Detection of a Biomarker for Alzheimer’s Disease from
Synthetic and Clinical Samples Using a Nanoscale Optical
Amanda J. Haes,†,§Lei Chang,‡William L. Klein,*,‡and Richard P. Van Duyne*,†
Contribution from the Department of Chemistry, Northwestern UniVersity, 2145 Sheridan Road,
EVanston, Illinois 60208-3113, and Department of Neurobiology and Physiology,
Northwestern UniVersity, 2205 Tech DriVe, EVanston, Illinois 60208
Received September 28, 2004; E-mail: firstname.lastname@example.org; email@example.com
Abstract: A nanoscale optical biosensor based on localized surface plasmon resonance (LSPR)
spectroscopy has been developed to monitor the interaction between the antigen, amyloid-? derived diffusible
ligands (ADDLs), and specific anti-ADDL antibodies. Using the sandwich assay format, this nanosensor
provides quantitative binding information for both antigen and second antibody detection that permits the
determination of ADDL concentration and offers the unique analysis of the aggregation mechanisms of
this putative Alzheimer’s disease pathogen at physiologically relevant monomer concentrations. Monitoring
the LSPR-induced shifts from both ADDLs and a second polyclonal anti-ADDL antibody as a function of
ADDL concentration reveals two ADDL epitopes that have binding constants to the specific anti-ADDL
antibodies of 7.3 × 1012M-1and 9.5 × 108M-1. The analysis of human brain extract and cerebrospinal
fluid samples from control and Alzheimer’s disease patients reveals that the LSPR nanosensor provides
new information relevant to the understanding and possible diagnosis of Alzheimer’s disease.
The development of highly sensitive and selective biological
sensors for the monitoring of disease biomarkers is an important
motivation for nanoscience research. The localized surface
plasmon resonance (LSPR) nanosensor has been demonstrated
to be an effective platform for the quantitative detection of
biological and chemical species.1-8The signal transduction
mechanism of the LSPR nanosensor is based on its sensitivity
to local refractive index changes near the surfaces of substrate-
confined, size- and shape-controlled, silver and gold nano-
particles.9-11The LSPR of noble metal nanoparticles arises
when electromagnetic radiation induces a collective oscillation
of the conduction electrons of the individual nanoparticles9,10,12-18
and has two primary consequences: (1) selective photon
absorption which allows the optical properties of these nano-
particles to be monitored with UV-vis spectroscopy and (2)
the enhancement of the electromagnetic fields surrounding the
nanoparticles which is responsible for all surface-enhanced
spectroscopies. The reader is referred to recent reviews14,16,19-21
for a detailed description of the physics behind LSPR spectros-
copy. The LSPR spectrum is measured by either transmission
extinction spectroscopy11,22or dark-field light scattering spec-
troscopy.23,24The extinction spectra of the nanoparticles exhibit
easily measured wavelength shifts that correspond to small
changes in the refractive index within the electromagnetic fields
surrounding the nanoparticles. It is well established that the
extinction spectra of Ag and Au nanoparticles synthesized using
nanosphere lithography (NSL) have refractive index sensitivities
†Department of Chemistry.
‡Department of Neurobiology and Physiology.
§Current address: Naval Research Laboratory, Washington D.C.
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Published on Web 00/00/0000
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of ∼200 nm/RIU3,5and a sensing volume that can be tuned by
controlling nanoparticle size, shape, and composition.3,4
The need for ultrasensitive detection methods for biological
and chemical sample screening is an important issue in disease
diagnosis and mechanistic understanding. For example, Alz-
heimer’s disease (AD), a progressive neurodegenerative disease
for which there is neither a cure nor a good clinical diagnostic,
is the leading cause of dementia in people over age 65 and
affects over four million Americans. Genetic, biochemical, and
animal model studies strongly suggest a central role for
amyloid-? (A?) in the pathogenesis of AD.25A? is a 42-amino
acid peptide that was first discovered as the monomeric subunit
of the large insoluble amyloid fibrils of AD plaques. In the past
several years, it has been found that, in addition to plaques, A?
will also self-assemble into small soluble oligomers, termed
ADDLs, (amyloid-derived diffusible ligands), and that ADDLs
will cause neurological dysfunctions relevant to memory.26-28
Two comprehensive reviews of current data have concluded that
early stages of the disease are likely to be caused by the synaptic
impact of such soluble oligomeric assemblies of A?.29,30Despite
the fact that several molecules including A?1-40, A?1-42, tau,
phosphorylated tau, and seeded A? forms or their combination
have been suggested as potential AD biomarkers,31-33at the
present time, there is no definite clinical diagnosis for AD other
than autopsy. Recently, it has been demonstrated that ADDLs
are present at significantly elevated levels in autopsied brain
samples from humans with AD.34The association of ADDLs
with disease mechanisms and brain pathology suggests that a
sensitive means to detect ADDLs in body fluid could provide
a definitive molecular basis for the laboratory diagnosis of AD.
