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Localized surface plasmon
resonance biosensors
Jing Zhao1,
Xiaoyu Zhang1,
Chanda Ranjit Yonzon1,
Amanda J Haes1 &
Richard P Van Duyne1†
†Author for correspondence
1Northwestern University,
Department of Chemistry,
2145 Sheridan Road,
Evanston, IL 60208-3113,
USA
Tel.: +1 847 491 3516;
Fax: +1 847 491 7713;
E-mail: vanduyne@
chem.northwestern.edu
Keywords: Alzheimer’s
disease, biosensing,
electrochemistry, localized
surface plasmon resonance,
multiplexed sensing,
nanosphere lithography, silver
nanoparticles, solution-phase
nanoparticles
10.2217/17435889.1.1.219 © 2006 Future Medicine Ltd ISSN 1743-5889
R EVIEW
In this review, the most recent progress in the development of noble metal nano-optical
sensors based on localized surface plasmon resonance (LSPR) spectroscopy is summarized.
The sensing principle relies on the LSPR spectral shifts caused by the surrounding dielectric
environmental change in a binding event. Nanosphere lithography, an inexpensive and
simple nanofabrication technique, has been used to fabricate the nanoparticles as the LSPR
sensing platforms. As an example of the biosensing applications, the LSPR detection for a
biomarker of Alzheimer’s disease, amyloid-derived diffusable ligands, in human brain
extract and cerebrospinal fluid samples is highlighted. Furthermore, the LSPR sensing
method can be modified easily and used in a variety of applications. More specifically, a
LSPR chip capable of multiplex sensing, a combined electrochemical and LSPR protocol and
a fabrication method of solution-phase nanotriangles are presented here.
The last 20 years have witnessed a rapid growth arrays can be measured with UV-visible extinction
in the development of the highly selective and spectroscopy [16–20]. Since the LSPR of the nano-
sensitive optical nanobiosensors for medical particles is highly dependent on the local environ-
diagnosis and monitoring of diseases, drug dis- ment, λmax will shift when adsorbates bind to the
covery and the detection of environmental pol- nanoparticles. The LSPR shift induced by the
lutants and biological agents [1]. Among the adsorbates, ∆λmax, is given by Equation 1:
many advantages of optical biosensors, sensitiv-
ity and real-time detection of biomolecular ∆λ max = λ max 〈 after〉 – λ max 〈 before〉
interactions allow them to be applied widely.
Optical sensing techniques are based on various Figure 1 is a representation of the biosensing
sensing transduction mechanisms, for example, strategy. First, nanoparticles are fabricated on
chemiluminescence [2,3], fluorescence [4], light substrates and functionalized with a self-assem-
absorption and scattering [5,6], reflectance [7], bled monolayer (SAM). Next, the SAM-coated
surface plasmon resonance (SPR) [8–10] and nanoparticles are chemically modified with
Raman scattering [11–13]. receptors that will specifically bind to the target
This review will focus on optical sensors based molecules. Finally, the sensing surface is exposed
on localized surface plasmon resonance (LSPR) of to the target molecules. The refractive index of
triangular nanoparticles. This nanoparticle-based the surrounding environment changes on bind-
optical sensing technique is effective for quantita- ing to the receptors, inducing a shift in the
tive detection of chemical and biological nanoparticle LSPR, λmax. This wavelength shift
targets [5,10,14,15]. The sensing principle employed is monitored by UV-Visible spectroscopy. Over-
in these experiments relies on the high sensitivity all, LSPR nanosensors retain the high selectivity,
of the LSPR spectrum of noble metal nanoparti- label-free operation, capability to probe complex
cles to adsorbate-induced changes in the dielectric mixtures without purification and multiple
constant of the surrounding environment. LSPR detection modes (extinction and resonance
is one of the signature optical properties of noble Rayleigh scattering) characteristic of or analo-
metal nanoparticles, which arises when the inci- gous to sensors based on SPR spectroscopy.
dent photon frequency is resonant with the collec- From the instrumentation perspective, LSPR
tive oscillation of the conduction electrons in the nanosensors can be implemented using
metal nanoparticles. The LSPR is dependent on extremely simple, small, light, robust, low-cost
the size, shape, interparticle spacing and dielectric equipment for unpolarized, UV-visible extinc-
properties of the material, as well as the dielectric tion spectroscopy in transmission or reflection
properties of the local environment that sur- geometry. The instrumental simplicity of the
rounds the nanoparticles. The LSPR LSPR nanosensor approach is expected to
