U.S. patent application number 09/810035 was filed with the patent office on 2003-08-21 for self-assembled metal colloid monolayers.
Invention is credited to Baker, Bonnie E., Natan, Michael J..
Application Number | 20030157732 09/810035 |
Document ID | / |
Family ID | 26699218 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157732 |
Kind Code |
A1 |
Baker, Bonnie E. ; et
al. |
August 21, 2003 |
Self-assembled metal colloid monolayers
Abstract
A biosensor based on complexes between biomolecule receptors and
colloidal Au nanoparticles, and more specifically, colloid layers
of receptor/Au complexes that can be used to detect biomolecule
analytes through measuring of binding-induced changes in electrical
resistance or surface plasmon resonance. Also disclosed is a method
for detecting and analysing carrier-borne chemical compounds with
Raman spectroscopy using an improved SERS substrate. Further
disclosed is an improved method for detecting compounds in solvents
using capillary electrophoresis in conjunction with Raman
spectroscopy.
Inventors: |
Baker, Bonnie E.;
(Collegeville, PA) ; Natan, Michael J.; (State
College, PA) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Family ID: |
26699218 |
Appl. No.: |
09/810035 |
Filed: |
March 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09810035 |
Mar 16, 2001 |
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09254142 |
Jan 12, 2000 |
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09254142 |
Jan 12, 2000 |
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PCT/US97/15581 |
Sep 4, 1997 |
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PCT/US97/15581 |
Sep 4, 1997 |
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08769970 |
Dec 19, 1996 |
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60025064 |
Sep 4, 1996 |
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Current U.S.
Class: |
436/531 ;
436/104; 436/126; 436/171 |
Current CPC
Class: |
G01N 33/54373 20130101;
G01N 33/587 20130101; B82Y 30/00 20130101; G01N 21/658 20130101;
B82Y 15/00 20130101; G01N 33/5438 20130101; Y10T 436/163333
20150115; C12Q 1/02 20130101; G01N 33/553 20130101; Y10T 436/196666
20150115 |
Class at
Publication: |
436/531 ;
436/104; 436/126; 436/171 |
International
Class: |
G01N 033/545; G01N
021/62 |
Claims
What is claimed is:
1. A process for detecting and quantifying an amount of a chemical
compound in a carrier, said process comprising: placing a sample
comprising said chemical compound in said carrier on a
surface-enhanced Raman scattering (SERS)-active surface; subjecting
said sample to Raman spectroscopy; and analyzing data generated by
said Raman spectroscopy and determining a concentration of said
compound in said sample from said data wherein said SERS-active
surface is prepared by a method comprising the steps of: coating a
substrate with a bifunctional organic film to impart to said
substrate a functionality that allows for bonding of metal colloid
particles; and contacting the organic film coated substrate with a
solution of colloid metal particles to bind said particles to
functional groups on said organic film thereby forming a monolayer
or submonolayer of said particles.
2. The process of claim 1, wherein said substrate is selected from
the group consisting of glass, quartz, alumina, tin oxides and
metals.
3. The process of claim 1, wherein functionality is imparted either
by covalent or non-covalent bonding.
4. The process of claim 1, wherein said metal colloid monolayers or
submonolayers comprise a colloid of a metal particles selected from
the group consisting of silver, gold, mixtures of silver and gold,
and silver/gold bimetallic particles.
5. The process of claim 1, wherein the size of said metal particles
lies within the range of 3-100 nm.
6. The process of claim 1, wherein said contacting of the organic
film with a solution of metal colloid particles is done by
immersing the coated substrate into a solution of colloid metal
particles causing the particles to bind to functional groups of the
organic film coated substrate.
7. The process of claim 1, wherein said bifunctional organic film
is an organosilane, poly(allylamine) hydrochloride or a biotin.
8. The process of claim 7, wherein said coating step involves
surface-initiated polymerization of a bifunctional alkoxysilane
having the formula (RO).sub.3Si(CH.sub.2).sub.3A, wherein pendent
functional group A has a high affinity toward metal surfaces.
9. The process of claim 3, wherein said metal particles are coated
with a protein.
10. The process of claim 9, wherein said substrate is coated with
biotin and then contacted with a colloidal metal derivatized with a
protein and streptavidin or avidin, thereby forming a colloid based
biocompatible surface held together by non-covalent
interactions.
11. The process of claim 1, wherein said chemical compound is
selected from the group consisting of pesticides,
disinfection-generated pollutants and environmental toxins and said
carrier is water.
12. The process of claim 11, wherein said chemical compound is a
pesticide, and said pesticide is an organophosphorous
pesticide.
13. The process of claim 11, wherein said chemical compound is a
disinfection-generated pollutant and said pollutant is a haloacetic
acid.
14. The process of claim 1, wherein said compound is a compound
having a Raman-active mode, and said carrier is air.
15. A process for detecting and quantifying an amount of a chemical
compound in a carrier, said process comprising: placing a sample
comprising said chemical compound in said carrier on a
surface-enhanced Raman scattering (SERS)-active surface; subjecting
said sample to Raman spectroscopy; and analyzing data generated by
said Raman spectroscopy and determining a concentration of said
compound in said sample from said data wherein said SERS-active
surface is prepared by a method comprising the steps of: coating a
substrate selected from the group consisting of glass, quartz,
alumina, tin oxides and metals, with a bifunctional organic film
selected from the group consisting of organosilanes,
poly(allylamine) hydrochloride and biotin, to impart to said
substrate a functionality that allows for bonding of metal colloid
particles, to form an organic film coated substrate; and immersing
the organic film coated substrate in a solution of colloid metal
particles to bind said particles to functional groups on said
organic film thereby forming a monolayer or submonolayer of said
particles,
16. The process of claim 15, wherein said colloid metal particles
are selected from Au and Ag.
17. The process of claim 15, wherein the size of said metal
particles lies within the range of about 3 to about 100 nm.
18. The process of claim 15, wherein said metal particles are
coated with a protein.
19. The process of claim 18, wherein said substrate is coated with
biotin and then contacted with a colloid metal coated with a
protein and streptavidin thereby forming a colloid based
biocompatible surface held together by non-covalent
interactions.
20. The process of claim 15, wherein said compound is a pesticide,
and said carrier is water.
21 The process of claim 20, wherein said pesticide is an
organophosphorous pesticide.
22. The process of claim 12, wherein said organophosphorous
pesticide is selected from the group consiting of methyl-parathion,
diazinon, cyanox, formthion, dimethoate, fonofoxon,
chlorfenvinphos, trichlorofon, and mixtures thereof.
23. The process of claim 21, wherein said organophosphorous
pesticide is selected from the group consiting of methyl-parathion,
diazinon, cyanox, formthion, dimethoate, fonofoxon,
chlorfenvinphos, trichlorofon, and mixtures thereof.
24. The process of claim 1, wherein said colloidal metal particles
are monodisperse Au particles having a particle size of about 3 to
about 100 nm, sufficiently spaced on said substrate to be only
weakly SERS enhancing, overcoated with Ag.
25. The process of claim 24, wherein the Ag overcoating is formed
using autometallography.
26. The process of claim 1 wherein said data derived from said
Raman spectroscopy is analyzed to determine a concentration of said
compound in said sample.
27. The process of claim 15, wherein said colloidal metal particles
are monodisperse Au particles having a particle size of 3-100 nm,
sufficiently spaced on said substrate to be only weakly SERS
enhancing, overcoated with Ag.
28. The process of claim 27, wherein the Ag overcoating is formed
using autometallography.
29 A biosensor for quantifying the presence of a biomolecule
analyte, said biosensor comprising a complex of a receptor that
binds to said biomolecule analyte and colloidal Au nanoparticles,
said complex being surface confined on a substrate to form at least
one layer of receptor/colloidal Au nanoparticle complexes on said
substrate.
30. The biosensor of claim 29, wherein said nanoparticles have a
particle diameter between about 3 nm and about 100 nm.
31. The biosensor of claim 30, wherein said complex is formed by
exposing a surface of an Au colloidal monolayer formed on said
substrate to said receptor.
32. The biosensor of claim 29, wherein said complex is formed by
forming an receptor/Au nanoparticle complex colloid and surface
confining said complex on said substrate.
33. The biosensor of claim 29, wherein said Au nanoparticles act as
a discontinuous conductor and said receptor functions as an
insulator that surrounds the particles of said conductor, wherein
swelling of said insulator, caused by a complexing of said receptor
with a biomolecule antigen increases insulator volume and decreases
conductor volume fraction.
34. The biosensor of claim 33, wherein a coverage of said
receptor/Au nanoparticle complex on said substrate is adjusted
whereby, when no biomolecule analyte is present, and said swelling
does not occur, said conductor concentration is greater than a
percolation threshold value, and when said biomolecule analyte is
present and said swelling occurs, said conductor concentration is
reduced to a value below said percolation threshold value.
35. The biosensor of claim 29, wherein a plurality of analyte/Au
nanoparticle complex layers are formed on said substrate.
36. A device for determining the presence and concentration of a
biomolecule analyte, said device comprising; the biosensor of claim
29; means for applying a voltage across said biosensor: means for
measuring an electrical characteristic of said biosensor; and means
for converting the measured electrical characteristic to a
biomolecule analyte concentration.
37. The device of claim 36, wherein said electrical characteristic
is resistance.
38. A process for detecting and quantifying an amount of a
biomolecule analyte, in a carrier, said process comprising forming
a biosensor comprising a complex of a receptor that binds to said
biomolecule analyte and colloidal Au nanoparticles, said complex
being surface confined on a substrate to form at least one layer of
a receptor/colloidal Au nanoparticle complex on said substrate;
wherein said Au nanoparticles act as a discontinuous conductor and
said receptor functions as an insulator that surrounds the
particles of said conductor, wherein swelling of said insulator,
caused by a complexing of said receptor with a biomolecule analyte
increases insulator volume and decreases conductor concentration, a
coverage of said receptor/Au nanoparticle complex on said substrate
being adjusted whereby, when no biomolecule analyte is present, and
said swelling does not occur, said conductor concentration is
greater than a percolation threshold value, and when said
biomolecule analyte is present and said swelling occurs, said
conductor concentration is reduced to a value below said
percolation threshold value; exposing said biosensor to said
carrier containing said biomolecule analyte; applying a voltage
across said biosensor; measuring an electrical characteristic of
said biosensor; and converting the measured electrical
characteristic to a biomolecule analyte concentration.
39. A process for detecting and quantifying an amount of a
biomolecule analyte, in a carrier, said process comprising forming
a biosensor comprising a layer of a receptor/colloidal Au
nanoparticle complex surface confined on a substrate, said layer
having an initial thickness insufficient to achieve surface plasmon
resonance upon application of a voltage across said biosensor;
first exposing said biosensor to said carrier containing said
biomolecule analyte, and; subsequently exposing said biosensor to
an additional amount of said receptor/colloidal Au nanoparticle
complex, whereby, when analyte biomolecules are present in said
carrier, said analyte biomolecules in said carrier bind to said
receptor of said complex surface confined to said substrate upon
exposure to said biosensor and to said receptor of said additonal
amount of said complex upon subsequent exposure of said biosensor
to said additional amount of said receptor/colloidal Au
nanoparticle complex, increasing the thickness of said layer
whereby said layer has a final thickness sufficient to achieve
surface plasmon resonance upon application of a voltage across said
biosensor, said method further comprising; applying a voltage
across said biosensor; measuring an electrical characteristic of
said biosensor; and converting the measured electrical
characteristic to a biomolecule analyte concentration.
40. The process of claim 39, wherein said electrical characteristic
is resistance.
41 An improved method for detecting low concentrations of at least
one compound in a solvent using capillary electrophoresis, said
method comprising: providing a surface enhanced Raman scattering
(SERS)-active surface on a substrate; placing a first end of a
capillary tube in a solvent reservoir containing said solvent and
placing a second end of said capillary tube on said SERS-active
surface: applying an electric current between said solvent
reservoir and said SERS-active surface to cause said solvent to
elute through said capillary tube onto said SERS-active surface;
moving said second end of said capillary tube across said
SERS-active surface whereby different compounds in said solvent are
deposited at different positions on said SERS-active surface;
subjecting said different compounds to Raman spectroscopy; and
analyzing data generated by said Raman spectroscopy and determining
a composition of said different compounds in said solvent; wherein
said SERS-active surface is prepared by a method comprising the
steps of: coating a substrate with a bifunctional organic film to
impart to said substrate a functionality that allows for bonding of
metal colloid particles; and contacting the organic film coated
substrate with a solution of colloid metal particles to bind said
particles to functional groups on said organic film thereby forming
a monolayer or submonolayer of said particles.
42. The method of claim 41, wherein said substrate is electrically
conductive and said electric current is applied between said
solvent reservoir and said substrate.
43. The method of claim 41, wherein said second end of said
capillary is metallized and said electric current is applied
between said solvent reservoir and said second end of said
capillary.
44. The method of claim 41, wherein said SERS-active surface on
which said compounds are eluted is analyzed by rastering said
surface across a focused laser and recording a resulting Raman
spectra.
45. A metal colloid monolayer comprising a plurality of colloidal
Ag clad Au nanoparticles surface confined on a substrate, said
monolayer having a gradient of nanoparticle density in a first
direction, and a gradient in particle size in a second direction
perpendicular to said first direction.
46. The metal colloid monolayer of claim 45, wherein said
nanoparticles are bound to said substrate by bonding with
functional groups of a bifunctional organic film.
47. The metal colloid monolayer of claim 46, wherein said
bifunctional organic film is selected from the group consisting of
an organosilane, poly(allylamine) hydrochloride and biotin.
48. The metal colloid monolayer of claim 45, wherein said substrate
is selected from the group consisting of glass, quartz, alumina,
tin oxides and metals.
49. The metal colloid monolayer of claim 45, wherein said
nanoparticles have a size of about 3 nm to about 100 nm.
50. A metal colloid monolayer comprising a plurality of colloidal
Ag clad Au nanoparticles surface confined on a substrate, said
monolayer having a gradient of nanoparticle density in a first
direction, and a gradient in particle size in a second direction
perpendicular to said first direction formed by: coating a
substrate with a bifunctional organic film; immersing the coated
substrate into a colloidal Au solution such that a first leading
edge of said substrate is immersed in said colloidal Au solution
for a longer period of time relative to a first trailing edge to
provide a monolayer of Au nanoparticles having a decreasing level
of coverage from said first leading edge to said first trailing
edge; withdrawing said substrate from said colloidal Au solution;
rotating said substrate through an angle of about 90.degree.;
immersing the substrate provided with the monolayer of Au
nanoparticles in an Ag.sup.+ solution such that a second leading
edge of said substrate is immersed in said Ag.sup.+ solution for a
longer period of time relative to a second trailing edge to provide
a monolayer of Au nanoparticles having a decreasing level of Ag
cladding thickness from said second leading edge to said second
trailing edge; and withdrawing said substrate from said Ag.sup.+
solution.
51. The metal colloid monolayer of claim 50, wherein said
nanoparticles are bound to said substrate by bonding with
functional groups of a bifunctional organic film.
52. The metal colloid monolayer of claim 51. wherein said
bifunctional organic film is selected from the group consisting of
an organosilane, poly(allylamine) hydrochloride and biotin.
53. The metal colloid monolayer of claim 50, wherein said substrate
is selected from the group consisting of glass, quartz, alumina,
tin oxides and metals.
54. The metal colloid monolayer of claim 50, wherein said Au
nanoparticles, prior to cladding, have a uniform size of about 3 nm
to about 10 nm.
55. A method of determining optimal surface characteristics of a
metal colloid monolayer comprising a plurality of colloidal Ag clad
Au nanoparticles surface confined on a substrate for use in an
analytical procedure, said method comprising: coating a substrate
with a bifunctional organic film; immersing the coated substrate
into a colloidal Au solution such that a first leading edge of said
substrate is immersed in said colloidal Au solution for a longer
period of time relative to a first trailing edge to provide a
monolayer of Au nanoparticles having a decreasing level of coverage
from said first leading edge to said first trailing edge;
withdrawing said substrate from said colloidal Au solution;
rotating said substrate through an angle of about 90.degree.;
immersing the substrate provided with the monolayer of Au
nanoparticles in an Ag.sup.+ solution such that a second leading
edge of said substrate is immersed in said Ag.sup.+ solution for a
longer period of time relative to a second trailing edge to provide
a monolayer of Au nanoparticles having a decreasing level of Ag
cladding thickness from said second leading edge to said second
trailing edge; withdrawing said substrate from said Ag.sup.+ to
form a monolayer having a gradient of nanoparticle density in one
direction, and a gradient in particle size in another direction;
using the gradated monolayer for an analytical procedure; and
analyzing the results achieved at different regions of said
gradated monolayer to determine a nanoparticle coverage and
particle size that provides an optimal result.
56. The method of claim 55, wherein said monolayer is a surface
enhanced Raman scattering response substrate.
57. The method of claim 55, wherein said monolayer is a biosensor
for detecting the presence of a biological ligand.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to self-assembled metal
colloid monolayers, methods of preparation, and use thereof.
[0002] In surface enhanced Raman scattering (SERS), million-fold
enhancements in Raman scattering can be obtained for molecules
adsorbed at suitably rough surfaces of Au, Ag, and Cu. Although
many approaches have been reported, preparation of well-defined,
stable SERS substrates having uniform roughness on the critical 3
to 100 nm scale has proven difficult. Because colloidal Au can be
synthesized as monodisperse solutions throughout most of this size
regime, and because molecules adsorbed to closely spaced colloidal
Au and Ag exhibit enhanced Raman scattering, these particles are
excellent building blocks for SERS-active substrates. The key issue
is whether colloidal Au and Ag particles can be organized into
macroscopic surfaces that have a well-defined and uniform
nanometer-scale architecture. Indeed, controlling nanostructure is
currently a central focus throughout materials research. Progress
in self assembly of organic thin films on metal surfaces [C. D.
Bain and G. M. Whitesides, Angew. Chem. Int Ed. Engl. 28, 506
(1989); A. Ulman, An Introduction to Ultrathin Organic Films, from
Langmuir-Blodgett to Self-Assembly (Academic Press, Boston, 1991)]
led us to explore the reverse process: self assembly of colloidal
Au and Ag particles onto supported organic films. As detailed
below, this approach has yielded surfaces that are SERS-active,
characterizable at both the macroscopic and microscopic levels,
highly reproducible, electrochemically addressable, and simple to
prepare in large numbers. Moreover, these substrates have a surface
roughness that is defined by the colloid diameter (which is
tunable) and an average interparticle spacing that is continuously
variable. As such, self-assembled Au, Ag and Ag-coated colloid
monolayers are likely to have extraordinary utility for SERS.
[0003] In the nearly twenty years since the discovery of surface
enhanced Raman scattering (SERS) of molecules adsorbed at roughened
Ag electrodes, and the accompanying theoretical work demonstrating
the need for surface roughness, there have been numerous reports of
new architectures for SERS substrates. See, for instance. Liao. P.
F.; Bergman, J. G.; Chemla, D. S.; Wokaun, A.; Melngailis, J.;
Hawyrluk. A. M.; Economou, N. P. Chem. Phys. Lett. 1981, 82, 355-9;
Creighton. J. A.; Blatchford. C. G.; Albrecht, M. G. J. Chem. Soc.,
Faraday Trans. 2 1979, 75. 790-8: Blatchford, C. G.; Campbell, J.
