Barcoded metal nanowires: Optical reflectivity and patterned fluorescence

Article abstract of DOI:10.1021/jp034139i

Metallic barcodes have been reported recently in which the size and location of distinguishable metal segments (e.g., Au and Ag) are used to encode information [Nicewarner-Pe?a et al., Science 2001, 294, 137-141]. Barcode readout is accomplished by conventional brightfield reflectance optical microscopy. Herein we report the wavelength-dependent optical reflectivity of individual stripes in metallic barcodes, and how this wave-length-dependence impacts the intensity of fluorescence from sandwich immuno- and hybridization assays performed on the particle surface. The encoded particles used in this study were striped nanowires on the order of 4-8 μm in overall length, with individual stripes typically on the order of 1-2 μm, and diameters ～320 nm. Reflectivity measurements were made for several metals (Ag, Cu, Co, Ni, Pd, and Pt) relative to Au, which was used as an internal standard. Despite the subwavelength diameters of these nanowires, good agreement was found between experimentally determined reflectivities and bulk metal values. Under some conditions, fluorescence intensity patterns corresponding to the underlying metal segments could be observed. We find that the ratio of fluorescence intensities on different metal segments correlate with the metal reflectivity ratios at the excitation and emission wavelengths for the dye. Surface roughness and chemical effects may also play a role for some metals. We have shown that by choice of the underlying metal, particle striping patterns can be accentuated or hidden in the fluorescence image. This is demonstrated in a triplexed DNA hybridization assay.

Full text of DOI:10.1021/jp034139i

7360
J. Phys. Chem. B 2003, 107, 7360-7367
Barcoded Metal Nanowires: Optical Reflectivity and Patterned Fluorescence†
Sheila R. Nicewarner-Pe n˜ a, Anthony J. Carado, Kristen E. Shale, and Christine D. Keating*
Department of Chemistry, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802
ReceiVed: January 17, 2003; In Final Form: April 3, 2003
Metallic “barcodes” have been reported recently in which the size and location of distinguishable metal segments
(e.g., Au and Ag) are used to encode information [Nicewarner-Pe n˜ a et al., Science 2001, 294, 137-141].
Barcode readout is accomplished by conventional brightfield reflectance optical microscopy. Herein we report
the wavelength-dependent optical reflectivity of individual stripes in metallic barcodes, and how this wave-
length-dependence impacts the intensity of fluorescence from sandwich immuno- and hybridization assays
performed on the particle surface. The encoded particles used in this study were striped nanowires on the
order of 4-8 µm in overall length, with individual stripes typically on the order of 1-2 µm, and diameters
∼
320 nm. Reflectivity measurements were made for several metals (Ag, Cu, Co, Ni, Pd, and Pt) relative to
Au, which was used as an internal standard. Despite the subwavelength diameters of these nanowires, good
agreement was found between experimentally determined reflectivities and bulk metal values. Under some
conditions, fluorescence intensity patterns corresponding to the underlying metal segments could be observed.
We find that the ratio of fluorescence intensities on different metal segments correlate with the metal reflectivity
ratios at the excitation and emission wavelengths for the dye. Surface roughness and chemical effects may
also play a role for some metals. We have shown that by choice of the underlying metal, particle striping
patterns can be accentuated or hidden in the fluorescence image. This is demonstrated in a triplexed DNA
hybridization assay.
Introduction
length), and investigate fluorescence intensity as a function of
underlying metal identity and fluorophore excitation/emission
Metal particles are known for their interesting and often useful
optical and electrical properties. Observed properties are
dramatically impacted by size, shape, and interparticle spacing.
For example, metal nanospheres tens of nanometers in diameter
spectra. In fluorescence-based assays employing striped nanow-
ires of this type, we had previously observed that the striping
1
-3
15,21
pattern was visible in the fluorescence image in some cases.
Here, we show that the striped fluorescence observed on
barcoded nanowires correlates with the wavelength-dependent
reflectivity of the underlying metals combined with the spectral
properties of the fluorophore used.
1
-3
are intensely colored, due to surface plasmon absorbance,
while bulk metals are highly reflective.4,5 The extinction spectra
of small metallic nanowires exhibit two plasmon resonance
features, corresponding to excitations along their long and short
axes.6 Such particles can be prepared by chemical reduction
For Au/Ag and Au/Pt striped nanowires, fluorophore surface
coverage is very similar on the different metal segments. It is
therefore possible, by appropriate choice of fluorophore and of
metals for barcode stripes, to either accentuate or obscure the
underlying striping pattern in fluorescence images. This is
demonstrated in a triplexed DNA hybridization assay on
barcoded Au/Ag nanowires, where three simultaneous sandwich
hybridization assays are conducted on the nanowires, with
fluorophores chosen to either visualize or obscure the particle
striping pattern.
-11
12,13
methods
or by electrodeposition within the pores of alumina
6-9,14
or polycarbonate templates.
As nanowire length and/or
width increases, scattering and reflectance become more intense.
Nanowires several micrometers in length have been prepared
by templated electrodeposition;14 optical microscopy has been
used to image these wires for diameters from 320 down to 30
1
5-20
nm.