extinction maximum (λmax) of the nanoparticle greatly facilitate field-portable environmental or
Nanomedicine (2006) 1(2), 219–228 219
REVIEW – Zhao, Zhang, Ranjit Yonzon, Haes & Van Duyne
Figure 1. Representative experimental set-up and procedure
for localized surface plasmon resonance (LSPR) sensing.
Sensor
chip Glass
buffer
Light source
Detector
Flow
PC
Ka, surf Ka, surf
Nanomedicine
Transmission UV-visible spectroscopy is used to monitor the optical absorption
(LSPR) of Ag nanoparticles. All spectra collected are macroscopic measurements
with unpolarized light. The probe beam diameter was approximately 4 mm. A
home-built flow cell was used to control the external environment of the Ag
nanoparticle substrates. The schematic illustration at the bottom of the
figuredisplays the surface chemical modification of the LSPR sensor. First,
surface-confined Ag nanoparticles (see atomic force microscopy, inset) are
fabricated using nanosphere lithography. Next, a mixed self-assembled
monolayer consisting of thiol-terminated receptor-binding molecules and
surface passivating molecules is immobilized. Then, ligand-specific receptors are
covalently attached. Finally, the ligand of interest is exposed to the surface [14].
point-of-service medical diagnostic applications.
A more comprehensive comparison of the two
methods is available in a recent review [21].
Well-defined and uniform nanostructures are
critical to the reproducibility of LSPR spectra.
We used nanosphere lithography (NSL) (Figure 2),
an inexpensive and simple method to fabricate
large arrays of nanoparticles with well controlled
size and shape [22,23]. NSL fabrication begins with
the self-assembly of size-monodisperse polysty-
rene nanospheres to form a deposition mask.
Then, metal is deposited through the nanosphere
masks using thermal or electron beam evapora-
tion. After the removal of polystyrene nano-
spheres, well ordered 2D triangular nanoparticle
arrays remain on the substrates. By changing the
nanosphere diameter (D) and the deposited metal
thickness (dm), nanoparticles with different in-
plane width (a), out-of-plane height (b) and
interparticle spacing can be produced.
A comparison analysis of the nanoparticle
shape on LSPR sensitivity revealed that the
LSPR shift induced from hexadecanethiol was
220
larger for NSL-fabricated nanotriangles than for
nanohemispheres [24]. Similarly, randomly
deposited substrates from gold colloid suffer
similar disadvantages as LSPR sensors, relative
to NSL-derived substrates [25,26]. Additionally,
both the experimental and theoretical
studies [24] have supported the fact that nanotri-
angles exhibit larger total LSPR shifts and can
detect molecules larger distances away from the
nanoparticle surface, indicating the larger elec-
tromagnetic field strength and sensing volume
than those of nanohemispheres.
Previously, we studied how the LSPR λmax of
NSL-fabricated Ag triangular nanoparticles
depends on the refractive index of the sur-
rounding environment by exposing the nano-
particles to different solvents. The sensitivity of
the nanoparticles that make up the LSPR nano-
biosensor to the local refractive index (m) is
found to be approximately 200 nm RIU-1 [16]
for bare Ag nanotriangles with a = 100 nm and
b = 50 nm, while the thiolate SAM
[CH3(CH2)15SH]-modified nanotriangles yield
m = 150 nm RIU-1 [5].
Similar wavelength shifts are observed when
molecules bind to the nanotriangle surface,
changing the surrounding local refractive index
while leaving the bulk refractive index essen-
tially unchanged. This effect is the basis for
detecting binding of proteins to the sensors.
Quantification of specific proteins is achieved
by functionalizing the nanotriangles with anti-
bodies to the target proteins. Model systems,
such as biotin/streptavidin [14], biotin/anti-
biotin [15] and dinitrophenyl (DNP)/anti-DNP
[27], have been studied using the LSPR sensors.
Upon binding of the biological targets, the
LSPR resonant wavelength shifts towards the
longer wavelength region. The magnitude of
the LSPR shift response for a given system is
determined by the coverage of the target ana-
lytes on the nanoparticle surface. For systems
consisting of nanoparticles with the same
refractive index sensitivity and target molecules
with the same refractive index change per mole-
cule binding, the detection limit is determined
by the binding constant between the capture
ligand on the surface and the target molecules
in solution [14,15].
Figure 3 shows a representation of the LSPR
response to the trilayer system SAM/DNP/anti-
DNP. The LSPR response R (= ∆λmax) can be
expressed by Equation 2 [14]:
R = m ( n eff – n ext )
Nanomedicine (2006) 1(2)
 Localized surface plasmon resonance biosensors – REVIEW