R.; Creighton, J. A. Surf Sci. 1982, 120, 435-55; Tran, C. D. Anal.
Chem. 1984, 56, 824-6; Soper, S. A.; Ratzhlaff, K. L.; Kuwana, T.
Anal. Chem. 1990, 62, 1438-44; Sequaris, J.-M.; Koglin, E.
Fresenius J. Anal. Chem. 1985, 321, 758-9; Aroca, R.; Jennings, C.;
Kovacs, G. J.; Loutfy, R. O.; Vincett, P. S. J. Phys. Chem. 1985,
89, 4051-4; Moody, R. L.; Vo-Dinh, T.; Fletcher, W. H. Appl.
Spectrosc. 1987, 41, 966-70; Ni, F.; Cotton, T. M. Anal. Chem.
1986, 58, 3159-63; Yogev, D.; Efrima, S. J. Phys. Chem. 1988, 92,
5761-5; Goudonnet, J. P.; Bijeon, J. L.; Warmack, R. J.; Ferrell,
T. L. Phys. Rev. B: Condensed Matter 1991. 43, 4605-12; Murray, C.
A.; Allara, D. L. J. Chem. Phys. 1982. 76, 1290-1303; Brandt, E. S.
Appl. Spectrosc. 1993, 47, 85-93; Alsmeyer, Y. W.; McCreery, R. L.
Anal. Chem. 1991, 63, 1289-95; Mullen, K.; Carron, K. Anal. Chem.
1994, 66, 478-83; Beer. K. D.; Tanner, W.; Garrell, R. L. J.
Electroanal. Chem. 1989, 258, 313-25; Dawson, P.; Alexander, K. B.;
Thompson, J. R.; Haas III, J. W.; Ferrell, T. L. Phys. Rev. B:
Condens. Matter 1991, 44, 6372-81; Roark, S. E.; Rowlen, K. L.
Appl. Spectrosc. 1992, 46, 1759-61; Roark, Shane E.; Rowlen, K. L.
Chem. Phys. Lett. 1993. 212. 50; Roark, Shane E.; Rowlen, K. L.
Anal. Chem. 1994, 66, 261-70; Walls, D.; Bohn, P. J. Phys. Chem.
1989, 93, 2976-82; Dutta, P. K.; Robins, D. Langmuir 1991, 7,
2004-6; Sheng, R.-S.; Zhu, L.; Morris, M. D. Anal. Chem. 1986, 58,
1116-9.
[0004] These surfaces span a wide range of assembly principles and
encompass similarly broad levels of complexity. Examples of
SERS-active surfaces include electrochemically-roughened
electrodes, microlithographically-prepared elliptical Ag posts,
aggregates of colloidal Au or Ag particles--both in solution and
associated with chromatographic media, evaporated thin films,
Ag-coated latex particles, substrates prepared by chemical
reduction of Ag.sup.+, and liquid Ag films. The motivation for this
work stems from several intrinsically attractive aspects of SERS as
a vibrational spectroscopy-based structural tool and/or analytical
method: million fold signal enhancements compared to solution Raman
spectra, adsorption-induced fluorescence quenching, a lack of
interference from H.sub.2O, and molecular generality. However,
while SERS has been invaluable for certain narrowly defined
applications, most spectroscopists would agree that the technique
has not lived up to its enormous potential.
[0005] The problem has been the inability of any previous surface
to meet all, or even most, of the essential criteria that would
define a truly useful SERS substrate: strongly enhancing,
reproducible, uniformly rough, easy to fabricate, and stable over
time. Biocompatibility is also extremely important, insofar as
previous studies demonstrating partial or full protein denaturation
upon adsorption to SERS-active substrates [Holt, R. E.; Cotton. T.
M. J. Am. Chem. Soc. 1989, 111 , 2815-21; Lee, N.-S.; Hsieh, Y.-Z.;
Morris, M. D.; Schopfer, L. M. J. Am. Chem. Soc. 1987, 109,
1353-63] have proven to be a major setback to the use of SERS in
biological systems. Other desirable characteristics include
electromagnetic tunability (i.e. the ability to control the
wavelength where optimal enhancement occurs, so as to match the
substrate to the photon source), electrochemical addressability--to
control the extent of adsorption and the redox state of adsorbed
species, a lack of surface "activation" steps, and a low cost per
substrate. This last feature is particularly important for
applications involving large numbers of routine measurements, such
as in environmental monitoring, airport security, and clinical
medicine.
SUMMARY OF THE INVENTION
[0006] Detailed embodiments of the present invention are disclosed
herein. However, it is understood that certain preferred
embodiments are merely illustrative of the invention which may be
embodied in various forms and applications. Specific compositional
and functional details disclosed herein are not meant to be
interpreted as limiting, but merely as support for the invention as
claimed and as appropriate representations for teaching those
skilled in the art to variously employ the present invention in any
appropriate embodiment.
[0007] We report here a new approach to SERS substrates that meets
all of the criteria delineated above. Our strategy involves
assembly of colloidal Au, Ag. or other suitable metal particles
into macroscopic two-dimensional arrays on polymer-immobilized
substrates (FIG. 1). In the first, covalent approach, reactive
hydroxyl/oxide groups are generated on a substrate. For many
substrates (glass, metal, etc.), such functional groups are already
present in high concentration. A second step involves
surface-initiated polymerization of bifunctional organosilanes such
as (RO).sub.3Si(CH.sub.2).sub.3A. The alkoxysilane forms covalent
attachments to the surface via hydrolysis. The pendant functional
group A (FIG. 1B), chosen for its high affinity toward noble metal
surfaces, extends out into solution. In the final step, the
polymer-derivatized substrate is immersed into a solution of
colloidal Au particles, where surface assembly spontaneously
occurs. An alternate approach based on high affinity binding of
streptavidin to biotin can also be used (FIG. 1C). See Wilchek. M.;
Bayer, E. A. Anal. Biochem. 1988, 171, 1 and Anzai, J.; Hoshi, T.:
Osa, T Trends Anal. Chem. 1994, 13, 205-10. Here, a biotinylated
surface is reacted with a colloidal Au-streptavidin conjugate to
form a colloid-based surface held together by non-covalent
interactions.
[0008] With molecular self-assembly on metal substrates [Nuzzo, R.
G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481-4483; Bain, C.
D.; Whitesides, G. M. Angew. Chem. Int. Ed. Engl. 1989, 28,
506-512] now established as an important route to controlling
interfacial properties, it should be pointed out that the
approaches delineated in FIG. 1 define what is essentially the
inverse process: self-assembly of well-defined particulate metal
films on organic substrates. The term self-assembly refers to our
finding that interparticle spacing is governed by interparticle
repulsive forces. We have explored this chemistry in detail by
varying substrate, polymer, colloid diameter, and reaction
conditions. Moreover, the electrochemical characteristics of
colloid-based surfaces, the kinetics of surface formation, and the
electromagnetic properties of composite particles have been
investigated. We also describe here the basic steps involved in
surface assembly and characterization, as well as experimental
verification of SERS activity. What distinguishes this work are the
following features: macroscopic surfaces of controlled and uniform
roughness can be prepared by self-assembly, the resulting
substrates are compatible with biomolecules, and the surfaces
exhibit a high degree of durability/stability over time.
[0009] Our construction protocol for SERS-active Au and Ag colloid
monolayers exploits the simplicity of self assembly from solution
and the affinity of noble metal surfaces for certain organic
functional groups (FIG. 1). In our case, these moieties are present
by virtue of organic films either polymerized or deposited on the
surface of macroscopic (0.8 cm.times.2 cm) substrates. Immersion of
the functionalized substrate into a dilute solution of monodisperse
colloidal Au or Ag particles leads to colloid immobilization. This
solution-based process is extremely general. encompassing numerous
permutations of insulating and conducting substrates [glass.
quartz, plasma-treated Teflon, Formvar, indium-doped SnO.sub.2
(ITO), and Pt], organic films [hydrolyzed mono-. di- and
trialkoxysilanes containing the functional groups CN, NH.sub.2,
2-pyridyl. P(C.sub.6H.sub.5).sub.2, and SH, as well as
carboxyl-terminated C.sub.18 organothiol self-assembled
monolayers], and colloids [5 to 70 nm in diameter for Au, and 5 to
20 nm in diameter for Ag and Au/Ag composites]. Our work has
focused on Au and Ag particles, but with the right functional group
A, a wide variety of colloidal particles could constitute building
blocks for well-defined macroscopic surfaces.
[0010] Solution-based surface assembly also eliminates preparative,
geometric, and operational constraints associated with most
previously described SERS substrates. Thus, one liter of 17 mM,
12-nm diameter colloidal Au, which can be stored indefinitely at
room temperature, can be used to prepare 2,000 0.5-cm.sup.2
surfaces with only a 1% decrease in colloid concentration.
Importantly, these substrates can be assembled sequentially or
simultaneously. Surfaces in novel geometries that extend the
utility of SERS can now be derivatized, including one face of a 5
ml-volume spectroelectrochemical cell, large glass sheets several
centimeters on a side, and the inside of a 20-mm inner diameter
glass capillary. Moreover, once constructed, no further activation
steps (such as electrochemical oxidation-reduction cycles or
particle aggregation) are required to initiate SERS activity. It
should be noted that the particles are tightly bound and the
thermodynamic stability of these surfaces is very high: exchange
with molecules in solution containing the functional group A does
not occur.
[0011] This preparation method differs greatly from electrochemical
roughening of electrodes and metal vapor deposition, the most
common routes to solid SERS substrates. Each of these protocols
yields surfaces with polydisperse roughness on the nanometer scale.
This problem is circumvented by the methods of FIG. 1. Since the
size of the colloid precursor can be easily varied and controlled,
the defining roughness of the surface is pre-determined. Not only
can the roughness be tuned according to experimental needs, but the
roughness is uniform--all particles are of the same size and
dimensions. This is of particular importance in SERS where
enhancement at the surface is directly correlated to nanometer
scale roughness.
[0012] Two-dimensional colloid self assembly also differs from the
numerous methods for preparation of SERS-active substrates
involving colloidal particles [Creighton, J. A.; Blatchford, C. G.;
Albrecht, M. G. J. Chem. Soc., Faraday Trans. 2 1979, 75 790-8;
Blatchford, C. G.; Campbell, J. R.; Creighton, J. A. Surf. Sci.
1982, 120, 435-55; Tran, C. D. Anal. Chem. 1984, 56, 824-6; Soper,
S. A.; Ratzhlaff. K. L.; Kuwana, T. Anal. Chem. 1990, 62, 1438-44;
Sequaris, J.-M.; Koglin, E. Fresenius J. Anal. Chem. 1985, 321,
758-9; Ahern, A. M.; Garrell, R. L. Langmuir 1991, 7, 254-61;
Angel, S. M.; Katz, L. F.; Archibald, D. D.; Honigs, D. E. Appl.
Spectrosc. 1989, 43, 367-72; Clarkson, J.; Campbell, C.;
Rospendowski. B. N.; Smith, W. E. J. Raman Spectrosc. 1991, 22,
771-775]. In those methods, there is a single size of particle, but
since there is no control over interparticle interactions.
aggregates of ill-defined dimensions are often formed. In this
work, strong covalent or non-covalent bonds to the substrate reduce
the surface mobility of the nanoparticles and prevent the
spontaneous coalescence of particles on the surface. Thus, the
initial size uniformity is maintained.
[0013] For several reasons, keeping the particles physically
separated is a critical component to our assembly strategy. (1) The
intrinsic biocompatibility of individual colloidal Au particles is
maximized: aggregates begin to approximate larger surfaces where,
for Au, protein denaturation is a serious concern. (2) The
resulting surfaces are more straightforwardly characterized than
particle aggregates. (3) It is known both from theory and
experiment that closely-spaced but physically separated particle
arrays can be strongly enhancing [Inoue, M.; Ohtaka, K. J. Phys.
Soc. Jpn. 1983, 52, 3853-64; Chu, L.-C.; Wang, S.-Y. J. Appl. Phys.
1985, 57, 453-9; Chu, L.-C.; Wang, S.-Y. Phys. Rev. B: Condens.
Matter 1985, 31, 693-9]. As the interparticle spacing increases
toward .lambda., nearly all of the SERS effect is lost: completely
isolated small colloidal Au particles are weakly enhancing. In our
view, the relatively small loss in enhancement for non-contacting,
closely-spaced particles is more than offset by an increased ease
of characterization, improved biocompatibility, and demonstrated
improvements in stability (vide infra). FIG. 1D depicts various
regimes for colloid immobilization that could result from using the
strategy delineated above. In surface A, the particles are
isolated, but too far apart to be strongly enhancing. In B, the
particles are close enough to see the SERS effect, but still
isolated (thus retaining the biocompatibility properties of
individual particles). Surface C represents a close-packed colloid
monolayer, while D represents particle multilayers approximating a
bulk surface. The latter surface can be prepared from a monolayer.
Our goals are to prepare and characterize surfaces like B, C and D,
and to use them to solve a variety of problems in analytical,
biological, environmental, clinical and inorganic chemistry.
[0014] To this end, the ease of fabrication and handling of Au
colloid monolayers is very significant. Large numbers of samples
can be prepared simultaneously, with no restrictions on the size or
shape of the substrates, and without the need for even moderately
sophisticated equipment (i.e. no potentiostat, no vacuum deposition
apparatus). Furthermore, with transparent substrates, the optical
properties of the SERS-active surface can be monitored directly.
This means that a reasonably accurate prediction of enhancement
factors can be made a priori. Indeed, as described below, uv-vis is
our basic characterization tool.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIGS. 1A-1B show assembly strategies for Au and Ag colloid
monolayers; X=A=CN, NH.sub.2, 2-pyridyl, P(C.sub.6H.sub.5).sub.2,
and SH.
[0016] FIG. 1C demonstrates one embodiment of the present invention
based on high affinity binding of streptavidin to a biotin.
[0017] FIG. 1D depicts various degrees of substrate surface
coverage by metal particles.
[0018] FIG. 2A illustrates the ultraviolet-visible kinetics of a Au
colloid monolayer on a glass substrate coated with
3-aminopropyltrimethoxysilane.
[0019] FIG. 2B illustrates the SERS kinetics of a Au colloid
monolayer on a glass substrate coated with
3-aminopropyltrimethoxysilane.
[0020] FIG. 3A is a TEM image of a Au colloid monolayer prepared on
a SiO.sub.x-coated formvar surface.
[0021] FIG. 3B shows the SERS spectrum of BPE adsorbed onto the
derivatized TEM grid used to produce FIG. 3A.
[0022] FIG. 4 shows an electrochemical potential dependence of SERS
intensity of the 1006cm.sup.2 band of pyridine on a Ag colloid
monolayer on Pt and on bulk Ag.
[0023] FIG. 5 shows the optical and SERS spectra before and after
deposition of Ag onto 18 nm diameter colloidal Au monolayer.
[0024] FIG. 6A shows optical spectra for solutions of isolated and
aggregated 13 nm colloidal Au particles in H.sub.2O.
[0025] FIG. 6B illustrates the diversity of optical properties
attainable through the method of the present invention.
[0026] FIG. 7 illustrates the optical stability of the Au
colloid-based monolayers of the present invention.
[0027] FIG. 8 shows the optical spectrum for a MP-biocytin coated
quartz slide containing a monolayer of Au derivatized with BSA and
streptavidin.
[0028] FIGS. 9A-9B show TEM micrographs of a colloidal Au surface
prepared by the method of the present invention.
[0029] FIG. 9C shows a TEM micrograph of a formvar-coated TEM grid
which was floated in colloidal Au for 2 days (not the method of the
present invention).
[0030] FIG. 10A shows a SERS spectrum of BPE drop-coated on a
colloid Au monolayer on a functionalized TEM grid, as well as the
SERS spectrum of the adsorbate-free surface.
[0031] FIG. 10B shows a SERS spectrum of BPE drop-coated on a
colloid Au monolayer on a HO.sub.2C(CH.sub.2).sub.16SH/AU/Cr/Si
substrate, as well as the SERS spectrum of the adsorbate-free
surface.
[0032] FIGS. 11-13 are TEM images of the colloidal particles
prepared in accordance with example 1.
[0033] FIG. 14 is the solution optical spectrum of the seed nuclei
of example 1. FIGS. 15-16 are uv-vis spectra of the larger
particles derivatized on glass substrates and of the larger
particles in solution as described in example 1.
[0034] FIG. 17 shows a TEM image of the colloidal surface in
example 2 after the first layer of colloidal particles has been
applied.
[0035] FIG. 18 shows an optical spectrum of the colloid surface in
example 2 after the first layer of colloidal particles has been
applied.
[0036] FIG. 19 shows an optical spectrum of the colloid surface in
example 2 after the second layer of colloidal particles has been
applied.
[0037] FIG. 20 shows non-covalent colloidal approaches to protein
coated metal surfaces.
[0038] FIG. 21 is a comparison of the optical properties of
covalent and non-covalent attachment.
[0039] FIG. 22 shows the changes in optical spectra upon
electrochemical reduction of Ag ions onto Au colloid monolayers in
accordance with example 4.
[0040] FIG. 23 shows theoretical modeling of the expected change in
optical spectra of Ag-clad Au colloid monolayers for increasing
deposits of Ag.
[0041] FIG. 24 shows AFM characterization of these surfaces
described in example 4.
[0042] FIG. 25 shows optical spectra as a function of
electrochemical potential (E) for a surface with high Au
coverage.
[0043] FIG. 26 shows the changes in optical properties accompanying
chemical deposition of Ag onto preformed Au colloid monolayers.
[0044] FIG. 27 shows the uv-vis optical properties of a surface
coated with colloidal metal particles that were stabilized with
PEG.
[0045] FIG. 28 shows the optical spectrum of a substrate coated
with poly(allylamine) hydrochloride and immersed in a solution of
colloidal Au.
[0046] FIG. 29 shows the optical spectrum of a colloid monolayer on
SnO.sub.2 electrode;
[0047] FIGS. 30a-c show a tapping-mode AFM images of Au colloid
multilayer film preparation on APTMS-derivatized glass using 12 nm
Au particles, wherein:
[0048] FIG. 30a shows an image of a submonolayer;
[0049] FIG. 30b shows an image of a submonolayer after 2 exposures
to 2-mercaptoethanol/Au colloid; and
[0050] FIG. 30c shows an image of the submonolayer(bottom left)
after 5 exposures to 2-mercaptoethanol/Au colloid, images of FIGS.
30 a-c each being 1 mm .times.1 mm with 0-100 nm z-scale.
[0051] FIG. 31 shows Au particle coverage dependence (in
particles/cm.sup.2) of uv-vis/near-IR transmission spectra of Au
colloid multilayers (prepared by successive, repeated immersion of
a glass slide derivatized with APTMS and a 12-nm diameter Au
colloid monolayer into (a) 4 mM 2-mercaptoethanol and (b) 17 nM, 12
nm-diameter Au);
[0052] FIG. 32 show the percent transmission at 1500 nm (+) and
2500 nm (O) as a function of Au colloid coverage for a Au colloid
multilayer. These data were obtained from FIG. 1 of the
manuscript.
