode
(Nikoobakht, B. and El-Sayed, M. A., Chem. Materials 2003, 15,
1957; Chang, S. S., Shih, C. W., Chen, C. D., Lai, W. C., and Wang,
C. R. C., Langmuir 1999, 15, 701). The longitudinal mode has lower
energy or redder absorption than the transfer mode. This is also
true for aggregated systems in which there are multiple resonances
within each given cluster of particles (Grant, C. D., Schwartzberg,
A. M., Norman, T. J., and Zhang, J. Z., J. Am. Chem. Soc. 2003,
125, 549; Quinten, M. J., Cluster Sci. 1999, 10, 319; Quinten, M.,
Applied Physics B-Lasers and Optics 2000, 70, 579; Quinten, M. and
Kreibig, U. Applied Optics 1993, 32, 6173; Norman, T. J. Jr. Grant,
C. Magana, D. Cao, D. Bridges, F. Liu, J. van Buuren, T. and Zhang,
J. Z., J. Phys. Chem. B 2002, 106, 7005; Norman, T. J., Grant, C.
D., Schwartzberg, A. M., and Zhang, J. Z., Opt. Mat. 2005, 27,
1197; and Kreibig, U. Optical properties of metal clusters;
Springer: Berlin; New York, 1995; Vol. 25). Therefore, controlling
size and shape of these metal nanostructures allows control of
their optical properties that have potential applications in
nanophotonics and sensing.
[0018] As an effort to engineer so-called "hot spots" of large
enhancement in single particles, Lee et al. produced nano-crescent
structures by depositing silver over latex beads on a surface, then
dissolving away the bead (Lu, Y., Liu, G. L., Kim, J., Mejia, Y.
X., and Lee, L. P., Nano Lett. 2005, 5, 119). These hollow spheres
are open-ended with a sharpened edge that greatly enhances the EM
field. This engineered "hot-spot" approach yields improved SERS
enhancements over core/shell systems and is of a similar
homogeneity due to the highly consistent latex beads available. For
applications requiring extremely small probe size, however, both
nano-crescents and core shell systems are relatively large.
[0019] A system of particular interest where probe size is of
utmost importance is intracellular studies (Chithrani, B. D.,
Ghazani, A. A., and Chan, W. C. W., Nano Lett. 2006, 6, 662-668).
It has been found that while particles larger than 100 nm can enter
a cell, they do not do so readily and may interrupt some cellular
functions. Similarly, particles that are too small, less than 20
nm, will diffuse out of the cell, rendering them useless. The ideal
is a structure that can be tuned in size between 20 nm and 100 nm
depending on the application.
[0020] Nanotubes of all shapes and sizes have become an area of
increasing interest for applications ranging from filtration to
electrical interconnects. (See, in particular, Holt, J. K. et al.,
Science 312, 1034-1037 (2006); Hinds, B. J. et al., Science 303,
62-65 (2004); Zhang, M. et al. Science 309, 1215-1219 (2005); and
Huang, Y. et al., Science 294, 1313-1317 (2001).) The application
of these structures is almost unlimited, however, as is the case
with most synthesized structures of this scale, nanoscopic
manipulation is challenging. While carbon nanotubes have been the
predominant structure of interest, lately there has been an effort
to utilize gold and silver nanotubes or nanowires for these
purposes as their conductivity and material properties are thought
to be superior (Siwy, Z. et al., J. Am. Chem. Soc. 127, 5000-5001
(2005); Kohli, P., Wharton, J. E., Braide, O. & Martin, C. R.
J., Nanosci. Nanotechnol. 4, 605-610 (2004)). Generally these metal
structures are produced by a physical or electroless deposition
technique, and while this produces well defined structures, their
shape and size is entirely dependent on the template on which they
are made, limiting the size and practical application of these
structures (Wiley, B., Sun, Y. G., Mayers, B. & Xia, Y. N.,
Chemistry-a European Journal 11, 454-463 (2005); Wiley, B. et al.,
M. R. S. Bull., 30, 356-361 (2005); Sun, Y. G. & Xia, Y. N.
Advanced Materials 16, 264-268 (2004); and Lee, M., Hong, S. C.
& Kim, D., Appl. Phys. Lett., 89 (2006)).
[0021] There is therefore a need in the art for use in the chemical
and biomedical analytical industries and the electronic
communications industries to provide more sensitive compositions
and devices that are inexpensive to manufacture and easy to
use.
BRIEF DESCRIPTION OF THE INVENTION
[0022] The present invention provides nanostructures comprising
hollow metal nanospheres or nanoshells and nanotubes for use as
chemical sensors, conduits for fluids, and electronic conductors.
The nanostructures can be used in microfluidic devices, for
detecting chemicals inside or outside a biological membrane, such
as a cell membrane or a viral coat, for transporting fluids between
devices and structures in analytical devices, for conducting
electrical currents between devices and structure in analytical
devices, and for conducting electrical currents between biological
molecules and electronic devices, such as microchips.
[0023] In one embodiment the present invention provides a chemical
sensor comprising gold nanoshells (hollow gold nanospheres; HGN),
the nanoshells having a mean particle size of between about 20 nm
and about 100 nm diameter. In one preferred embodiment the mean
diameter is between about 20 nm to about 70 nm. In another
preferred embodiment, the mean diameter is between about 22.8 nm
and about 50 nm diameter. In one embodiment, the invention provides
a chemical sensor for chemical and biological sensing applications,
particularly those requiring near-IR absorption.
[0024] The HGNs have an interior wall surface diameter and an
exterior wall surface diameter thereby defining the wall thickness.
The invention further provides HGNs having tunable interior and
exterior and wherein the peak of the surface plasmon band
absorption is between about 550 nm and about 820 nm. In one
embodiment the mean wall thickness of the HGNs is between about 2.4
nm and about 7.3 nm. In a preferred embodiment the mean wall
thickness is about 5 nm.
[0025] In another preferred embodiment, the chemical sensor has a
surface wherein the surface can induce surface enhanced Raman
scattering (SERS).
[0026] In still another preferred embodiment, the chemical sensor
further comprises at least one detecting molecule, wherein the
detecting molecule is bound to the surface. In a more preferred
embodiment the detecting molecule is selected from the group
consisting of proteins, peptides, antibodies, antigens, nucleic
acids, peptide nucleic acids, sugars, lipids,
glycophosphoinositols, and lipopolysaccharides.
[0027] In a yet more preferred embodiment the detecting molecule is
an antibody. In another preferred embodiment, the detecting
molecule is an antigen.
[0028] In another embodiment, the invention provides a chemical
sensor further comprising at least one semiconductor quantum dot.
In a preferred embodiment the semiconductor quantum dot further
comprises a linker molecule, the linker molecule selected from the
group consisting of a thiol group, a sulfide group, a phosphate
group, a sulfate group, a cyano group, a piperidine group, an Fmoc
group, and a Boc group.
[0029] In a still further embodiment, the invention provides a
chemical sensor comprising at least one semiconductor quantum dot
wherein the semiconductor quantum dot further comprises a detecting
molecule, wherein the detecting molecule is bound to the
semiconductor quantum dot. In a more preferred embodiment, the
detecting molecule is selected from the group consisting of
proteins, peptides, antibodies, antigens, nucleic acids, peptide
nucleic acids, sugars, lipids, glycophosphoinositols, and
lipopolysaccharides.
[0030] In a more preferred embodiment, the detecting molecule is an
antibody. In the alternative, a more preferred embodiment comprises
a chemical sensing device wherein the detecting molecule is an
antigen.
[0031] Another embodiment of the invention provides a method for
detecting an analyte in a sample using a chemical sensor, the
method comprising the steps of: i) providing a sample; ii)
providing a semiconductor quantum dot comprising a linker molecule
(LM-SQD); iii) conjugating the analyte in the sample with the
LM-SQD thereby producing an analyte-LM-SQD conjugate; iv) providing
a chemical sensor comprising a plurality of particles, each
particle comprising: a shell having at least one surface and
wherein the shell comprises a gold molecular species, the shell
surface further comprising a detecting molecule; v) incubating the
analyte-LM-SQD conjugate with the chemical sensor for a
predetermined time period; and vi) measuring the extent of binding
between the analyte-LM-SQD conjugate and the chemical sensor;
thereby detecting the analyte in the sample. In one embodiment the
sample is selected from the group consisting of mammalian cells,
vertebrate cells, invertebrate cells, plant cells, fungal cells,
mold cells, archaeal cells, bacterial cells, viruses,
bacteriophages, and the like. In another embodiment the sample is
selected from the group consisting of blood fluids, lymph fluids,
hemolymph fluids, pulmonary surfactant fluids, peritoneal fluids,
gastric fluids, xylem fluids, phloem fluids, and the like. In yet
another embodiment the sample is selected from the group consisting
of fluvial fluids, marine fluids, atmospheric precipitate fluids,
waste-water fluids, agricultural run-off fluids, fluids comprising
hydrocarbons, fluids contaminated by hydrocarbons, aerosol fluids,
aqueous fluids, non-aqueous fluids, and the like.
[0032] The invention also provides a method of using the chemical
sensor as disclosed herein for measuring cellular processes. These
embodiments are merely exemplary of the invention, which
encompasses any small nanostructures having SERS activity as
disclosed herein.
[0033] In another embodiment the invention provides a method for
detecting an analyte that is a cancer marker. In one embodiment the
cancer marker is an antibody. In one embodiment of the invention
the detecting molecule in the chemical sensor is an antigen that
binds to a cancer marker antibody with an affinity (K.sub.a) of at
least 10.sup.6 l/mole. In a more preferred embodiment the K.sub.a
is at least 10.sup.8 l/mole. In another preferred embodiment the
analyte is a phospholipid. In a most preferred embodiment the
phospholipid is lysophosphatidic acid (LPA).
[0034] The invention further provides a synthetic nanotube, the
synthetic nanotube being substantially hollow and having dimensions
of between about 20 nm and about 100 nm in mean diameter and at
least between about 0.1 .mu.m and 4 .mu.m in mean length. In a more
preferred embodiment the mean diameter is between about 30 nm and
80 nm. In a more preferred embodiment the mean length is between
about 4 .mu.m and about 50 .mu.m, for example, about 6 .mu.m, about
8 .mu.m, about 10 .mu.m, about 15 .mu.m, about 20 .mu.m, about 25
.mu.m, about 30 .mu.m, about 40 .mu.m, and about 50 .mu.m, and any
other length therebetween.
[0035] In one embodiment the synthetic nanotube has a wall of mean
dimension of between about 2.4 nm and about 7.3 nm across. In a
preferred embodiment the wall has a mean dimension of about 5
nm.
[0036] In one preferred embodiment the synthetic nanotube comprises
a metal selected from the group consisting of gold, silver,
platinum, copper, aluminum, palladium, cadmium, iridium, rhodium,
and the like.
[0037] In another embodiment, the invention provides a conduit for
conducting fluids, the conduit comprising the synthetic nanotube as
disclosed herein.
[0038] In yet another embodiment, the invention provides an
electronic conductor, the electronic conductor comprising the
synthetic nanotube as disclosed herein.