Segmented nanowires composed of adjacent metallic
1
5,17-20
stripes can also be prepared by this method.
We recently reported the use of striped metal nanowires
several micrometers long and ∼320 nm in diameter as “bar-
coded” particles for possible applications in multiplexed bio-
Materials and Methods
15
analysis. The metal striping pattern produced during synthesis
is used to encode information such as the identity of a
biomolecule bound to the nanowire surface; this pattern can be
Materials. Gold (Orotemp 24-1 troy oz/gal), silver (Silver
1025-4.5 troy oz/gal), nickel (Nickel S), copper (Cu U), and
palladium (Pallaseed VHS 8 g/qt) plating solutions for nanowire
synthesis were purchased from Technic, Inc. Pt and Co were
electrodeposited from solutions prepared in house. Pt was
electroplated from 0.01 M H2PtCl6 in 0.5 M H2SO4. Co was
electroplated from 0.36 M CoSO4‚7 H2O, 0.19 M Na3citrate,
and 0.1 M citric acid adjusted to pH 5 with 10.0 M NaOH. Ag
wire (99.999%) and Ag shots for evaporation onto the backside
of alumina membranes were purchased from Acros. A mini
centrifuge with maximal relative centrifugal force (RCF): 2000g
was purchased from VWR Scientific Products. Immersion oil
(nD ) 1.5150) for microscopy was purchased from Cargille
Laboratories, Inc. Au finder grids were purchased from Electron
1
5,18
read out via simple reflectance microscopy.
Most of the
literature on metal particles describes absorbance and scattering
for much smaller nanowires or spheres; little has been reported
on the optical properties of nanowires in this size range or having
this level of compositional complexity.
Herein we describe the reflectivity for a variety of metals
incorporated as 1 to 2 micrometer stripes in barcoded nanowires
(overall dimensions ∼320 nm in diameter and 4-8 µm in
†
Part of the special issue “Arnim Henglein Festschrift”.
*
Corresponding author. E-mail: keating@chem.psu.edu.
1
0.1021/jp034139i CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/31/2003

Barcoded Metal Nanowires
J. Phys. Chem. B, Vol. 107, No. 30, 2003 7361
TABLE 1: Oligonucleotide Sequences Used in This Work
sequence number
sequence (5′ to 3′)
description
Ia
5′-biotinsaaa AAA AAC TCC TTG CGC ACG T
CGT GCG TCA AAA ATT ACG TGC GGA AGG AGT T
AAT TTT TGA CGC ACG-3′-fluora
5′-biotinsaaa AAA ACG TTG TCT GAT GCG TCA
ACA CAG ACG TAC TAT CAT TGA CGC ATC AGA CAA CGT
ATG ATA GTA CGT CTG TGT-3′-fluora
22-mer capture sequence 1
Ib
31-mer complementary to I
Ic
fluorophore labeled 15-mer for detection of Ib
24-mer capture sequence 2
36-mer complementary to II
fluorophore labeled 18-mer for detection of IIb
21-mer capture sequence 3
31-mer complementary to III
IIa
IIb
IIc
IIIa
IIIb
IIIc
IV
5′-biotinsaaa AAA CGA TAA CGG TCG GTA
TAT GGC CAG CTC CCG TAC CGA CCG TTA TCG
CGG GAG CTG GCC ATA-3′-fluora
fluorophore labeled 15-mer for detection of IIIb
12-mer noncomplementary control
CGC ATT CAG GAT
a
Two different fluorophores were used for oligonucleotide labeling: F ) 6-carboxyfluorescein (6-FAM), λex ) 495 nm, λem ) 520 nm; R )
rhodamine-X (ROX), λex ) 588 nm, λem ) 608 nm. Note that these λex and λem values are essentially identical to those of FITC and Texas Red,
respectively, which were used to label antibodies in this work.
Microscopy Sciences (200 mesh). The following chemicals were
purchased from Calbiochem: R-rabbit IgG (H&L chain) mono-
specific for RIgG heavy and light chains conjugated to FITC
or Texas Red, and R-rabbit IgG (Fc specific); both isolated from
goat. Bovine serum albumin (BSA), R-globulin and protease-
free, was purchased from Jackson Immuno Research Labora-
tories, Inc. Bis(3-aminopropyl)-terminated poly(ethylene glycol)
by treatment with 3 M NaOH for 30 min, and the resulting
nanowire suspension was centrifuged at 2000g for 30 s to collect
the nanowires. The nanowires were resuspended and rinsed in
H2O several times to remove residual base and salts. They were
then washed at least three times in ethanol, and stored in this
solvent until ready to use.
Optical Microscopy. Data were acquired on a Nikon TE-
300 inverted microscope equipped with a Hamamatsu ORCA
12 bit high-resolution, 6.7 µm pixel, digital cooled CCD camera
(1024 × 1024). Nanowires were imaged using a CFI Plan fluor
100× oil immersion lens (N.A. ) 1.3), and Image-Pro Plus
software. The excitation source for both fluorescence and
reflected light images was a 100 W Hg lamp. Bioassay samples
in aqueous solution were mounted between a glass slide and
cover slip using a Secure-Seal spacer (Molecular Probes).