[0053] FIG. 33 shows a log plot of DC resistance versus Au particle
coverage for Au colloid multilayers. See FIG. 29 for details of
sample preparation.
[0054] FIG. 34 shows Au particle coverage (in particles/cm.sup.2)
vs the number of immersions into 12-nm colloidal Au in which one
immersion corresponds to a monolayer.
[0055] FIG. 35 shows a diagram of a device for determining the
presence of an antigen protein.
[0056] FIG. 36 shows the coupling of a colloid-based SERS with
capillary electrophoresis.
[0057] FIG. 37 illustrates a technique for forming a self-assembled
metal colloid monolayer that exhibits continuous nanometer-scale
gradient of mixed-metal composition and surface architecture. Such
gradients are useful for optimizing surface characteristics of the
metal colloid monolayer for a given application.
[0058] FIG. 38 shows UV-vis waves of 9 different Au/Ag coatings of
a stepwise gradient combinatorial surface. The sample was submerged
into Au in 3 steps of 0.83 cm, with 50 minute pauses, turned
90.degree., and lowered into an Ag+solution in 3 steps of 0.67 cm,
with 10 minute pauses resulting in a maximum Au exposure time of
2.5 hrs, and Ag exposure time of 30 min. Au and Ag exposure times
for spectrum .omega. are analogous to the exposures for the
morphology represented by AFM image C in FIG. 42.
[0059] FIG. 39 shows SERS response of spatially discrete areas on a
2.5 cm.times.2.0 cm continuous gradient combinatorial surface (4
hrs for Au, and 20 minutes for Ag. respectively). Each box
represents a SERS spectrum collected in a 0.2 cm.times.0.2 cm area
on the sample; each spectrum's x-axis is Raman shift (1300
cm.sup.-1to 1650 cm.sup.-1) and y-axis is intensity (0 counts per
second (cps) to 35000 cps). The numbers located in each box
indicate the background subtracted intensity of the 1610 cm.sup.-l
ring stretch mode of p-NDMA, and box locations in space correspond
to the acquisition position on the sample; bold vertical lines
indicate a 0.2 cm jump in position. Particle coverage increases
from left to right. and particle size increases from top to bottom.
Experimental conditions, [p-NDMA] =0.3 mM, 20 mW of 530.9 nm
excitation, 1 cm.sup.-1 step, 1-s integration, 5 cm.sup.-1 band
pass.
[0060] FIG. 40 shows background subtracted SERS intensity map in
counts per second (cps) for the 1610 cm.sup.-1 symmetric ring
stretch of p-NDMA on a large scale continuous gradient
combinatorial surface, 0-12 hr for Au, 0-30 min for Ag. Each box
represents one SERS spectrum in a 0.1 cm.times.0.1 cm area, total
sample size is 2 cm.times.2 cm. Experimental conditions; [p-NDMA]
=2.5 mM, 25 mW of 647.1 nm photons focused at the sample; 1
cm.sup.-1 step, 1 s integration, 5 cm.sup.-1 band pass.
[0061] FIG. 41 shows background subtracted SERS intensity in counts
per second (cps) for the 1168 cm.sup.-1 phenyl-nitroso stretch of
p-NDMA as a function of sample position. Each shaded box represents
one SERS spectrum collected in a 0.1 cm.times.0.1 cm area. Regions
A-D correspond to AFM images in FIG. 39. Experimental conditions:
[p-NDMA] =2.5 mM; 30 mW of 647.1 nm. 2 mm diameter spot at the
sample; 1 cm.sup.-1 step, 1 s integration, 5 cm.sup.-1 band
pass.
[0062] FIG. 42 shows AFM images (500 nm.times.500 nm) of
MPTMS-derivatized glass slides with varying coverages of 12-nm
diameter colloidal Au and varying quantities of deposited Ag.
Images A-D correspond to regions delineated in FIG. 38. Z-axis
(vertical) gray scale: black=0 nm. white=35 nm.
[0063] FIG. 43 shows wavelength-dependent SERS maps from a single
sample of a Au/Ag continuous gradient colloidal Au on
3-mercaptopropylmethyl-dim- ethoxysilane (MPMDMS)/glass.
Intensities have been normalized to 30 mW of 514 nm excitation.
Each square represents 1 spectrum in a 2 mm.times.2 mm area, with
the peak intensities calculated from the 1610 cm.sup.-1 band of
trans-4,4'-bis-pyridylethylene (BPE). Experimental conditions: 30
minutes Au exposure, 40-minute Ag exposure. [BPE] =10 mM; two
collections of a 2-s integration, 5 cm.sup.-1 band pass.
[0064] FIGS. 44A. 44B and 44C shows wavelength dependent SERS maps
from three samples of continuous combinatorial gradients based on
14- and 28-nm diameter colloidal Au (Au colloid/MPMDMS/glass).
Exposure times on the right-hand image (Au exposure=180 minutes,
5.times. diluted colloid, Ag exposure=40 minutes) were based on the
experimental findings for the left-hand image (30 minutes Au
exposure, 40-minute Ag exposure). Experimental conditions are the
same as in FIG. 43.
[0065] FIG. 45 is a bar graph showing the number of particles per
square micron as derived from analysis of AFM images (Au
collod/MPMDMS/glass). Numbers on the bars represent the mean
particle height (in nm) for the images as obtained via a section
analysis.
[0066] FIG. 46A shows the optical spectra of 14-nm diameter
colloidal Au clad with Ag. Each spectrum has the same exposure time
to Ag, but different amounts of Au particles with
Au.sub.3>Au.sub.2>Au.sub.1. The samples comprised monolayers
of Au colloid on MPMDMS-coated glass slides.
[0067] FIG. 46B shows the optical spectra of 14-nm colloidal Au
clad with Ag. The spectrum of each of FIG. 46A and FIG. 46B has the
same exposure time to Au, but different durations of Ag exposure
with Ag.sub.3>Ag.sub.2>Ag.sub.1. The samples comprised
monolayers of Au colloid on MPMDMS-coated glass slides.
[0068] FIGS. 47A and 47B show SERS spectra for the samples shown in
FIG. 46A and 46B, respectively. Experimental conditions: [BPE]=10
mM; 30 mW of 647.1-nm excitation; two collections of a 2- s
integration, 5 cm.sup.-1 band pass.
[0069] FIGS. 48A. 48B and 48C. show AFM images (1 .mu.m.times.1
.mu.m, with 80 nm Z-scale) for the samples with varying Ag
coverages described in FIGS. 46A and 46B, and FIGS. 47A and
47B.
[0070] FIGS. 49A, 49B and 49C show AFM images (1 .mu.m.times.1
.mu.m, with 80 nm Z-scale) for the samples with varying Au
coverages described in FIGS. 46A and 46B, and FIGS. 47A and
47B.
[0071] FIG. 50A shows the optical spectra of Ag-clad, 28-nm
diameter colloidal Au monolayers on MPMDMS/glass
(Ag.sub.3>Ag.sub.2>Ag.sub.1- ).
[0072] FIG. 50B shows SERS spectra for Ag-clad. 28-nm diameter
colloidal Au monolayers on MPMDMS/glass . Experimental conditions
for FIGS. 50A and 50B:[BPE]=10 mM; 30 mW of 647.1-nm excitation;
two collections of a 2-s integration. 5 cm.sup.-1 band pass.
[0073] FIGS. 51 A. 51B and 51C show AFM images (1 .mu.m.times.1
.mu.m, with 80 nm Z-scale) for the samples described in FIGS. 50A
and 50B.
DETAILED DESCRIPTION OF THE INVENTION
[0074] Below we describe data regarding the preparation,
characterization, and applications of self-metal colloid
monolayers. Experimental examples are included.
[0075] Two lines of evidence demonstrate that immobilized particles
are located solely at the surface of, and not embedded within, the
organic film. (i) Colloidal particles are very tightly attached to
the polymer (when stored in water, no particle dissociation occurs
after 1 year), yet monolayer formation does not occur on polymers
with pendant methyl or methoxy groups. These data indicate that
multiple specific covalent interactions between polymer functional
groups (which are oriented toward the solution) and the particle
surface are necessary for immobilization. (ii) Although SERS
spectra for adsorbates from solution are easily obtained (see
below), the SERS spectra of organosilane polymer films underneath
Au monolayers are quite weak. This contrasts with published SERS
studies of colloid/polymer mixtures [P. Matejka. B. Vlckova. J.
Vohlidal, P. Pancoska, V. Baumruk, J. Phys. Chem. 96. 1361 (1992);
P. C. Lee and D. Meisel, Chem. Phys. Lett. 99, 262 (1983)], and
demonstrates that the surface of immobilized metal particles is
accessible to solvent. In accord with this finding is our
observation that the optical spectrum of Au colloid monolayers on
transparent substrates depends on the dielectric constant of the
surrounding medium.
[0076] The optical properties of colloidal Au and the nature of
self assembly offer an unprecedented opportunity to monitor surface
evolution in real time. The time course of Au colloid monolayer
formation on a glass slide coated with polymerized
3-aminopropyltrimethoxysilane (APTMS) is shown in FIG. 2. Binding
of 12nm diameter Au particles to amine groups on the surface is
indicated by an absorbance feature at 520 nm, the location of the
Mie resonance for isolated small Au particles. As the particle
coverage increases, interparticle spacing becomes small compared to
the incident wavelength and a new feature corresponding to a
collective particle surface plasmon oscillation grows in at
.about.650 nm. This feature is responsible for the pronounced SERS
activity of collections of colloidal Au particles. Accordingly,
when a colloid monolayer in various stages of formation is placed
in a solution containing the adsorbate
trans-1,2-bis(4-pyridyl)ethylene (BPE), the SERS intensity for the
ring stretch at 1610 cm.sup.-1 closely tracks the magnitude of the
absorbance at 650 nm. Immersion time is one of four routes we have
found to alter the rate or extent of surface formation, the others
being choice of organosilane functional group (rate of surface
formation for SH>NH.sub.2>>CN), colloid concentration, and
the presence or absence of an adsorbate on the colloidal
particle.
[0077] FIG. 2A shows kinetics of Au colloid monolayer formation by
ultraviolet-visible (uv-vis). A series of uv-vis spectra of Au
colloid-functionalized glass slides in H.sub.2O obtained with an
HP-8452A spectrophotometer. Cleaned (4:1
H.sub.2SO.sub.4:H.sub.2O.sub.2, 70.degree. C.) rectangular glass
slides (0.9 mm.times.25 mm) were placed into a dilute solution of
3-aminopropyltrimethoxysilane (APTMS) (0.3 ml: 3 ml of CH.sub.3OH)
for 12 hours and rinsed with CH.sub.3OH upon removal. The
polymer-coated slides were then immersed in a 17 nM solution of
12-nm diameter colloidal Au particles (wavelength maximum=520 nm)
[Garrell, R. L. Anal. Chem. 1989, 61, 401A-11A; Tran. C. D. Anal.
Chem. 1984, 56, 824-67,20]. At each time indicated (and at several
others not shown), the slide was removed from the Au colloid
solution, and an optical spectrum was recorded in H.sub.2O,
followed by a SERS spectrum in 4 mM BPE in 95:5 H.sub.2O:CH.sub.3OH
(20 mW 632.8 nm, Spex 1403 double monochromator, Hamamatsu R928
photomultiplier tube, bandpass=7 cm.sup.-1, scan rate=1
cm.sup.-1s.sup.-1, integration time=1 s). FIG. 2B shows kinetics of
Au colloid monolayer formation by SERS. SERS intensity for the 1610
cm.sup.-1 band versus absorbance at 650 nm. Other bands in the BPE
SERS spectrum evolve with identical kinetics.
[0078] This high degree of control over surface formation has
important ramifications for reproducibility, a long-standing
complication in SERS research. For example, when BPE was adsorbed
to eight identical Ag colloid monolayers on glass, the greatest
variation in integrated peak intensity for the 1610 cm.sup.-1 band
was less than 8%. Similarly, for five different locations on a
single substrate, the greatest difference was only 5%. As these
values incorporate intrinsic errors associated with variation in
laser power and sample positioning, the actual sample
reproducibility is significantly better. This reproducibility
extends to the nanometer scale, where Au and Ag colloid monolayers
have been imaged using transmission electron microscopy (TEM),
field emission scanning electron microscopy (FE-SEM), and atomic
force microscopy (AFM). A representative TEM image of an Au colloid
monolayer prepared on an SiO.sub.x-coated Formvar surface is shown
in FIG. 3. The Au particles are confined to a single layer, and the
vast majority of particles are isolated from each other, unlike
previous TEM studies of SERS-active Au and Ag colloids.
Furthermore, the large field of view available with TEM allows us
to conclude that particle aggregation has been eliminated over the
entire sample. Similar conclusions obtain from large-field FE-SEM
images and from multisite tapping-mode AFM images of Au-modified
glass surfaces. The AFM image from a glass slide coated with
3-aminopropylmethyldimethoxysilane indicates a roughness of 1 to 3
nm, notwithstanding a few isolated locations where the polymer
roughness approaches 8 to 10 nm. This roughness scale is typical
for organosilane films on glass or quartz. Immobilization of 12-nm
colloidal Au particles to a coverage equivalent to that shown for
180 to 210 min in FIG. 2 yields a surface with features 12 to 20 nm
high and 20 to 30 nm wide. The increased dispersion in particle
size relative to TEM results from convolution of the true particle
size with the AFM tip size, but are nevertheless of sufficient
quality to conclude that the surface is composed of a monolayer of
separated particles, in agreement with FE-SEM images on similar
substrates. Importantly, we have demonstrated that the spacing
obtained on these colloid-based surfaces is sufficient to yield
SERS enhancement. The bottom panel of FIG. 3B shows the SERS
spectrum of BPE adsorbed onto the derivatized TEM grid pictured in
the top panel. For comparison, the Raman scattering spectrum of an
equivalent amount of BPE deposited onto an unmodified
SiO.sub.x-coated TEM grid is also shown. The intensity difference
in these two samples clearly demonstrates the enhancing properties
of colloid-based surfaces.
[0079] FIG. 3A shows an image from a Formvar-coated Cu TEM grid
which had been sputter-coated with a thin layer of SiO.sub.x (Ted
Pella, Inc.), treated for 2.5 hours in neat
3-cyanopropyldimethylmethoxysilane, rinsed exhaustively with
CH.sub.3OH upon removal, and immersed for 12 hours in colloidal Au
(12 nm diameter) [Garrell, R. L. Anal. Chem. 1989. 61. 401A-11A:
Tran, C. D. Anal. Chem. 1984, 56, 824-6]. Imaging was performed on
a JEOL 1200 EXII instrument operated at 80 kV accelerating voltage.
The area depicted is 0.28 mm.sup.2 and is representative of the
sample surface. FIG. 3B shows a SERS spectrum (upper) of 5 ml of 1
mM BPE drop-coated onto the surface of the derivatized TEM grid
(100 mW, 647.1 nm, 5 cm.sup.-1 bandpass, 2 cm.sup.-1 step, 2 s
integration). For comparison, an identical quantity of BPE was
drop-coated onto an underivatized SiO.sub.x grid; the Raman
spectrum from this sample is shown (1 cm.sup.-1 step, 1 s
integration).
[0080] Another important feature of film-supported metal colloid
monolayers is that the particles are subject to electrochemical
potentials applied to underlying conductive substrates.
Consequently, like SERS-active electrodes, Ag colloids immobilized
on Pt exhibit an electrochemical potential-dependent SERS intensity
for adsorbed pyridine (FIG. 4). Identical maximas for the two
surfaces in the intensity versus potential plots suggests that the
voltage drop across the polymer film is minimal. Voltammetry at
colloid-based surfaces also resembles that at macroscopic
electrodes. The first reduction wave for methyl viologen
(MV.sup.2+) is markedly rectified at an organosilane-coated Pt
electrode (FIG. 4, inset) but returns upon immobilization of Au
particles. The slightly broadened peak-to-peak separation is
expected for an array of closely spaced microelectrodes.
Considering the demonstrated biocompatibilitv of 5 to 20 nm
diameter Au particles, the ability to make electrochemical
measurements at Au colloid monolayers suggests possible
electrode-based biosensor applications.
[0081] FIG. 4 shows an electrochemical potential dependence of SERS
intensity of the 1006 cm.sup.-1 band of pyridine on an Ag colloid
monolayer on Pt and on bulk Ag [Albrecht, M. G.; Creighton. J. A.
J. Am. Chem. Soc. 1977, 99, 5215-7]. The monolayer was prepared as
follows: Clean Pt foil was placed into neat APTMS for 4 hours.
After rinsing with triply distilled H.sub.2O and air-drying, the
polymer-coated foil was dipped in Ag colloid solution [Soper, S.
A.; Ratzhlaff, K. L.; Kuwana, T. Anal. Chem. 1990. 62, 1438-44] for
1 hour. The derivatized foil was then rinsed with triply distilled
H.sub.2O and air-dried. In the absence of colloidal Ag, no pyridine
SERS spectra were observed at any potential. See FIG. 2 for
spectral acquisition parameters. Inset: Voltammograms (100 mV/s,
N.sub.2 atmosphere) of 5 mM MV.sup.2+ in 0.1 M Na.sub.2SO.sub.4 on
three surfaces: unmodified Pt, Pt coated with surface-polymerized
3-mercaptopropylmethyl-dimethoxysilane (MPMDMS), and Pt coated with
MPMDMS and derivatized with 15-nm diameter Au particles (5 hours in
neat silane, rinsed, 4 hours in colloidal Au).
[0082] Interparticle spacing in preformed Au monolayers can be
further reduced by chemical deposition of an Ag coating; increased
interparticle coupling because of decreased spacing and concomitant
changes in dielectric properties lead to a dramatic increase in
SERS activity. The optical and SERS spectra before and after
deposition of Ag onto 18 nm diameter colloidal Au are shown in FIG.
5. Initially, relatively large interparticle spacing is indicated
by the absence of a collective particle surface plasmon band in the
ultraviolet-visible and by a weakly enhanced SERS spectrum for
adsorbed para-nitrosodimethylaniline (p-NDMA). Silver deposition
causes a large increase in extinction at all wavelengths as well as
a shift in .lambda.max from 520 to 386 nm. The shift in energy of
and increased extinction at .lambda.max concur with expectations
based on a computer algorithm for predicting the optical properties
of isolated coated particles [C. Bohren and D. R. Huffman.
Absorption and Scattering of Light by Small Particles (Wiley, New
York, 1983)]; best agreement between the experimental and model
data is reached with a 4-nm Ag coat (to make 26-nm diameter
particles) [The optical constants for Au and Ag were taken from R.
H. Morriss and L. F. Collins, J. Chem. Phys. 41, 3357 (1961). These
values were fit to exponential curves to generate continuous values
between 300 and 700 nm]. The exceptional SERS activity (enhancement
factor =105) [an enhancement factor (EF) of 5.7.times.10.sup.5 was
calculated for the Ag-coated surface by comparing the ratios of
background-corrected intensities for a SERS spectrum and a solution
spectrum in units of counts s.sup.-1 watt.sup.-1 molecule.sup.-1,
and averaging the EF values obtained for six different common
peaks--low signal/noise precluded calculation of accurate EFs for
the as prepared Au sample] of these substrates reflects
optimization of the Ag coating thickness for this particular
particle size and spacing of colloidal Au--even greater
enhancements may be possible with other combinations.