[0039] The invention further provides a method for synthesizing a
nanotube, the nanotube comprising a metal, the method comprising
the steps of (i) combining an aqueous solution of Co.sup.2+ salt
with an aqueous solution of citrate salt thereby forming a first
mixture (ii) degassing the first mixture; (iii) purging at least
once with nitrogen gas; (iv) adding an aqueous solution of
NaBH.sub.4 thereby reducing the Co.sup.2+ to Co.sup.0, and thereby
forming a second mixture comprising Co.sup.0 particles, the step of
adding being in the presence of an induced magnetic field and
wherein the presence of the induced magnetic field aligns the
Co.sup.0 particles; (v) agitating the second mixture until hydrogen
evolution is substantially complete; (vi) adding the second mixture
comprising aligned Co.sup.0 particles to an aqueous solution of
Au.sup.3+ salt; (vii) allowing the Au.sup.3+ to be reduced to
Au.sup.0 and the Co.sup.0 oxidized to Co.sup.2+, and wherein the
Au.sup.0 is deposited adjacent to the aligned Co.sup.0 thereby
creating a nanotube comprising Au.sup.0, the method thereby
synthesizing a nanotube. In one preferred embodiment the nanotube
comprises a metal selected from the group consisting of gold,
silver, platinum, copper, aluminum, palladium, cadmium, iridium,
and rhodium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0041] FIG. 1 shows an exemplary synthesis procedure for HGNs.
[0042] FIG. 2 illustrates the different tunable colors of the HGNs
having combinations of different dimensions.
[0043] FIG. 3 illustrates the UV-visible electronic absorption
spectra of different HGNs.
[0044] FIG. 4 shows a comparison of SERS spectrum consistency
between HGNs and silver nanoparticles/aggregates. Shown are the
single particle SERS spectrum of MBA on HGNs (red trace, top) and
silver aggregates (blue trace, bottom). The inset is a histogram of
the relative intensity of the two most intense peaks of MBA at 1070
cm.sup.-1 and 1590 cm.sup.-1of 150 HGNs (red bars) and 150 silver
aggregates (blue bars).
[0045] FIG. 5 illustrates a correlation between confocal SERS (a)
and TEM (b).
[0046] FIG. 6 is a representative low resolution TEM of HGNs.
Examining 150 particles from such images, the mean size is found to
be 30.+-.4.5 nm.
[0047] FIG. 7 is a high resolution TEM of an individual HGN of
diameter 29.1 nm with approximately 5 nm wall thickness. Twinning
in the HGN wall demonstrates its polycrystalline nature. A TEM of
the whole HGNs is inset.
[0048] FIG. 8 illustrates the ensemble average solution absorption
spectrum of an as prepared solution (black trace, top), and the
Rayleigh scattering spectrum of a single HGNs (red trace, middle)
immobilized on a glass coverslip in air. Plotted against the right
axis is a histogram of the peak wavelength in the scattering
spectra (.quadrature..sub.max) of 100 particles (average
621.+-.10.6 nm) (blue bars, bottom). The absorption spectrum is
shifted in intensity for clarity. Inset: Rayleigh scattering
spectra of two silver aggregates.
[0049] FIG. 9 is a TEM image of individual HGNs on a holey carbon
TEM grid of which, the SERS spectrum has been measured (inset). TEM
image was overlapped with confocal Raman images to co-locate the
SERS active particles shown. Light region of the image is a hole in
the film. Red circle marked "Focal Area" represents the approximate
diameter of the laser focal area used to measure the SERS response
of the sample.
[0050] FIG. 10 represents each black point as the intensity of the
pH sensitive 1430 cm.sup.-1 peak of 20-30 particles at different pH
normalized to the pH insensitive 1590 cm.sup.-1 peak, while the red
crosses are the individual data points. Error bars represent
standard deviation of the measurements. The fit line is a guide to
the eye.
[0051] FIG. 11 illustrates particle size as a function of citrate
and sodium borohydride concentration. All particle sizes are
determined by examining TEM images of the resulting gold structures
and represent the measurement of at least 200 particles. Reported
sizes are the particle diameter.
[0052] FIG. 12 illustrates histograms showing the size dispersion
of cobalt nanoparticles produced by slow and fast addition of
cobalt chloride. Solid lines are best fits demonstrating particle
dispersion. Particles sizes determined by measuring low resolution
TEM images.
[0053] FIG. 13 show transmission electron micrographs of the HGNs.
FIG. 13A is a high resolution TEM of a single, 30 nm HGN. The wall
thickness is approximately 4 nm and large areas of crystallinity
are clearly visible. FIGS. 13B-E are low resolution TEM images of
particles of 71.+-.17 nm (B), 50.+-.5 nm (C), 40.+-.3.5 nm (D), and
28.+-.2.3 nm.
[0054] FIG. 14 illustrates the UV-visible absorption spectra of
nine HGN samples with varying shell diameters and wall
thicknesses.
[0055] FIG. 15 shows plasmon absorbance maximum wavelength
(.lamda..sub.max) as a function of shell thickness and shell
diameter. Each point represents an individual set of experiments
and represents the average measured lengths.
[0056] FIG. 16 illustrates spectral dependence on volume of added
gold salt. Gold solutions were diluted to 10 ml with water before
30 ml of a cobalt solution made by the fast cobalt addition method
with 100 .mu.l sodium borohydride and 600 .mu.l of citric acid.
Average particle size is 35.+-.2 nm.
[0057] FIG. 17 illustrates a comparison of ensemble averaged
absorption and single particle Rayleigh scattering of 30.+-.2.6 nm
HGNs.
[0058] FIG. 18 illustrates the ensemble averaged surface enhanced
Raman scattering spectrum of mercaptobenzoic acid on the HGNs.
[0059] FIG. 19 is a high resolution TEM of an HGN formed from a
slightly oxidized cobalt particle.
[0060] FIG. 20 illustrates TEMs of the gold nanotubes. FIG. 20a is
a low resolution TEM image of gold nanotubes. Red line indicates
the path of a single .about.4 m tube. FIG. 20b is a high resolution
TEM image of a large section of one tube illustrating the
continuity and consistency of the samples. FIG. 20c is a high
resolution TEM image of one section of the gold tube showing its
continuous nature. FIG. 20d is a more detailed high resolution TEM
image of the tube showing gold lattice fringes indicating its
poly-crystalline nature.
[0061] FIG. 21 is a schematic of gold tube formation templated with
Co nanopartciles aligned by an external magnetic field. Top portion
of figure: alignment of cobalt nanoparticles along magnetic field
lines. Middle portion: Gold is reduced onto the surface of aligned
cobalt particles. Bottom portion: Cobalt is further oxidized by
dissolved oxygen leaving a hollow structure.
[0062] FIG. 22 illustrates the UV-visible absorption spectra of 50
nm diameter solid gold nanoparticle solution, 40 nm diameter
nanotube with .about.5 nm shell thickness solution, and 60 nm
diameter HGN with 3 nm shell thickness solution.
[0063] FIG. 23 illustrates how the terminal amino acid residues in
different peptides affect the SERS spectrum. The SERS spectra
change when the terminal amino acid residue is changed thereby
indicating a possible relationship between SERS spectrum and
distance between the terminal residues. Y=tyrosine; P=proline, and
W=tryptophan.
[0064] FIG. 24 illustrates cross-sectional representations
different hollow nanosphere or nanoshell structures. The same
structures can be considered for nanotubes in cross-section.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The embodiments disclosed in this document are illustrative
and exemplary and are not meant to limit the invention. Other
embodiments can be utilized and structural changes can be made
without departing from the scope of the claims of the present
invention.
[0066] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a particle" includes a plurality of such particles, and a
reference to "a surface" is a reference to one or more surfaces and
equivalents thereof, and so forth.
[0067] The invention disclosed herein is based on the galvanic
replacement of cobalt with gold, a procedure shown to produce
considerably more homogeneous hollow spheres than those synthesized
with silver (Liang, H. P.; Wan, L. J.; Bai, C. L.; Jiang, L. J.,
Phys. Chem. B 2005, 109, 7795-7800). The process starts with a
cobalt nanoparticle that is synthesized as a template for the
growth of hollow gold nanospheres (HGNs). Utilizing the difference
in redox potential between cobalt and gold, it is possible to
reduce gold ions while oxidizing the cobalt nanoparticles. Because
this reaction takes place entirely at the surface of the cobalt
particle, the shape and size of the resulting hollow structure is
completely dependent on the original template. Moreover, this
process is able to produce SERS active nanoparticles that are
significantly smaller than traditional nanoparticle structures used
for SERS, providing a sensor element that can be more easily
incorporated into cells for localized intracellular
measurements.
[0068] We provide the successful SERS application of HGNs with
improved optical and structural homogeneity over other SERS
substrates that are highly desired and important for size sensitive
biological sensing applications. The consistency of particle shape
and size is reflected in the optical properties that lead to a
tenfold increase in SERS spectral consistency over aggregated
silver nanoparticles commonly used in SERS applications. SERS from
single HGNs was achieved, the first such measurement on hollow
nanostructures. Finally, pH sensing as a model system was
demonstrated showing an approximate doubling of resolution and a
ten-fold increase in precision over previous nano-sized pH SERS
probes. This clearly represents a new detecting platform and a
major step forward in potential biological sensing
applications.
[0069] Since the early work by Turkevich et al. and later Frens et
al., it has been understood that in a standard colloidal gold
synthesis using the hot citrate reduction of chloroauric acid, the
particle size may be controlled by the concentration of citrate.
Citrate stabilizes the initially formed nuclei and the more citrate
is present, the more nuclei will be stabilized. However, when
trying to apply this logic to the aqueous synthesis of cobalt
nanoparticles, it is a significantly more challenging task. (See
Turkevich, J.; Stevenson, P. C.; Hiller, J., Discussions of the
Faraday Society 1951, 11, 55; Frens, G., Nature Physical Science
1973, 241, 20.)
[0070] Due to the stability of the cobalt salt, the reduction
cannot be done by citrate alone and a stronger reducing agent is
required. In this case sodium borohydride is used to reduce the
salt, and citrate is present only as a capping agent.
[0071] Nearly monodisperse HGNs with tunable interior and exterior
diameter have been synthesized by sacrificial galvanic replacement
of cobalt nanoparticles. By carefully controlling particle size and
wall thickness, it is possible to tune the peak of the surface
plasmon band absorption between 550 nm and 820 nm. Cobalt particle
size is tunable by simultaneously changing the concentration of
sodium borohydride and sodium citrate, the reducing and capping
agent, respectively. The thickness of the gold shell can be varied
by carefully controlling the addition of gold salt. With successful
demonstration of ensemble as well as single HGN surface enhanced
Raman scattering, these HGNs have shown great potential for
chemical and biological sensing applications, especially those
requiring nanostructures with near IR absorption.
[0072] In this application we present the synthetic route necessary
to control the particle size of the cobalt nanoparticles, which is
reflected in the resultant HGN diameter. The inner diameter, or
wall thickness, can be controlled by the concentration of gold salt
used, leading to complete control of the optical properties of
particles ranging from 20 nm to 70 nm. For a particular diameter
and wall thickness, the absorption band is relatively narrow due to
the near monodisperse distribution, as determined by single
nanosphere scattering spectrum. These HGNs have been further
demonstrated to be excellent SERS substrates with excellent
consistency measured based on single HGN SERS spectrum.
[0073] The size of the particles can be in the range from between
about 20 nm to about 100 nm, about 25 nm to about 85 nm, about 35
nm to about 70 nm, and about 50 nm in diameter. The dimensions of
the wall of the particle, that is the wall thickness, is in the
range form about 2.4 nm to about 10 nm, from about 2.4 nm to about
7.3 nm, and about 5 nm thick.
[0074] An exemplary method for synthesizing HGNs is illustrated on
FIG. 1. FIG. 2 illustrates the different color spectra associated
with HGNs of different combinations of shell thickness and outer
shell diameter. FIG. 3 exemplifies the UV-visible electronic
absorption spectra of HGNs having a variety of combinations of
shell thickness and outer diameter FIG. 5 compares a confocal image
(labeled a) with that of a TEM (labeled b) of a sample of
nanospheres. The inset photomicrographs show a high resolution
image of Particle A and of Particle B; the graphs below show the
SERS spectrum associated with Particle A or Particle B which can be
discretely distinguished. As used herein, the terms "nanoshells"
and "hollow nanospheres" are interchangeable.