Reflectance optical microscopy images were acquired using a
Nikon bright field reflectance filter set containing a 50/50 beam
splitter. Band-pass filters at 430, 490, 520, 580, and 600 ( 5
nm (Omega Optical) were used for wavelength selection. Un-
derivatized striped rods used for reflectivity studies were
dropped onto glass slides, dried under a light stream of argon,
and coated with a drop of immersion oil followed by a coverslip
for imaging.
For fluorescence images, samples were kept in the dark prior
to imaging, and fluorescence images were taken before reflec-
tivity images to prevent photobleaching. All exposure times were
less than 1 s. Fluorescence images were obtained using Nikon
filter cube sets. Two green and two red fluorophores were used
in this work. Antibodies were labeled with either fluorescein
isothiocyanate (FITC) or Texas Red (TR), while oligonucle-
otides were labeled with either 6-carboxyfluorescein (6-FAM)
or rhodamine-X (ROX). FITC and 6-FAM have nearly identical
excitation and emission maxima (λex and λem, respectively);
fluorescence images for these probes were acquired using a
Nikon FITC cube containing the following filters, excitation:
465-495 nm, dichroic: 505 nm, and emission: 515-555 nm.
Texas Red (TR), and rhodamine-X (ROX) images were acquired
using a Nikon “wide green” filter cube containing the following
filters, excitation: 515-555 nm, dichroic: 565 nm, and
emission: g 590 nm.
[amino PEG] was purchased from Aldrich. NeutrAvidin was
purchased from Pierce. Calf thymus DNA was purchased from
Sigma. Oligonucleotides were purchased from Integrated DNA
technologies, Inc (IDT). All H2O was distilled and purified to
at least 18.2 MΩ through a Barnstead Nanopure system.
Nanowire Synthesis. Anodisc alumina membranes (25 mm
diameter, Whatman) were used as templates for the synthesis
of striped nanowires. The diameters of the pores were nominally
2
00 nm; however, SEM measurements of the membranes
indicate that the pores widen in the membrane channel giving
average diameters of 318 ( 50 nm.1 These data are supported
by transmission electron microscopy analysis of nanowires after
removal from the templates. Synthesis of striped nanowires was
performed on the basis of a modification of previously described
4,22
6
-9,22,23
18
methods
and has been described in detail elsewhere.
In brief, the backside (the side attached to the supporting ring)
of an alumina membrane was coated with ∼500 nm of Ag by
thermal evaporation. The membrane was then placed into an
electrochemical cell where an additional ∼1 µm of Ag was
electrodeposited onto the evaporated Ag film. This was done
to ensure complete coverage of membrane pores and to prevent
leakage during electrodeposition. Following Ag deposition, the
membrane was removed from the cell, reversed, and placed back
into the cell, pore side up, so that more Ag could be electrode-
2
posited (-0.55 mA/cm for 20 min) into the membrane. This
step is included due to template irregularities near the membrane
surface (smaller, interconnected pores); this region is filled with
sacrificial Ag prior to nanowire deposition. By changing the
plating solution at defined intervals for the metal of interest,
the desired striping pattern was achieved. A variety of nanowire
striping patterns were used in this work, including several
different Au/Ag patterns, Au-X-Au where X ) Co, Cu, Ni,
Pt, or Pd, and Ni-Au-Ag. In general, widths as determined
from TEM were ∼320 nm and overall lengths ranged from 4
to 8 µm, with each stripe ∼1 µm or longer. Exact particle and
stripe dimensions varied from batch to batch within this range;
within-batch standard deviations in nanowire length were on
the order of 10%. When synthesis was complete, the cell was
disassembled, and the membrane was removed and thoroughly
rinsed with H2O. The silver backing was dissolved by the
addition of 4 M HNO3 onto the evaporated Ag side of the
membrane for 5 min after which the membrane was rinsed
extensively with H2O. The alumina membrane was dissolved
Electron Microscopy. Field emission-scanning electron
microscopy images (FE-SEM) were acquired for uncoated
nanowire samples using secondary electron detection on a LEO
1
8,24
1530 FE-SEM.
Transmission electron microscopy (TEM)
images were acquired on a JEOL 1200EXII located at the PSU
Life Sciences Electron Microscope Facility.
Sandwich Immunoassay Procedure. An aliquot of nano-
wires (∼100 µL) was removed from the ethanolic stock solution,
and rinsed twice in H2O (1 mL each rinse), and once in 50 mM
Na phosphate buffer pH 7.2 (1 mL). Nanowires were derivatized

7362 J. Phys. Chem. B, Vol. 107, No. 30, 2003
Nicewarner-Pe n˜ a et al.
Figure 1. Wavelength-dependent reflectivity for bulk metals. Data
plotted from ref 25.
with the capture antibody (R-RIgG Fc specific) by suspension
into 500 µL of a 0.05 mg/mL solution. This was allowed to
proceed for 1 h at room temperature, after which the samples
were rinsed with 1 mL of the 50 mM Na phosphate buffer pH
7
.2. Following direct adsorption of the capture antibody,
nanowires were further derivatized with bis-aminated poly-
ethylene glycol) (amino PEG) using a 1% solution of amino
(
PEG in the 50 mM Na phosphate buffer. Following adsorption
of amino PEG for 15 min., the nanowires were rinsed with 50
mM Na phosphate buffer three times (1 mL each rinse).