An ultrasensitive method for ADDLs detection potentially could
emerge from LSPR nanosensor technology, providing an
opportunity to develop the first clinical lab diagnostic for AD.
In this study, we combine LSPR nanosensor technology with
A? oligomer specific antibodies to determine if ADDLs are a
potential biomarker for AD. In comparison with other biosensing
approaches, the LSPR nanosensor allows for the analysis of
biological species in a surfactant-free environment. This is
extremely important because the absence of a surfactant allows
for biological species to be analyzed in their native state (not
one influenced by the presence of physiologically unnatural
molecules). In addition, the LSPR nanosensor is demonstrated
to be sufficiently sensitive for the detection of ultralow
concentrations of ADDLs in biological samples. By optimizing
the size of the nanoparticles, the sensing distance of the
nanoparticles extends ∼35 nm from the surface. This sensing
distance is required for the implementation of a sandwich assay
for the target ADDL molecules. Quantitative detection models
for both the ADDL and the second anti-ADDL antibody
response are developed to extract information regarding the
affinity constants between the antigen and antibody species and
to aid in the analysis of human samples. Human brain extracts
and cerebrospinal fluid (CSF) samples from control and diseased
patients exhibit drastically different responses, which indicate
the presence of elevated ADDL concentrations in the samples
from diseased patients in comparison to the control patients.
These preliminary results indicate that LSPR nanosensor
technology is useful as a screening method of human samples
for disease diagnosis as well as a more general approach to a
mechanistic understanding of diseases and drug target interac-
Methods and Materials
Materials. 11-Mercaptoundecanoic acid (11-MUA) and 1-octanethiol
(1-OT) were purchased from Aldrich (Milwaukee, WI). Phosphate-
buffered saline (PBS) solutions of 10 and 20 mM, pH 7.4, was
purchased from Sigma (St. Louis, MO). 1-Ethyl-3-[3-dimethy-lami-
nopropyl]carbodiimide hydrochloride (EDC) was obtained from Pierce
(Rockford, IL). Absolute ethanol was obtained from Pharmco (Brook-
field, CT). Methanol was purchased from Fisher Scientific (Pittsburgh,
PA). Silver wire (99.99%, 0.5-mm diameter) was purchased from D.
F. Goldsmith (Evanston, IL). Tungsten vapor deposition boats and
chromium rods were acquired from R. D. Mathis (Long Beach, CA).
Polystyrene spheres of 390 nm ( 19.5 nm were received as a suspension
in water from Duke Scientific (Palo Alto, CA). Millipore cartridges
(Marlborough, MA) were used to purify water to a resistivity of 18.2
MΩ cm-1. Triton X-100 was purchased from Aldrich (Milwaukee, WI).
Ruby red muscovite mica substrates were purchased from Asheville-
Schoonmaker Mica Co. (Newport News, VA).
Synthetic ADDL and Anti-ADDL Antibody Preparation. A?1-42
peptide (California Peptide Research, Napa, CA) was used to prepare
synthetic ADDLs according to published protocols.26An aliquot of
A?1-42was dissolved in anhydrous DMSO to a concentration of 22.5
mg/mL (5 mM), pipet mixed, and further diluted into ice-cold F12
medium (phenol red free) (BioSource, CA) at a ratio of 1:50. The
mixture was quickly vortexed, incubated at 6-8 °C for 24 h, and
centrifuged at 14 000g for 10 min. The oligomers were then collected
from the supernatant. The concentration of synthetic ADDLs was
determined by a micro-BCA assay (Pierce, Rockford, IL). A similar
protocol was followed for vehicle preparation except no A? peptide
was added. Antibodies targeting ADDLs in the LSPR immunoassay
(polyclonal M71 and monoclonal 20C2) were generated and character-
ized as previously described.35
CSF and Brain Extract Preparation. CSF samples were provided
by Dr. John Lee, Loyola University Medical School, Maywood, IL.