SERS Detection Applications for Sensing and Imaging
[0075] Raman spectroscopy is a powerful optical technique for
detecting and analyzing molecules. Its principle is based on
detecting light scattered off a molecule that is shifted in energy
with respect to the incident light. The shift, called Raman shift,
is characteristic of individual molecules, reflecting their
vibrational frequencies that are like figure prints of molecules.
As a result, the key advantage of Raman spectroscopy is its
molecular specificity while its main limitation is the small signal
due to low quantum yield of Raman scattering. One way to enhance
the Raman signal is to tune the excitation wavelength to be on
resonance with an electronic transition, so called resonance Raman
scattering. This can usually produce an enhancement on the order of
10.sup.2-10.sup.3. Another technique to enhance Raman scattering is
surface enhancement by roughened metal surfaces, notably silver and
gold, that provides an enhancement factor on the order of
10.sup.6-10.sup.8. Similar or somewhat larger enhancement factors
(.about.10.sup.8-10.sup.10) have been observed for metal, mostly
silver, nanoparticles.
[0076] In the last few years, it has been shown that an even larger
enhancement (.about.10.sup.10-10.sup.15) is possible for aggregates
of metal nanoparticles, for example, comprising silver and/or gold.
The largest enhancement factor of 10.sup.14-10.sup.15 has been
reported for rhodamine 6G (R6G) on single silver nanoparticle
aggregates. This huge enhancement is thought to be mainly due to
significant enhancement of the local electromagnetic fields of the
nanoparticle aggregates that absorb strongly the incident
excitation light for the Raman scattering process. With such large
enhancement, many important molecules that are difficult to detect
with Raman normally can now be easily detected. This provides many
interesting and new opportunities for detecting and analyzing
molecules using SERS with extremely high sensitivity and molecular
specificity.
[0077] SERS can also be developed into a molecular imaging
technique for biomedical and other applications. Exciting Raman
imaging equipment may be usable for SERS imaging. SERS can provide
an enhanced signal and thereby significantly shortened data
acquisition time, making the technique practically useful for
medical or other commercial and industrial applications including,
but not limited to, chip inspection or chemical monitoring.
Antigen/Antibody Detection with Metal and Semiconducting
Nanoparticles
[0078] Fluorescent nanoparticles (semiconductor quantum dots, SQDs)
have been used recently as fluorescent biological markers and have
been found to be extremely effective. They offer advantages
including higher stability, stronger fluorescence, tunability of
color, and possibility of optical encoding based on different sized
or colored SQDs.
[0079] HGNs of the invention can be used to detect an analyte. Such
an analyte can be, for example, but not limited to, an antigen, an
antibody, a biochemical metabolite, an organic compound, a compound
or element having biological activity, or the like.
[0080] SERS is also useful for detecting other cancer biomarkers
that can interact or bind to the HGN surface. For example, Sutphen
et al. have recently shown that lysophospholipids (LPL) are
potential biomarkers of ovarian cancer (Sutphen et al., (2004)
Cancer Epidemiol. Biomarker Prev., 13: 1185-1191). Based on the
molecular structure of LPL molecules, a favorable interaction
between LPL molecules with HGN through electrostatic interaction
can occur at the appropriate pH. In the case of the SERS experiment
using a polyclonal Ab, the strongest interaction with HGN occurs at
the isoelectrostatic pH, i.e. pH at which the HGN has equal number
of positive and negative charges. The pH is varied to adjust the
charge on the HGN to determine the optimal pH or charge for strong
interaction with LPL.
[0081] By conjugating fluorescent nanoparticle QDs to antigens and
mixing the Ag-QD conjugate with a HGN-Ab composition, quenching of
fluorescence upon binding of the antigen/antibody pair can be
observed. The Ag and/or the Ab can be conjugated to the QD or HGN
using a linker molecule (LM). A decrease in fluorescence can
indicate the presence of the antibody for that particular antigen
to which the fluorescing QDs have been attached. Depending on which
antigen is utilized a wide array of antibodies can be detected.
This can allow for the rapid detection of cancers or diseases that
currently can take days or weeks to diagnose. Likewise, the scheme
can work as well if antibody is attached to a fluorescent QD and
the respective antigen to a metal nanoparticle. Metal particles
have no florescence with visible excitation. The fluorescence
quenching by metal nanoparticles can be more effective than
quenching by larger QDs. This approach is sensitive and specific.
The distance between the metal nanoparticle and QD is important for
this to work (for example, the distance can be less than 2 nm). The
interaction between the two components can be adjusted to achieve
the maximum quenching effect.
Detection of Tumor Markers
[0082] Surface-enhanced Raman scattering using silver nanoparticles
was applied to detect various forms of lysophosphatidic acid (LPA)
to examine its potential application as an alternative to current
detection methods of LPA as biomarkers of ovarian cancer.
Enhancement of the Raman modes of the molecule, especially those
related to the acyl chain within the 800-1300 cm.sup.-1 region, was
observed. In particular, the C-C vibration mode of the
gauche-bonded chain around 1100 cm.sup.-1 was enhanced to allow the
discrimination of two similar LPA molecules. Given the molecular
selectivity of this technique, the detection of LPA using SERS may
eliminate the need for partial purification of samples prior to
analysis in cancer screening.
[0083] Lysophosphatidic acid (LPA), originally known for its role
as an intermediate in intracellular lipid metabolism, has now been
recognized as an important multifunctional biological mediator that
can elicit cellular responses including mitogenic and antimitogenic
effects on the cell cycle, actin skeleton regulation, and cellular
motility (see Tigyi et al., (1994) Proc. Nat. Acad. Sci. 91:
1908-1912; van Corven et al., (1989) Cell 59: 45-54; Ridley and
Hall, (1992) Cell 70: 389-399; and Zhou et al., (1995) J. Biol.
Chem. 270: 25549-25556). The involvement of LPA in inducing cell
proliferation, migration and survival implicates it in the
initiation and progression of malignant disease, and has been
proposed as a sensitive biomarker for ovarian cancer (see Xu et al.
(1998) JAMA 280: 719-723; Mills and Moolenaar (2003) Nature Rev. 3:
582-591; Fang et al. (2004) J. Biol. Chem. 279: 9653-9661; and
Sutphen et al. (2004) Cancer Epidemiol. Biomark. Prey. 13:
1185-1191).
[0084] Typically, the detection of LPA has been conducted using
chromatography and mass spectroscopy assays that require a partial
purification of the samples using thin layer chromatography (TLC)
prior to analysis. Although this method is effective, an
underestimation of LPA concentration can result during the recovery
process due in part to the varying mobility of the LPA salts (free
acid, sodium and calcium salts) when subjected to chromatography by
TLC. The low stability of LPA also calls for fast and sensitive
detection techniques.
[0085] A powerful optical detection technique based on
surface-enhanced Raman scattering (SERS) offers a unique
combination of high sensitivity and molecular specificity. With
SERS, the Raman signal of a molecule is increased by many orders of
magnitude as a result of strong enhancement of the excitation light
through the resonance of the metal's surface electrons called the
surface plasmon (see Moskovitz (1985) Rev. Modern Physics 57:
783-828; Otto et al., (1992) J. Phys. Condense Matter 4: 1143-1212;
and Campion and Kambhampati, (1998) Chem. Soc. Rev. 27: 241-250).
SERS has been successfully used in the detection and analysis of a
large number of chemicals and biological molecules (see Albrecht
and Creighton, (1977) J. Am. Chem. Soc. 99: 5215-5217; Nie and
Emory (1997) Science 275: 1102-1106; Keating et al., (1998) J.
Phys. Chem. B 102: 9414-9425; Kneipp et al., (1998) Phys. Rev. E
57: R6281-R6284; and Schwartzberg et al., (2004) J. Phys. Chem. B
108: 19191-19197).
SERS Application for Detection and Analysis of Semiconductor
Nanoparticles
[0086] Another application of SERS based on the gold nanoparticle
system is for measuring Raman spectrum of semiconductor
nanoparticles (QDs). Similar to molecules, normal Raman signals are
very small and thus Raman spectrum is challenging to measure. SERS
as an enhanced Raman technique for measuring Raman for
semiconductor nanoparticles have not been reported before. The
surface chemistry of the metal nanoparticles and the semiconductor
QDs must be compatible for this to work. The sulfur species on the
surface of the HGNs are ideal for II-VI SQDs to bind, enabling SERS
detection of the SQDs. This provides a powerful method for
detecting and analyzing semiconductor nanoparticles.
[0087] The material and methods described heretofore have
additional properties and uses. As such, we herein disclose an
aqueous solution phase synthesis of continuous gold nanotubes that
are controllable in shape and size, currently up to 5 .mu.m in
length, by magnetic field manipulation and synthetic parameters.
Because of the ease with which magnetic fields may be manipulated,
precise placement should not only be possible, but relatively
simple as compared to other methods. This is the first step in
producing controllable, well-defined, chemically stable structures
for any application that requires hollow, electrically conducting
one-dimensional nanomaterials.
Use of Nanostructures in Miniature Electrical Circuits
[0088] The nanostructures in the form of nanotubes can be used in
the production of miniature electronic circuits, with applications
in the microelectronics industries for producing very small
circuits for memory chips, for creating electrical circuits used
with biological media, such as proteins, including cell surface
receptor proteins, antibodies; photosensitive compounds, such as
chlorophyll and related compounds; xanthocyanins; compounds having
oxidoreductase activity, including, but not limited to, cytochromes
and related compounds, haemoglobin, myoglobin, and the like, and
fluorescent compounds.
Synthesis of Nanotubes Using Magnetic Alignment of Metal
Particles
[0089] In previous works we and others have shown that it is
possible to produce highly uniform hollow gold nanospheres (HGN)
using synthesized cobalt nanoparticles in aqueous solution as
sacrificial electroless deposition templates (Schwartzberg, A. M.,
Olson, T. Y., Talley, C. E. and Zhang, J. Z. J. Phys. Chem. B 110,
19935-19944 (2006)). In this reaction Au.sup.3+ is reduced to
Au.sup.0 by Co.sup.0 nanoparticles via the following mechanism:
3Co.sup.0+2Au.sup.3+.fwdarw.3Co.sup.2++2Au.sup.0
[0090] Because only two gold atoms are generated for every three
cobalt atoms oxidized, there is a net loss of volume, resulting in
gold nanostructures of approximately 2/3 the volume of the original
Co nanoparticle. Since the gold shell grows inward from the surface
of the cobalt particle, a void remains at the center of the final
gold nanostructure that is filled, most likely, by water and
various ionic species. The shape and size of these hollow
structures therefore depends strongly on the original cobalt
nanostructure.
[0091] We have found that the size and shell thickness of HGNs can
be rather simply controlled by synthetic methods (Schwatzberg
(2006) supra). In attempts to increase particle size, and thereby
red-shift the resulting HGN plasmon into the near IR, it was found
that reducing the concentration of sodium citrate, the particle
stabilizing agent, and increasing the concentration of the cobalt
salt results in a controllable aggregation of the cobalt
nanoparticles. By carefully varying the citrate concentration we
were able to induce varying states of aggregation from complete
flocculation to partial crosslinking. Surprisingly, in the presence
of a relatively strong magnetic field, a magnetic stir-plate or the
like, upon reaction with the gold salt, these weakly aggregated
cobalt nanoparticles were found to produce long, well organized
gold tubes as in FIG. 20a. While the alignment of cobalt
nanoparticles has been observed in the past, this is the first time
the phenomenon has been used to create extended gold nanostructures
(Puntes, V. F., Krishnan, K. M. and Alivisatos, A. P., Science 291,
2115-2117 (2001); Salgueirino-Maceira, V. and Correa-Duarte, M. A.,
J. Mat. Chem. 16, 3593-3597 (2006); and Salgueirino-Maceira, V.,
Correa-Duarte, M. A., Hucht, A. and Farle, M., J. Magnetism Magnet.