Nonspecific sites on the capture antibody were blocked using a
1
7
% solution of BSA in the 50 mM Na phosphate buffer pH
.2. Blocking of the capture antibody was allowed to proceed
for 15 min at room temperature, after which the nanowires were
rinsed two times in the 50 mM Na phosphate buffer (1 mL each
rinse), re-suspended into 500 µL of a 0.05 mg/mL solution of
the analyte, RIgG, and allowed to react for 60 min at room
temperature. Samples were rinsed with 1 mL of the 50 mM Na
phosphate buffer. Nanowires were then re-suspended into ∼300
µL of the fluorescently labeled detection antibody, R-RIgG H&L
FITC or Texas Red conjugate. Incubation with the detection
antibody was allowed to proceed for 60 min at room temper-
ature, after which the nanowires were rinsed three times in 500
µL of the 50 mM Na phosphate buffer. During adsorption of
capture antibody, blocking steps, and incubation with the analyte
and detection antibodies, samples were placed onto a vortexer,
equipped with an attachment for holding eppendorf tubes.
Samples were gently shaken in order to keep the nanowires
suspended. Samples were also sonicated briefly (∼10-15 s)
between rinsing steps to ensure good re-suspension of the
nanowires and displacement from the eppendorf tube wall.
Sandwich DNA Hybridization Assay. Aliquots (600 µL)
from each of three different barcoded nanowire patterns Ag-
Ag-Au-Ag-Ag (1), Au-Ag-Au-Au-Au (2), and Au-
Ag-Au-Ag-Au (3) were removed from the stock solutions
stored in ethanol. The nanowires, each placed in different
eppendorf tubes, were rinsed with 1 mL of H2O three times,
then with 1 mL of 50 mM Na phosphate buffer pH 7.2, after
which they were re-suspended into 1 mL of 0.05 mg/mL of
NeutrAvidin (NA) in 50 mM Na phosphate buffer. The NA
was allowed to adsorb onto the nanowire surface for 2 h at room
temperature during gentle shaking. Following adsorption of NA,
the nanowires were allowed to react with 500 mL of a 1%
Figure 2. Ratio of reflected intensities for various metals vs Au for
bulk materials (4) and striped particles (b) at 430 nm (A), 520 nm
(B), and 600 nm (C). Experimental values were determined by
reflectance optical microscopy.
solution of amino PEG in 50 mM Na phosphate buffer for an
additional 15 min at room temperature, then rinsed twice with
1 mL of the 50 mM Na phosphate buffer. To provide specific
capture chemistry for a solution-phase oligonucleotide (the
analyte), nanowires of type 1, 2, and 3 were derivatized with a
unique biotinylated oligo sequence Ia, IIa, and IIIa, respectively
(Table 1 shows the DNA sequences used in this work). The
nanowires were re-suspended into 1 mL of a 2 mM solution of
the biotinylated oligo in 0.3 M NaCl/10 mM Na phosphate
buffer pH 7.0. Binding of the biotinylated oligo with NA was
allowed to proceed for 2 h at room temperature with gentle
shaking. Following attachment of the biotinylated oligo, the
samples were rinsed with 1 mL of the 0.3 M NaCl/10 mM Na
phosphate buffer twice. Nanowires from all three reactions were
mixed together and then separated into eight different aliquots

Barcoded Metal Nanowires
J. Phys. Chem. B, Vol. 107, No. 30, 2003 7363
TABLE 2: Ratio of Fluorescence and Reflectivity on
Striped Particles
observed
intensity ratio
bulk
a
intensity ratiob
Pt/Au Wires
FITC fluorescencec
reflectivity @ 490 nm
reflectivity @ 520 nm
0.98 ( 0.05
1.28 ( 0.11
0.94 ( 0.07
1.42
0.96
Texas Red fluorescencec
reflectivity @ 580 nm
reflectivity @ 600 nm
0.60 ( 0.30
0.72 ( 0.08
0.69 ( 0.07
0.71
0.71
Ag/Au Wires
FITC fluorescenceb
reflectivity @ 490 nm
reflectivity @ 520 nm
2.21 ( 0.34
2.21 ( 0.23
1.51 ( 0.13
2.08
1.44
Texas Red fluorescencec
reflectivity @ 580 nm
reflectivity @ 600 nm
1.00 ( 0.06
1.17 ( 0.24
1.04 ( 0.08
1.01
1.00
Ni/Au wires
FITC fluorescenceb
reflectivity @ 490 nm
reflectivity @ 520 nm
0.48 ( 0.43
1.05 ( 0.10
0.88 ( 0.05
1.31
0.91
Figure 4. Optical reflectivity for striped rods with Co, Au, Ni, and
Ag stripes. (A) Optical reflectivity image of an Au-Co-Au nanowire
with 430 and 600 nm illumination. The Co segment is more reflective
than the Au segment in the blue, but less reflective in the red. (B)
Optical reflectivity image of Ni-Ag-Au wires with 430 and 600 nm
illumination. The three metals can be distinguished by intensity at 430
nm and by their change in relative intensity when illuminated with
Texas Red fluorescencec
reflectivity @ 580 nm
reflectivity @ 600 nm
0.52 ( 0.13
0.65 ( 0.07
0.67 ( 0.04
0.68
0.68
a
Ratios are reported as M/Au, where M ) Pt, Ag, or Ni. Reported
reflectivity and fluorescence ratios are the average values ( standard
deviation from approximately 50 particles, with intensities measured
6
00 nm light. Scale bars are 5 µm.