[0083] FIG. 5 shows the effect of Ag coating on the uv-vis and SERS
spectra of preformed Au colloid monolayers. The initial substrates
were prepared as in FIG. 2, except that the organic film was formed
from reaction with 2-(trimethoxysilyl)ethyl-2-pyridine (PETMS) for
24 hours. Silver coating was performed by immersing Au colloid
monolayers into a 1:0 mixture of LI Silver enhancer and initiator
solutions (Nanoprobes Inc. Stony Brook. NY) for 13 min. The SERS
spectra were of 0.5 mM p-NDMA solutions in CH.sub.3OH. Optical
spectra (inset) were measured in H.sub.2O. Instrumental parameters
were described in FIG. 2. When Ag is deposited from the plating
solution onto a PETMS-derived polymer on glass in the absence of
colloidal Au, no SERS intensity could be observed for the same
p-NDMA solution. irrespective of coating time.
[0084] More detailed characterization of these surfaces follows.
The top panel of FIG. 6 shows optical spectra for solutions of
isolated and aggregated 13-nm diameter colloidal Au particles in
H.sub.2O. The unaggregated sol, which has a particle concentration
of 17 nM, has a .lambda.max of 520 nm. The physical nature of this
surface plasmon mode, which gives colloidal Au its characteristic
intense burgundy color, is well-understood, as are its dependence
on particle size and shape. When the interparticle repulsive forces
are sufficiently screened by molecular adsorption, irreversible
aggregation occurs and generates a new red-shifted feature in the
optical spectrum centered between 600-800 nm. The intensity and
.lambda.max of this feature scale with the extent of aggregation,
with large aggregates exhibiting increased extinction and
red-shifted peaks. This "aggregated" band results from coupling of
surface plasmons between closely-spaced particles. It has been
amply demonstrated, both theoretically and experimentally, that the
SERS-activity of aggregated colloidal Au arises from this
interparticle coupling. In aggregated sols, the particles are
physically connected, but it is important to note that direct
contact is not needed to observe collective plasmon modes: as long
as the spacing between particles is small compared to the
wavelength of light, these collective plasmon modes can be
observed. Uv-vis is thus particularly well-suited for analyzing our
samples, since the optical spectra of Au colloid monolayers on
transparent substrates is easily measured. The same cannot be said
of most SERS substrates with a notable exception being those
prepared by Roark et al. [Roark, S. E.; Rowlen, K. L. Appl.
Spectrosc. 1992, 46, 1759-61; Roark, Shane E.; Rowlen, K. L. Chem.
Phys. Lett. 1993, 212. 50; Roark, Shane E.; Rowlen, K. L. Anal.
Chem. 1994, 66, 261-70]. Moreover, colloid self-assembly provides a
means of tuning surface optical properties through control of
interparticle spacings.
[0085] The diversity of optical properties attainable through
self-assembly of colloidal Au is illustrated in the bottom panel of
FIG. 6. Use of two different organosilanes and two sizes of
colloidal Au particles yields four distinct surfaces, as evidenced
by different optical spectra. In comparison to the data in the top
panel, it is clear that interparticle coupling is not as pronounced
as for aggregated colloidal solutions. Using 13-nm diameter
particles at 0.15 monolayer coverage, there are roughly
1.times.10.sup.11 particles in a 1 cm.sup.2 monolayer which, using
a 15-nm slab thickness, are in a volume of 15.times.10.sup.-10
liters. This translates to a surface concentration of
1.times.10.sup.-4 M, versus 17 nM in solution. Despite this 4
order-of-magnitude increase in concentration (one that cannot be
maintained in solution without aggregation), the particles remain
distinct; this lack of surface aggregation is additional strong
evidence for specific interactions between the surface of Au and
the polymer functional groups and the high stability suggests that
multiple linkages must be present. These data are reinforced by the
absence of Au or Ag immobilization on polymers derived from
trimethoxypropylsilane, which lacks a high-affinity functional
group. The key point is that the polymer-particle interaction, an
adjustable parameter, controls the particle density, which in turn
dictates the optical properties.
[0086] FIG. 6A shows absorbance spectra for solutions of Au colloid
(13 nm diameter). Unaggregated Au has a .lambda.max at 520 nm,
while aggregated Au exhibits a second, red-shifted absorbance
centered at 700 nm. The sol was aggregated via addition of a small
volume of concentrated NaCl solution. FIG. 6B shows absorbance
spectra of quartz slides derivatized for 14 h in neat silane and
for 24 h in colloidal Au: (A) APTMS, 30 nm Au; (B) APTMS, 13 nm Au;
(C) MPMDMS, 30 nm Au; (D) MPMDMS, 13 nm Au.
[0087] Thus, for surfaces with the same polymer and containing a
single size of particle, difference in optical properties must be
attributed to differences in coverage (and therefore, average
interparticle spacing). For example, spectra B and D are of
immobilized 13 nm-diameter Au on quartz substrates derivatized with
amino (APTMS)- and sulfhydryl (MPMDMS)-functionalized siloxane
polymers, respectively. Relatively strong interparticle coupling is
found in B, as evidenced by the presence of the collective surface
plasmon absorbance feature, but is absent (or significantly weaker)
in D. Since the area probed by the uv-vis beam is constant, and
since the Au particle coating is homogenous over the entire
surface, the stronger interparticle coupling results from an
increased particle density. Whether this difference is attributable
to a higher concentration of pendant functional groups in
surface-confined APTMS than for MPMDMS, an increased affinity of Au
for amine over sulfhydryl, or some other factor is under
investigation.
[0088] These surfaces are fundamentally different from those
prepared by evaporation of drop-coated colloidal Au solutions.
Evaporated substrates exhibit complete colloid aggregation,
sometimes to the extent of producing films that to the eye look
like bulk Au. In contrast, the protocol described herein involves
no bulk aggregation on the surface. Furthermore, with adequate
rinsing between the polymer formation and colloid derivatization
steps, there is no aggregation of particles in solution; immersion
of the polymer-functionalized substrate into a colloidal Au
solution, and subsequent removal of the colloid-derivatized
surface, does not appreciably change the optical spectrum of the
colloidal Au solution. We have also shown that colloid
immobilization is not a sedimentation reaction by performing
derivatizations upside down. Thus, immobilization of colloids on a
polymer-functionalized glass substrate suspended upside down in
solution yields colloidal surfaces indistinguishable from those
obtained by complete immersion. Similarly, polymer-coated TEM grids
can be derivatized with Au by flotation on aqueous colloidal
solutions.
[0089] Once attached, the binding of colloidal Au to derivatized
surfaces is extremely strong and essentially irreversible. There is
very little change in the optical spectrum of an Au colloid-based
monolayer after storgage for 7 months in H.sub.2O (FIG. 7).
Ag-based surfaces are also very durable, with no loss of particles
over 2 years. For Ag, a shift of .lambda.max from 396 to 420 nm may
reflect particle aging, as has previously been shown for Ag
colloids [Henglein, A. J Phys. Chem. 1993, 97, 5457-71 and
references therein. An alternative explanation for the shift is
that bacterial growth in solution, against which no precautions
were taken, leads to adsorption of protein on the particle surface
(see FIG. 3)]. More importantly, these surfaces are rugged enough
to survive exposure to appreciable concentrations of aggregating
agents. Thus, exposure of an MPMDMS-based Au colloid surface to 5
mM mercaptoethanol does not alter the optical spectrum, indicating
that particle aggregation has not taken place (data not shown). It
is significant that the same concentration of aqueous
mercaptoethanol instantaneously aggregates colloidal Au and Ag in
solution. The high durability of these substrates is further
manifested by their resistance toward ligand exchange: solution
RS.sup.- does not displace surface RS.sup.- /Au bonds. Indeed, neat
mercaptoethanol is needed to effect particle removal. Likewise,
immersion of an Au-coated substrate into a solution of H.sub.2O at
75.degree. C. for a period of one hour had no effect on the optical
spectrum. Equivalent stabilities are found for surfaces based on
NH.sub.2-Au linkages.
[0090] FIG. 7A shows absorbance spectra for a glass slide
derivatized for 24 h in APTMS (diluted 1:4 with CH.sub.3OH) and for
11 days in colloidal Au (13 nm). FIG. 7B shows absorbance spectra
for a glass slide derivatized for 14 h in neat MPMDMS and for 1
week in colloidal Ag. After the initial optical spectra were
recorded, slides were stored in H.sub.2O until the final spectra
were taken.
[0091] Surfaces based on non-covalent interactions (FIG. 1) possess
optical properties completely analogous to those prepared by
covalent attachment (FIG. 8). An important aspect of these data and
of the concept described in FIG. 1 is that the biological activity
(i.e. biotin binding) of streptavidin adsorbed on colloidal Au is
necessarily retained: unmodified Au particles and particles coated
completely with a protein that doesn't specifically bind biotin
(i.e. BSA) do not lead to surface formation in the presence of
biotinylated substrates. The retention of biological function
contrasts sharply with streptavidin adsorbed at bulk Au surfaces,
for which biological activity is compromised [Ebersole, R. C.;
Miller, J. A.; Moran, J. R.; Ward. M. D. J. Am. Chem. Soc. 1990,
112, 3239-41]. The use of colloidal Au as a histochemical and
cytochemical marker is based on the tendency of proteins adsorbed
to small Au particles to retain their biological function. A major
advantages of these surfaces, then, is their biocompatibility.
Because they are composed of isolated colloidal particles, the
behavior of the surface mirrors the behavior of particles in
solution. The creation of macroscopic metal surfaces with high,
nanometer-scale biocompatibility is important for biosensor
applications, and reinforces the importance of maintaining some
interparticle spacing, for only under these conditions can single
particle behavior toward biomolecules be assured. Of course, the
biomolecule itself may be the spacer . A final comment on FIG. 1
concerns the use of a coating protein to completely isolate Au
particles. For the data in FIG. 8, BSA was used, meaning that each
particle had multiple BSA molecules adsorbed for each streptavidin
bound. (Note the peak shift of roughly 20 nm for .lambda.max,
reflecting a change in local dielectric constant of proteins
relative to H.sub.2O.) However, this choice is arbitrary; it is
possible to prepare particle-based Au surfaces where each particle
is pre-coated with a protein of interest.
[0092] Transmission Electron Microscopy
[0093] Direct evidence concerning the morphology and interparticle
spacing comes from transmission electron microscopy studies of
colloidal Au bound to polymers on TEM grids. These were prepared
using commercially available formvar-coated Cu TEM grids possessing
a thin sputter-coated overlayer of SiO.sub.2. Careful treatment of
these fragile surfaces with organosilane followed by colloidal Au
yielded surfaces that could be directly imaged. FIG. 9A shows two
magnifications of a surface derivatized in this manner with 13-nm
colloidal Au. The areas shown in these micrographs are roughly 4.0
mm.sup.2 for the top panel and 0.2 mm.sup.2 for the bottom panel,
and are representative of the entire sample. FIGS. 9, 9A & 9B
show TEM micrographs of a colloidal Au surface prepared by
derivatizing an SiO.sub.x-coated TEM grid for 2.5 h in neat CPDMMS
and for 12 h in 13 nm Au colloid. Areas depicted are approximately
as follows: (top) 4.0 mm.sup.2, (bottom) 0.2 mm.sup.2.
[0094] Examination of these images verifies several critical
aspects of the strategy delineated by FIG. 1: (1) there is a single
two-dimensional submonolayer of colloidal Au; (2) the particles are
closely spaced but not aggregated in two dimensions: (3) the
particle coverage is uniform over areas macroscopic compared to the
particle size; (4) the roughness is uniform and defined solely by
the particle diameter; and (5) there appears to be a limitation to
the number of particles that can be bound per unit area, with only
15%-20% of the surface covered. The observed distribution of
particles extends over macroscopic areas, i.e. 3 mm.times.3 mm, the
size of the TEM samples that we typically prepare.
[0095] It is well-known that aggregation of colloidal Au produces
fractal clusters and strings. In the Creighton group's
groundbreaking work on colloid SERS, two-dimensional strings of
SERS-active particles were imaged by TEM [Jeanmaire, D. L.; Van
Duyne, R. P. J. Electroanal. Chem. 1977, 84, 1-20]. Such species
are not seen here, and the small percentage of dimers and trimers
are invariably found in colloidal Au solutions as prepared. Weitz
and co-workers have characterized the fractal dimension of
aggregated Au and correlated it to SERS activity [Weitz, D. A.;
Lin, M. Y. Surf. Sci. 1985, 158, 147-64]. Again, these large
aggregates are not seen in images of carefully prepared surfaces.
By way of contrast, FIG. 9C depicts a TEM image of colloidal Au on
a non-functionalized, formvar-coated grid. Three-dimensional
clusters of particles are clearly present in addition to isolated
particles; the inability to achieve a good focus further signifies
the existence of multiple layers of colloidal particles. Such
species are not observed on the grids from which the data in FIGS.
9A and 9B was extracted. Rather, observation of closely-spaced,
predominantly unaggregated colloidal particles confirms the
arguments made above based on optical spectroscopy. The fact that
all the colloidal particles are confined to nearly a single plane,
as evidenced by good focus over large areas, suggests that for
these surfaces, the roughness of the underlying organosilane film
and/or substrate is comparable to the particle diameter or smaller.
In accord with this notion, several studies of organosilane polymer
films on smooth surfaces indicate a thickness <20 .ANG.
[Dressick, W. J.; Dulcey, C. S.; Georger, J. H., Jr.; Calabrese, G.
S.; Calvert, J. M. J. Electrochem. Soc. 1994, 141, 210-20;
Karrasch, S.; Dolder, M.; Schabert, F.; Ramsden, J.; Engel, A.
Biophys. J. 1993, 65, 2437-46; Nakagawa, T.; Ogawa, K.; Kurumizawa,
T. Langmuir 1994, 10, 525-9].
[0096] The tendency toward even spacing between particles observed
in FIG. 8 results from electrostatic factors. It is known that
colloidal particles are negatively charged and thus naturally repel
one another, and that aggregation occurs only under conditions
where this interparticle repulsion is screened. Within this
framework, the protocol described here is self-assembly, in that
long range order arises from secondary interactions between
individual particles, as opposed to particle-surface
interactions.
[0097] Surface Enhanced Raman Scattering (SERS)
[0098] One of the principle objectives of assembly of macroscopic
metal surfaces exhibiting controlled roughness is to prepare
well-defined, reproducible SERS-active substrates. The optical
spectra show that the particle spacing is small compared to 1,
suggesting that these particle arrays should be SERS-active. FIG.
10A shows the SERS spectrum of 5 nmol of BPE drop-coated onto a
colloid monolayer on a functionalized TEM grid, as well as the SERS
spectrum of the adsorbate-free surface. These data are extremely
significant because they were obtained on the same type of surface
imaged by TEM in FIG. 8. In the absence of adsorbate, no major
features are observed in the Raman spectrum, indicating SERS from
the polymer underlayer is weak. Typically, a low energy mode is
observed for the S-Au vibration from MPMDMS-derived films, but
little else is easily discerned.
[0099] FIG. 10A shows SERS spectrum of 5 ml of 1 mM BPE drop-coated
onto an SiO.sub.x coated TEM grid derivatized with MPMDMS and 10 nm
colloidal Au. Excitation source: 647.1 nm, 100 mW; 2 cm.sup.-1
step, 2 s integration; 5 cm.sup.-1 bandpass. The bottom spectrum
was taken of the substrate surface prior to adsorption of BPE. The
surface topography was identical to that depicted in FIG. 2. FIG.
10B shows SERS spectrum of BPE (10 ml, 1 mM in CH.sub.3OH)
drop-coated onto a colloidal Au (10 nm diameter) monolayer prepared
on a HO.sub.2C(CH.sub.2).sub.18SH/Au/Cr/Si substrate (upper). Prior
to adsorption of BPE, a background spectrum of the colloidal Au
substrate was run (lower). Excitation source: 647.1 nm; 150 mW
(BPE), 100 mW (background); 2 cm.sup.-1 step, 1 s integration; 5
cm.sup.-1 bandpass.
[0100] It is important to understand the factors responsible for
the observed SERS behavior of these substrates: why do we see BPE
and not the polymer underlayer? Most SERS studies of polymers show
a number of polymer-related bands. However, in those studies, the
polymers completely surrounded the colloidal particles, while in
this work, if the idealized geometry in Scheme I is reasonable (as
the TEM data in FIG. 8 suggest is the case), only a small fraction
of the colloid surface contacts polymer. For a particle with
diameter=2r, the fraction of a sphere's surface area covered by a
polymer with x nm vertical flexibility (over a horizontal distance
2r) is equal to: 1 cos - 1 [ ( r - x ) / r ] p ( 1 )
[0101] In our system, using r=6 nm and x=1 nm (a vast overestimate,
considering the length of the --CH.sub.2CH.sub.2CH.sub.2A tail is
itself<1 nm), the fraction of total surface area exposed to
polymer is 0.186. In other words, the ratio of adsorbate molecules
at monolayer coverage to polymer tails is at minimum 5:1, and more
likely closer to 10:1.
[0102] Another factor influencing the SERS enhancement is the Raman
scattering cross-section. BPE is an exceptionally strong scatterer,
while alkanes yield very weak Raman spectra. Bryant et al, have
measured SERS spectra of octadecanthiols on Au foil: using a CCD.
very long (10 min) integration times were required. A third
consideration is the magnitude the electric fields responsible for
the electromagnetic enhancement. The largest fields are expected to
occur in the plane of the particles, not in the plane normal to the
substrate, i.e. Raman spectra of molecules adsorbed in this region
are enhanced to a greater extent than those adsorbed elsewhere, and
from our calculations above, only BPE can access this region.
Finally, chemical enhancement effects in SERS certainly favor
observation of enhanced Raman scattering from the
nitrogen-containing BPE versus an alkane. The combination of these
factors all favor observation of BPE SERS, and help explain the
consistently observed finding that, over the region between
400-1700 cm.sup.-1, very weak or no SERS spectra are seen for
underlying films. A benefit of these substrates is thus the lack of
background spectra, simplifying the data acquisition process. On
the other hand, these substrates, like many others previously
described, may not be sufficiently enhancing to measure Raman
spectra for weak scatterers or poor adsorbates.
[0103] Because the Raman intensity of BPE adsorbed onto
organosilane-coated glass slides is too small for us to measure, we
crudely estimated how enhancing these surfaces are by comparing the
solution concentration [x] of BPE needed to yield the same normal
Raman spectrum as we obtained for a BPE concentration [y] in the
presence of an Au colloid monolayer. Typically,
x/y.gtoreq.10.sup.4. This number is in line with enhancements
measured at roughened Au electrodes. The spectra yielding these
enhancement factors (data not shown) are less than a factor of ten
more intense than the data in FIG. 10A. Thus, enhancement factors
of >1000 can routinely be obtained from arrays of closely spaced
but non-contacting particles. Importantly, identical spectra are
obtained on substrates in which BPE was adsorbed from solution; in
fact, our experiments are routinely carried out in this
fashion.
[0104] Below are experimental details associated with colloid
monolayer preparation:
[0105] Materials.