Premortem samples were obtained via lumbar puncture and kept frozen
until used. Tissues of cortex of AD (pathology diagnosis based on Braak
& Braak, CERAD, and NIA/Reagan Institute Criteria) and age-matched
control brains were obtained from Northwestern University Alzheimer’s
Brain Bank and stored at -80 °C before using. Brain extracts were
prepared following the literature.34One hundred milligrams of tissue
was homogenized in 1 mL Ham’s F12 medium (phenol-free BioSource,
Camarillo, CA) containing protease inhibitors (complete mini-EDTA
free tablet, Roche, Indianapolis, IN) on ice using A Tissue Tearor
(Biospec Products, Bartlesville, OK). After centrifugation at 20 000g
for 10 min, the supernatant was centrifuged at 100 000g for 60 min.
(25) Selkoe, D. J.; Hardy, J. Science 2002, 298, 963.
(26) Lambert, M. P.; Barlow, A. K.; Chromy, B. A.; Edwards, C.; Freed, R.;
Liosatos, M.; Morgan, T. E.; Rozovsky, I.; Trommer, B.; Viola, K. L.;
Wals, P.; Zhang, C.; Finch, C. E.; Krafft, G. A.; Klein, W. L. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 6448.
(27) Walsh, D. M.; Klyubin, I.; Fadeeva, J. V.; Cullen, W. K.; Anwyl, R.; Wolfe,
M. S.; Rowan, M. J.; Selkoe, D. J. Nature 2002, 416, 535.
(28) Chromy, B. A.; Nowak, R. J.; Lambert, M. P.; Viola, K. L.; Chang, L.;
Velasco, P. T.; Jones, B. W.; Fernandez, S. J.; Lacor, P. N.; Horowitz, P.;
Finch, C. E.; Krafft, G. A.; Klein, W. L. Biochemistry 2003, 42, 12749.
(29) Mattson, M. P. Nature 2004, 430, 631.
(30) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
(31) Hampel, H.; Mitchell, A.; Blennow, K.; Frank, R. A.; Brettschneider, S.;
Weller, L.; Moller, H. J. J. Neural Transmission 2004, 111, 247.
(32) Pitschke, M.; Prior, R.; Haupt, M.; Riesner, D. Nature Medicine 1998, 4,
(33) Seubert, P.; Vigopelfrey, C.; Esch, F.; Lee, M.; Dovey, H.; Davis, D.; Sinha,
S.; Schlossmacher, M.; Whaley, J.; Swindlehurst, C.; McCormack, R.;
Wolfert, R.; Selkoe, D.; Lieberburg, I.; Schenk, D. Nature 1992, 359, 325.
(34) Gong, Y.; Chang, L.; Viola, K. L.; Lacor, P. N.; Lambert, M. P.; Finch, C.
E.; Krafft, G. A.; Klein, W. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
(35) Lambert, M. P.; Viola, K. L.; Chromy, B. A.; Chang, L.; Morgan, T. E.;
Yu, J. X.; Venton, D. L.; Krafft, G. A.; Finch, C. E.; Klein, W. L. J.
Neurochem. 2001, 79, 595.
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Protein concentration of 100 000g supernatant was determined by
standard BCA assay. Fractions were stored at -80 °C. Gloves were
worn at all times when handling all biological materials.
Nanoparticle Fabrication. Nanosphere lithography (NSL) was used
to create monodisperse, surface-confined Ag nanotriangles.2,4Polysty-
rene nanospheres (diameter ) 390 nm + 19.5 nm, Duke Scientific)
were diluted as a 1:1 solution with Triton X-100 and methanol (1:400
by volume). Approximately 4 µL of this solution was drop-coated onto
the freshly cleaved mica substrates2and allowed to dry, forming a
monolayer in a close-packed hexagonal formation, which served as a
deposition mask. The samples were mounted into a Consolidated
Vacuum Corporation vapor-deposition chamber. A Leybold Inficon
XTM/2 quartz crystal microbalance was used to monitor the thickness
of the metal being deposited. For all experiments, 25 nm of Ag (D. F.
Goldsmith) was evaporated onto the samples. Following metal deposi-
tion, the samples were sonicated for 3-5 min in ethanol (Pharmco) to
remove the polystyrene nanosphere mask, creating Ag triangular
nanoparticles on the mica substrate.