Equation 3 describes the measured LSPR

Figure 2. Ag nanoparticle fabrication procedure using
nanosphere lithography. response ∆R = Rlayer2–Rlayer1 [15,28]:
∆ R = m θ ( n target – n ext ) [ exp ( – 2d SAM ⁄ l d ) ]
[ 1 – exp ( – 2d target ⁄ l d ) ]
1. Clean substrate 2. Drop coat
3. Dry
where θ is the fractional coverage of the target
molecules, ntarget is the refractive index of the tar-
get molecules of detection, ld is the characteristic
electromagnetic field decay length, dSAM is the
6. Representative atomic
approximate thickness of the SAM and dtarget is
force microscopy the effective target layer thickness.
5.Lift off spheres 4. Deposit material
image of substrate Equation 4 describes maximum LSPR response,
∆Rmax, which occurs when θ = 1:
Ag
∆ R max = m ( n target – n ext ) [ exp ( – 2d SAM ⁄ l d ) ]
[ 1 – exp ( – 2d target,max ⁄ l d ) ]

1500 nm For the case where 2dtarget « ld, the ∆R/∆Rmax

Nanomedicine
response reduces to the Langmuir adsorption
isotherm (for a more detailed explanation, please
refer to [15]) as described in Equation 5:
where m is the sensitivity of the nanoparticles to K a, surf [ target ]
∆R
the local refractive index, next is the bulk refrac- ---------------- = -----------------------------------------------
-
∆R max 1 + K a, surf [ target ]
tive index of the external medium and neff is the
effective refractive index of the trilayer structure
The binding constant Ka,surf can be calculated
above the Ag nanoparticle sensor surface.
using Equation 5. LSPR sensors enable: the study of
the binding of the target molecules to the nanopar-
Figure 3. The plot of localized surface plasmon ticle surface and calculation of the binding con-
resonance response (∆R) versus anti-dinitrophenyl stant; the estimation of the detection limit of the
(DNP) concentration. LSPR sensor; and the saturation response of the
LSPR sensor to the binding of the target molecules.
The limit of detection of the LSPR sensor can be
Ag estimated where the LSPR shift induced by the
binding of the target molecules is three-times the
Glass
peak-to-peak wavelength shift noise of the baseline.
50 The LSPR sensors can detect 1 pM streptavidin [14]
$Rmax = 46.6 nm and approximately 700 pM anti-biotin [15] to bioti-
Ka, surf = 9.21 × 106 M-1 nylated Ag nanoparticles and <7.5 nM anti-
40 LOD < 7.5 nM
DNP [27] to dinitrophenylated Ag nanoparticles
30 with almost no nonspecific binding effects.
$R (nm)

20 Detection of a biomarker of
Alzheimer’s disease
10 Encouraged by the success of streptavidin, anti-
biotin and anti-DNP sensors, we attempted to
0 use the LSPR nanobiosensors to detect an
Alzheimer’s disease (AD) biomarker [29]. AD is
-10 the most common form of dementia, a group of
10-12 10-10 10-8 10-6 10-4 conditions that gradually destroy brain cells and
[AntiDNP] (M) lead to the progressive decline in mental func-
tion. It is estimated that 4.5 million Americans
The solid line is the calculated fit to the experimental data points using have AD [52]. The most widely accepted
equations 2–4. The calculated maximum response (∆Rmax) is 46.4 nm, the hypothesis for the progression of AD is the
binding constant at the nanoparticle surface (Ka,surf) is 9.21 x 106 M-1 and the ‘amyloid hypothesis’, which assigns a central
LOD is less than 7.5 nM. Error bars display the total spread of the data. role to abnormal processing of amyloid precur-
DNP: Dinitrophenyl; LOD: Limit of detection.
sor protein. This abnormal processing yields a

www.futuremedicine.com 221
REVIEW – Zhao, Zhang, Ranjit Yonzon, Haes & Van Duyne

Figure 4. Demonstration of a localized surface plasmon pathology [31]. During recent years, research has
resonance sandwich assay at high and low amyloid-derived shown that, in addition to plaques, Aβ will
diffusable ligand concentrations. aggregate into small oligomers, called amyloid-
derived diffusable ligands (ADDLs) [30–34].
ADDLs cause neurological dysfunction relevant
A to memory and have been recognized as poten-
ADDLs
tial AD biomarkers. A sensitive method to
detect ADDLs in body fluid could provide a
Ka, surf Ka, surf definitive molecular basis for a laboratory diag-
nostic for AD. A sensitive ADDL test would
also monitor disease progression and effective-
B 0.24 ness of medication designed to reduce ADDL
2 722.7
load. We have developed a LSPR nanobiosensor
1 718.5 3 730.3
0.22 to detect ADDLs using a (anti-ADDL anti-
body/ADDLs/anti-ADDL antibody) sandwich
Extinction

0.20 assay [29].