Mat. 303, 163-166 (2006)).
[0092] Such a surprising occurrence would not have been predicted
and is clearly an example of superior unexpected results when
compared with what is known in the prior art.
[0093] The highlighted tube in FIG. 20a is over 4 .mu.m long and
upon close inspection, the location of each individual cobalt
particle that oxidized to form the gold tube is clear. This leads
to the almost peapod or intestine-like structure of the tube and it
is clear that the cobalt particles are aligning into a "string of
pearls" type structure. From low resolution TEM images it is not
entirely clear if each section of the "intenstine" is divided by
walls, or if the hollow portion of the tube is continuous. High
resolution TEM, however, conclusively shows that in most cases the
whole tube is hollow and polycrystalline in nature, that is,
nanotubes, not rods or wires (see FIGS. 20b, c, and d). We thought
initially that these may be aggregates of gold nanoshells, which
would be easier to explain. However, closer examination by HRTEM
shows that this is not the case since the hollow part of the
structure is connected throughout the whole tube (FIGS. 20b and c)
and the lattice fringes of each "section" extend into the next
indicating simultaneous growth (FIG. 20d). Therefore, the nanotube
structure is clearly not simply aggregates of gold nanoshells.
Furthermore, the apparent one-dimensional (or linear) structure is
inconsistent with randomly aggregated structures that tend to be
three-dimensional. The linear structure suggests that something is
directing the formation of the nanotube structure in an ordered
manner.
[0094] One possible clue to explaining the formation of the Au
nanotubes comes from the magnetic property of the seeding Co
nanparticles and the application of an external magnetic field to
drive a magnetic stir bar during synthesis. To test this, we then
intentionally examined the effect of an external magnetic field on
the synthesis process and the final product. We have found that the
magnetic field has a strong influence on the formation of the
nanotubes. Without an external magnetic field we observed little or
no nanotube formation. This led us to suggest that the mechanism of
growth depends on the ordering of the Co nanoparticles by a
magnetic field. The initial finding of nanotubes was the result of
an unintended use of a magnetic stir bar and plate that uses a
strong magnetic field that caused alignment of the Co
nanoparticles. Even though Co nanoparticles are magnetic and could
align by themselves, this self-alignment is apparently
insignificant at room temperature. When the magnetic field is
strong enough, the Co nanoparticles align into chains along the
field lines by the applied external magnetic field, as illustrated
in FIG. 21. Reduction of Au salt into Au metal with these aligned
Co chains resulted in the formation of the Au nanotube. The Co
nanoparticles are close enough that the Au salt cannot access the
interstitial space between the particles resulting in a structure
with a completely hollow core.
[0095] It is possible not only to control the cobalt particle
before reacting with gold. By performing the reaction in an oxygen
free environment and using only enough gold to oxidize part of the
cobalt particles, we were able to leave some portion of the
magnetic particles at the core of the gold tube. The tube is then
magnetically controllable and can be aligned in whatever way
desired by magnetic field manipulation. By merely exposing the
tubes to oxygen, the cobalt was completely oxidized within seconds
and dissolved into solution, leaving a completely hollow tube.
[0096] Given the nature of the tube synthesis there are always
pores that allow gold ions to permeate in to react with the cobalt
and to allow oxidized cobalt to diffuse out. It is important to
note that for applications requiring pore free tubes, it is simple
to backfill the holes after the oxidation of cobalt is complete. By
adding additional gold salt in the presence of sodium citrate, a
seed mediated growth mechanism will induce reduction of gold at the
tube surface, specifically at sharp features such as pores. With
this process it is possible to form smooth surfaced tubes and, with
sufficient gold salt, grow the wall thicker if desired. The
potential applications of this type of controllable, conducting
nanostructure are numerous.
[0097] In addition, we have found that the average length of the
tubes is dependent on the amount of initial cobalt aggregation
chemically induced. By decreasing the amount of sodium citrate it
is possible to lengthen the tubes. With this, we have additional
control of the structures. Length and diameter can be controlled
chemically, wall thickness can be controlled by the amount of gold
added, and order and placement can be controlled magnetically. It
should be noted that tubes are not formed when sufficient citrate
is present to prevent aggregation. This indicates that, while the
particles may align in the presence of the magnetic field, they are
not close enough to prevent the Au salt from penetrating the
junction of the particles to reduce at their surfaces. The
synthesis has been reproduced by different researchers in our lab
independently with very similar results. The electromagnetic field
can be generated, for example, using a magnetic stirbar and a
rotating magnet system well-known to those of skill in the art. In
addition, an electromagnetic field can be induced using a generator
or the like that induces an electromagnetic field in the vicinity
of the experimental particles to be aligned. Other physical means
for inducing an electromagnetic field are well-known to those of
skill in the art.
[0098] Associated with the novel Au nanotubular structure are some
interesting optical properties. Generally, gold nanostructures with
asymmetric axes, such as nanorods, will exhibit multiple plasmon
absorption bands. A red shifted longitudinal band and a transverse
band to the blue associated with the long axis and the short axis
of the structure, respectively. A third mode perpendicular to the
wall of the tube is likely too blue to be observed due to the
thinness of the wall. In the absorption spectra of these
structures, however, there is only one band despite the presence of
two possible axes of plasmon oscillation (FIG. 22). There are two
possible explanations for this. First, because of their length, the
longitudinal mode is red shifted far into the near IR and is not
visible in the range in which we are looking. More likely, however,
there is no surface plasmon absorption observed on the long axis
because it is on the micron length scale and any electron
oscillation is no longer surface confined and is more bulk-like.
Therefore, the absorption peak present in FIG. 22 must be due to
oscillations around the circumference of the tube only. This is
further reinforced by the spectral position of the nanotube band
between the thinly shelled 60 nm HGNs and solid gold nanoparticles.
With an average diameter of 40 nm and wall thickness of .about.5 nm
the circumference of the tube should yield a plasmon absorption red
shifted from the HGN presented.
[0099] The mean length of the nanotube can be, for example, between
about 0.1 .mu.m and about 50 .mu.m, such as 0.1 .mu.m, 0.2 .mu.m,
0.5 .mu.m, 0.75 .mu.m, 1.0 .mu.m, 1.5 .mu.m, 2.0 .mu.m, 3.0 .mu.m,
4.0 .mu.m, 5.0 .mu.m, 7.5 .mu.m, 10 .mu.m, 15 .mu.m, 20 .mu.m, 25
.mu.m, 30 .mu.m, 35 .mu.m, 40 .mu.m, 45 .mu.m, 50 .mu.m, or any
length therebetween. The length can be measured using electron
microscopy and standard metrics well known to those of skill in the
art. The mean wall thickness can be, for example, 0. 5 nm. 1.0 nm,
1.5 nm, 2.0 nm, 2.4 nm, 2.6 nm, 3.0 nm, 4 nm, 5 nm, 6 nm, 7 nm, 7.3
nm, 8 nm, 9 nm, and 10 nm, or any thickness therebetween The mean
diameter of the nanotube can he, for example, 10 nm, 20 nm, 30 nm,
40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, and 100 nm, or any
diameter therebetween. The mean diameter can be measured from the
image on an electronmicrograph. The mean diameter can be measured
over a portion of the entire nanotube.
[0100] In summary, we herein present a new method for producing
chemically stable and electrically conducting nanotubes. The
length, diameter, and wall thickness of the nanotubes can be
controlled chemically while their position and structural alignment
can be controlled magnetically. This method affords the possibility
of fabricating a variety of easily manipulated, useful linear
nanotubular structures for different applications.
Synthesis of Biological Molecules
Chemical Synthesis of Peptides
[0101] Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in a
batchwise or continuous flow process which sequentially adds
.alpha.-amino- and side chain-protected amino acid residues to an
insoluble polymeric support via a linker molecule. A linker
molecule such as methylamine-derivatized polyethylene glycol is
attached to poly(styrene-co-divinylbenzene) to form the support
resin. The amino acid residues are N-.alpha.-protected by acid
labile Boc (t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin.
[0102] Trifluoroacetic acid or piperidine are used to remove the
protecting group in the case of Boc or Fmoc, respectively. Each
additional amino acid is added to the anchored residue using a
coupling agent or pre-activated amino acid derivative, and the
resin is washed. The full-length peptide is synthesized by
sequential deprotection, coupling of derivatized amino acids, and
washing with dichloromethane and/or N,N-dimethylformamide. The
peptide is cleaved between the peptide carboxy terminus and the
linker group to yield a peptide acid or amide. These processes are
described in the Novabiochem 1997/98 Catalog and Peptide Synthesis
Handbook (San Diego Calif. pp. S1-S20). Automated synthesis may
also be carried out on machines such as the ABI 431A peptide
synthesizer (ABI). A protein or portion thereof may be purified by
preparative high performance liquid chromatography and its
composition confirmed by amino acid analysis or by sequencing
(Creighton (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York N.Y.).
[0103] In particular, a purified antigen may be used to produce
antibodies or to screen libraries of pharmaceutical agents to
identify those that specifically bind an antigen. Antibodies to an
antigen may also be generated using methods that are well known in
the art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies (i.e., those which inhibit dimer formation)
are especially preferred for therapeutic use.
[0104] For the production of polyclonal antibodies, various hosts
including goats, rabbits, rats, mice, humans, and others may be
immunized by injection with an antigen or with any fragment or
oligopeptide thereof that has immunogenic properties. Rats and mice
are preferred hosts for downstream applications involving
monoclonal antibody production. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface-active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable. (For review of
methods for antibody production and analysis, see, for example,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.)
[0105] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to an antigen have an amino
acid sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 14 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of antigen amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
Antibodies
[0106] Monoclonal antibodies to an antigen may be prepared using
any technique that provides for the production of antibody
molecules by continuous cell lines in culture. These include, but
are not limited to, the hybridoma technique, the human B-cell
hybridoma technique, and the EBV-hybridoma technique. (See, for
example, Kohler et al. (1975) Nature 256: 495-497; Kozbor et al.
(1985) J. Immunol. Methods 81: 31-42; Cote et al. (1983) Proc.
Natl. Acad. Sci. 80: 2026-2030; and Cole et al. (1984) Mol. Cell
Biol. 62: 109-120.)
[0107] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for an antigen. Affinity is expressed as an
association constant, K.sub.a, which is defined as the molar
concentration of antigen-antibody complex divided by the molar
concentrations of free antigen and free antibody under equilibrium
conditions. The K.sub.a determined for a preparation of polyclonal
antibodies, which are heterogeneous in their affinities for
multiple antigen epitopes, represents the average affinity, or
avidity, of the antibodies for an antigen. The K.sub.a determined
for a preparation of monoclonal antibodies, which are monospecific
for a particular antigen epitope, represents a true measure of
affinity. High-affinity antibody preparations with K.sub.a ranging
from about 10.sup.9 to 10.sup.12 l/mole are preferred for use in
immunoassays in which the antigen-antibody complex must withstand
rigorous manipulations. Low-affinity antibody preparations with
K.sub.a ranging from about 10.sup.6 to 10.sup.7 l/mole are
preferred for use in immunopurification and similar procedures
which ultimately require dissociation of antigen, preferably in
active form, from the antibody. (See Catty (1988) Antibodies,
Volume I: A Practical Approach, IRL Press, Washington, D. C.; and
Liddell and Cryer (1991) A Practical Guide to Monoclonal
Antibodies, John Wiley & Sons, New York, N.Y.)