Ultraviolet-Visible Extinction Spectroscopy. Macroscale UV-
vis extinction measurements were collected using an Ocean Optics
(Dunedin, FL) S2000 fiber optically coupled spectrometer with a CCD
detector. All spectra in this study are macroscopic measurements
performed in standard transmission geometry with unpolarized light.
The probe diameter was approximately 1 mm. A home-built flow cell5
was used to control the external environment of the Ag nanoparticle
Nanoparticle Functionalization. Immediately following nanosphere
removal, the samples were incubated for 24-48 h in 3:1 1 mM 1-OT/1
mM 11-MUA solution (in ethanol). Next, the samples were rinsed
thoroughly with ethanol and dried with N2. Following SAM function-
alization, the samples were incubated in a 100 nM anti-ADDL antibody
solution in 10 mM PBS. Covalent linking was ensured via the presence
of 100 mM EDC during this 1-h incubation period. Exposing the sample
to varying concentrations of ADDL in 10 mM PBS for 30 min was
the first detection step of the immunoassay. The assay was completed
via the incubation of the sample in 100 nM anti-ADDL antibody in 10
mM PBS for 30 min. Experiments were performed using a monoclonal
antibody for the second antibody; however, no notable differences were
observed in the LSPR shift response data. For this reason, only
polyclonal antibodies were used for these sandwich assays.
Nonspecific interactions were monitored by exposing the SAM
functionalized nanoparticles to a 250 nM anti-ADDL antibody solution
(no EDC coupling agent) for 1 h. For the sandwich assay experiments
involving the human samples, following SAM modification, the samples
were incubated in a 100 nM anti-ADDL/100 mM EDC solution in 10
mM PBS for 1 h. Next, the sample was exposed to varying concentra-
tions of human brain extract or CSF for 30 min. Finally, the sample
was exposed to 100 nM ADDL solution for 30 min. Samples were
rinsed with cycles of 10 mM PBS and 20 mM PBS (with 0.1% Tween
20) following each functionalization step to ensure the removal of
nonspecifically bound species. For the binding curves in Figure 4A
and 4B, each data point represents averaged experimental results from
a minimum of two fresh samples.
Atomic Force Microscopy. Atomic force microscope (AFM) images
were collected using a Digital Instruments Nanoscope IV microscope
and Nanoscope IIIa controller operating in tapping mode. The resulting
AFM linescan analysis reveal that the bare triangular nanoparticles have
∼90-nm perpendicular bisectors and ∼25-nm heights.
Detection of ADDLs Using an Antibody Sandwich Assay.
For the first time, a sandwich assay has been accomplished using
the LSPR nanosensor technology. Figure 1A shows a schematic
representation of the nanoparticle structure and chemical func-
tionalization for the sandwich assay. Briefly, triangular Ag
nanoparticles (perpendicular bisectors ) 90 nm, heights ) 25
nm) are synthesized on mica substrates and functionalized with
antibodies specific for ADDLs. Next, the nanoparticle surface
is exposed to varying concentrations of synthetic ADDLs.
Finally, the nanosensor is exposed to a fixed concentration of
anti-ADDL antibody for 30 min to complete the assay. A
polyclonal anti-ADDL antibody was used in both instances of
By functionalizing the Ag nanoparticles with a self-assembled
monolayer (SAM), the stability of the samples is greatly
increased and consistent red shifts2are produced upon incubation
in given concentrations of ADDLs and the second anti-ADDL
antibody. Because it is hypothesized that the formation ADDLs
may be implicated in the early symptoms associated with AD,
an assay that targets these molecules at sub-nanomolar concen-
trations is desired. A sandwich assay that relies on the specific
interactions between ADDLs and their antibodies is the most
Figure 1. Design and experimental setup for the LSPR biosensor for the detection of ADDLs using a sandwich assay. Transmission UV-vis spectroscopy
is used to monitor the optical properties (LSPR) of Ag nanoparticles. The schematic illustration displays the sandwich assay and surface chemistry of the
LSPR nanosensor. First, surface-confined Ag nanoparticles (see AFM inset, nanoparticle width ) 90 nm, nanoparticle height ) 25.0 nm) are synthesized
using NSL on mica substrates. Next, a SAM layer consisting of a mixed monolayer of OT and MUA passivates the nanoparticles for nonspecific binding
and activates the nanoparticles for the attachment of the first anti-ADDL antibody, respectively. The first anti-ADDL antibody is covalently attached to the
nanoparticles via incubation in 100 mM EDC/100 nM anti-ADDL antibody solution for 1 h. Samples are then incubated in varying concentrations of ADDLs
for 30 min. Finally, to enhance the LSPR shift response of the ADDLs, the samples are incubated in a 100 nM anti-ADDL solution for an additional 30 min.