In this review, a successful demonstration of
0.18 the LSPR nanbiosensors in detection and diag-
nosis, which is the detection of ADDLs for AD,
0.16 is focused on. Since the LSPR nanobiosensing
method is highly compatible with other sensing
0.14 methods, several other novel techniques will
640 680 720 760 800 also be described, which can be combined with
Wavelength (nm)
the current LSPR sensing methods to improve
the sensing ability and broaden the potential
C
0.24
field of applications.
ADDLs are peptides with molecular weights
0.22
of between 17 and 42 kDa [33]. The relatively
1 741.1 2 743.6
small mass is sufficient to produce a small but
3 747.0 detectable change in LSPR λmax upon ADDL
Extinction

0.20
0.18
0.16
0.14
640 680 720 760 800
Wavelength (nm)
(A) Schematic illustration of the experimental procedure. The first anti-ADDL
antibody is covalently attached to the nanoparticles with a mixed monolayer of 1-
OT and 11-mercaptoundecanoic acid. Samples are then incubated in ADDL
solution and finally in an anti-ADDL solution. After every stage, the sample was
dried in N2 gas and the LSPR spectrum was required. (B) LSPR spectra for each
step of the sandwich assay for 10 pM ADDL. Ag nanoparticles after modification
with 100 nM anti-ADDL antibody (B-1), after exposure to 10 pM ADDL (B-2) and
100 nM anti-ADDL antibody (B-3). (C) LSPR spectra for each step of the sandwich
assay for 100 fM ADDL. Ag nanoparticles after modification with 100 nM anti-
ADDL antibody (C-1), after exposure to 100 fM ADDL (C-2) and 100 nM anti-
ADDL (C-3). All spectra were collected in a N2 environment.
ADDL: Amyloid-derived diffusible ligands; LSPR: localized surface
plasmon resonance.
peptide fragment called β-amyloid (Aβ) [30]. Aβ
is a peptide that aggregates by stages into the
amyloid plaques that are one hallmark of AD
binding to the receptor-functionalized nanopar-
ticle surface. In order to amplify the signal and
improve the detection limit, the bound ADDLs
were further labeled with anti-ADDL in a sand-
wich assay that has been used to detect ADDLs
from both synthetic and human samples.
Figure 4A shows a schematic illustration of the
procedure for nanoparticle functionalization and
the sandwich assay. Briefly, nanoparticles are fab-
ricated using NSL and prefunctionalized with a
mixture of 11-mercaptoundecanoic acid (11-
MUA) and 1-octanethiol (1-OT). 11-MUA and
1-OT molecules form a mixed SAM on the nan-
oparticles. Anti-ADDL antibodies are covalently
linked to the SAM-coated nanoparticles with 1-
ethyl-3-[3-dimethy-aminopropyl] carbodiimide
hydrochloride (EDC), coupling the amine
groups on the antibody to the carboxyl groups
on 11-MUA [14,15]. Then, the nanoparticles are
exposed to an ADDL solution and finally incu-
bated in an additional anti-ADDL antibody for
30 min. By varying the concentration of ADDLs
in solution, the response of the LSPR to the
binding of ADDLs can be monitored systemati-
cally. Figures 4B & 4C show representative results

222 Nanomedicine (2006) 1(2)

 Localized surface plasmon resonance biosensors – REVIEW

Figure 5. Demonstration of the localized surface plasmon Furthermore, two types of human samples
resonance (LSPR) sandwich assay for two types of (human brain extract and cerebrospinal fluid
human samples. [CSF]) from a control patient and an AD patient
were analyzed using the LSPR sensor.
Figures 5A & B show the LSPR sandwich assay
A Brain extract – AD B CSF – AD
0.16
responses of human brain extract from the AD
0.48 2 809.1 patient (results shown in Figure 5A) and an age-
2 758.3 0.14 1 780.6 matched control patient (results shown in
3 824.5
Extinction