[0106] The following materials were obtained from Aldrich:
HAuCl.sub.4.3H.sub.2O, AgNO.sub.3, trisodium citrate dihydrate,
trans-1,2 bis(4-pyridyl)ethylene (BPE), and
trimethoxy-propylsilane. The following organosilanes were obtained
from Huls America. Inc. and used as received:
(3-aminopropyl)trimethoxysilane (APTMS).
(3-cvanopropyldimethyl)methoxysi- lane (CPDMMS),
(3-mercaptopropylmethyl)dimethoxysilane (MPMDMS), and 3
-cyanopropyltriethoxysilane (CPTES). Concentrated HCl, HNO.sub.3,
and H.sub.2SO.sub.4 were purchased from J. T. Baker Inc. and 30%
H.sub.2O.sub.2 was obtained from VWR. CH.sub.3OH
(spectrophotometric grade) was obtained from EM Science; all
H.sub.2O was 18 MW, distilled through a Barnstead Nanopure water
purification system. Streptavidin. bovine serum albumin (BSA), and
3-(N-maleimidopropionyl)biocytin (MP-biocytin) were purchased from
Sigma. BPE was recrystallized several times from a mixture of
H.sub.2O and CH.sub.3OH; the other materials were used as received.
Substrates were obtained as follows: glass and quartz microscope
slides from Fisher Scientific and Technical Glass Products,
respectively; SiO.sub.x-coated TEM grids from Ted Pella, Inc.; and
self-assembled monolayers (SAMs) of HS(CH.sub.2).sub.18CO.sub.2H on
Au foil from literature procedures [Bain, C. D.; Troughton, E. B.;
Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem.
Soc. 1989, 111, 321-351.].
[0107] Colloid Preparation.
[0108] All glassware used in these preparations was thoroughly
cleaned in aqua regia (3 parts HCl, 1 part HNO.sub.3), rinsed in
triply-distilled H.sub.2O, and oven-dried prior to use. Au colloids
were prepared according to Frens [Frens, G. Nature Phys. Sci. 1973,
241, 20-2] or Sutherland [Sutherland, W. S.; Winefordner, J. D. J.
Colloid. Interface Sci. 1992, 48, 129-41] with slight
modifications. The following stock solutions were prepared from
triply-distilled H.sub.2O that had been filtered through a 0.8 mm
membrane filter (Gelman Scientific): 1% HAuCI.sub.4, 38.8 mM sodium
citrate, and 1% sodium citrate. Other solutions were made fresh as
needed using triply-distilled, filtered H.sub.2O. Two typical Au
preparations and one Ag preparation are described below.
[0109] Preparation I: Using a 1 L round bottom flask equipped with
a condenser. 500 ml of 1 mM HAuCl.sub.4 was brought to a rolling
boil with vigorous stirring. Rapid addition of 50 ml of 38.8 mM
sodium citrate to the vortex of the solution resulted in a color
change from pale yellow to burgundy. Boiling was continued for 10
minutes; the heating mantle was then removed, and stirring was
continued for an additional 15 minutes. After the solution reached
room temperature, it was filtered through a 0.8 mm Gelman Membrane
filter. The resulting solution of colloidal particles was
characterized by an absorption maximum at 520 nm. Transmission
electron microscopy (TEM) indicated a particle size of 13 nm.+-.1.7
nm (100 particles sampled). Preparation II: In a 1 L round bottom
flask equipped with a condenser, 500 ml of 0.01% HAuCl.sub.4 was
brought to a boil with vigorous stirring. To this solution was
added 7.5 ml of 1% sodium citrate. The solution turned blue within
25 s; the final color change to red-violet occurred 70 s later.
Boiling continued for an additional 10 min., the heating source was
removed, and the colloid was stirred for another 15 min. TEM data
indicated an average diameter of 18 nm.+-.4.6 nm (89 particles
sampled). Particle diameter was varied by adding larger or smaller
amounts of sodium citrate to decrease or increase the particle
size.
[0110] Ag colloid was prepared according to Lee and Meisel [Lee, P.
C.; Meisel, D. J. Phys.Chem. 1982,, 86. 3391-3395]. Using a heating
plate and a 1 L flask, a solution of 90 mg AgNO.sub.3 in 500 ml of
triply distilled H.sub.2O was brought to boiling with rapid
stirring. To this solution was added 10 ml of 1% sodium citrate.
Boiling continued for 30 min, after which time the flask was
removed from the heat source, and the solution was diluted with
triply distilled H.sub.2O to obtain a final volume of 420 ml.
[0111] All colloids were stored at room temperature in dark bottles
and were generally used within 1-2 months after preparation.
Samples for particle sizing by TEM were prepared by drop coating 10
ml of the colloid onto a formvar-coated Cu grid and allowing the
sample to dry. Average sizes were determined by measuring diameters
along a consistent axis throughout the sample.
[0112] Protein-Colloid Conjugates.
[0113] Streptavidin-labelled Au particles were prepared using
modifications of literature protocols [Liesi, P.; Julien, J.-P.;
Vilja, P.; Grosveld. F.; Rechanrdt. L. J. Histochem. Cytochem.
1986, 34, 923]. To 25 ml of colloidal Au (preparation I) were added
0.725 ml of streptavidin (0.34 mg/ml in triply distilled H.sub.2O)
and 0.241 ml of BSA (7.24 mg/ml in triply distilled H.sub.2O). The
protein-Au conjugates were observed to sediment within 24
hours.
[0114] Surface Derivatization.
[0115] Substrates were cleaned prior to derivatization as follows:
glass and quartz, cut to dimensions of approximately 2 cm.times.0.7
cm, were cleaned for 10 minutes in a bath consisting of 4 parts
H.sub.2SO.sub.4 to 1 part 30% H.sub.2O.sub.2 at 60.degree. C. The
samples were rinsed in spectrophotometric grade CH.sub.3OH and
stored in this solvent until needed. SiO.sub.x-coated TEM grids
were cleaned in an ozone plasma for 30 min using a home-built
instrument. Cleaning often preceded use of the grids by several
weeks: during this period, the grids were stored in TEM grid
holders in air.
[0116] Derivatization of glass and quartz substrates with
alkoxysilanes was accomplished in the following manner: Clean
substrates were submerged into vials of silane diluted 1 part to 4
parts with spectrophotometric grade CH.sub.3OH. After a period of
24 h, the substrates were removed and rinsed profusely with
CH.sub.3OH to remove unbound monomer from the surface. At this
point, silanized substrates were stored in CH.sub.3OH until needed.
Prior to derivatization with colloidal Au, the substrates were
rinsed with H.sub.2O; they were then immersed in vials of colloidal
Au for 24 h. A final H.sub.2O rinse concluded the derivatization
process. Similarly, carboxyl-terminated SAMs prepared on Au-coated
silicon substrates were immersed in colloidal Au solutions for
several days. The substrates were stored in H.sub.2O until needed
for analysis.
[0117] Due to their inherent fragility and small size, greater care
was required for the derivatization of TEM grids. Specifically, the
SiO.sub.x-coated TEM grids were immersed in neat silane for 3 h,
followed by extensive methanol rinses and a H.sub.2O rinse. The
rinsing was accomplished by pipetting solvent across the grid
surface, or by swishing the grid back and forth in a vial of
solvent. Effort was made to minimize the solvent flow perpendicular
to the grid face in order to better preserve the formvar film.
Finally, the grids were floated on a colloid solution for 12 h.
Samples were rinsed with H.sub.2O and allowed to air dry on filter
paper prior to analysis.
[0118] Sample Preparation.
[0119] Two methods were employed for mounting the substrates for
SERS detection. The first method involved mounting the substrate
via double-sided tape to a black mount positioned in the laser beam
(TEM grids, SAM substrate). In the second, the substrate (glass or
quartz) was supported in the front of a quartz cuvette by means of
a teflon block whose height was only 1/3 that of the sample. This
cuvette could be filled with solvent or empty. The cuvette rested
in a snug, home-built cuvette holder. Both sample configurations
were mounted on a stage such that the sample position could be
adjusted in all three dimensions. For measurements carried out in
air, solutions of BPE in CH.sub.3OH were drop-coated onto the
substrate surface and allowed to evaporate; alternatively, the
cuvettes were placed in cuvettes containing known concentrations of
BPE.
[0120] Instrumentation.
[0121] SERS spectra were obtained with a Coherent Kr.sup.+ ion
laser model 3000 K. operated at 647.1 nm in TEM.sub.00. Spectral
scanning and detection were accomplished through the use of a Spex
Model 1404 scanning double monochromator with a pair of 1800
grooves/mm gratings and a thermoelectrically-cooled cooled
Hamamatsu R928 photomultiplier tube housed in a Products for
Research casing. Monochromator entrance and exit slits were
typically set at 700 mm. and center slits were set at 1400 mm to
yield an effective band pass of 5 cm.sup.-1. Grating movement and
spectral acquisition were controlled using the DM3000 software
provided by Spex. Plasma lines were filtered out of the incident
laser beam through the use of a band pass filter (Ealing
ElectroOptics) or a pre-monochromator tuned to the 647 nm line
(Optometrics). The laser beam was focused onto the substrate sample
at an angle of <30.degree. from the surface normal. Scattered
radiation was collected and collimated with a Minolta 50 mm camera
lens (f#1.2) and focused through a polarization scrambler (Spex)
onto the entrance slits of the monochromator.
[0122] Absorption spectra were obtained using a Hewlett-Packard
8452A diode array spectrophotometer (2 nm spectral resolution, 1 s
integration time). Again, substrates in quartz cuvettes were
maintained in an upright position through the use of a teflon
block. Transmission electron microscopy was performed on a JEOL
Model 1200 EXII instrument operating at 80 kV accelerating voltage;
the images were not manipulated, altered, or enhanced in any
way.
[0123] Below are eight (8) further examples of colloid monolayer
experimental protocols:
EXAMPLE 1
Surfaces Made from Seeded Colloidal Au Particles.
[0124] Glass slides (2.5 cm.times.0.8 cm.times.1 mm) were cleaned
in a mixture of HCl:HNO.sub.3 (3:1). Slides were rinsed in H.sub.2O
and CH.sub.3OH prior to derivatization for 18h in a solution of
aminopropyltrimethoxysilane (diluted 1:5 in CH.sub.3OH). The
derivatized surfaces were rinsed extensively in CH.sub.3OH and
H.sub.2O prior to immersion in solutions of colloidal are described
below. After 24 hours, the colloid derivatization was complete.
[0125] Au nuclei ("seeds") were prepared by adding 1 ml of 1%
Na.sub.3 citrate to a vigorously stirring solutions of 0.01%
HAuCl.sub.4. After 1 min., 1 ml of a solution of composition 0.075%
NaBH.sub.4 and 1% Na.sub.3 citrate was added. Reaction continued
for five minutes. The solution of nuclei was stored at 4.degree. C.
until needed.
[0126] The first seeded colloid was prepared by refluxing 1 ml of
1% HAuCL.sub.4.multidot.3H.sub.2O with 100 ml of 18 M.OMEGA. water
with vigorous stirring. 0.4 ml of 1% Na.sub.3Citrate and 30 .mu.l
of the above described nuclei was added rapidly and boiled for an
additional 15 minutes followed by cooling to RT. The resulting
colloid was stored in a dark bottle.
[0127] A second seeded colloid was prepared by an identical method
using 15 .mu.l of nuclei instead of 30 .mu.l.
1 Colloid Major Minor Std. Dev. # of Part. Nuclei 2.64 2.03 1.04
131 Seeded #1 52.7 43.7 5.24 70 Seeded #2 93.4 68.0 20.0 16
[0128] FIGS. 11-13 are TEM images of the particles. FIGS. 14 is the
solution optical spectrum of the seed nuclei. FIGS. 15-16 are the
uv-vis spectra of the larger particles derivatized on glass
substrates and of the larger particles in solution.
EXAMPLE 2
Preparation of 2-Layer Colloid Surfaces.
[0129] Once a colloid monolayer is formed, it is possible to
produce a multi-layered material by introducing a chemical linking
agent and a second layer of particle. Possible linkers include
4,4'-bis-pyridylethylene, 4,4'bipyridyl, p-xylenedithiol, and
mecaptoethylamine. Experimental protocol for preparation of a
typical surface follows.
[0130] Glass slide surfaces, approximately 2 cm.sup.2, were washed
first in a solution of HCl:HNO.sub.3 (3:1) followed by an H.sub.2O
rinse and cleaning in a mixture of 30%
H.sub.2O.sub.2:H.sub.250.sub.4 (1:4). The cleaned slides were
placed in about 3 mL of a 1:10 solution of
aminopropyltrimethylsiloxane (APTMS) in methanol for 1 hour. The
slides were rinsed with water and placed in about 3 mL of a 7.0
10.sup.-12 M colloid solution overnight. These particles had the
following shape: 65.8 nm major axis, and 49.3 nm minor axis. The
slides were again rinsed with water, and stored in water. A TEM
image of the particles is shown in FIG. 17. The resulting optical
spectrum is shown in FIG. 18. This surface was immersed for 5
minutes in 1 mM mercaptoethylamine in water and rinsed. This
surface was then immersed in a 17 nM solution of 12 nm colloidal Au
particles for 15 minutes. FIG. 19 is the resulting optical
spectrum.
EXAMPLE 3
Protein Coated Au Colloid Monolayers
[0131] Quartz surfaces which had been cleaned in a mixture of
H.sub.2SO.sub.4:H.sub.2O.sub.2 (4:1) were derivatized in neat
silane solution for 2 days. After rinsing these surfaces in
spectrophotometric grade CH.sub.3OH, they were placed in a solution
of 3-(N-maleimdopropionyl-biocytin (0.31 mg/ml in 0.05 M Tris
buffer containing 0.1% BSA) for 18 h. Slides were rinsed in
H.sub.2O and placed in solutions of the Au probe described below.
The protein-Au probes had been aged for 1 hour prior to coating the
slides; slides remained in solution overnight and were removed when
the colloidal particles sedimented. Little to no coating was
achieved in the first 5 h of exposure to the Au probes; optical
spectra taken after the colloid sedimented indicated Au coating on
all of the silane/biocytin surfaces employed, with .lambda.max=550
nm.
[0132] Protein-coated Au probes were prepared as follows: To 25 ml
of a citrate-prepared Au colloid (12 nm diameter, 17 nM) was added
0.725 ml of streptavidin (0.34 mg/ml in triply distilled H.sub.2O)
and 0.241 ml of BSA (7.24 mg/ml in triply distilled H.sub.2O).
Within 4 hours, this solution showed some aggregation/sedimentation
of particles, but particles were easily resuspended with shaking
the solution. However, within 24 h, the particles had sedimented
completely and could not be resuspended.
[0133] Quartz surfaces which had been cleaned in a mixture of
H.sub.2SO.sub.4:H.sub.2O.sub.2 (4:1) were derivatized in neat
silane solution for 2 days. After rinsing these surfaces in
spectrophotometric grade CH.sub.3OH, they were placed in a solution
of 3-(N-maleimidopropionyl)-biocytin (0.31 mg/ml in 0.05 M Tris
buffer containing 0.1% BSA) for 18 h. Exposure of this surface to a
streptavidin-coated Au colloid resulted in surface formation.
[0134] FIG. 20 shows the strategies available for non-covalent
attachment of colloids to surfaces, and how these strategies can be
utilized to bind biomolecules to metals in a biocompatible fashion.
FIG. 21 compares the coverages by covalent and non-covalent
methods.
Example 4
Detailed Experimental Protocols for the Production of Ag-Coated Au
colloid Monolayers by Chemical or Electrochemial Reduction.
[0135] Materials.
[0136] The following materials were obtained form Aldrich:
HAuCl.sub.4.3H.sub.2O. Na.sub.2SO.sub.4, trisodium citrate
dihydrate, pyridine, and N,N-dimethyl-4-nitrosoaniline (p-NDMA).
The following organosilanes were obtained from Hulls America, Inc.:
2-(diphenylphosphino)-ethyltriethoxysilane (DPPETES),
3-aminopropyltrimethoxysilane (APTMS),
mercaptopropylmethyldimethoxysilan- e (MPMDMS) and
2-(trimethoxysilyl)ethyl-2-pyridine (PETMS). KCl, HCl, and
HNO.sub.3 were obtained from J. T. Baker.
Ru(NH.sub.3).sub.6Cl.sub.3 was obtained from Johnson Matthey. Li
Silver was obtained from Nanoprobes, Inc. Methanol
(spectrophotometric grade) was obtained from EM Sciences. All
chemicals were used as received. All H.sub.2O was 18 M.OMEGA.,
distilled through a Barnstead Nanopure water purification system.
Single component Ag epoxy was obtained from Epoxy Technologies, Inc
and white expoxy was epoxi-patch from The Dexter Corporation. Glass
microscope slides were obtained from Fisher Scientific, quartz
slides from Technical Glass Products, and In-doped SnO.sub.2 from
PPG Industries, Inc.
[0137] Colloid Preparation.
[0138] Colloidal Au particles of 18 nm.+-.1 nm and 12 nm were
prepared as described previously. The sols were characterized by
optical spectroscopy.
[0139] Surface Derivitization.
[0140] Glass and quartz microscope slides were cut to 25 mm.times.9
mm prior to cleaning in a bath of 4 parts H.sub.2SO.sub.4 to 1 part
30% H.sub.2O.sub.2 at 60.degree. C. After rinsing in H.sub.2O
slides were cleaned in a bath of 3:1 HCl:HNO.sub.3. Slides were
rinsed in H.sub.2O and stored in CH.sub.3OH until needed. Clean
surfaces were placed in 2% (by volume)
2(diphenylphosphino)ethyltriethoxysilane or
2-pyridylethyltriethoxysilane in CH.sub.3OH. Solutions were shaken
vigorously and reacted for 24 hours. Substrates were then removed
from solution and rinsed thoroughly in CH.sub.3OH, then rinsed and
stored in water. Silanized surfaces were immersed colloidal Au for
24 hours. Surfaces were then removed, rinsed several times in
H.sub.20 and stored in H.sub.2. In-doped SnO.sub.2 was cut to an
approximate area of 3.0 cm.sup.2. Copper wire was adhered to the
SnO.sub.2 with Ag epoxy. The copper wire was encased in glass
tubing and sealed with white Epoxy-Patch The contact area was
covered with a white epoxy. The electrodes were sonicated in neat
reagent grade acetone, soap solution, and H.sub.2O for
approximately 15 minutes each, then were soaked in approximately 3
M NaOH for over 2 hours. Clean electrodes were placed in aqueous
APTMS (1% by wt.) for 5 minutes. Silanized electrodes were then
rinsed with H.sub.2O and dried for 48 hours. The electrodes were
placed in the Au colloid solution for 2 hours. Colloid-coated
electrodes were then rinsed and stored in H.sub.2O. The other
SnO.sub.2 samples were placed in neat MPMDMS for approximately 5
hours. The substrate was rinsed with CH.sub.3OH and air dried. The
polymer-coated substrate was then placed into fresh Au colloid for
at least 8 hours for high coverage SnO.sub.2. The Au colloid-coated
substrate was removed, rinsed with triply-distilled H.sub.2O, and
air dried.
[0141] Electrode Characterization.