Detection of a Biomarker Using an Optical Biosensor
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straightforward method to analyze and detect these molecules.
To perform this sandwich assay, implementation of the opti-
mization of nanoparticle size and sensing distances were
employed.4That is, the nanoparticles were fabricated to have
electromagnetic fields or an effective sensing distance that
extends ∼35 nm away from their surface.4
Representative sandwich assays for the direct detection of
high and low concentrations of ADDLs are displayed in Figure
2. In this study, anti-ADDL antibody was specifically linked to
the SAM functionalized nanoparticles over a 1-h period. Next,
the nanosensor is exposed to a 10 pM ADDL (Figure 2A)
solution for 30 min. During the ADDL incubation, the extinction
maximum of the sample shifts from 718.5 nm (Figure 2A-1) to
722.7 nm (Figure 2A-2), a 4.2-nm shift. Next, this shift is
amplified by exposing the sample to an additional anti-ADDL
antibody for 30 min resulting in an additional 7.6-nm wavelength
shift or a total LSPR shift of 11.8 nm. When this sandwich
assay is repeated with 100 fM ADDL (Figure 2B), the LSPR
extinction maximum shifts 1.9 nm (from 741.7 to 743.6 nm)
upon exposure to ADDLs. An additional 3.4-nm shift is
observed upon incubation in the second antibody for a total
LSPR shift of 5.3 nm. The samples were checked for nonspecific
binding after step 1, and no nonspecific binding was observed.
Elimination of Nonspecific Binding Using Mica Substrates.
The development of a successful sensor protocol requires the
demonstration of minimal to no nonspecific binding. In our
previous studies for implementing the LSPR nanosensor tech-
nology with an immunoassay specific for targeting antibodies
of AD markers,1the LSPR nanosensor response was dominated
by nonspecific interactions at low target molecule concentra-
tions. The source of this undesirable response was attributed to
a molecular interaction with the Cr adhesion layer. While silane
passivation decreased this undesirable result, it did not totally
eliminate the problem. Presently, we have made great strides
to overcome the nonspecific binding observed with the Cr
containing nanoparticles. First, mica substrates have been
substituted in place of the glass substrates and the use of Cr as
an adhesion layer has been eliminated. The adhesion of silver
to mica is sufficiently great that the nanoparticles remain
surface-bound throughout an experiment.6Additionally, as seen
in Figure 3A, when a SAM functionalized nanoparticle surface
is exposed to 250 nM anti-ADDL solution for an hour without
the use of a coupling agent (EDC), no shift in the LSPR
wavelength is observed. This indicates that no antibody is
randomly attaching to the nanoparticle sample and that the
nonspecific interactions that had previously plagued the LSPR
nanosensor shift responses have been eliminated.
A second nonspecific binding experiment is performed to
analyze the influence of the biological vehicle (see Materials
and Methods for description) on the LSPR response of an
antibody functionalized nanoparticle sample (Figure 3B). To
test for this nonspecific interaction, the LSPR nanosensor is first
specifically functionalized with anti-ADDL antibodies using an
EDC coupling agent (λmax ) 798.4 nm, Figure 3B-1). Next,
the sensor surface was exposed to the biological vehicle for 30
min revealing a slight blue-shift to 797.4 nm, Figure 3B-2.
Finally, the sensor surface was exposed to 100 nM anti-ADDL
Figure 2. Demonstration of the LSPR sandwich assay at high and low
concentrations. (A) LSPR spectra for each step of the sandwich assay for
10 pM ADDL. Ag nanoparticles after modification with (A-1) 100 nM anti-
ADDL antibody, λmax) 718.5 nm, (A-2) 10 pM ADDL, λmax) 722.7 nm,
and (A-3) 100 nM anti-ADDL, λmax ) 730.3 nm. (B) LSPR spectra for
each step of the sandwich assay for 100 fM ADDL. Ag nanoparticles after
modification with (B-1) 100 nM anti-ADDL antibody, λmax) 741.7 nm,
(B-2) 100 fM ADDL, λmax) 743.6 nm, and (B-3) 100 nM anti-ADDL,
λmax) 747.0 nm. All spectra were collected in N2.