b
at three locations per metal within each particle. Intensities listed are
c
expected ratios based on bulk metal from Figure 1. FITC λex ) 494
buffer pH 7.0). Analyte solutions were spiked with 2 mM of
oligonucleotide sequence IV and 0.5 mg/mL calf thymus DNA
to decrease nonspecific interactions between noncomplementary
oligos. The first hybridization was allowed to proceed for 4 h
at 40 °C, after which, the samples were rinsed with 1 mL of
the hybridization buffer twice. Each of the samples was then
separated into half for a total of sixteen reactions (this was
necessary so that each reaction could be hybridized with both
a 6-FAM labeled and ROX labeled detection oligo). The samples
were re-suspended into 200 mL of a 2 mM mixture of all three
detection oligos, Ic-R or Ic-F, IIc-R or IIc-F, and IIIc-R or
IIIc-F that was spiked with 2 mM IV and 0.5 mg/mL calf
thymus DNA. This second hybridization was allowed to proceed
for 4 h at 40 °C. Following the second hybridization, the samples
were rinsed in 1 mL of 1% SDS/0.3 M NaCl/10 mM Na
phosphate buffer pH 7.0 solution. Then, 1 mL of 0.1% SDS/
c
nm, λem ) 518 nm. Texas Red λex ) 587 nm, λem ) 602 nm.
0
1
.3 M NaCl/10 mM Na phosphate buffer, and a final rinse of
mL 0.3 M NaCl/10 mM Na phosphate buffer. During
adsorption of NA and blocking with amino PEG, samples were
placed onto a vortexer, equipped with an attachment for holding
eppendorf tubes. Samples were gently shaken in order to keep
the nanowires suspended. During hybridizations, samples were
attached to a horizontal rotator (to keep the particles suspended
in solution), which could be placed into a water bath. Samples
were also sonicated briefly (∼10-15 s) between rinsing steps
to ensure good re-suspension of the nanowires and displacement
from the eppendorf tube wall.
Figure 3. Ag degradation in underivatized Au-Ag-Au striped
particles. FE-SEM images of as-prepared (A), and of the same particles
Results and Discussion
2
after aging for 14 days in H O (B). Similar effects were observed for
particles stored dry in air. (C) Particles stored in ethanol for over 1
year prior to FE-SEM imaging. Scale bars are 2 µm.
Optical Characterization of Striped Particles. The barcode
patterns of striped nanowires are visualized by the differential
reflectivity of adjacent metal stripes. We have reported that the
reflectivity ratio at 430 nm illumination for segments within
striped nanowires closely matches that for bulk metal.15 Here,
we further characterize the optical reflectivity of Au, Co, Ni,
Pt, Cu, Ag, and Pd nanowire segments at several wavelengths.
For this study, we prepared striped nanowires with 320 ( 30
nm diameter and several micrometers total length. To facilitate
for reaction to various combinations of the oligonucleotide
analytes, Ib, IIb, and IIIb. An amount of 400 µL of a 2 mM
solution of each analyte combination was used during the first
hybridization step. For the control sample, which did not contain
any analytes, 400 mL of hybridization buffer was used
(hybridization buffer was 0.3 M NaCl/10 mM Na phosphate

7364 J. Phys. Chem. B, Vol. 107, No. 30, 2003
Nicewarner-Pe n˜ a et al.
in H2O. The Ag segments become rough and pitted over time
as a result of oxidation, while Au segments are unchanged.
Similar results are observed for Ag-containing nanowires stored
dry in air. In contrast, nanowires stored in ethanol are stable
indefinitely; Figure 3C shows an FE-SEM image of Au-Ag-
Au particles after 1 year in ethanol. Cu segments are much more
susceptible to oxidation; damage is observable via optical
microscopy after only 24 h storage in H2O (data not shown).
Cu segments could be protected by storage in ethanol or dry
under argon. Note that for the remainder of experiments
described here, no special efforts were taken to avoid air
oxidation; nanowires are routinely stored in ethanol prior to use,
and in buffered H2O after derivatization with biomolecules.