[0142] A 5 mM Ru(NH.sub.2).sub.6Cl.sub.3 in 0.1 M Na.sub.2SO.sub.4
was degassed with N.sub.2 for over 15 minutes prior to analysis. An
SCE was used as a reference electrode and Pt gauze was used as the
counter electrode in all measurements.
[0143] Sample Preparation.
[0144] Li Silver Reduction.
[0145] Ag was reduced onto the colloid-coated slides by immersing
the substrates in a solution of equal volumes of LI Silver enhancer
and initiator solutions. Reaction time was varied between 5 and 30
minutes. Surfaces were then rinsed and stored in H.sub.2O.
[0146] Ag Deposition.
[0147] An electrolyte solution of 1 mM Ag.sub.2SO.sub.4 and 0.1 M
Na.sub.2SO.sub.4 was degassed with N.sub.2 for at least 15 minutes
prior to the electrochemical deposition. A Pt gauze electrode was
used as a counter electrode. A cyclic voltammogram was first done
with the electrode scanning from 1.0 V to 0.0 V vs SCE at 50
mV/sec. A constant potential was applied to the electrode until a
predetermined number of Coulombs had been counted. The cyclic
voltammogram and the reduction of Ag were both done in the same
solution under a blanket of N.sub.2. The electrodes were then
removed, UV-Vis spectra were collected, and the electrodes were
stored in H.sub.2O.
[0148] Surface Characterization.
[0149] SERS of 0.5 M pyridine/0.1 M KCl solution on the electrodes
were performed. The potentials were obtained with a Pt counter
electrode and a SCE reference electrode and were varied from 0.0 V
to -0.9 V. SERS of the Li Silver was done on 0.5 mM p-NDMA in
CH.sub.3OH. Experiments were performed with 632.8 nm excitation,
7.5 cm.sup.-1 bandpass, 0.5 cm.sup.-1 steps and 1 second
integration.
[0150] Instrumentation.
[0151] SERS spectra were obtained with a Spectra-Physics Model 127
HeNe ion laser operated at 632.8 nm. Raman spectra were acquired
with a Spex Model 1403 scanning double monochromatic with a pair of
1800 groove/mm gratings and thermoelectrically-cooled Hamamatsu
photomultiplier tube housed in a Products for Research casing.
Monochromator entrance and exit slits were set for a spectral
bandpass of 7.5 cm.sup.-1. Spectral acquisition and grating
movement were controlled via the DM3000 software provided by Spex.
Plasma lines were filtered out of the incident beam through a band
pass filter (Ealing ElectroOptics.) Incident light was focused at
an angle.about.<30.degree. to the surface normal. Scattered
radiation was collected and collimated with a Minolta 50 mm camera
lens (f/#1.2) and focused through a polarization scrambler (Spex)
onto the entrance slits of the monochromator. Substrates were
supported by Teflon block (1/3 of sample height) in a quartz
cuvette. The cuvette holder rested on a home-built stage adjustable
in all three dimensions.
[0152] Optical spectra were obtained using a Hewlett-Packard 8452A
diode array spectrophotometer with a 2 nm spectral resolution and 1
s integration time. Samples were supported in the cuvettes by use
of the teflon block described above.
[0153] The cyclic voltammograms of the Ru(NH.sub.3).sub.6Cl.sub.3
were done on a Cypress Systems CS87 potentiostat. The cyclic
voltammograms and the Ag deposition were carried out using an
EG&G PAR 173/175 combination programmer/potentiostat, and
recorded by a NGI Servogor 790 XY recorder. Ag deposition was
monitored using a Linseis L6512B strip chart recorder. The
potential during the Raman scans was held constant by a BAS CV27
potentiostat.
[0154] Atomic Force Microscopy (AFM) was performed on a Digital
Instruments Nanoscope III Multimode AFM in tapping mode. The
samples were cut to approximately 1 cm.times.1 cm and allowed to
air dry. The scan size was 1.00 .mu.m, and scan rate was 0.9988 Hz.
X and Y axes are 1 .mu.m with 0.2 .mu.m divisions, and all the
z-axis are 300 nm.
[0155] FIG. 22 show the changes in optical spectra upon
electrochemical reduction of Ag ions onto Au colloid monolayers.
FIG. 23 shows theoretical modeling of the expected changes, showing
that the Ag is selectively deposited on the Ag surface. FIG. 24
shows AFM characterization of these surfaces. FIG. 25 shows optical
spectra as a function of electrochemical potential (E) for a
surface with high Au coverage (from 0 to -1 V vs. SCE),showing that
these surfaces are stable under electrochemical conditions. FIG. 26
shows the changes in optical properties accompanying chemical
deposition of Ag onto preformed Au colloid monolayers.
EXAMPLE 5
[0156] Large colloids can be stabilized by addition of
polyelthyleneglycol (PEG). This does not preclude derivatization on
surfaces. For example, colloidal solutions were stabilized by the
addition of fresh 1% PEG (mw 20,000 Fluka) until the final PEG
concentration was 0.1%. The colloidal solution was then centrifuged
in glass test tubes at 300.times.G for 15 Minutes to pellet the
colloid. The supernatant was removed and the colloid was
resuspended in distilled H.sub.2O. This resuspended colloid was
then adsorbed on to silanized surfaces as described elsewhere. The
resulting surface was characterized by uv-vis in FIG. 27.
EXAMPLE 6
Preparation of 2.5 nm Au Particles on Thin Layers of Inorganic
Oxides Coated with Organic Polymers.
[0157] Indium-doped SnO.sub.2, cut to dimensions of 1 cm by 1 cm.
was cleaned as follows: surfaces were sonicated for 15 min each in
soapy water, 3 changes of triply distilled H.sub.2O, and acetone.
After the surfaces were rinsed in H.sub.2O, they were soaked in 3 M
NaOH for 1.5 h. Substrates were stored in H.sub.2O until needed for
further derivatization. The SnO.sub.2 was derivatized with
alternating layers of K.sub.2Nb.sub.6O.sub.17 and poly(allylamine)
hydrochloride (PAH) as decribed elsewhere [Keller et al, JACS, 116,
8817]. PAH-coated surfaces were immersed in a solution of colloidal
Au (.times.nm .+-.y nm) for 28 h. Substrates were then rinsed and
stored in H.sub.2O. The optical spectrum in FIG. 28 shows the
presence of Au particles on the surface.
EXAMPLE 7
Preparation of a 12 nm Au Colloid Monolayer on SnO.sub.2 Electrode
Preparation
[0158] Pyrolytically deposited Sb doped SnO.sub.2 (R=100 ohm
cm.sup.2) on glass 3 mm thick was purchased from Delta Technology
Lmtd. The SnO.sub.2 on glass was cut into pieces of the size 2
cm.sup.2, piranah washed and sonicated in triply distilled H.sub.2O
for 15 min. Contact to the SnO.sub.2 was made with Ag epoxy. Cu
wire encased in 3 mm glass tubing was used as the lead. The Ag
epoxy was covered with white epoxi patch (Dexter Corporation).
Electrodes were sonocated in triply distilled H.sub.2O for 15 min.
prior to use.
[0159] The clean electrodes were placed in 3 M NaOH for five hours,
rinsed in H.sub.2O, placed in 1% (by weight) aqueous APMDES for
10-15 minutes, and then rinsed with H.sub.2O. The electrodes were
allowed to dry at room temperature for 2 days. The electrodes were
placed in a solution of Au colloid for 3 hours, 20 min., rinsed
with H.sub.2O, and stored in H.sub.2O. An optical spectrum is shown
in FIG. 29.
[0160] The Au and Ag colloid-based surfaces have many of the best
attributes of previously described SERS substrates (uniform
particle size, electrochemical addressability, and large
enhancement factors), and a combination of other features few
surfaces can match (ease of characterization, no geometric
constraints, low cost, and very high intra- and inter-sample
reproducibility). When the substrate is optically transparent in
the visible, uv-vis can be used to assess particle coverage and
spacing. These substrates are strongly active for SERS using 647.1
nm excitation, as are those prepared on glass and quartz slides.
These advantages, coupled with the ability to vary particle size,
spacing, and the nature of the monolayer support suggest a rich use
for these materials in fundamental and applied studies. For
example, no theoretical model correlating SERS activity with
particle size or spacing has ever been quantitatively tested; metal
colloid monolayers should allow such experiments to be carried out.
The flexibility and control available through this method are
noteworthy. Particle size, particle-polymer interactions, and the
physical and chemical properties of the underlying substrate can
all be manipulated to control the nanoscale architecture produced.
The solution-based assembly protocol makes substrate fabrication
routine; it also removes virtually all constraints on substrate
size/shape. Therefore. Au colloid monolayers can be prepared on
substrates that allow facile characterization, including
macroscopic glass/quartz slides and TEM grids. Most importantly,
SERS can now be considered for applications in which preparation of
multiple high-quality substrates was previously viewed as an
insurmountable barrier.
[0161] Additional Applications of the Inventive Metal Colloid
Monolayers
[0162] A Method for the Detection and Analysis of a Chemical
Compound Dispersed in a Carrier
[0163] The improved SERS substrates, as described above enable
Raman spectroscopy to be used as a practical method for detecting
and analysing carrier-borne chemical compounds, specifically
water-borne pesticides, herbicides and other agrochemicals. In
addition, they can be used to detect disinfection-generated
pollutants in water, such as haloacetic acids. They can also be
used to detect, in air, any vapor-phase compounds possessing
Raman-active modes, such as explosives, fragrances and the like, in
extremely low concentrations.
[0164] Background
[0165] The widespread use of pesticides in modem agriculture leads
inexorably to their appearance in water, and such contamination
continues to be an significant threat to water quality.
Accordingly, determination of pesticide levels in aqueous samples
is a necessary and ubiquitous analytical procedure, and there is an
increasing need for rapid, simple, reliable, and pollution-free
methods of determining these pesticide levels. Organophosphorous
pesticides (OPPs), while not as persistent as other organic
compounds used in agriculture, are nevertheless monitored quite
carefully, in large part due to their extreme toxicity. Indeed, the
National Pesticide Survey (NPS) included 43 different OPPs or OPP
degradation products, and left out numerous more because of their
instability.
[0166] A tremendous amount of published work is concerned with
analysis of these compounds, using a compendium of analytical
methods. One method previously used relied on the determination of
nitrogen/phosphorous (N/P)-containing pesticides in ground water by
GC with an N/P detector, using methyl t-butyl ether as the final
extraction solvent. For obvious reasons, there is tremendous
pressure to reduce or completely eliminate the use of organic
solvents in the liquid-liquid extraction (LLE) step. Recently
discussed methods for doing so include thin-layer chromatography,
gas chromatography (with nitrogen-phosphorous, flame photometric,
or mass spectrometric detection), liquid chromatography (with
electrochemical, uv diode array, or fluorescence detection), and
inhibition of cholinesterase biosensors. In most cases, there is an
emphasis on solid-phase extraction (SPE) methods using disks or
cartridges, simultaneously eliminating the need for LLE and
providing a sample concentration mechanism; in recent reports, the
SPE step has been fused to the separation step to yield "on-line"
methods. It should be noted, however, that humic acids, clays, and
pH have been shown to impact SPE extraction efficiency for several
organopesticides. Moreover, several OPPs have been shown to
completely degrade on SPE precolumns at 4.degree. C. A reasonable
alternative to SPE is accelerated solvent extraction, a technique
that reduces the volumes of conventional liquid solvents by the use
of increased temperature and pressure.
[0167] A second thrust, relevant to the entire field of pesticide
analysis, is the recent proliferation of portable instrumentation.
The rationale for development of these instruments is the ability
to make analytical measurements on-site, a critical component of
the emerging national water quality monitoring program. These new
instrument and protocols place a premium on "operational
simplicity", with the basis of the analytical method typically
resting on a single measured parameter, and sometimes even a yes-no
response.
[0168] A good example of this approach is a recently introduced
portable analysis kit for a number of pesticides, including
chlorpyrifos, based on a competitive immunoassay, a technique in
which labeled and unlabeled analytes compete for binding sites on
immobilized antibodies-which in this particular technique are
conjugated to magnetic particles. Separation of the magnetic
particles, followed by a spectrophotometric assay of an enzyme
linked to the labeled analyte [enzyme-linked immunoassay (ELISA)],
gives an absorbance proportional to the amount of label. Using a
calibration curve, this can be correlated to the concentration of
unlabeled analyte in the sample. A similar immunoassay for
parathion has also been reported that does not require its removal
from hexane.
[0169] There are a number of serious drawbacks to these approaches:
(i) it is well-recognized that competitive immunoassays are not
trivial to carry out, and unlike sandwich immunoassays, in which
the signal is proportional to analyte concentration, competitive
immunoassays yield signals inversely proportional to analyte
concentration; and (ii) different immunoassays kits are necessary
for each analyte to be detected; no more than two uv-vis-based
ELISAs can be carried out in the same test tube, and multianalyte
detection by any method is difficult. Moreover, enzyme labeled
analogs and antibodies exist for only a small fraction of OPPs in
use. In addition, (iii) the extreme similarity in OPP structure
(see Table II) suggests that very high specificity in monoclonal
antibodies is not possible, i.e. one OPP could act as an
interferant in an assay for another OPP, and (iv) other
interferences, which are often present in real-world samples,
cannot be identified in single parameter measurements. Finally, (v)
even if none of these problems existed, the shelf life and
stability of protein-based analytical reagents are generally quite
short. The net effect is that while development of immunological
approaches to on-site measurements of pesticides is being widely
pursued, the measurements must necessarily be carried by people
with technical expertise; the possibility of using this method to
determine water quality on individual farms by individual farmers,
a clear goal of the national water quality monitoring program, is
slight.
[0170] It would therefore be advantageous to develop a direct
method for quantitation of OPPs and other water-borne pestisides or
agrochemicals based on surface enhanced Raman scattering (SERS), as
this ultrasensitive spectroscopic technique is optimally suited for
analysis of dilute aqueous solutions of organic compounds. Like its
vibrational counterpart, infrared spectroscopy, Raman spectroscopy
yields molecule-specific information relating to the identity of
atoms, bond strengths, and bond angles. In other words, a Raman
spectrum provides a characteristic fingerprint for a given
molecule. Few other spectroscopic techniques can match the high
information content of vibrational spectroscopy. This is to be
contrasted with methods based on fluorescence and
absorbance--immunoassays, for example--in which a single datum is
often used to ascertain analyte concentration. Raman spectroscopy
has a great advantage over other spectroscopic techniques for
analysis of aqueous solutions; H.sub.2O is a very poor Raman
scatterer, in contrast to the nearly insurmountable interference
H.sub.2O generates in infrared spectroscopy. Raman spectroscopy has
not been previously used in the analysis of water-borne pesticides,
however, because Raman scattering is inherently a very weak
phenomenon, with an intrinsic sensitivity 3-4 orders of magnitude
too low to detect OPPs in the submicromolar concentration range.
The improved results achieved with the SERS substrates formed in
accordance with the present invention enable Raman spectroscopy to
be used as a practical method for detecting and analysing
water-bone pesticides.
[0171] The attraction of Raman spectroscopy as a technique for
pesticide detection, and OPP detection in particular, is clearly
elaborated in Table I, which lists the strong or medium Raman
vibrations between 400 cm.sup.-1 and 1700 cm.sup.-1 for 8 OPPs
(whose structures are illustrated in Table II). Each of the eight
species has a unique spectrum, and each has at least one strong
vibration that is spectrally well-resolved from the others. Thus,
each compound in this table can be identified directly, without any
separation required. In contrast to TLC, GC, and HPLC, this is a
direct measurement in the truest sense of the word, since all
compounds are detected simultaneously on the basis of their
molecular structure. Among the many OPPs available for study, this
particular set of compounds was focused on only because SERS
spectra already exist: It is stressed that this same technology is
applicable to any finite set of OPPs and to other pesticides and
other classes of compounds, such as haloacetic acids, the toxic
byproducts of chemical disinfection. Table III shows the unique
Raman bands for haloacetic acids and chlorinated phenols.
2TABLE I Raman Peak Locations for 8 Organophosphorous Pesticides
1
[0172]
3TABLE II Structures for 8 Organophosphorous Pesticides 2 3 4 5 6 7
8 9
[0173]
4TABLE III Raman Spectra of 9 Holoacetic Acids TBAA DBAA BAA TCAA
DCAA CAA 2-CP 2.4-DCP 2.4.6-TCP A B C C C C B B C 193 vs 217 vs 16
280 vs 343 vs 378 vs 979 vs 10 930 vs 412 vs 13 vs 449 vs 489 s 563
vs 614 vs 19 656 vs 20 667 vs 677 vs 680 vs 654 vs 081 vs 27 vs 729
vs 24 775 vs 25 797 vs 795 vs 26 321 vs 27 835 vs 28 556 vs 29 307
vs 30 906 vs 903 s 31 927 vs 32 1031 vs 1392 s 34 1405 s
Abbreviations: TBAA = tribromoacetic acid: DBAA = dibromoacetic
acid: BAA = bromoacetic acid : TCAA = trichloroacetic acid: DCAA =
dichloroacetic acid: CAA = chloroacetic acid: 2-CP =
2-chlorophenol: 2.4-DCP = 2.4 dichlorophenol: 2.4.6 =
2.4.6-trichlorophenol: vs = very strong: s = strong. Roman data for
bromochloroacetic acid are not available. Darkened boxes refer to
vibrations that are spectrally distinct, and whose presence can be
used to measure the concentration of a particular HAA in a mixture.
Note that (a) for some compounds, there are multiple unique bands.
of which could be used as a diagnostic: and (b) there are other.
much weaker bands in each spectrum.
[0174] It is important to note that a double monochromator and
photomultiplier tube detector was used to aquire the data in Table
I (in 1987) and Table III (in 1989). The instrumentation available
today not only provides greater throughput (with a single
monochromator/holographic notch filter combination) but also
improved signal/noise (with CCD detectors). Thus, the detection
limits for the work proposed herein will be superior to the
nanogram levels previously reported.
[0175] SERS-based detection has additional advantages relative to
HPLC and GC. both of which have significant setup time, involve
machinery that is difficult to make portable, and typically require
operator training. In other words, these "direct" methods are not
necessarily "simple". . . With pre-formed substrates, SERS data are
extremely easy to acquire, comparable to measuring absorbance.
[0176] Another significant advantage to Raman spectroscopy over GC
and HPLC is the information content per molecule. GC and HPLC
techniques identify compounds based on their viscoelastic
properties and mobilities, and yield little information: a
retention time, or in the case of diode array uv-vis detection for
HPLC, an optical spectrum. Unfortunately, these properties cannot
always be directly correlated to molecular structure, particularly
for compounds with such similar atomic connectivity. Raman
spectroscopy offers the advantage of multi-peak identification,
with the signal for each OPP having numerous components. In complex
matrices, where there is an increased likelihood of interferences
and/or impurities, the ability to directly identify a compound
using several distinct peaks (Table I) is a huge advantage. This
property is especially important for compounds like OPPs that
degrade rapidly; characteristic vibrations can be used to identify
degradation products, without knowing the degradation chemistry.
Similarly, characteristic frequencies can be used to identify other
classes of pesticides. In contrast, an unknown compound cannot be
identified by GC or HPLC solely on the basis of its retention time.