Extinction
1 751.6 Figure 5B). During the process, the brain extracts
0.44 3 762.3 0.12
from the AD and control patient are exposed to
0.40 0.10 NSL-fabricated anti-ADDL Ag nanoparticles for
0.08
30 min, then to the second anti-ADDL antibody
0.36 for 30 min. The LSPR shifts from 751.6 to
0.06 758.3 nm during the brain extract incubation for
640 680 720 760 800 840 880 650 700 750 800 850 900 950
Wavelength (nm)
the AD patient, that is, a 6.7 nm shift; while the
Wavelength (nm)
LSPR shifts from 782.3 to 782.4 nm for the con-
trol patient, that is, a 0.1 nm shift. After the incu-
C Brain extract – control D CSF – control
0.36 0.16 bation with the second antibody, an additional
2 782.4 1 759.7 2 762.6 4.0 nm shift is observed for the AD patient while
0.32 1 782.3 3 782.9 0.14 3 766.9 a shift of only 0.5 nm is observed for the control
Extinction
Extinction

0.28 0.12 patient. Although further trials are needed to ver-

0.24 0.10
0.20 0.08
0.16 0.06
650 700 750 800 850 900 650 700 750 800 850 900
Wavelength (nm)
Wavelength (nm)
(A) LSPR spectra for each step of the sandwich assay for human brain extract
from an AD patient. Ag nanoparticles after modification with anti-amyloid-
derived diffusable ligand (ADDL) antibody (A-1), brain extract (A-2) and anti-
ADDL antibody (A-3). (B) LSPR spectra for each step of the sandwich assay for
human brain extract from a control patient. Ag nanoparticles after modification
with anti-ADDL antibody (B-1), brain extract (B-2) and anti-ADDL (B-3). (C) LSPR
spectra for each step of the sandwich assay for cerebrospinal fluid (CSF) from an
AD patient. Ag nanoparticles after modification with anti-ADDL antibody (C-1),
CSF (C-2) and anti-ADDL antibody (C-3). (D) LSPR spectra for each step of the
sandwich assay for CSF from a control patient. Ag nanoparticles after
modification with anti-ADDL antibody (D-1), CSF (D-2) and anti-ADDL (D-3). All
spectra were collected in N2.
AD: Alzheimer’s disease.
of LSPR of the nanoparticles before and after
exposure to ADDLs and the second antibody.
Figure 4B depicts that, after incubation in 10 pM
ADDLs, the LSPR λmax in N2 shifts 4.2 nm to a
longer wavelength. After incubation in the sec-
ond anti-ADDL antibody, the LSPR shifts fur-
ther, that is, an additional 7.6 nm shift or a total
LSPR shift of 11.8 nm in N2. The addition of
the second antibody significantly amplified the
LSPR response (by a factor of almost three), thus
improving the limit of detection of ADDLs. The
same experiments have been repeated, changing
the ADDL concentration to 100 fM, which
induces a 5.3 nm shift (Figure 4C).
ify the increased ADDL concentrations in the AD
patient, the results are promising.
Brain extract is only obtained postmortem. To
observe ADDL concentrations in living patients,
CSF from both AD and control patients have been
tested following the same LSPR sandwich assay
method. Dramatic differences in LSPR shift have
been observed from the two patients. After incuba-
tion in CSF and the second anti-ADDL antibody, a
total LSPR shift of 43.9 nm is observed for the AD
patient (Figure 5C) and a 7.2 nm shift for the control
patient (Figure 5D). Thus, we can conclude that this
LSPR nanobiosensor using the sandwich assay is
the first demonstration of effective detection of
ADDLs from human samples.
Fabrication of a multiplexed sensor
A multiplexed assay allows detection of multiple
analytes in the same sample, which is useful for
screening multiple diseases or examining relation-
ships between multiple analytes. To develop an
efficient diagnostic assay, it is crucial that the sen-
sor is capable of detecting and tracking the target
analytes, while minimizing the number of required
sensors. A multiplexed sensing chip has several
advantages, including rapid and parallel detection
of multiple targets on a single chip, minimized
sample to sample variation, lower cost per chip,
reduced analyte(s) volume and reduced time [9].
Concanavalin A (Con A), a mannose-specific
plant lectin, was chosen to test a LSPR multiplexed
carbohydrate sensor [10,35–37]. NSL was employed
to fabricate nanoparticles with two different
heights (35 and 75 nm) on a single substrate.