The data in Figure 2 suggest the possibility of optically
differentiating more than two metals in a single nanowire. While
contrast between Ag and Au under blue illumination is the
highest of any metal pair, other pairs of metals can be readily
distinguished. Images of Au-Co-Au and Ni-Ag-Au nano-
wires are shown in Figure 4 (additional examples and wave-
lengths are available as Supporting Information). With 430 nm
illumination, Co segments appear brighter than adjacent Au
segments, while at 600 nm Au segments appear brighter, as
expected based in Figures 1 and 2. Since the reflectivities of
Co, Ni, Pd, and Pt are all quite similar (Figure 1), any of these
metals can be incorporated as stripes in Au particles to give
similar images. When Co, Pd, Pt, or Ni is incorporated in
nanowires containing both Au and Ag, all three metals can be
distinguished by imaging at 430 and 600 nm. While it is possible
to identify all three metals by carefully evaluating reflectance
intensities at a singe wavelength (e.g., 430 nm), the use of a
second wavelength, i.e., 600 nm, enables unambiguous identi-
fication of the third metal. This can be seen in the bottom panels
of Figure 4, which show an Ni-Ag-Au particle imaged at 430
and 600 nm. At 430 nm, Ag is brightest, followed by Ni and
then Au. At 600 nm, Au and Ag segments have the same
reflectivity, and the Ni segment can be readily identified. We
have imaged multi-metal striped nanowires, e.g., Pt-Pd-Ni-
Ag-Au, in which up to four of the different metal segments
Figure 5. Fluorescence and reflectance optical microscopy images (top
panels) and line profiles (bottom panels) of a sandwich immunoassay
performed using FITC labeled R-RIgG on Au-Ag-Au particles (A)
and on Au-Pt-Au particles (B). The particle striping pattern can be
seen in the fluorescence image of A, while not in B.
quantitative comparison with bulk values, Au was used as an
internal standard. Nanowires were synthesized such that the
metal of interest was placed between two Au segments with
the striping pattern Au-X-Au (where X denotes the metal of
interest).
The wavelength-dependent reflectance for the bulk metals
used (Au, Ag, Co, Pd, Pt, Cu, and Ni) is shown in Figure 1,
plotted from values in ref 25. Experimentally determined
reflectivity ratios for nanowire segments are plotted in Figure
15
were distinguished at one wavelength. Given the current error
bars on segment reflectivity, four intensity levels (i.e., metals)
in a single particle is impractical for multiplexing applications;
however, improvements in nanowire synthesis, particularly if
coupled with multiwavelength interrogation, may ultimately
make four-metal striping patterns a realistic option.
Patterned Fluorescence on Striped Particles. One applica-
tion for barcoded nanowires is as encoded, suspendable micro-
1
5,26
2
along with values for the corresponding bulk metals. Reflec-
supports for multiplexed bioanalysis.
For this application,
tivities are plotted at 430, 520, and 600 nm as the ratio of
intensity for the metal of interest to that of an adjacent Au
segment, averaged from g50 particles of each striping type.
Ratios for additional wavelengths are given in Table 2.
it is necessary not only to encode information (i.e., the type of
bioassay being performed on each nanowiresfor example, the
sequence of DNA on the particle surface); it must also be
possible to determine when a binding event has occurred. The
most straightforward way to detect and/or quantitate bioassays
on barcoded nanowires is using fluorescent tags in a sandwich-
type format. We had previously noted that nanowire striping
patterns could be observed in fluorescence images for on-particle
immuno- or DNA hybridization assays. This effect can be seen
in Figure 5A, which shows a sandwich immunoassay on Au-
Ag-Au nanowires. In this assay (lefthand panel of Scheme 1),
a FITC labeled anti-rabbit immunoglobin (R-RIgG) was used
for detection of RIgG binding to nanowire-bound antibodies
specific for the Fc region of RIgG. The left panel of Figure 5
is a representative fluorescence optical microscopy image, while
the middle and right panels are reflectance images taken using
band-pass filters to reveal the metal striping pattern for this
Agreement between 320 nm diameter, micrometer long metal
stripes and bulk material is quite good; in most cases,
experimentally obtained and bulk values are within one standard
deviation. Irregularities in surface nanostructure contribute to
the standard deviations in reflectivities for all metals. These
features cannot be directly observed via optical microscopy but
can be seen in electron micrographs. For Ag/Au at 430 nm, the
error bars are quite large, presumably due to variations in surface
roughness resulting from oxidation. Because Ag segments
oxidize in air, the prior history of samples (age and storage
conditions) can be important to their reflectivity. Figure 3 shows
FE-SEM images of bare Au-Ag-Au nanowires (A) freshly
prepared, and (B) the same nanowires after storage for 14 days
1
5

Barcoded Metal Nanowires
J. Phys. Chem. B, Vol. 107, No. 30, 2003 7365
SCHEME 1: Schematic of Sandwich Assays Used in
a
This Work
a(Left) For immunoassays, the striped particles are first derivatized
withacaptureantibody,a,thatisspecificfortheanalyte,b,whichisalsoan
antibody. Following incubation of the capture antibody with the ana-
lyte, the fluorescently labeled detection antibody, c, was added. (Right)
For hybridization assays, particles are derivatized with NeutrAvidin
(NA, shown here as an X), followed by reaction with a biotinylated
capture sequence, a, that is specific for a solution-phase analyte, b.
Fluorescently labeled oligonucleotides, c, were then added for detection.
nanowire. The bright region in fluorescence correlates with the
location of Ag metal in the nanowire, as can be seen from the
reflectivity at 430 nm. The Ag/Au intensity ratio for the
fluorescence image is similar to that for the reflectivity image
taken at 520 nm, which is close to λem for this fluorophore.