It should be noted that phenylamide pesticides have been
quantitated by Resonance Raman scattering in solution, but this
requires derivatization with an intensely absorbing azo dye
derivatives. Such an approach would not be practical for OPPs,
which lack the appropriate chemical functionality.
[0177] A final advantage to SERS is that it is an extremely general
method; if analyte adsorption can be induced, it can be used for
any water analysis without any changes in equipment or
configuration. As water quality takes on increased significance in
society, and as the number of different compounds in water that
must be monitored increases, this is an important
consideration.
[0178] SERS behavior of colloid monolayers can further be
controlled by adjusting the intrinsic properties thereof. It is
known, for example, that the dielectric constant of Ag make it more
enhancing than Au for visible light excitation. However, over the
10-100 nm scale Au colloids are more monodisperse than Ag. Thus, in
a preferred embodiment of the invention, the strengths of each
metal are exploited by using autometallography, a technique whereby
Ag.sup.+ is selectively reduced onto pre-existing Au particle
surfaces. Thus, submonolayers of monodisperse colloidal Au,
sufficiently separated so as to be only weakly SERS enhancing, are
overcoated with Ag. The resulting particles are larger and
therefore exhibit reduced interparticle separation. Particle arrays
formed thereof possess Ag-like optical properties and enhancement
factors. Testing with Ag-coated Au confirms that the SERS intensity
achieved with such particles are within a factor of two of the
signal obtained with pure Ag colloid, and within a factor of ten of
the best roughened Ag electrodes.
[0179] These results indicate that these surfaces possess
exceptional enhancement characteristics.
[0180] While SERS is capable of providing an enormous stream of
data, it will be necessary to rapidly convert the data into a
usable form. One way to do so is to use artificial neural networks
(neural nets) for this task. A growing body of work describes the
use of neural nets for spectral recognition, quantitation, or
classification. Applications to Mossbauer, laser-induced
fluorescence, and mass spectra are notable. A particularly
intriguing implementation appears in commercial instrumentation for
odor detection.
[0181] Neural nets have previously been used in several capacities
with regard to pesticide physical properties. These include an
estimation of soil partition coefficients based on chemical
structure, a model for half-lives, and an assessment of toxicity.
More importantly, experiments have been carried out to classify
pesticide IR spectra into organophosphorous and
non-organophosphorous classes with neural networks. The same group
also used a three-layered neural network for automated
interpretation of pesticide IR spectra. These results, coupled with
demonstrated recent successes of neural networks for multiparameter
spectral analysis, indicate that the proposed neural net-based
identification and quantitation of OPPs is possible.
[0182] Establishment of a non-separation based analytical method on
samples containing mixtures requires three stages of
experimentation. In the first, detection limits are established for
each analyte individually. In the second, detection limits are
established for mixtures. Finally, signal calibration is required
for mixtures with differing concentrations.
[0183] Measurement and optimization of individual detection limits
is a straightforward task. The detection limit is defined according
to convention as the amount of sample producing a signal/noise
ratio of 2. A tremendous advantage of Raman over separation-based
methods is the removal of flow-induced limits on integration times.
In other words, since the analyte is not moving, one can integrate
the Raman spectrum for arbitrarily long times. For CCD detectors,
background noise is virtually nonexistent, but practical
limitations on integration times are about 15 minutes.
Nevertheless, this number should be compared to just the few
seconds of integration time available for a chromatographic
detector.
[0184] There are two fundamentally different approaches to
measuring SERS spectra of OPPs, each with different detection
limits. In Method A, the solution is made basic to insure complete
deprotonation of OPPs. A small aliquot (50 .mu.L) is applied to an
Ag/Au colloid monolayer on a substrate of 1.0.times.1.0 cm
dimensions. The hydrophilic nature of these surfaces allows an
acceptable degree of wetting; the resulting high surface
area/volume ratio leads to rapid evaporation of excess water.
Alternatively the substrate can be mildly heated to promote
evaporation. The sample is then placed in front of the laser beam
and the SERS spectrum is measured. In Method B, a glass slide
containing a colloid monolayer is immersed in the treated H.sub.2,
and the SERS spectrum is acquired in situ. Below are estimates for
the detection limits for each method.
[0185] Method A:
[0186] Assumptions
[0187] (1) Detection limit=10.sup.-5 monolayers of adsorbate
[0188] (2) Sampling area=focused laser spot size.apprxeq.2.0 mm x
2.0 mm
[0189] (3) Molecular area.apprxeq.40 .ANG..sup.2/molecule
[0190] These assumptions are completely reasonable. (1) Single
monolayers of adsorbates can be detected by Raman spectroscopy
using CCD detectors on unenhanced surfaces. With an enhancement
factor of 10.sup.6, it therefore follows that 10.sup.-6 monolayers
can be detected on SERS-active surfaces using a CCD. Thus, a
detection limit of 10.sup.-5 monolayers is conservative. (2)
Focusing a laser through a microscope objective allows Raman
spectra to be obtained from spot sizes approaching .mu. m in
diameter--thus the 4 mm.sup.2 sampling area is simple to achieve.
(3) The value of 40 .ANG..sup.2/molecule translates to
2.5.times.10.sup.14 molecules/cm.sup.2, a commonly cited value for
monolayer coverage of small adsorbates on surfaces.
[0191] Sample Calculation 2 Let MNDAM = the minimum number of
detectable adsorbate molecules = 0.00001 monolayers .times. 0.04 cm
2 sampled .times. 1 molecule / 40 2 .times. 10 16 2 / cm 2 = 1
.times. 10 8 molecules
[0192] Only the actual volume above the sampled area is relevant to
the detection limit, and this is the sampled area/total area
multiplied by the total volume, i.e. (0.04 cm.sup.2/1
cm.sup.2).times.50 .mu.L=2 .mu.L.
[0193] The presence of 10.sup.8 molecules in a volume of 2 .mu.L
corresponds to a concentration of 83 pM. For a species with MW=200
(around the average MW for the 8 OPPs under consideration), this is
equivalent to 16.6 ng/L. Assuming all 8 OPPs are present in equal
concentration, the detection limit will be increased by a factor of
9. Thus, a simultaneous detection limit of 149 ng/L=0.15 .mu.g/L is
anticipated. This number is equivalent to detection limits reported
for GC, but without any sample pre-concentration. In other words,
direct adsorption to SERS substrates eliminates the need for sample
concentration using SPE or liquid-liquid extraction. This is a
significant advantage to this method.
[0194] Method B:
[0195] Assumptions
[0196] Whereas Method A has the advantage of effectively adsorbing
every molecule in a given volume of solution through increased
evaporation, in Method B one is constrained by K. the equilibrium
constant for adsorption. This parameter defines the surface
coverage for a given solution concentration (c). In addition to the
assumptions delineated above, the calculation for in situ detection
limit will assume Langmuirian adsorption (i.e. no interaction
between particles). Thus, q/(1-q)=Kc, where q is fractional surface
coverage. In the absence of concrete data regarding attraction or
repulsion between particles, it is a reasonable approximation to
assume a Langmuirian adsorption isotherm. A value of K=10.sup.5 on
Ag is likely an underestimate for two reasons: first, OPPs are
sparingly soluble, which dramatically increases their affinity for
surfaces; and second, most of the compounds in Table II contain
sulfer, which binds very strongly to Ag and Au.
[0197] Sample Calculation
[0198] Using q=10.sup.-5,
c=q/[K(1-q)]=10.sup.-5/[10.sup.5(0.99999)]=1.tim- es.10.sup.-10 M.
With MW =200, and all 8 OPPs present, this corresponds to 0.18
.mu.g/l, a value similar to that for method A, abut again without
the need for pre-concentration.
[0199] It is important to note that Method B essentially
corresponds to an in situ method, and as such, has important
ramifications. A protocol that consists of withdrawing 2 mls of
sample, placing it into a uv-vis cuvette, adding a colloid-coated
glass slide, and pressing a button on a computer console is
exceptionally attractive. If an internal standard is desired (vide
infra), this could be added to the cuvette. Furthermore, if the
colloid monolayer technology could be transferred to an optical
fiber (a likely scenerio, given the success with oxidic surfaces
such as glass, quartz. SnO.sub.2, and Al.sub.2O.sub.3), true in
situ monitoring can be achieved. Raman spectroscopy using optical
fibers is well-established, as is its use to monitor industrial
processes; it would be invaluable to monitor speciation of
pesticides in real time.
[0200] Inherent to the inventive methods are four possible
problems: non-systematic intensity variations, the possibility of
interferences, the need for adsorption of analyte, and surface
saturation-induced nonlinearities in signal. However, each of these
can be straightforwardly addressed.
[0201] Addition of an internal standard eliminates any concerns
regarding surface and/or intensity variation. Any one of hundred of
compounds possessing a Raman spectrum distinct from those of the
OPPs would suffice. So that adsorption properties would be similar,
structural similarity is desirable; thus, fluorinated phenols are a
logical choice. Small volumes of known concentrations would be
added prior to measurement.
[0202] The most likely interferences in SERS are adsorption of
inorganic anions (HO.sup.--, HCOO.sup.--, etc.) Their influence can
be minimized by acidifcation; at pH 5, [HO.sup.--]=10.sup.-9. Thus
a simple route to identify (and eliminate) undesired adsorbates is
to acquire Raman spectra at several pH's. Halide anions are bound
more tightly, but each have only one band in the Raman spectrum, so
that their net effect is essentially limited to a reduction of
available OPP binding sites. If this leads to significant problems,
it can be overcome by introducing an element of molecular
selectivity at the Au/Ag interface. The introduction of organothiol
self-assembled monolayers (SAMs) onto SERS-active surfaces
increases selectivity for the desired analytes, and that partition
from solution into the SAM film can be quantitated. A similar
strategy can be adopted with regard to the inventive method using
HSCH.sub.2CH.sub.2OH or HSCH.sub.2CH.sub.2NH.sub.3.sup.+.
Monolayers of the first molecule should preferentially hydrogen
bond with the P=S or P=O functional group relative to H.sub.2O,
HO.sup.--, or halide. Note that direct analyte adsorption is not
necessary for SERS: enhancement by the so-called electromagnetic
effec--which accounts for 10.sup.4 of the 10.sup.6 overall
enhancement--is still quite substantial 10 .ANG. from the
surface.
[0203] A legitimate concern is the presence of organic carboxylate
impurities. These can be avoided in the preparation of colloid
monolayers, but may be present in treated waters (a disadvantage to
non-separation based methods). Fortunately, if such compounds are
present, they can be easily identified by their Raman spectra.
Considering the purity of the waters under consideration, it is
anticipated that this will not be a serious problem.
[0204] All 8 OPPs will strongly adsorb to Ag, but do not adsorb to
equal extents. If two species are present in equal concentration in
solution, but one adsorbs with greater affinity, the true
concentration of the poorly adsorbed species can easily be masked.
Fortunately, the structures of the OPPs fall into two
narrowly-defined categories (phosphotriesters and
phosphothiotriesters). Within each structural class, the
equilibrium constants will vary little. Any non-zero differences
between the equilibrium constants for adsorption can be measured
and factored in. This process is further described below in the
discussion of mixture analysis.
[0205] A final potential problem is saturation of the
surface-binding sites: at high OPP concentrations, this could lead
to a non-linear response. Inaccuracies can be avoided by diluting
the original OPP-containing solution by 10-100 when peak
intensities exceed 50000 counts per second. If there is no
appreciable decrease in signal intensity, the surface is likely
saturated. A good procedure for dealing with this is to discard the
substrate, dilute by a factor of 500-1000, and repeat the
measurement with a new substrate. In general, it is desirable to
run each sample at three slightly different concentrations under
non-saturating conditions, particularly since the measurement
process itself is so simple. This will yield more statistically
valid data.
[0206] To best understand the complexities of mixtures, the ranges
of K for each class of OPP should be determined. Using a
microcalorimeter, adsorption isotherms can be generated, from which
K can be calculated. With values for K in hand, mixtures are
prepared, and the measured intensities compared to those predicted.
If they are identical, then by measuring all K's, it is possible to
know the exact surface distribution of a prepared mixture a priori.
More likely is the case where the predicted and observed differ, in
which case it will be necessary to generate calibration curves
manually. This requires substantial effort, and can constitute a
critical component of the protocol.
[0207] Alternatively, the method of standard addition can be used
instead of calibration curves. In this approach, known amounts of
OPPs are added to samples containing unknown; the x-intercept that
results from the linear plot of signal vs. amount added gives the
concentration of unknown.
[0208] Thus, as described above, surface enhanced Raman scattering
(SERS), coupled to a neural net can be used to identify and
quantify water-borne pesticides and other compounds, such as
haloacetic acids, with detection limits below 0.2 .mu./L. This can
be accomplished without the need for extraction or separation. This
method makes use of Au, or preferably Ag-clad Au colloid
monolayers, used in combination with SERS instrumentation. In
contrast to existing methods, the method of the invention is
remarkably simple to carry out, and provides direct spectral
identification for each compound. The inventive technique can be
used alone, or interfaced to existing separation methods. Further,
the inventive method makes on site detection of water-borne
pesticides possible.
[0209] Colloid Monolayer-Based Detection in Separation
Experiments
[0210] SERS can also be used as a detection method in a separation
experiment. A default protocol would thus be to do GC or HPLC with
SPE, adsorbing the eluate directly onto colloid monolayers. Using a
micromanipulator to raster the surface across the solvent exit port
will thus preserve the chromatogram spatially on the SERS
substrate, which can then be addressed in a site-by-site fashion by
SERS. For HPLC, this is a technically trivial task. The task is
slightly more complex for GC; nevertheless, a SERS-based GC
detector has been described. In any case, coupling HPLC or GC with
SERS eliminates any problems mixtures engender, while considerably
improving the detection limits currently available for each.
[0211] SERS can also be used in conjunction with capillary
electrophoresis (CE). The basic idea behind SERS detection in CE is
to spread the eluate onto an Au or Ag (or Ag-coated Au colloid
monolayer on conducting SnO.sub.2 and then interrogate the surface
using microRaman. FIG. 30 illustrates this approach. An electric
field is applied between one solvent reservoir and a conductive
substrate, such as a doped SnO.sub.2 surface. Alternatively, the
tip of the capillary can be metallized, and the electric field
applied thereto, in which case a non-conductive substrate can be
used. One end of the capillary is placed in the solvent reservoir,
and the other end is contacted to the Au colloid monolayer. The
substrate is then rastered by the capillary. As compounds A, B, and
C elute, they are deposited at different spots on the surface.
Elimination of diffusion and forced adsorption can be
simultaneously effected by solvent removal, and MPMDMS
(mercaptopropylmethyldimethoxysilane)-based Au colloid surfaces can
be heated to 70.degree. C. with no effects on optical or SERS
behavior. Once immobilized the substrate is rastered across a
focused laser and the spectra are recorded using a CCD. Like
recently-popularized direct uv fluorescence detection schemes for
CE, no pre- or post-column derivatization is needed. However, this
method is not restricted to uv fluorophores and works for all
Raman-active molecules.
[0212] It has previously been demonstrated that a capillary can be
rastered while in operation and that Au and Ag colloid monolayers
have a high degree of reproducibility from area to area and within
a given sample. Therefore, several runs can be carried out on a
given substrate. Alternatively, the sample can be discarded and
another used--the value of the Au on a microscope-slide sized
substrate is a fraction of one cent. In other words, these
substrates are disposable. SERS has previously been demonstrated
using a microRaman apparatus with attomole mass sensitivity, and
detector sensitivity has improved considerably since then.
[0213] A possible pitfall in the device shown in FIG. 30 is the
presence of non-uniform fields on the colloid monolayer surface,
resulting in analyte migration. If this occurs, it can be
circumvented in two ways. The simplest approach is to make
electrical contact to both ends of the SnO.sub.2 substrate,
eliminating any potential drops across the surface. Another
alternative is to metallize the outer surface of the capillary end
and make contact to it.
[0214] A conservative estimate of the detection limit can be
calculated using a 5 .mu.m .times.5 .mu.m spot size and a SERS
signal/noise (S/N) ratio of 1000:1. Raman microprobes allow
focusing down to spot sizes down to 1 .mu.m.times.1 .mu.m, and with
the CCD detector now available, a S/N of >5000:1 is attainable.
With a monolayer coverage of 10.sup.14 molecules/cm.sup.2, one
calculates 25 .mu.m.sup.2.times.10.sup.14
molecules/cm.sup.2.times.1 cm.sup.2/10.sup.8 .mu.m.sup.2=25
.times.10.sup.6 molecules in the laser beam for a full monolayer.
Factoring in a detection limit where S/N=2. this corresponds to
50,000 molecules.
[0215] Surface-Confined Protein/Au Complexes and the use Thereof as
Biosensors
[0216] Studies indicate a future trend in the medical field will
lead to an increased use of implantable "biochips", responding to
biosensor analysis of health-related biological markers via
controlled release of surface-confined drugs. Before such
technology can be practically used, however, dramatic advances are
needed in biosensor architecture: the distinct fabrication
chemistries and different readouts (photons, current, frequency,
etc.) of the present devices preclude integration into miniature,
multi-sensor arrays. What is lacking is a uniform mechanism for
transducing a ligand binding event or a solution concentration into
a measurable quantity. In view of this need, as well as for the
need to replace immunoassays with more rapid methods. Applicants
have developed a series of biosensors based on complexes between
biomolecules and colloidal Au nanoparticles. It has been found that
appropriately sized colloidal Au nanoparticles can be used as
non-denaturing photon and electron "antennae" for biomolecules,
allowing biomolecule/Au colloid conjugates to be used as building
blocks for a variety of sensing mechanisms. More specifically, this
aspect of the invention is directed to biomolecule/Au complexes
that can be used to detect biological ligands (either small
molecules or proteins) through measuring of binding-induced changes
in electrical resistance, using the previously described
self-assembled metal colloid layers.
[0217] Discontinuous conductors surrounded by insulators are an
imporant class of materials that include evaporated thin metal
films and particle-loaded polymers. At low conductor concentrations
(p), these materials do not conduct electricity; at high p,
conductivity approaches bulk values. At a certain critical
concentration (p.sub.c, the percolation threshold), dramatic
increases in ac and dc conductivity are observed. In this threshold
region, small changes in insulator volume (which decrease conductor
concentration) lead to large changes in film resistance.
Accordingly, small molecules that induce swelling in polymers can
be detected by measuring the resistance changes in
polymer/conducting particle composites. Similarly, volume changes
in percolating Au/antibody films, induced by antigen:antibody
complex formation can be detected by simple resistance measurements
which can then be converted to analyte concentration data. Related
implementations include detection of receptors using arrays of Au
particles harboring surface-confined ligand, and detection of
oligonucleotides using surface-confined complementary
fragments.
[0218] As was previously noted, Au colloid-based macroscopic
surfaces are simple and inexpensive to prepare, stable, amenable to
assembly in large numbers and in any geometry, extremely
reproducible, capable of further modification (i.e. Ag deposition
to yield Ag-clad Au), SERS-active, and electrochemically
addressable. Further, surface confined protein/Au complexes retain
bioactivity, a property of the utmost importance for potential
sensor applications.