www.futuremedicine.com 223
REVIEW – Zhao, Zhang, Ranjit Yonzon, Haes & Van Duyne
Figure 6. Localized surface plasmon resonance (LSPR) spectra
illustrating multiplexing Ag nanosensor carbohydrate-
sensing chip.
A C
B D E Con A to the mannose-functionalized Ag nano-
Ka, surf
+
Concanavalin A
D E
C
B Electrochemical tuning of NSL nanoparticles
Extinction
500 600 700 800 900
Wavelength (nm)
Ag nanobiosensor consisted of nanosphere lithography (NSL)-fabricated Ag
nanoparticles with the same in-plane width but two different out-of-plane
heights: 35 and 75 nm and, thus, two different LSPR λmax. (A) Schematic
illustration of selective Con A binding to the mannose-functionalized portion of
the carbochip. (B) Ag nanoparticles (height = 75 nm) after 5 mM mannose
modification, λmax = 677.7 nm, ∆λmax = 5.0 nm. (C) Ag nanoparticles (height =
75 nm) after exposure to 19 µM Con A, λmax = 682.7 nm. (D) Ag nanoparticles
(height = 35 nm) after 5 mM galactose modification, λmax = 724.5 nm. (E) Ag
nanoparticles (height = 35 nm) after exposure to 19 µM Con A,
∆λmax = 724.6 nm, ∆λmax = 0.1 nm.
These nanoparticles were spatially separated and
also had a different LSPR maximum owing to
their different heights. A mixture of tri(ethylene
glycol)-terminated alkanethiol- and maleimide-
terminated alkanethiol were immobilized on these
nanoparticles. Furthermore, the 75 and 35 nm
nanoparticles were functionalized with mannose-
thiol and galactose-thiol, respectively, using a sepa-
rator. Finally, the chip was exposed to 19 µM
Con A (Figure 6A). Upon exposing Con A to man-
nose-functionalized nanoparticles, the LSPR λmax
red shifted 5 nm (Figure 6B & C). By contrast, galac-
tose-functionalized nanoparticles showed no
224
response to Con A (Figure 6D & E). The experi-
ments showed that the affinity of Con A to a man-
nose-functionalized surface is much higher than
that to a galactose-functionalized surface. Addi-
tionally, to verify that the response seen on the
LSPR sensor was due to the specific binding of
sensor, several nonspecific binding studies were
performed. Neither erythrina crystagalli nor
bovine serum albumin bound to the mannose-
functionalized Ag nanosensor [10]. The results
demonstrate that the multiplexed LSPR chip is
capable of screening multiple targets.
LSPR measurements are compatible with a variety
of detection platforms. For example, LSPR can be
combined with electrochemistry by fabricating
NSL nanoparticles on indium tin oxide (ITO)-
coated glass [38]. This new type of transparent and
conducting material allows us to conduct both
electrochemical and LSPR measurements simulta-
neously. The experimental setup is shown in
Figure 7. An ITO substrate with Ag nanoparticles
was used as a working electrode in a traditional
three electrode cell. Extinction spectra of the Ag
nanoparticles were measured using a fiber-coupled
spectrometer in transmission geometry. As an ini-
tial proof-of-concept experiment, we electrochemi-
cally oxidized the Ag nanoparticles on the ITO
surface and monitored their in situ LSPR spec-
trum, which is extremely sensitive to nanoparticle
geometric change. Figure 7A shows a single cyclic
voltammetric sweep performed on the Ag nano-
particles/ITO electrode (solid line). Additionally, a
parallel measurement was carried out on a smooth
Ag electrode (dashed line in Figure 7A). Comparing
the two cyclic voltammograms, we can conclude
that the oxidation of silver nanoparticles starts at a
less positive potential, indicating that a portion of
nanoparticles are more easily oxidized than bulk
silver [39–42]. During the oxidation, Ag0 in the nan-
oparticles is oxidized to Ag+ and released into solu-
tion, which decreases the size of nanoparticles and
results in a blue shift of LSPR (Figure 7B). It is also
worth noting that the NSL-produced nanoparti-
cles on ITO are very robust under the electrochem-
ical oxidations. By contrast, previously reported
nanostructures cannot remain on ITO surfaces
under positive electrochemical potentials [43].
Electrochemical techniques have been well
established and developed to detect nucleic acids
and proteins [44,45]. Inspired by this, the detec-
tion of electrochemically active protein based on
electro-LSPR is currently under investigation.
Nanomedicine (2006) 1(2)
 Localized surface plasmon resonance biosensors – REVIEW
Figure 7. Schematic illustration of the
experimental setup of electrochemically
modified localized surface plasmon
resonance (LSPR).
Potentiostat
Ag particles
on ITO
Light source
Detector
A B
80
Ag film
Extinction
Current (µA)
40 Ag PPA
0 This review has briefly summarized the very
-40
-80
0.40 0.32 0.24 800 900 1000
Potential (volt vs Ag/AgCl) Wavelength (nm)