When the same sandwich assay was repeated with Au and Pt
as the underlying metals, much more uniform fluorescence was
observed as shown in Figure 5B. The metal striping pattern,
visible in the reflectivity image with 430 nm illumination, is
not visible at 520 nm. These data indicate that the observed
phenomena could be related to metal reflectivity near the
fluorophore λem and/or λex. Other possibilities include differences
in fluorophore surface coverage on the different metals, selective
fluorescence quenching by Au segments, or roughness-induced
enhancement on Ag segments. In the sandwich immunoassays
performed here (Scheme 1A), the capture antibody is attached
to the nanowire surface by direct, nonspecific adsorption. We
would not expect dramatic differences in coverage between Au,
Ag, and Pt surfaces under these conditions.
Figure 6. Fluorescence and reflectance optical microscopy images (top
panels) and line profiles (bottom panels) of a sandwich immunoassay
performed using TR labeled R-RIgG on Au-Pt-Au particles (A) and
on Au-Ag-Au particles (B). The particle striping pattern can be seen
in the fluorescence image of A, while not in B.
and/or fluorophores with high quantum yields. In our syste-
m,only high quantum yield fluorophores are used, and the
morphology of the particles (320 nm diameter, ∼6 µm long
cylinders) is not expected to give unusually high field amplifica-
tions. Thus, we do not expect a dramatic contribution from SEF
in this system. However, the fluorophores are located in the
near field (ca. 15 nm from the metal surface) and may
experience different electromagnetic field enhancements above
different stripes related to metal reflectivity.
To investigate the observed phenomenon further, the same
sandwich assay was performed using TR labeled R-RIgG on
the same Au-Pt striped nanowires, Figure 6A. This time,
fluorescence was brighter on the Au segments as can be seen
in the left panel. Again, the observation that fluorescence was
brighter on certain metal segments correlated well with the
reflectivities of the underlying metals. For this selection of
metals (Au and Pt), Au is more reflective than Pt near the λex
and λem of TR. When the TR labeled immunoassay is performed
on Au/Ag/Au nanowires, where the reflectivities are matched
at λem, the striping pattern cannot be seen in the fluorescence
image (Figure 6B). Together, these data suggest that striped
fluorescence can be predicted on the basis of the relative
reflectivities of the underlying metals.
Fluorescence near metal surfaces is a well-studied phenomen-
on.2
7-29
Two competing interactions, quenching and local field
amplification (or enhancement), are observed. Quenching results
from nonradiative energy transfer from the excited-state mol-
-3
ecule to the metal surface, and varies as d where d ) distance
from the metal surface.30 Generally, quenching becomes im-
portant for distances very close to the metal surface, usually
2
8,31,32
<
ca. 10 nm.
The local field amplification also drops off
with distance from the surface, but less steeply; thus, fluores-
cence intensities are increased for molecules on the order of
8,31
1
0-20 nm from the surface.2 Optimal metal-molecule sep-
aration distances for surface-enhanced fluorescence (SEF) de-
pend on the experimental system; a wide range of values has
2
7-29,33-36
been reported.
For the assays used in this work
(Scheme 1A), fluorophores were located far enough from the
metal surface to minimize quenching (i.e., there were ∼3 layers
of antibodies between the fluorophore and the nanowire surface
which places the fluorophore ca. 15 nm from the metal).
Surface-enhanced fluorescence intensities depend not only
on distance from the surface, but also on the morphology of
3
7-39
the metal surface
and the quantum yield for the fluoro-
phore.4 Maximal enhancements are achieved in regions of high
electromagnetic field amplification (such as occur near nanoscale
surface features), and/or for fluorophores with low quantum
yields. Smaller SEF effects are observed for smooth surfaces
0
Fluorescence and reflectance intensity ratios are compared
4
1
quantitatively in Table 2. If the observed uniform FITC
fluorescence on Pt and Au segments is related to similar metal
reflectivities near the λem of FITC, which occurs at 518 nm,

7366 J. Phys. Chem. B, Vol. 107, No. 30, 2003
Nicewarner-Pe n˜ a et al.
reflectivity ratio for Ag/Au at 520 nm. However, the measured
FITC ratio is 2.21 ( 0.34, which is 1.5× higher. This may be
due in part to additional electromagnetic field enhancement of
FITC fluorescence by Ag at this wavelength due to surface
roughness or to the greater reflectivity for Ag vs Au at λex. Data
for TR fluorescence indicate that any increase in Ag surface
roughness does not significantly increase its surface area.
Uniform TR fluorescence (ratio ) 1.00 ( 0.06) is observed on
Ag/Au rods, in agreement with nearly identical reflectivities at
6
00 nm (ratio ) 1.04 ( 0.08).
Much larger deviations from expected fluorescence intensities
are observed for nanowires composed of Au and Ni. The general
trend of brighter fluorescence from Au vs Ni segments for both
FITC and TR matches the reflectivity data at λem for these
fluorophores. However, in both cases the observed fluorescence
ratios are considerably lower than the reflectivity ratios at λex
or λem. This may indicate less efficient antibody adsorption on
the Ni surface, perhaps due to surface oxidation of the Ni.