[0108] Metal nanostructures are currently studied for a wide
variety of biomedical applications including contrast imaging,
ultrasonic imaging, thermal destruction of specific cancer cells,
and laser tissue welding. All applications of this type rely on the
optical and physical properties associated with metal
nanoparticles, nominally of gold. Much of this work has focused on
gold nanoshells due to their near IR optical absorption where
tissue transmission is at its peak, making in-vivo applications
feasible.
[0109] One of the most exciting of these applications is thermal
destruction of cancer cells. The nanostructures are selectively
attached to cancer cells in a tumor by a passive mechanism that has
been termed an "enhanced permeability and retention effect". The
tumor mass is then illuminated with near IR laser light which
passes harmlessly through the tissue, but is absorbed strongly by
the aggregates, causing them to heat drastically, killing only the
cancerous cells. (See O'Neal et al., (2004) Cancer Lett. 209:
171-176, herein incorporated by reference in its entirety.) This
technology has been utilized with gold-silica nanoshells further
comprising "stealthing" polymers, such as poly(ethyleneglycol) and
derives thereof, or liposomes; however this can be done better with
HGNs of the present invention.
[0110] The nanostructures disclosed herein can be formed and shaped
into a desired shape, such as a sphere, a cylinder, a rod, a rod, a
cone, a pyramid, or other shape, not limited to regular shapes, and
can be deposited upon a substrate at a desired density using means
well known to those of skill in the art. (See, for example, Fan et
al., (2005) J. Vac. Sci. Technol. 8: 947-953; Chaney et al., (2005)
Appl. Phys. Lett. 87: pub. no. 031908.)
[0111] Nearly monodisperse HGNs of tunable interior and exterior
diameter have been synthesized by sacrificial galvanic replacement
of cobalt nanoparticles. We have been able to control the position
of the surface plasmon band between 550 and 820 nm by carefully
controlling particle size and wall thickness. Cobalt particle size,
the sacrificial template that controls the resulting HGN size, is
tunable by simultaneously changing the concentration of sodium
borohydride and sodium citrate, the reductant and capping agent
respectively. This varies from all previously reported aqueous
syntheses of cobalt particles. We also show that by controlling the
addition of gold carefully the thickness of the gold shell can be
varied. These HGNs have been further demonstrated to be excellent
SERS substrates in terms of spectral consistency. They are
promising for chemical and biological sensing applications,
particularly those requiring near IR absorption.
Effects of Oxygen on HGN Formation.
[0112] Cobalt is extremely sensitive to oxygen, especially in
aqueous solution. If the solution is not properly de-oxygenated, or
if air is allowed to enter the reaction vessel the results can be
disastrous. While it is still possible to perform the reduction of
gold salt on partially oxidized cobalt particles, it produces very
poor results. The physical result of this is shown in FIG. 19.
While the oxidized cobalt will dissolve in the solution, it does
not oxidize homogeneously which results in malformed HGNs.
Optically, this has extremely deleterious results greatly
broadening the absorption band due to the random nature of the
oxidation. When solutions are badly oxidized, the percentage of
these types of particles tends to increase.
EXAMPLES
[0113] The invention will be more readily understood by reference
to the following examples, which are included merely for purposes
of illustration of certain aspects and embodiments of the present
invention and not as limitations.
Example I
Synthesis of Hollow Gold Nanospheres
[0114] HGNs were synthesized by first producing cobalt
nanoparticles as templates. 100 ml of 18 MS water, 500 .mu.l of 0.1
M aqueous sodium citrate (Aldrich), and 100 .mu.l of 0.4 M aqueous
CoCl.sub.2 (Aldrich) was degasses with nitrogen for 1 hour in a
well sealed three neck flask. To this, 300 .mu.l of a freshly
prepared 1 M aqueous sodium borohydride (Aldrich) solution was
added quickly. Hydrogen gas begins to form immediately and the
solution turns from colourless to brown. The solution is allowed to
stir under nitrogen for an additional 45 minutes to allow the
sodium borohydride to completely react. While maintaining nitrogen
flow, a 0.1 M aqueous chloroauric acid (Sigma-Aldrich, St. Louis,
Mo.) solution is added in 50 .mu.l aliquots to a final volume of
500 .mu.l. The solution changes from brown, to red-purple, and is
finally a deep blue color. Silver particles were synthesized by the
method of Lee and Meisel (Lee, P. C.; Meisel, D., J. Phys. Chem.
1982, 86, 3391-3395).
Example II
Synthesis of Cobalt Nanoparticles
[0115] Cobalt nanoparticles were synthesized with the utmost
attention paid to cleanliness and exclusion of air. All glassware
was cleaned with alconox glassware detergent, then aquaregia to
ensure the removal of all adsorbates, and then washed repeatedly
with ultra-pure water. To ensure completely air free solutions, all
solutions were vacuumed on a Schlink line until gas evolution
ceased, then bubbled with ultra-pure argon for ten minutes. This
process was repeated twice to remove as much oxygen as possible
from the reaction vessel.
[0116] Fast addition of cobalt chloride. 100 ml of water was placed
into a three neck flask with 100-800 .mu..mu.l of a 0.1 M solution
of sodium citrate or citric acid and deairated. To this, 100-800
.mu.l of a freshly made 1M sodium borohydride solution was added.
With rapid magnetic stirring, 100 .mu.l of a 0.4 M-0.6 M cobalt
chloride solution was added. Hydrogen immediately evolves and the
solution changes from pale pink to brown/gray indicating the
reduction of Co (II) into cobalt nanoparticles. This solution was
allowed to react for between 15 and 60 minutes (under constant
argon flow) depending on sodium borohydride concentration until
hydrogen stopped evolving, indicating complete hydrolysis of the
reductant. The addition of sodium borohydride and cobalt chloride
was also performed in reverse order.
[0117] Slow addition of cobalt chloride. 75 ml of water was placed
in a 500 ml three neck flask with 400 .mu.l of a 0.1 M solution of
sodium citrate. 25 ml of water with 100 .mu.l of 0.4 M cobalt
chloride was placed in a 250 ml three neck flask. These two
solutions were deairated. To the 500 ml three neck flask, 300
.mu.l-400 .mu.l of a freshly prepared 1M sodium borohydride
solution was added. Using a cannula and argon gas to pressurize the
250 ml flask, the cobalt chloride solution was added dropwise at
approximately 10 ml/minute. During this addition, the solution
slowly changes from colorless to brown/gray signifying cobalt
particle formation. This solution was allowed to react for 25
minutes to completely hydrolyze the sodium borohydride.
Example III
Gold Shell Growth
[0118] Due to the ease with which sodium borohydride is able to
reduce the gold salt it is imperative that it be completely
hydrolyzed before introducing gold. The presence of sodium
borohydride is monitored by halting stirring and inspecting the
solution for bubbles indicating the continuing hydrolysis of the
reductant. It is only when bubbling has ceased completely that gold
may be added.
[0119] High concentration addition. Upon insuring complete
hydrolysis of the sodium borohydride the flow of argon is increased
and a 0.1 M solution of chloroauric acid is added at 50
.mu.l/addition to a total volume between 150 .mu.l and 450 .mu.l.
Between each addition 30 to 60 seconds are allowed to pass to
ensure complete mixing. Upon completion of gold addition, the argon
flow is stopped and the vessel is opened to ambient conditions
under rapid stirring to oxidize any remaining cobalt metal left in
solution.
[0120] Low concentration addition (retaining Co at core). Using a
cannula, 30 ml of the sodium borohydride free cobalt nanoparticle
solution is transferred to an argon-purged graduated cylinder. This
is then rapidly added to a vortexing 10 ml solution of chloroauric
acid. The gold solution contains between 20-60 .mu.l of cloroauric
acid diluted to 10 ml. To retain the cobalt core this solution may
be kept under argon flow, however, by exposing the solution to air
the cobalt is completely oxidized leaving only water and dissolved
salts at the core of the HGN. Samples with remaining cobalt cores
retain a brown color, while oxidized samples change to between
purple and green colored depending on amount of gold added and size
of the particle.
Example IV
Single Particle SERS/Luminescence and Bulk SERS
[0121] Single particle SERS and Rayleigh scattering were performed
on a home built confocal microscope system described previously
(Schwartzberg, A. M., Grant, C. D., Wolcott, A., Talley, C. E.,
Huser, T. R., Bogomolni, R., and Zhang, J. Z., J. Phys. Chem. B
2004, 108, 19191-19197.) with the addition of transmitted light
dark field illumination (NA 1 to 1.4). For SERS experiments and
imaging, a Zeiss Apochromat 100 X, 1.4 NA oil emersion objective
was used. Typically the sample was integrated for 30 seconds with a
total power of 100 .quadrature.W from a helium-neon laser (632.8
nm, Melles Griot). Rayleigh scattering experiments were performed
with a Zeiss Apochromat 100 X, 0.7 NA oil emersion objective.
[0122] Samples for SERS and Rayleigh scattering were prepared by
immobilizing the particles on glass coverslips with
trimethoxy-[3-(methylamino)propyl]silane (APS) (Aldrich).
Coverslips were cleaned prior to the silanization step by
sonication in a 2% solution of Hellmanex, followed by 18 M.OMEGA.
water. They were then submerged in 5 mM aqueous solution of APS to
deposit the tethering molecules. After one to two minutes, the
coverslips were rinsed with water, dried under nitrogen, and 40
.mu.I of the as prepared particle solution was placed on one
surface. After several seconds exposed to the solution, it was
rinsed with water then blown dry with nitrogen. For samples
prepared for SERS studies, the HGNs coated surface was then treated
with MBA by applying 40 .mu.l of a 1 mM ethanoic solution for 60
seconds. The sample was then rinsed with ethanol and dried under
nitrogen.
[0123] While questions remain about the nature of the enhancement
at the HGNs surface, it is the consistency of their optical
response that ultimately determines their reproducibility in sensor
applications. To characterize the optical response, we began by
examining the Rayleigh scattering spectra of many individual HGNs
(FIG. 8, red trace, middle) and the ensemble average solution
absorption (FIG. 8, black trace, top), both are indicative of
plasmon resonance position and structure. Similar to other shell
structures, a single peak is observed, nominally indicating
non-aggregated HGNs (Nehl, C. L., Grady, N. K., Goodrich, G. P.,
Tam, F.; Halas, N. J., and Hafner, J. H., Nano Lett. 2004, 4,
2355-2359). The spectral width of the individual particle is not
significantly different from that of the ensemble averaged
solution, which is a result of the sample consistency. The shift of
the peak position in the scattering spectrum as compared to that of
the ensemble average solution absorption spectrum is due to a
change in refractive index of the HGNs surrounding from air to
water. This is known to affect the plasmon resonance of particles
of this type. (See Nehl, C. L. et al., (2004) supra; Grady, N. K.,
Goodrich, G. P., Tam, F., Halas, N. J., and Hafner, J. H. Nano
Lett., 2004, 4, 2355-2359. Sun, Y. G. and Xia, Y. N., Anal. Chem.
2002, 74, 5297-5305.)
[0124] The Rayleigh scattering spectra of 100 HGNs were taken and
compiled (FIG. 8, bottom) into a histogram showing an average
maximum scattering intensity (.lamda..sub.max) of 621.+-.10.6 nm.
While there is some particle-to-particle variation it should not
have an overly strong effect on the SERS response of the particles
as the shift is less than 10% of the homogeneous line width. Any
shift should have a minimal effect on the absorption cross-section
at the excitation or Raman scattering wavelengths. This is clearly
not the case with silver aggregates such as those shown in the
inset of FIG. 8. The scattering of individual silver aggregates
shifts drastically, by hundreds of nanometres, and many show
multiple peaks.