(A) Single scan cyclic voltammograms of Ag
film/ITO (dashed line, dm = 50 ± 5 nm) and Ag
nanoparticle arrays on an ITO electrode (solid line)
in 0.1 M NaClO4 aqueous solution. Scan rate =
0.1 V/s. The arrow shows the initial scan direction.
(B) Localized surface plasmon resonance spectra
during the electrochemical oxidation and
corresponding atomic force microscopy images of
Ag nanoparticles on ITO.
ITO: Indium tin oxide.
Synthesis of solution-phase
triangular nanoparticles
The techniques discussed here are all based on sur-
face-bound nanoparticles. Another important type
of nanoparticle widely in use for biosensing is solu-
tion-phase nanoparticles. Solution-phase nanopar-
ticles can be dispersed into macrostructures, such as
cells [46,47] and tissue [47] samples, thus allowing for
the development of in vivo biological detection
methods. A novel method has been presented to
fabricate solution-phase nanoparticles with well
controlled geometry by releasing NSL-fabricated
surface-bound nanoparticles into solution [48]. The
experimental procedure is illustrated in Figure 8A.
First, the nanoparticles are produced by NSL on
glass and incubated in alkanethiol ethanol solution
for 48–96 h immediately after the removal of the
nanospheres. The alkanethiol molecules form a
www.futuremedicine.com
SAM on the nanoparticles. The formation of this
SAM is critical to the successful release of isolated
nanoparticles, as it forms a physical barrier against
aggregations. The corresponding atomic force
microscopy (AFM) image is shown in Figure 8B-1.
The nanoparticles are then released from the sub-
strate into solution by sonication in ethanol for
3–5 min. Upon the addition of alkanedithiol, the
nanoparticles are asymmetrically linked and form
dimers. The corresponding TEM images are shown
in Figures 8B-2 & 8B-3. The extinction spectra of the
nanoparticles on substrate and in solution are dis-
played in Figure 8B. There is a dramatic change in
the LSPR spectra when the nanoparticles are
released into solution and linked to form dimers.
This finding opens up the possibility of using
the solution-phase NSL particles to detect a small
number of sandwiched biological targets and to
develop nanobiosensors in living systems.
Conclusions
recent development of NSL-fabricated nanopar-
ticle-based LSPR biosensors. They are very sensi-
tive to changes in the local dielectric
environment and can provide sufficient informa-
tion on the binding constants and orientations of
the target molecules to the nanoparticle surface.
The LSPR nanobiosensors are sufficiently sen-
sitive to detect ultralow concentrations of biologi-
cal analytes. Therefore, they have been used to
detect ADDLs: an AD biomarker. A sandwich
assay composed of antibody/ADDL/antibody was
developed to amplify the LSPR response, thus
improving the limit of detection, which is as sensi-
tive as 100 fM ADDLs. Human brain extract and
CSF fluid samples from an AD patient and an
age-matched control patient have been tested
using the LSPR sensor. After exposing the anti-
body-functionalized nanoparticles to the brain
extract and then the second antibody, the total
LSPR shift induced by the AD patient brain
extract was 6.7 nm, while that induced by the
control patient brain extract was 0.1 nm. More-
over, after exposing the nanoparticles to the CSF
fluid and then the second antibody, the total
LSPR shift induced by the AD patient sample was
43.9 nm, while that induced by the control
patient sample was 7.2 nm. The significantly dif-
ferent LSPR responses from the human samples of
the AD and control patients are the first step
towards a molecular diagnostic test for AD.
The nanoparticle-based LSPR platform is eas-
ily adapted to other readout and fabricating
techniques for a wide range of applications. For
225
REVIEW – Zhao, Zhang, Ranjit Yonzon, Haes & Van Duyne
Figure 8. Demonstration of the
synthetic procedure of solution-phase
nanoparticles using nanosphere
lithography and the corresponding
optical and structural characterizations
of the nanoparticles.
A Sonicate Add
in ethanol dithiol
Substrate
1 2
B
0.12 1
3
Extinction
0.10
0.08
0.06 2 hypothyroidism. In addition, the LSPR tech-
0.04
400 600 800 1000
Wavelength (nm)
(A) Schematic illustration of the synthetic
procedure of the solution-phase nanoparticles.
(A-1) Surface-bound Ag nanoparticles fabricated
by nanosphere lithography and modified with a
self-assembled monolayer (SAM) of alkanethiol.
(A-2) Releasing the Ag nanoparticles into solution.
(A-3) Asymmetrically linked solution-phase Ag
nanoparticles with alkanedithiol.
(B) Corresponding localized surface plasmon
resonance spectra and atomic force microscopy
and transmission electron microscope images of