In summary, the data in Table 2 suggest that the observed
fluorescence striping patterns can be predicted based on the
relative reflectivities of the underlying metals. Since metal-
fluorophore spacing was held constant in our experiments, and
since data are acquired as ratios of intensity for fluorophores
above adjacent segments, we cannot definitively attribute the
observed effects to SEF phenomena. Indeed, a similar result
might be expected for fluorophores located in the far field, where
fluorescence patterning could arise not from increased local
fields but rather from a wavelength-dependent mirror effect from
the underlying substrate.
Figure 7. Fluorescence (left panel) and a reflectance optical microscopy
image (right panel) of a multiplexed DNA hybridization using 6-FAM
labeled detection oligos. The panels in A show the detection of a single
analyte from solution while B shows the simultaneous detection of two
analytes from solution. The particle striping pattern is clearly visible
in the fluorescence image indicating that only the fluorescence image
could be used to provide both analyte identity and presence. Scale bars
are 5 µm.
DNA Hybridization on Striped Nanowires. Patterned
fluorescence has both advantages and disadvantages in bio-
analysis. For example, it could be advantageous for streamlining
bioassay analysis in that one image could be used for both
analyte detection and nanowire identification. That is, instead
of acquiring two images (a fluorescence image to determine
presence of the analyte and a reflectivity image to reveal striping
pattern), only one image would be needed to provide both
analyte presence and striping pattern (which codes for a
particular analyte). In addition, having segments with differing
fluorescence intensities could potentially extend the dynamic
range for detection. On the other hand, quantification is
simplified by uniform brightness over an entire nanowire, and
sensitivity is optimized by choosing λex and λem to match high
reflectivity regions for striping metals.
We have carried out multiplexed DNA hybridizations in
which three different nanowire striping patterns of Au and Ag
composition were used, each coding for a unique analyte (in
this case a different oligonucleotide sequence). In parallel
experiments, 6-FAM tagged and ROX tagged detection oligo-
nucleotides were used for each analyte sequence. These fluo-
rophores have nearly identical λex and λem as the FITC and TR
dyes used in the immunoassays described above. Scheme 1B
illustrates the sandwich hybridization assay format used, in
which three strands of DNA are involved in each of the three
simultaneous analyses. The three unique capture sequences (Ia,
IIa, and IIIa-a in the righthand panel of Scheme 1) were
attached to nanowires of one of the three striping patterns: type
1 ) Ag-Ag-Au-Ag-Ag (binary code 11011), type 2 ) Au-
Ag-Au-Au-Au (01000), and type 3 ) Au-Ag-Au-Ag-
Au (01010). Following derivatization, the nanowires were mixed
together and hybridized with a mixture of the 3 analytes (Ib,
IIb, and IIIb-b in Scheme 1). For detection, each possible
combination of analyte mixture was then hybridized to a mixture
of all three detection oligos, both FITC labeled and ROX
Figure 8. Fluorescence (left panel) and a reflectance optical microscopy
image (right panel) of a multiplexed DNA hybridization using ROX
labeled detection oligos. The panels in A show the detection of a single
analyte from solution, while B shows the simultaneous detection of
two analytes from solution. Particle striping pattern is not visible in
the fluorescence image, indicating that both the fluorescence and
reflectivity images are needed to provide analyte presence and identity,
respectively. Scale bars are 5 µm.
then the expected ratio from the fluorescence intensity and the
reflectivity intensity should be nearly identical. Indeed, that is
what is observed. The intensity ratio for FITC is 0.98 ( 0.05,
while the measured reflectivity ratio for Pt/Au at 520 nm is
0
.94 ( 0.07. The increased fluorescence intensities for fluoro-
phores located on more reflective metals is consistent with
increased local fields above these metals.
Following this reasoning, the expected ratio for FITC on Au
and Ag segments would be ∼1.50 on the basis of the observed

Barcoded Metal Nanowires
J. Phys. Chem. B, Vol. 107, No. 30, 2003 7367
labeled. The detection sequences (Ic-R and Ic-F, IIc-R and IIc-
F, and IIIc-R and IIIc-F) were each complementary to the
second half of the analyte. Control experiments were also
performed in which the analyte was omitted; the results showed
virtually no background (data not shown). The results of the
hybridization experiments are shown in Figures 7 and 8, in
which a fluorescence image and a reflectivity image are shown
for the detection of one or more analyte sequences in solution.
To aid in the identification of the striping pattern, nanowires
have been labeled (i.e., 1, 2, or 3) in both the fluorescence and
reflectivity images. Clearly, the striping pattern can be seen in
the fluorescence image of Figure 7, however, not in Figure 8.
Moreover, fluorescence is predominately observed from the
intended particles (left panels of Figures 7 and 8), indicating
that the specific capture sequences on the three nanowire types
were able to selectively bind to their target oligo. These results
demonstrate that multiplexed DNA hybridizations can be
performed on barcoded nanowires such as these and that the
reflectivity image was sufficient for the identification of the
analyte oligo, as only one fluorophore was used in each
experiment. Furthermore, the data also shows the possibility of
using only the fluorescence image to determine both the presence
of the analyte in solution and the identity of the analyte.
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The wavelength-dependent reflectivity for striped metal
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Acknowledgment. We thank Prof. Vin Crespi for helpful
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Supporting Information. This material is available free of charge
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