[0125] The homogeneous scattering properties of the HGNs suggest a
consistent SERS response. To assess this, we compared the spectra
of 150 HGNs and 150 standard silver particles coated with the same
model analyte, 4-mercaptobenzoic acid (MBA). Representative SERS
spectra of MBA bound to HGNs (red trace, top) and solid silver
particles (blue trace, bottom) are shown in FIG. 4. The histograms
in the inset show the statistical representation of the signal
homogeneity of the silver (blue) and HGNs samples (red). To compare
the two samples without the influence of absolute intensity
fluctuations, two peaks were chosen to normalize the results. Peaks
at 1070 cm.sup.-1 and 1590 cm.sup.-1, both ring breathing modes,
were chosen because these are the most intense peaks. Also, they
are spaced 500 cm.sup.-1 apart where any variation due to plasmon
shift should become readily apparent. From the histograms in the
inset of FIG. 4, it is clear that the HGNs (red) have a
significantly narrower distribution than the silver nanoparticles
(blue). All peak ratios of the 150 HGNs fall within 0.9 and 1.1,
representing statistical distribution of 5% by standard deviation,
while the ratios for silver particles range from 0.5 to 1.7, or
45%. This is nearly a tenfold increase in consistency,
demonstrating that these HGNs are a clear and significant
improvement over the silver nanoparticle aggregates previously
reported.
[0126] Considering the importance of probe size, especially for
intracellular sensing, it is imperative to confirm that single HGNs
are sufficient to generate the observed SERS spectra, and not
merely HGNs aggregates. All single particle experiments were
performed with this in mind. By immobilizing particles to the
surface of glass coverslips while taking steps to avoid aggregation
we hope to minimize signal due to aggregated HGNs. To prove that
enhancement can originate from individual HGNs, however, a more
detailed and involved study was required. Single particles treated
with the sample analyte MBA on indexed TEM grids were co-located by
TEM and confocal Raman imaging. An area was located in which SERS
was observed, while no aggregates were present within several
hundred microns (FIG. 9). The diffraction limited focal area of the
laser is indicated by the red circle, approximately 350 nm. The
strong background of this spectrum is due to fluorescence of the
holy carbon film on the TEM grid. While this fluorescence affects
the appearance of the overall spectrum, it has little effect on the
SERS spectrum of MBA that contains characteristic peaks, e.g. at
1070 cm.sup.-1 and 1590 cm.sup.-1. This is experiment clearly
demonstrates that SERS can and does originate from individual
hollow nanostructures.
[0127] The probe molecule used, MBA, was chosen for its utility as
a model system in SERS pH sensing. SERS has the potential to become
a valuable alternative approach to intracellular sensing compared
to fluorescent dyes, because of its high sensitivity and molecular
specificity. Even with resonant probe molecules, SERS provides
robust signals that are not prone to rapid photodecomposition. In
the following, we demonstrate that isolated functional gold
nanostructures provide a highly consistent and reproducible SERS
response for pH detection with a direct comparison to the
aggregated silver colloids presented in earlier work (Talley, C.
E., Jusinski, L., Hollars, C. W., Lane, S. M., and Huser, T., Anal.
Chem. 2004, 76, 7064-7068). The response of MBA coated HGNs was
taken at 7 different pH points with 20-30 particles sampled
individually for each data point. The results are shown in FIG. 10
and represent a pH calibration curve. In FIG. 10, the SERS
intensity ratio between the 1430 cm.sup.-1 peak, due to COO.sup.-
stretching mode and most sensitive to pH changes, and the 1590
cm.sup.-1 ring breathing mode, which is insensitive to pH, is
graphed as a function of different bulk pH. Error bars in the
intensity ratio correspond to the standard deviation of each
measurement and increase with signal intensity, as error is a
function of enhancement variability and will linearly increase as
the measured signal becomes larger. The percentage error is
relatively constant through the entire pH range at an average of
9.1.+-.2.4%. In a direct comparison to previous work, however, it
is clear that for pH sensing applications, the HGNs are a
significantly more precise probe than aggregated silver
nanoparticles which have an average error of 104.5.+-.71.6%
(Talley, C. E. et al., (2004) supra); Jusinski, L., Hollars, C. W.,
Lane, S. M., and Huser, T., Anal. Chem. 2004, 76, 7064-7068).
Again, this is a ten-fold improvement over the solid silver
particle system.
[0128] The most important feature to note in FIG. 10 is the narrow
distribution of relative intensities represented by the error bars
at each measured pH value. The pH resolution is dependent on signal
homogeneity of the probe. With nanoparticle aggregates, resolution
was limited to about 1 pH unit due to the large variability
resulting from the aggregated structures. With increased
homogeneity of the HGNs, however, the resolution is now increased
to 0.5 pH units or less, effectively doubling the sensitivity of
the probe undoubtedly due to sample homogeneity and the ability to
attain SERS from individual particles. In addition to this
improvement in pH resolution, the HGNs are sensitive to a much
broader pH range. While silver substrates yield a sensing region
from .about.pH 6.5-pH 8, these HGNs are responsive from .about.pH
3.5-pH 9. The reason for this wider pH sensitivity is not
immediately apparent, however, it is important to recognize that
with increasing particle homogeneity, the packing of the MBA
molecules at the particle surface will become more uniform. This
will result in stronger molecule-molecule interactions that may
have the effect of partially shielding some of the MBA from the
bulk solution. This can shift the kinetics of the protonation or
deprotonation process of the acid group, effectively expanding the
window of sensitivity. By increasing the active range, this
HGN-based probe is sensitive at most biologically relevant pH
ranges.
Example V
Transmission Electron Microscopy (TEM)
[0129] Low resolution TEM measurements were performed on a JEOL
model JEM-1200EX microscope and High resolution TEM was performed
on a Philips CM300-FEG at the National Center for Electron
Microscopy at Lawrence Berkeley National Laboratory.
[0130] Absorption measurements were taken on a HP 89532A
spectrometer. All spectra were fit with Igor Pro 5.0 using a
lorentzian function with chi square values less than 0.1. Particles
were sized with imageJ image processing software (Abramoff, M. D.,
Magelhaes, P. J., and Ram, S. J., Biophotonics Internat. 2004, 11,
36).
[0131] The homogeneity of the HGN samples is demonstrated in FIG.
6, which shows a representative low resolution transmission
electron micrograph (TEM) of HGNs. The HGNs have an average
diameter of 30 nm with a coefficient of variation of 14%. The
relatively narrow size distribution is a reflection of the cobalt
seed particles from which the HGNs were grown. The high resolution
TEM of an individual HGN, shown in FIG. 7 illustrates the
polycrystalline, and uniform nature of the HGNs. Twinning of the
lattice planes confirms that the shell is comprised of nanocrystals
that have been fused together upon growth. Due to the nature of
wall growth, from the outside in, and the flow of oxidized cobalt
out of, and gold ions into the HGNs, it is likely that pinholes in
the wall will remain. Computationally, it has been shown that
pinholes in hollow particle structures concentrate the evanescent
field that results from the excitation of surface plasmons (Hao,
E., Li, S. Y., Bailey, R. C., Zou, S. L., Schatz, G. C., and Hupp,
J. T., J. Phys. Chem. B 2004, 108, 1224-1229). They are, however,
difficult to detect by TEM measurement. This may be a factor
important to SERS enhancement and will be explored in depth in
future works.
Example VI
Effect of Cobalt Chloride, Sodium Borohydride and Sodium Citrate
Concentration on Particle Size
[0132] The goal of this study was to gain control of the cobalt
particle size by aqueous solution chemical methods. Previous work
on this system by Liang et al. focused more on the thickness of the
shell to control its optical properties (Liang, H. P., Wan, L. J.,
Bai, C. L., and Jiang, L. J., Phys. Chem. B 2005, 109, 7795). While
their work produced excellent results, further tunability is
necessary to make the system as useful as possible. Initial
attempts to reproduce the work of Liang et al. did not yield
satisfactory results. The particles obtained were inhomogeneous and
significantly smaller than the 60 nm reported. In fact, using as
close to precisely the same synthesis as possible, .about.25 nm
cobalt particles were obtained, however, with their method of gold
addition only inhomogeneous, gray solutions were observed. Upon
determining an improved method of gold addition, this yielded
excellent results for single particle SERS probes. However, there
are many applications that may benefit from larger particle size
and further red-shifted absorption, including SERS.
[0133] The other guiding hand in this work was provided by
Kobayashi et al., who first reported this cobalt particle
synthesis, but proceeded to cap the particles with silica shells to
protect them from oxygen (Kobayashi, Y., Horie, M., Konno, M.,
Rodriguez-Gonzalez, B., and Liz-Marzan, L. M., J. Phys. Chem. B
2003, 107, 7420). Kobayashi et al. found that as citrate
concentration was reduced, particle size increased. This is
consistent with colloidal gold and silver syntheses and is not an
unreasonable claim. For this application however, their trend did
not hold true. A significant difference between this work and that
of Kobayashi et al. is the time at which the reaction could be
halted. In their work, for large cobalt particles, they were forced
to add the silica growth reagents almost immediately upon reduction
of the cobalt salt. Any delay at low citrate concentration and the
solutions would become unstable and flocculate. In this work
however, if the gold solution is added too quickly, it is
immediately reduced by the remaining sodium borohydride instead of
the cobalt particles. This leads to an unfortunate mess of
nanoparticles. To achieve optimal particle growth a significant
amount of time must pass in order to allow the sodium borohydride
to completely hydrolyze before the gold can be added.
[0134] This being said, it is also important to note that even at
relatively high concentrations of citrate where the particles are
still stable after some time, there is little change in particle
size by merely altering the citrate concentration. There may be a
relatively simple explanation for this observation. Because the
particle stability is directly related to the concentration of
citrate there may have been an aggregation affect responsible for
the size increase observed previously. As citrate concentration is
reduced, we have observed that the rate of aggregation increased.
Therefore, when capping the particles immediately after reduction,
they are likely halting the aggregation at different stages
depending on citrate concentration. When concentration is low, a
larger aggregate will be formed before the silica can stabilize it,
at high concentration a smaller aggregate will be present. This may
be responsible for the lack of crystalline structure in the as
synthesized particles. By sintering them at high temperature, they
are likely fused into one crystalline particle.
[0135] Why then, does citrate not affect particle size as strongly
as previously thought? In the case of colloidal gold, the reduction
is done by the relatively weak reductant, citrate. This reaction is
slow which allows for thermodynamic processes to control the
formation of clusters. Only as many seed particles will be formed
in the reaction as can be stabilized by the cappant/reductant. This
means that the capping agent concentration will have a strong
affect on the number of seed particles and hence, particle size. In
the formation of cobalt particles however, a much stronger reducing
agent is required. As sodium borohydride is a significantly
stronger reductant than is technically required to reduce the
cobalt salt to cobalt metal, the reduction is extremely fast,
taking place in less than one minute as opposed to five to ten
minutes for the reduction of gold salt by citrate. Because of this,
kinetic processes dominate the formation of seed particles. The
number of seeds, and therefore the size of the resulting particle,
will be more dependent on the rate of the reduction.
[0136] The rate of reduction can be controlled in several ways.
Temperature plays a strong role in the rate of reaction, however,
little change in particle size was observed between particles
synthesized at 0.degree. C. and room temperature. A second way to
alter rate is by changing the solution pH. The reductive potential
of sodium borohydride is pH dependent. It is important to note at
this point that contrary to previous reports of this synthesis, we
use sodium citrate instead of citric acid. This is because the
reaction was found to be slower at the higher pH, and particle
homogeneity was superior in the neutral solution. Higher and lower
pH was also attempted by adjusting with HCl and NaOH. These
solutions, however, were unstable and immediately crashed out. This
is most likely due to the presence of excess ions, especially
Cl.sup.- which has a strong disrupting effect on aqueous colloidal
capping. Finally, altering the concentration of reductant was used
to change reaction rate. This was found to be the best method of
controlling particle size without drastically decreasing particle
homogeneity.