[0219] An important feature of the Au colloid-based substrates is
the control of feature size, spacing, and substrate coupling
available through self-assembly. Size control is simple, as
descibed above. Since solution-based surface formation is based on
particle diffusion, manipulation of interparticle spacing can be
achieved through a detailed understanding of the kinetics of
surface evolution. In addition, tremendous variation in coupling
between colloidal submonolayers and the underlying substrates is
available through modulation of the spacer film thickness.
[0220] These parameters are crucial in determining how metal
surfaces interact with proteins. Numerous studies using a variety
of spectroscopic techniques have shown that direct protein
adsorption on macroscopic metal surfaces leads to partial or
complete denaturation. The most likely, process for this
denaturation is through formation of thermodynamically favorable
protein-surface contacts that lead to reduced conformational
flexibility and, eventually, to unfolding. The denaturing
properties of macroscopic metal surfaces pose a serious problem for
biosensors, which typically require metals to transduce biological
signals.
[0221] It has been found that, unlike macroscopic metal surfaces,
small colloidal Au particles do not denature proteins as the high
curvature of the nanoparticles minimizes the surface area available
to the proteins. In fact, colloidal Au particles of .ltoreq.5 nm in
diameter are the same size as the proteins themselves. In contrast,
the diminished curvature of larger particles leads to approximately
the same characteristics noted with macroscopic Au surfaces, and
thus, it is generally accepted that larger Au particles, as well as
aggregates of smaller Au particles, have an increased tendency to
denature proteins.
[0222] The surface confined protein/Au complexes can be formed by
exposing the surface of a previously formed Au colloidal monolayer
to protein. Alternatively, a pre-formed protein/Au complex can be
immobilized from solution onto a substrate. The latter method is
preferable as it allows the particle:protein ratio and the
biological activity to be ascertained prior to immobilization. The
ability to prepare macroscopic, bioactive metal interfaces of well
defined structure is the key step to a uniform biosensor
architecture, and when coated with protein receptors, or
antibodies, such surfaces form the basis for a novel type of
biosensor based on ligand binding-induced conductivity changes.
[0223] While ligand-induced changes in the dielectric constant of
protein films have been proposed as a sensing mechanism, and
changes in solution conductivity are routinely used to measure cell
growth, the use of biopolymers as molecule-sensitive insulators in
composites with metal particles has not been previously suggested.
Such technology was heretofore unavailable as only Au nanoparticles
combine the high degrees of conductivity and biocompatibility
needed for operation of this type of biosensor, and until the
present discovery of a self-assembled colloidal Au monolayer, there
was no mechanism for the preparation of macroscopic, well-defined
two or three dimensional particle-based films having the degree of
coverage required for conductivity. The two dimensional particle
based films display a p.apprxeq.0. 16. The inventive method,
however, allows the interparticle spacing to be decreased (by, for
example, coating the Au particles with Ag ) to such a degree that a
p of about 0.60 is achieved. Alternatively, multilayers with p 0.4
to 0.6 can be formed. In the three dimensional regime, these are
conductive.
[0224] The varying of particle spacing has been found to have a
profound impact on the electronic and electrochemical properties of
surface-confined colloidal Au films. For example, in measuring the
sheet resistivity (measured by the four-point probe method) of a
12-nm diameter colloidal Au film on glass as a function of
absorbance at 520 nm, in which increased absorbances (which
correspond to decreased spacing) were effected by repetitive
exposure of the Au colloid-derivatized surfaces to
HOCH.sub.2CH.sub.2SH and colloidal Au in solution led to an abrupt
five order of magnitude drop in resistivity as the absorbance
increased from 0.4 to 0.6. Because this absorbance range
corresponds to relatively close packed monolayers (as opposed to
multiple layers), the data demonstrates that sensitive resistance
measurements on monolayers of protein/Au complexes can be
performed. Further, the insulator-conductor transition is extremely
sharp compared to those obtained with vapor-deposited thin Au
films, demonstrating that protein/Au complexes assembled to the
percolation threshold are extremely sensitive to volume
changes.
[0225] The colloidal Au monolayers described above have the
properties of a two dimensional system, surrounded by air. Because
three dimensional composites of conductive particles and insulating
polymers display a much lower P.sub.c, the use of Au colloid
multilayers is prefered to a monolayer arrangement. Therefore,
there is described below a method for the stepwise assembly of
conducting Au colloid multilayers from solution.
[0226] When a Au colloid monolayer on 3-aminopropyltrimethoxysilane
(APTMS)-coated glass is exposed to a molecule having two functional
groups that bind to or induce flocculation of colloidal Au
{2-mercaptoethanol, 2-mercaptoethylamine, HS(CH.sub.2).sub.6SH,
APTMS, polyvinylpyrrolidone, etc.}, immersion in colloidal Au leads
to additional particle binding. Repetitive dips (5-15 minutes for
the crosslinker, 5-60 minutes for the colloid) into bifunctional
organic crosslinker and colloidal Au solutions (with copious
rinsing between immersions) rapidly leads to formation of Au
colloid multilayers. Note that no interlayer registry of particles
is expected. This method is very general, and can be used with
colloidal Au nanoparticles of any diameter, although the lower
particle concentrations for standard preparations of both large and
small particles lead to longer derivatization times.
[0227] Tapping-mode atomic force microscopy (AFM) has been used to
monitor the growth of colloidal Au films. Images obtained after 2
and 5 exposures to 2-mercaptoethanol/colloidal Au indicate a porous
geometry, with neither close-packed coverage within a layer (in
accord with previous findings) nor registry between layers (FIG.
31). Importantly, these AFM images clearly indicate large areas of
uninterrupted particle contact after just two 2-mercaptoethanol/Au
treatments, suggesting the possibility of conductive paths.
[0228] Further support for metal-like attributes comes from
visible/near-infrared optical transmission spectra as function of
particle coverage for samples immersed between 1-12 times in
2-mercaptoethanol/12-nm Au. The spectra for low coverages match
those previously obtained for 2-D Au arrays or small Au aggregates
in solution: strongly absorbing in the visible and transparent in
the near-infrared (FIG. 32). As the particle coverage increases,
near-infrared transmission decreases markedly and becomes only
weakly wavelength dependent. Similar behavior is seen in evaporated
thin Au films, which also exhibit linear plots of transmittance vs.
p at 1500 nm or 2500 nm. The intersection point of these lines
(i.e. the point at which %T is wavelength-independent) corresponds
to P.sub.c In Au colloid multilayers, a similar plot shows no such
intersection (FIG. 33).
[0229] Nevertheless, at sufficiently high coverages, Au colloid
multilayers undergo an insulator-conductor transition. A plot of DC
resistance (plotted on a log scale) vs. particle coverage shows
that samples containing 15.times.10.sup.11 particles/cm.sup.2 are
insulating, with a resistance approaching 10.sup.8 .OMEGA..
Increasing particle coverage leads to an enormous decrease in
resistance; when the particle coverage is doubled to
30.times.10.sup.11/cm.sup.2, the resistance drops below 100
.OMEGA., a change of almost 6 orders of magnitude (FIG. 34).
Considering the extremely porous nature of these films, and the
presence of organic adsorbed 2-mercaptoethanol on the particles,
these films are remarkably conductive: the most conductive sample
(12 treatments) has a resistivity of 5.1.times.10.sup.-4
.OMEGA.-cm, a value roughly {fraction (1/200)} that of pure Au of
the sarne geometry. Au colloid multilayers on glass substrates are
thus more conductive than In-doped SnO.sub.2, for example, and can
be used for routine voltammetric measurements. Electron transfer
between particles likely occurs by activated hopping between
nearest neighbors, as previously observed for granular metal
films.
[0230] Interestingly, the Au volume fraction does not change with
increasing particle coverage, evidenced by a linear plot of
coverage vs. number of immersions in Au (FIG. 33). In other words,
each step in the multilayer assembly adds the same amount of Au;
the slope of the line (2.66.times.10.sup.11/cm) corresponds to
p=0.38. This value is below that needed for 2-D percolation, but
sufficient for 3-D percolation, suggesting that the dramatic
decrease in resistance results from establishment of multiple 3-D
percolation paths.
[0231] Devices for determining the presence and/or concentration of
a biomolecule, with a biosensor comprising a receptor, antibody, or
biocomplement-coated Au colloidal layer can be constructed in the
same manner as devices using the conventional discontinuous
conductor in insulator-based sensors. As shown in FIG. 36, such a
device could include, for example, the inventive biosensor 101,
means for applying a voltage across the biosensor 102, means for
measuring an electrical characteristric (e.g. resistance) of the
biosensor 103 and means for converting resitivity data to analyte
(antigen) concentration 104. An alternative, but equally viable
implementation of the device shown in FIG. 36 involves measurement
in air. The present technology is not limited to any specific class
of biomolecules and is applicable to biosensors for detecting any
biomolecule for which there is a corresponding binding partner
[0232] Binding of a colloidal Au particle monolayer to a preformed
Au colloid monolayer or multilayer changes the effective film
thickness, a fact that can be used to fabricate a biosensor based
on changes in surface plasmon resonance (SPR) signals.
[0233] To wit, a Au colloid monolayer or mulitlayer of insufficient
thickness to achieve resonance (at a given excitation
wavelength/angle of incidence) is coated with an antibody. This
surface is then exposed to analyte (antigen). After rinsing, the
sample is exposed to a Au colloid: antibody complex (the same
antibody as bound to the surface). Binding of the Au:antibody to
the surface-bound antigen leads to an increase in Au film thickness
leading to resonance. Thus, in contrast to a conventional SPR
method, which measures small, binding event-induced changes in
reflectivitv of a thin Au film under resonant plasmon excitation,
the binding event described above leads to an enormous increase in
reflectivity. This novel approach has several advantages. First,
the "sandwich" immunoassay approach is widely used in
immunochemical analytes, including enzyme-linked immunoassays
(ELISAs). Thus, there is little doubt that chemistry described
above will obtain. Second, this approach can be used to sense small
molecules, not routinely possible with current SPR technology.
Third, the sensing chemistry is general for any analyte B in an
A-B-C ternary complex. For example, A and C can be
non-complementary strands of DNA attached to surface-bound and
solution phase DNA, and B can be an oligonucleotide (to be sensed)
with ends that are complementary to A and C.
[0234] Optimization of the Surface Characteristics of the Metal
Colloid Monolayer
[0235] Preferably, for any specific application of the metal
colloid monolayers, the surface characteristics, i.e., Au particle
coverage (density), Au particle size, Ag cladding thickness, will
be optimized. To determine the optimal surface characteristics, a
solution-based combinatorial strategy for synthesis of surfaces
exhibiting nanometer-scale variation in mixed-metal composition and
architecture has been developed. With this method, continuous or
stepped gradients in the size and number density of surface
features can be generated simultaneously on different regions of a
single substrate. In order to demonstrate this technique, the
surface enhanced Raman scattering (SERS) response of a glass slide
upon which the coverage of colloidal Au and the extent of Ag
coating were varied and optimized. Over a 2 cm .times.2 cm sample
area, changes in enhancement factor (EF) spanning four orders of
magnitude were realized, and the changes in nanostructure
responsible for these effects were determined using atomic force
microscopy (AFM). The results show significant changes in SERS EFs
over small alterations in surface morphology, demonstrating the
power of solution-based combinatorial approaches for optimizing the
characteristics of mixed-metal materials.
[0236] The general combinatorial assembly approach is shown in FIG.
37. The number of colloidal Au particles bound to functionalized
surfaces can be predicted from immersion times in colloidal
solution using initial (time).sup.1/2 kinetics. Thus, when a
3-mercaptopropyltrimethoxysilane (MPTMS)-coated glass slide
attached to a motorized translation stage is immersed at a fixed
rate into a 17 nM aqueous solution of 12-nm diameter colloidal Au,
a gradient in particle coverage is generated in the direction of
immersion. After sample rotation by 90.degree., fixed-rate
immersion into a Ag.sup.+-containing solution designed to
specifically deposit Ag on Au leads to a gradient in particle size
over the new immersion direction. The resulting surface exhibits a
continuous variation in nanometer scale morphology as defined by
particle coverage and particle size that results from known,
repeatable immersion conditions. In preparing the gradient surface,
a stepper motor with a 2.degree. step yields a 3 .mu.m translation.
Over a 3 cm.times.3 cm region, it is therefore possible to generate
10.sup.8 different, 9 .mu.M.sup.2 nanostructures. Macroscopic
regions with uniform morphology can be prepared by very rapid
successive steps followed by a long delay. These stepwise gradients
are especially useful in examination of bulk changes in physical
properties. For example, optical changes linked to growth of the
characteristic Ag plasmon band at 400 nm with increasing Ag
coverage, evident to some degree in continuous gradient samples,
are clearly visible as square regions with uniform shading. FIG. 38
shows optical spectra of 9 different Au/Ag coatings, showing
evolution of the Ag plasmon mode.
[0237] To demonstrate the SERS screening approach, the SERS
response on spatially discrete areas of a 2.5.times.2.0 cm
continuous gradient substrate were determined; the results are
shown in FIG. 39. The data clearly show regions where the SERS
intensity was maximized. A second example in FIG. 40 shows similar
data plotted in a different way. Here, increasing SERS intensity is
denoted by increasing depth of shading on a grid, with the most
intense spectra corresponding to the darkest squares.
[0238] An example of combinatorial signal optimization was
determined as follows. SERS signal for adsorbed
p-nitrosodimethylaniline (p-NDMA) was measured over a 2 cm.times.2
cm sample exhibiting continuous gradients in Au coverage and Ag
cladding thickness. The immersion times for sample preparation (0-6
hr for the Au gradient, 8-22 min for the Ag gradient) were
established by determination of the most enhancing region from an
initial, large gradient SERS screen on a 2 cm.times.2 cm sample
(0-12 hr for Au, 0-30 min for Ag). FIG. 41 shows a spatial map of
the background-corrected SERS intensity for the phenyl-nitroso
stretch at 1168 cm.sup.-1. Intensity trends from 15 initial spots
at 0.4 cm intervals were used to locate the most SERS-active area
on the sample. Further interrogation at smaller intervals revealed
a region that was at least 10.sup.4-fold more enhancing than the
least SERS-active domains of the substrate. The location of this
region, a narrow diagonal band of moderate Au particle coverage and
Ag coating, is surprising, suggesting that maximum EFs are obtained
at relatively fixed interparticle spacing. Such spacing can be
achieved either by a thin coat of Ag on closely-spaced Au
particles, or with larger Ag coatings on particles exhibiting
slightly larger spacing.
[0239] AFM was used to determine the nanometer-scale morphology at
positions of interest (FIG. 42). In FIG. 42, the image labels A-D
correspond to representative morphologies at locations marked by
shaded squares in FIG. 41. Sampling positions were chosen to
highlight variation in Ag cladding thickness (i.e. to span the
vertical gradient) as opposed to variation in number density of
particles. Accordingly, differences in particle coverage in images
A, B and D are not significant, and represent positional
uncertainty in the horizontal (coverage) gradient. Immersion time
in Ag.sup.+increases in the order D>C>B>A (A=0 min Ag),
but SERS intensity follows the pattern C>B>D>A. Section
analyses reveal that mean particle size increases from A to C as
expected, but decreases in D. Two related effects contribute to
this result; the formation of small Ag particles between Ag clad Au
colloids, and the loss of monolayer structure (i.e. formation of
multilayers) upon extended Ag exposure. Evidently, interconnection
of closely-spaced particles to yield surfaces with a morphology
resembling roughened electrodes has a deleterious effect on
SERS.
[0240] The wavelength dependence of the SERS intensity is shown in
FIG. 43. The data show that the highest SERS enhancement factors
were obtained using 647-nm excitation, even though the v.sup.4
Raman effect favors a lower excitation wavelength. Using this
wavelength, the nanostructure was optimized over two iterations, as
shown in FIGS. 44A and 44B. These figures show position-dependent
SERS intensity maps. The results of the first map were used to
optimize the maximum signal in the second map. FIG. 44C is a SERS
intensity map generated using 28-nm diameter Au nanoparticles. FIG.
44C shows that the use of the larger core particles leads to an
increase in overall signal, as evidenced by the different intensity
scales.
[0241] A stepped gradient synthetic protocol was prepared that
allowed a 10.times.10 array of separate glass slides to be
simultaneaouly derivatized and characterized. Using this apparatus,
a 5.times.5 array was selected for further analysis. The 3D bar
graph shown in FIG. 45 depicts particle coverage and height
measurements from several AFM images collected from a 5.times.5
array of Ag-clad-Au samples. The Au particle diameter before
exposure to Ag was 14 nm. Immersion times were chosen using
previous combinatorial data as a guide. The Z-scale on the graph
shows the number of particles/.mu.m.sup.2, reflecting the
increasing Au particle coverage with exposure time. The number
written on each bar is the average particle height on the sample,
as determined by AFM section analysis. As expected, the particle
height increases with continued Ag plating solution exposure.
[0242] Representative AFM images, uv-vis spectra, and SERS spectra
from this data set are shown in FIGS. 46A and 46B, FIGS. 47A and
47B, FIGS. 48A, 48B and 48C and FIGS. 49A, 49B and 49C. For
Ag.sub.1, Ag.sub.2, and Ag.sub.3, the samples were exposed to
colloidal Au for the same time, therefore differences in optical
spectra and SERS intensity arise from different amounts of Ag
cladding. All three optical spectra exhibit a typical Mie
absorbance at .about.430 nm for Ag, and a collective plasmon band
(.about.650) indicating at least partial electronic coupling
between particles. Similarly, for Au.sub.1, Au.sub.2, and Au.sub.3,
coverage of Ag is fixed. The data show that changes in SERS
intensity from non-optimal Au colloid coverage are significantly
more dramatic than those seen by varying the Ag cladding.
[0243] In another example, images and spectra were collected from a
combinatorial sample series using 28-nm diameter colloidal Au. Each
sample was exposed to colloidal Au for the same amount of time.
Total particle coverage needed to achieve high SERS signals is
significantly lower when using larger colloid cores. Optical
spectra exhibit an Au Mie resonance at .about.530 nm in addition to
the collective plasmon and Ag bands found in the Au/Ag optical
spectra having smaller Au particles, as shown by FIGS. 50A and 50B.
As shown, SERS intensity is significantly increased with larger Au
cores (FIG. 50B). AFM for these samples are shown in FIGS. 51A, 51B
and 51C.
[0244] The ability to vary surface nanostructure combinatorially
from solution is extremely important for optimizing the surface
characteristics of self-assembled metal colloid monolayers for
SERS, surface plasmon resonance, sum frequency generation and
electrochemistry, where substrate composition and topography are
critical. The utility of this approach in materials synthesis is
further augmented by the widespread availability of dispersible
metal-containing nanoparticles and the numerous routes to metal
deposition from complexed metal cations. Moreover, recently
developed Raman imaging methods are intrinsically more efficient
than the point-by-point mapping method described herein. With
tunable filter imaging spectrometers and charge-coupled device
detectors, it should be possible to simultaneously probe up to
10.sup.6 sample structures.
[0245] Thus is described my invention and the manner and process of
making and using it in such full, clear, concise, and exact terms
so as to enable any person skilled in the art to which it pertains,
or with which it is most nearly connected, to make and use the
same.
* * * * *