Figure 3. LSPR spectra illustrating the lack of nonspecific binding for the
LSPR nanosensor. (A) Demonstration of nonspecific binding elimination
from anti-ADDL antibody to SAM-functionalized nanoparticles on mica.
(A-1) Ag nanoparticles after modification with 1:3 1 mM 11-MUA:1-OT,
λmax) 596.6 nm, and (A-2) Ag nanoparticles after exposure to 250 nM
anti-ADDLs (in the absence of EDC), λmax) 596.6 nm. (B) Demonstration
of the absence of nonspecific binding from a vehicle solution to anti-ADDL
antibody functionalized nanoparticles on mica. Ag nanoparticles after
modification with (B-1) 100 nM anti-ADDL antibody, λmax) 798.4 nm,
(B-2) vehicle, λmax ) 797.4 nm, and (B-3) 100 nM anti-ADDL, λmax )
797.1 nm. All spectra were collected in a N2environment.
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antibody for an additional 30 min. Once again, a slight blue-
shift in the LSPR extinction maximum to 797.1 nm (Figure 3B-
3) is observed. These small blue-shifts, which indicate that no
additional mass is being detected at the nanoparticle sensor chip,
can be attributed either to the removal of a small amount of
covalently bound anti-ADDL antibody from the substrate or to
slight variations in spectrometer noise. Considering nonspecific
binding and the peak-to-peak wavelength shift noise of the
baseline in repetitive experiments (∼0.3 nm), one can conser-
vatively estimate the smallest, reliable LSPR wavelength shift
as 3 times this value (∼0.9 nm).
Quantification of ADDL and Second Anti-ADDL Anti-
body Response. To thoroughly investigate the influence of
varying concentrations of ADDLs on the LSPR nanosensor
response, the shift induced by the direct binding of ADDLs onto
the anti-ADDL antibody functionalized nanoparticle surface has
been analyzed. To develop a more quantitative understanding
of the concentration-dependent response, only the shift from
the ADDLs and not the response from the second antibody is
included. The LSPR λmaxshift, ∆λmax) ∆R, versus [ADDL]
response curve was measured over the concentration range 1
fM < [ADDL] < 10 µM (Figure 4A). While the thermodynamic
affinity constant between antibodies and their specific antigens
is by no means large enough to be considered irreversible,
sample incubation and rinsing conditions have been optimized
to minimize nonspecific interactions while retaining the largest
possible LSPR shift response.
Figure 4A shows the experimental data for the ADDL shifts
plotted as the LSPR λmax shift, ∆R versus [ADDL]. The
experimental response curve for ADDLs has been quantitatively
interpreted in terms of a model that makes the following
assumptions: (1) 1:1 binding of a solution-phase multivalent
analyte (ADDL) with different sites but invariant affinities to
the surface-bound capture ligand (anti-ADDL antibody); (2) the
only operative nanoparticle sensing mechanism is the change
in the local refractive index caused by the adsorbed analyte
(ADDL); and (3) the measured LSPR λmaxshift response, ∆R,
is proportional to the thickness (and mass) of the adsorbed
analyte layer and its refractive index. The response curve can
be described by the following equation:6
∆R ) ∆Rmax[
where ∆R ) ∆λmax, LSPR λmaxshift for a given concentration,
∆Rmaxis the maximum LSPR response at high concentrations,
Figure 4. Quantitative response curves. (A) LSPR shift, ∆R or ∆λmax, versus [ADDL] response curve for the binding of ADDLs to an anti-ADDL antibody
functionalized Ag nanobiosensor. All measurements were collected in a N2environment. The solid line is the calculated value of ∆R using eq 1. The shaded
box indicates the limit of detection as determined from the spectrometer noise (3 times the smallest reliable wavelength shift measured). Error bars represent
the spread in the data. (B) Enhanced LSPR shift, ∆R or ∆λmax, versus [anti-ADDL antibody] response curve for the binding of second anti-ADDL antibody
to the ADDL functionalized Ag nanobiosensor. All measurements were collected in a N2environment. The solid line is the calculated value of ∆R using eq
2. The shaded box indicates the limit of detection as determined from the spectrometer (3 times the smallest reliable wavelength shift measured). Error bars
represent the spread in the data. The data are divided into three detection regions: (region 1) response for concentrations less than 10 pM, (region 2)
response for concentrations between 10 pM through 10 nM, and (region 3) response for concentrations greater than 10 nM.
1 + Ka,surf[ADDL]]
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