the nanoparticles after each fabrication step. All
spectra were collected in ethanol.
example, LSPR has been adapted to three differ-
ent applications. First, a single surface with two
different carbohydrate sensors was produced,
demonstrating a multiplexed carbohydrate
LSPR-sensing chip, which has been realized by
synthesizing nanoparticles with two different
heights, functionalized with mannose thiol and
galactose thiol. Target molecules, Con A, which
will specifically bind to mannose, are exposed to
the nanoparticles and the LSPR responses from
the two types of nanoparticles are distinct. Sec-
ond, nanoparticles have been fabricated on con-
ducting ITO substrates, which allows the
application of electropotential on the substrate
and measuring the LSPR at the same time. The
nanoparticle geometry was tuned electrochemi-
cally and characterized with AFM, and the corre-
sponding LSPR spectra have been monitored.
Finally, a novel fabrication technique was
226
developed to release the NSL-fabricated surface
bound nanoparticles into solution. The solution-
phase nanoparticles are then asymmetrically
linked to form dimers. The corresponding LSPR
spectra of the nanoparticle arrays, monomers
and dimers have been monitored.
Future perspectives
NSL-fabricated nanoparticles demonstrate a
great potential for applications in the field of
biomedical diagnostics. The nanoparticles have
3 tunable optical properties, which make them an
ideal LSPR-sensing platform. By functionalizing
the nanoparticle surface with the appropriate
receptor, the LSPR nanosensor can be used to
detect specific ligands. Such LSPR nanosensors
can be used as diagnostic tools for a variety of
diseases, such as AD, cancer and congenital
nique is capable of screening oligomerization-
blocking drugs for AD, as well as other diseases.
Moreover, to improve the sensing efficiency, a
multiplexed LSPR sensing chip for rapid and
parallel detection of multiple targets will be fab-
ricated by integration with microfluidic [49]
channels to lower the required analyte(s) volume
and to save time. Furthermore, electrochemical
potential can be applied to nanoparticles fabri-
cated on an ITO substrate while monitoring the
LSPR of these nanoparticles. Electrochemically
modified LSPR sensors are under investigation
for the detection of the electrochemically active
heme proteins, such as cytochrome c and P450.
Finally, the same principles that apply to nano-
particle arrays also apply to single nanoparticles.
The nanoparticle array-based biosensors can be
transitioned to single nanoparticles to improve
LSPR sensitivity. Releasing NSL-fabricated
nanoparticles in solution provides a novel
method to synthesize monodispersed nanoparti-
cles, which can, in turn, be used as a single nan-
oparticle-sensing platform, opening up the
possibility of detecting a low number of adsorb-
ate molecules. Previously, a single nanoparticle
was used to detect zeptomolar alkanethiols [6]
and lower than 100 nM streptavidin [14,50,51]. By
appropriate functionalization, the released nano-
particles can be dispersed into a living system to
develop in vivo LSPR sensors.
Acknowledgements
We gratefully acknowledge support from the National Science
Foundation (EEC-0118025, CHE-0414554 and
DMR-0076–97) and the Air Force Office of Scientific
Research MURI program (Grant F49620–02–1-0381).
Nanomedicine (2006) 1(2)
 Localized surface plasmon resonance biosensors – REVIEW

Executive summary
• Nanoparticle-based localized surface plasmon resonance (LSPR) biosensing is a broad platform
suitable for many different sensing architectures.

• LSPR of noble metal nanoparticles is highly sensitive to the adsorbate-induced changes in the
surrounding dielectric environment and, thus, can be used for biosensing.

• The nanosphere lithography (NSL) technique is an inexpensive method to produce well-ordered

nanostructures that are good biosensing platforms.

• The NSL-fabricated Ag nanoparticles were used to detect the biomarker of Alzheimer’s disease
(AD) – amyloid-derived diffusable ligands.

• The LSPR response has been tested using human samples from AD and control patients and the
results are significantly different.

• A multifunctionalized nanoparticle sensor has been fabricated to demonstrate multiplex sensing on a

single chip.

• Nanoparticles have been fabricated using NSL on a transparent conducting substrate. The
nanoparticle structure can be finely tuned by applying a potential to the substrate and the
corresponding optical properties have been monitored.

• A novel technique has been developed to release the NSL-fabricated nanoparticles from the substrate
into solution.

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