[0137] By decreasing the amount of sodium borohydride present, the
reaction time is increased substantially. This produces larger
particles that remain stable in solution. Table 1 shows the result
of varying sodium borohydride concentration by one quarter. The
particle size is increased by approximately 40%, however, this is
the practical limit of size tunability by this method. Lower
concentrations produce incredibly inhomogeneous results that are
often unstable. In order to form larger particles we must also
alter the sodium citrate concentration.
TABLE-US-00001 TABLE 1 Particle size is dependent upon sodium
borohydride concentration Volume 0.4M Volume 0.1M Volume 1M
Particle CoCl.sub.2 (.mu.l) Citrate (.mu.l) NaBH.sub.4 (.mu.l) Size
(nm) 100 400 400 31 .+-. 2 100 400 100 44 .+-. 5
All reactions were performed in 100 ml water. All particle sizes
are determined by examining the resulting gold particles. Reported
sizes are in diameter.
[0138] While the sodium borohydride reduction of metal salts is
largely kinetics driven, there are still some thermodynamic-type
processes controlling particle size. This is especially true as the
concentration of reductant is decreased and the reaction is slowed.
The reaction is now substantially more thermodynamically
controlled, making the variation in capping agent concentration
more effective in controlling particle size. By decreasing both
NaBH.sub.4 and citrate concentration we observed a drastic increase
in particle size, this is shown in the 3D plot in FIG. 11. The
trend appears to be linear, at least within the concentrations
shown here. At lower concentrations the particle sizes could be
substantially larger, however, because they crash out of solution
almost immediately this is not something we could test. We present
this as a general method of tuning the size of cobalt
nanoparticles. Using this plot, it is possible to predict roughly
what the final particle size will be at a given sodium borohydride
and sodium citrate concentration.
Example VII
The Influence of the Rate of Addition and Concentration of
CoCl.sub.2 on Particle Homogeneity
[0139] To increase particle homogeneity and size, a slow addition
of low concentration cobalt salt was attempted. It was thought that
this would artificially slow the rate of reaction. This, however,
was not the case, as is shown in Table 2.
TABLE-US-00002 TABLE 2 Rate of addition and concentration of cobalt
salt influences particle size. Rate of CoCl.sub.2 CoCl.sub.2
Concentration Volume 0.1M Volume 1M Particle Addition (M) Citrate
(.mu.l) NaBH.sub.4 (.mu.l) Size (nm) Fast 0.4 400 400 78 .+-. 2
Slow 0.4 400 400 31 .+-. 6 (diluted) Fast 0.5 400 400 50 .+-. 5
[0140] All particle sizes are in diameters. The cobalt chloride
solution used for the slow addition is diluted to 25 ml with
water.
[0141] While slightly larger particles were achieved, the
coefficient of variation increases from 7% to 18%. This is clearly
not an advantageous method of controlling particle size. The reason
for this great increase in variation is due to the continual
formation of seed particles as the cobalt is added. When examining
the particles it is obvious that some seeds are formed initially
and result in very large particles, while others are formed
throughout the addition and lead to small particles. This is clear
in FIG. 12 that shows histograms of particle size from slow and
fast addition of cobalt. Not only does this exemplify the
inhomogeneity of the slow addition sample, it also shows the
asymmetric formation of particles. While the fast addition yields a
nice, even sample, the slow addition yields a curve broadened and
asymmetrically shifted by the presence of large particles formed
early in the cobalt addition. This is clearly not the way to
increase particle size. By increasing the concentration of cobalt
while maintaining volume, however, we have found that particle size
changes drastically without excessively broadening particle
distribution, this is also shown in Table 2. While higher
concentrations of cobalt seem to induce flocculation, it may be
possible to better control this with careful changes in citrate
concentration.
Example VIII
Formation of Gold Shells
[0142] Along with the tunability of cobalt particle sizes we have
been able to produce a wide variety of sizes of the HGNs as shown
in FIG. 13. These are representative TEM images of the HGNs at
different sizes. FIG. 13A is a high resolution TEM of a 30 nm
particle, the lattice fringes of gold are clearly defined and show
that these particles are poly-crystalline with large single
crystalline areas. FIGS. 13B-F show the tunability of the samples,
from 70 nm to 28 nm. The largest particle sample in FIG. 13B
clearly demonstrates the inhomogeneity that seems to be inherent at
larger sizes.
[0143] Forming the gold shell seems to be an extremely simple
matter at first glance, however, under closer inspection it becomes
clear that there are many parameters that must be carefully
controlled in order to form high quality samples. As mentioned
above, attempting to recreate the previous works did not result in
good samples. Another method was needed to make homogeneous samples
of high optical and structural quality like those shown in FIG.
13.
[0144] High concentration gold addition. The general consensus on
homogeneous nanoparicle formation is that a low concentration of
reagents yields the best results. It is important to remember,
however, that in the addition of gold here, we are not forming a
normal colloidal nanoparticle system. All that determines particle
size and shape is the sacrificial template. For this reason the
high concentration addition of gold should not necessarily produce
poor results. After many attempts, it was found that by adding high
concentration (0.1 M) gold salt in small volumes yielded excellent
results. Adding the gold all at once gave poor results, as did
adding the solution dropwise. By using approximately 50 .mu.l per
addition over five to eight additions, spectrally narrow, highly
concentrated samples were achieved.
[0145] The explanation for this is a fairly simple one: it is a
matter of mixing. The reaction of gold salt with the cobalt
particle is very fast, happening almost instantaneously upon the
addition of the gold. There is also a secondary shell mediated
growth that takes place on a slightly longer time scale, where free
citrate in solution will reduce excess gold salt onto the formed
shells. This can result in significantly thicker shells when too
much gold is added. When a small amount of gold is introduced to
the stirred solution, all particles at the site of the addition
will immediately be oxidized completely in the presence of such
high concentration gold. If there is excess gold at this site, it
will diffuse through the solution being reduced onto the cobalt
particles until there is no more gold. If the volume of gold
solution is too low, i.e. dropwise, the immediate impact will be
relatively small but due to the small size of the droplet it will
dilute quickly. As the gold dilutes into the water, less and less
will be reduced onto the cobalt, resulting in a gradient of shell
thicknesses. Thickest at the site of addition and thinner shells
moving away from the concentration center. This leads to an
incongruous sample in which some shells are badly under-formed and
some are over-grown by seed mediated growth. An excellent example
of this over-growth is in FIG. 13C. The second particle from the
top has some slight over-growth that looks like small particle
stuck to the surface. When the concentration is excessive this
becomes a much more pronounced feature of the particle.
[0146] At the other end of the addition rate scale is the
all-at-once addition of the gold. This suffers similar problems to
the drop-wise addition, however, there is significantly more
over-growth, and less under-formed particles. We were able to
overcome this problem by using a middle of the road approach. By
using 50 .mu.I per addition the resulting particles were uniform
and we did not observe excessive over-growth. The choice of this
volume was not obvious and was only discovered by experimental
trials. This method does, however, have one major flaw. Because
such high concentrations are used, we were not able to readily
control the shell thicknesses. In theory, if the gold is added
correctly, the shell thickness should be a function of the amount
of gold added. This was achieved by using relatively large volumes
of low concentration gold.
[0147] Low concentration gold addition. It was determined early on
in this study that using low concentrations of gold would not
produce satisfactory results; however, this assessment was not
entirely correct. Several factors are required for the low
concentration addition of gold to work properly. The first is that
the solution should be mixed very well, as quickly as possible. If
the cobalt is added to the gold solution too slowly, most of the
gold will be utilized by a small number of particles, which will
lead to poor sample homogeneity. Second, the volume of the gold
salt to which the cobalt is added must be large enough that mixing
can happen very quickly. With low volumes of gold at higher
concentrations there is still a pronounced mixing problem, leading
to poor samples. This is the problem we observed in reproducing the
work of Liang et al. (Liang et al. (2005) supra). While the larger
volumes of gold produced reasonable results, using 5 ml or 8 ml of
gold salt gave widely varying results and consistency was a major
issue. Because mixing is the biggest issue in producing consistent
results, it was hypothesized that by holding the volumes of gold
and cobalt solutions constant, a more consistent result could be
obtained.
[0148] By diluting varying volumes of gold salt to 10 ml with water
and adding the cobalt as quickly as possible under rapid stirring
we were able to produce homogeneous HGN with tunable wall
thicknesses, similar to the work of Liang et al. (Liang et al.
(2005) supra). Shell thickness varies linearly with gold
concentration, indicating that homogeneous mixing is taking place,
as shown in Table 3. These are representative values from a single
sample and are consistent with all other data.
TABLE-US-00003 TABLE 3 Wall thickness as a function of the volume
of gold salt added Volume of Volume Volume Volume 0.1M 11AuCl.sub.4
0.1M 0.1M 1M Particle Dilluted to CoCl.sub.2 Citrate NaBH.sub.4
Size Wall 10 ml (.mu.l) (.mu.l) (.mu.l) (.mu.l) (nm) Thickness 25
100 600 100 40 .+-. 6 6.2 .+-. 0.6 35 100 600 100 40 .+-. 6 6.9
.+-. 0.8 60 100 600 100 40 .+-. 6 .sup. 8 .+-. 0.7
Example IX
Effect of Particle Size and Wall Thickness on Optical
Properties
[0149] One of the major intents of all this size tuning is the
control of the optical properties of the HGN. We have found that by
varying wall thickness and particle size it is possible to tune the
plasmon absorption across much of the visible spectrum as in FIG.
4. These spectra are representative of many experiments and show
the full range of tunability of this system. While the full width
half max (FWHM) of the spectra remains relatively unchanged from
500 to 750 nm at between 50 and 100 nm, the last two spectra are
fairly broadened to over 200 nm. This is likely due to the
formation of gold shells and rings. These are shells that have not
completely formed and are likely red shifted in absorption from the
complete shells. The weak shoulder at 700 nm may be due to the
presence of complete shells, while the peak is due to the rings. At
this time, however, it is not possible to determine the exact
affect of the presence of the rings.
[0150] By increasing particle size at a constant wall thickness the
absorption band will red-shift as the plasmon oscillation decreases
in energy. On the other hand, increasing wall thickness at constant
particle size will blue shift the absorption band. The band shifts
to higher energy because as the inner diameter of the HGN
decreases, it takes on more solid particle like properties. As
solid gold particles at these sizes have plasmon bands at
approximately 520 nm, the absorption will always shift in this
direction as wall thickness increases. This is predicted in the
work of Hao et al. and is shown experimentally here in FIG. 4 (Hao,
E., Li, S. Y., Bailey, R. C., Zou, S. L., Schatz, G. C., and Hupp,
J. T., J. Phys. Chem. B 2004, 108, 1224). This 3D plot shows the
effect of particle size and wall thickness on plasmon absorption.
Representing thirteen independent experiments, the trend is clearly
shown here. Because the work of Hao et al. is for particle of
different sizes than those made here, we are not able to directly
correlate their results to our data. However, we are currently
working on similar calculations that should determine if these
results match well to the theory.
[0151] Because wall thickness plays such an important role in the
position of the plasmon absorption, it is important to understand
how this corresponds to the amount of gold added to the solution.
FIG. 5 shows the non-normalized absorption spectra of three samples
made from a single batch of 35 nm cobalt nanoparticles. The highest
concentration sample, at 60 .mu.I of 0.1 M gold salt added absorbs
most strongly at 638 nm, is the most blue shifted of the three as
would be expected and has a wall thickness of 7.+-.0.8 nm. The
lower concentration samples at 35 ml and 25 ml are red shifted to
685 nm (wall thickness 5.6.+-.0.6 nm) and 702 nm (wall thickness
3.7.+-.0.6 nm) respectively. Interestingly, as the band shifts the
FWHM changes only slightly from 80 nm for the 60 .mu.I sample, to
91 nm for the 35 .mu.l sample to 82 nm for the 25 .mu.I sample.
This is not the trend one might expect given the propensity of
solid gold nanoparticles to broaden significantly in spectrum with
increasing size. This broadening is due to the introduction of new
multi-pole modes which are non-radiative and broader in energy than
the normal dipole plasmon mode (Payne, E. K., Shuford, K. L., Park,
S., Schatz, G. C., and Mirkin, C. A., J. Phys. Chem. B 2006, 110,
2150; Millstone, J. E., Park, S., Shuford, K. L., Qin, L. D.,
Schatz, G. C., and Mirkin, C. A., J. Am. Chem. Soc. 2005, 127,
5312). In fact, upon close examination of FIG. 13 it is clear that
with the exception of the last two spectra, the FWHM changes little
regardless of particle size or shell thickness. The explanation for
this is tied to the electron mean free path in gold. Because the
wall thickness is much less than this length, (.about.50 nm) longer
axes will dominate the plasmon oscillations and the multi-pole
modes which require large particles will be minimized.
Interestingly, this also explains why only one absorption band is
observed for this system, while nanorods, which also have multiple
axes of oscillation, will show two.
[0152] It may be noted that as the concentration of gold added
decreases, there is a decrease in optical density as well. This is
not a matter of particle concentration, since 10 ml of gold is
added to each sample, and the total number of HGNs is fixed to the
number of cobalt particles present in the original solution. This
is a function of absorption cross section of the HGNs due to the
different thicknesses of gold. As the wall grows thicker it will
have a larger absorption cross section.
Example X
Homogeneous Line Width and Inhomogeneous Broadening
[0153] To determine if, and to what extent the absorption spectrum
is broadened by inhomogeniety in the sample, we examined the
Rayleigh scattering spectra of the HGNs. While the FWHM of the
ensemble averaged solution of 30.+-.2.6 nm particles is 75 nm, the
single particle FWHM is 47 nm as shown in FIG. 17. This is a
broadening of 27 nm that shows that the samples are slightly
inhomogeneously broadened. This is to be expected to some point,
but is impressively small considering how sensitive these
structures are to variance in wall thickness and local environment
(Nehl, C. L., Grady, N. K., Goodrich, G. P., Tam, F., Halas, N. J.,
and Hafner, J. H., Nano Lett. 2004, 4, 2355; Sun, Y. G. and Xia, Y.
N. Anal. Chem. 2002, 74, 5297). The sensitivity to local
environment is clear upon examination of the spectral shift between
the ensemble averaged and scattering spectra. This is a shift of 14
nm and is consistent with all particles examined. The scattering
spectra were taken from particles immobilized on glass substrates
in air while the ensemble-averaged spectra were taken in aqueous
solution. The refractive index of the imbedding medium decreases
from 1.33 to 1 in going from water to air in these two scenarios.
This substantially changes the optical properties of the HGNs. A
decrease in refractive index has been shown to correspond to a red
shift, and explains our observations here.
Example XI
Surface Enhanced Raman Scattering
[0154] SERS experiments were performed on solutions of as prepared
HGNs with mercaptobenzoic acid (MBA) added to a final concentration
of 1 mM. At this concentration there was no spectral shift observed
which would indicate aggregation, therefore we can nominally say
that the resulting spectra are from non-aggregated or at least
minimally aggregated. This was confirmed in our previous work on
SERS of single HGNs that showed that enhancement is observable from
non-aggregated HGNs (Schwartzberg, A. M., Olsen, T. Y., Huser, T.
R., Zhang, J. Z., and Talley, C. E., Anal. Chem. 2006, 78,
4732-4736). Here we show the ensemble averaged SERS spectrum of MBA
in FIG. 18. In terms of enhancement, when compared in the SERS
intensity to aggregated Lee and Meisel silver particles, the
standard high enhancement SERS substrate, we achieve about 10% of
the signal. This is an excellent result for nominally
non-aggregated particles and significantly better than many current
single particle systems.
Example XII
Synthesis of Metal Nanotube
[0155] Hollow gold nanotubes were synthesized by an electroless
deposition on semi-ordered, aggregated cobalt nanoparticles. The
formation of cobalt nanoparticles and the electroless deposition of
gold has been reported previously, however, in order to form
nanotubes, reaction conditions are altered slightly. (See
Schwartzberg, A. M., Olson, T. Y., Talley, C. E. and Zhang, J. Z.,
J. Phys. Chem. B 110, 19935-19944 (2006); Liang, H. P., Wan, L. J.,
Bai, C. L. and Jiang, L., J. Phys. Chem. B 109, 7795-7800
(2005).)
[0156] Briefly, 100 ml of 18 M.OMEGA. purified water with 100 .mu.I
of a 0.5 M CoCl.sub.2 aqueous solution and 600 .mu.I of a 0.1 M
aqueous sodium citrate solution was degassed in a round bottom
three neck flask under vacuum and purged with nitrogen three times
to ensure an oxygen free environment. To this, 100 .mu.l of a 1 M
aqueous NaBH.sub.4 solution was added under vigorous magnetic
stirring. Hydrogen evolution was immediate and subsequently the
solution changed in color from light pink to light brown/gray. This
cobalt nanoparticle solution was allowed to stir under nitrogen for
35-40 minutes until hydrogen evolution ceased to insure that all
sodium borohydride was reacted. To form the gold nanotubes, 30 ml
of the stock cobalt nanoparticle solution was added to 10 ml of
rapidly mixing water with 25 .mu.l of 1 mM HAuCl.sub.4 solution.
This same procedure was also followed in the absence of magnetic
stirring to determine the effect of the magnetic field on the
nanotube formation. In this case manual swirling of the reaction
vessel was used.
[0157] Low resolution TEM measurements were performed on a JEOL
model JEM-1200EX microscope and High resolution TEM was performed
on a Philips CM300-FEG at the national center for electron
microscopy at Lawrence Berkeley National Laboratory.
Example XIII
Detection of Ab-GNP Binding Interaction Using a Secondary Ab
[0158] The effect of binding an antigen to its antibody is observed
by taking the Raman spectrum of the antibody before and after
exposure to the antigen through the use of SERS. To study the
applicability of this method, a primary antibody (SC2020, Santa
Cruz Biotechnology Santa Cruz CA) and a secondary antibody (SC1616,
Santa Cruz Biotechnology Santa Cruz CA) were used. SC2020 was
obtained at a concentration of 400 .mu.g/ml and diluted by a factor
of two with 20 mM HEPES buffer (pH 7.4). This solution was mixed
equal volume with a GNP solution that was also diluted by a factor
of two with 20 mM HEPES buffer. After twenty minutes of
interaction, a SERS spectrum was obtained. An equal amount of
SC1616 was added to the system and the SERS spectrum was obtained
again. The binding of the secondary antibody (SC1616) to the
primary antibody (SC2020) caused the SERS intensity of the
secondary antibody to increase by 20-50%. This method provides an
indirect means of detecting antigens in a system.
Example XIV
Detection of Tumour-Antigens in Bodily Fluids
[0159] A murine monoclonal antibody raised against the CA125
ovarian cancer marker (OC125; Bast et al., (1981) J. Clin. Invest.
68: 1331-1337; Cat. No. AB19551, AbCam Ltd., Cambridge, UK) is
incubated at a final concentration of 100 .mu.g/ml in HEPES buffer
(pH 7.4) with GNA as prepared above at a final concentration of 1
mg/ml for twenty minutes at ambient temperature. The mixture is
then washed four times with excess sample buffer, then stored at
4.degree. C. until use. A fraction is subjected to SERS to obtain
baseline values.
[0160] Fluid samples from individuals with diagnosed ovarian cancer
are incubated with SQD in the presence of a conjugating agent and
linker molecule for 20 minutes at ambient temperature. The mixture
is washed four times and resuspended in HEPES buffer (pH 7.4) to
produce SQD-Ag conjugate. A fraction is subjected to SQD
luminescence to obtain baseline values.
[0161] The SQD-Ag conjugate is added to OC125-GNA mixture in HEPES
incubation medium (pH 7.4) at ambient temperature for 8 hours.
Control samples are from individuals without diagnosed disease or
disorders. The samples are then washed four times with incubation
medium, resuspended in sample buffer, and then divided into two
fractions. One fraction is subjected to SQD luminescence. The other
fraction is subjected to SERS. Baseline values obtained earlier are
then compared with the values obtained under experimental
conditions.
Example XV
Production of Antigen Specific Antibodies
[0162] Antigen substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, for example, Harrington (1990) Methods
Enzymol. 182: 488-495) or other purification techniques is used to
immunize rabbits and to produce antibodies using standard
protocols. The antigen amino acid sequence is analyzed using
DNASTAR software (DNASTAR Inc., Madison Wis.) to determine regions
of high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Methods for selection of appropriate epitopes,
such as those near the C-terminus or in hydrophilic regions are
well described in the art. (See, for example, Ausubel et al. supra,
chapter 11.)
[0163] Typically, the oligopeptides are 15 residues in length, and
are synthesized using an Applied Biosystems Peptide Synthesizer
Model 431A using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich,
St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase
immunogenicity. (See, for example, Ausubel et al. supra.) Rabbits
are immunized with the oligopeptide-KLH complex in complete
Freund's adjuvant. Resulting antisera are tested for antipeptide
activity, for example, by binding the peptide to plastic, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG. In the alternative, a
non-peptide antigen is used and is conjugated to KLH.
Example XVI
Purification of Naturally Occurring Antigen Using Specific
Antibodies
[0164] Naturally occurring or recombinant antigen is substantially
purified by immunoaffinity chromatography using antibodies specific
for the antigen. An immunoaffinity column is constructed by
covalently coupling anti-antigen antibody to an activated
chromatographic resin, such as CNBr-activated Sepharose (Pharmacia
& Upjohn, Kalamazoo Mich.). After the coupling, the resin is
blocked and washed according to the manufacturer's
instructions.
[0165] Media containing antigen are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of antigen (for example, high ionic
strength buffers in the presence of detergent). The column is
eluted under conditions that disrupt antibody/antigen binding (for
example, a buffer of pH 2 to pH 3, or a high concentration of a
chaotrope, such as urea or thiocyanate ion), and antigen is
collected.
Example XVII
Identification of Molecules that Interact with Antigen
[0166] Antigen, or biologically active fragments thereof, are
labeled with [.sup.125I] Bolton-Hunter reagent. (See, for example,
Bolton and Hunter (1973) Biochem. J. 133: 529-539.) Candidate
molecules previously arrayed in the wells of a multi-well plate are
incubated with the labeled antigen, washed, and any wells with
labeled antigen complex are assayed. Data obtained using different
concentrations of antigen are used to calculate values for the
number, affinity, and association of antigen with the candidate
molecules.
[0167] Those skilled in the art will appreciate that various
adaptations and modifications of the just-described embodiments can
be configured without departing from the scope and spirit of the
invention. Other suitable techniques and methods known in the art
can be applied in numerous specific modalities by one skilled in
the art and in light of the description of the present invention
described herein. Therefore, it is to be understood that the
invention can be practiced other than as specifically described
herein. The above description is intended to be illustrative, and
not restrictive. Many other embodiments will be apparent to those
of skill in the art upon reviewing the above description. The scope
of the invention should, therefore, be determined with reference to
the appended claims, along with the full scope of equivalents to
which such claims are entitled.
* * * * *