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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J. AM. CHEM. SOC. XXXX, XXX,
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antibody. From the quantitative model, which underestimates
the LSPR response for ADDL detection in the sub-10 pM
region, the actual response observed here suggests that the
molecules have a higher molecular weight than the second class
of ADDLs detected. This is apparent from the larger than
predicted LSPR shifts observed in the direct ADDL response
analysis. The results in region 2 (10 pM to 10 nM) verify this
claim. The second class of molecules was revealed to have a
binding constant, Ka,surf,2, about 9.5 × 108M-1. A comparison
study using size exclusion chromatography (SEC) and ELISA
revealed that ADDLs are comprised of two major types of
molecules with different molecular weights (unpublished data).
This is consistent with studies by Bitan and Teplow who
detected different subpopulations in experiments with cross-
linking reagents.36The data in region 3 (10 nM to 10 µM)
reveals unusual behavior. The drop-off in the response was
initially thought to arise from the so-called “Hook effect”.37,38
However, this type of behavior should be eliminated when a
monoclonal antibody is used in place of the second polyclonal
antibody. When this assay is repeated with a monoclonal second
antibody, the hook effect is still observed (data not shown).
Consequently, this response cannot be attributed to the Hook
effect. Further work is in progress that seeks to understand this
phenomenon. Our working hypothesis regarding this deviation
is that at extremely high ADDL concentrations, small surface
ADDLs (with a weak affinity to the first antibody) begin to
interact and begin to behave as large ADDLs with higher
binding affinities to the second antibody thereby being pulled
off of the nanoparticles and exhibiting a reduction in second
Finally, this work presents the first analysis of endogenous
biological samples using LSPR detection. These extremely
promising results indicate that the surface chemistry of the LSPR
nanosensor has been designed for optimal analysis of complex
biological species. The first set of samples discussed here
confirms the previous observation that ADDLs are present in
elevated concentrations in AD brain in comparison to a control.34
Using the quantitative models, the magnitude of the shifts
indicate that the ADDL concentration is ∼1 pM in the diseased
brain while the signal from the control sample is undetectable
with the noise level (i.e., ∼0). Again, the magnitude of the CSF
shift indicates that a higher concentration of ADDLs or ADDL-
related molecules is present in the CSF AD sample in
comparison to the control. While the magnitude of the shift from
the CSF sample is much larger than predicted by the ADDL
response binding curve (Figure 4A), the second anti-ADDL
antibody response indicates that the CSF sample contains
ADDLs (possibly complexed to other molecules). This response
is much larger than that observed in the analysis of the AD
brain extract. At this time, we hypothesize that the oligomer
size in CSF versus brain extract is quite different, perhaps
influenced by the differing molecular milieus of CSF and brain
extracts. Thus, it is most relevant to assess the two types of
samples separately. Even though these studies must be regarded
as preliminary given the small number of samples analyzed,
they are, nonetheless, very exciting. They indicate that the LSPR
nanobiosensor can be used to study human samples and may
aid in the understanding of the mechanism and diagnosis of
The success of this LSPR nanosensor was directly related to
the synthesis and isolation of target biomolecules. By integrating
this technology with the newly modified amyloid hypothesis,
previously unavailable information has been revealed regarding
the nature of ADDLs. All previous biophysical characterization
of the oligomerization and fibrillogensis of these molecules
typically required concentrations so high as to be irrelevant to
in vivo conditions. The LSPR nanosensor has been demonstrated
to be a powerful tool for studying the oligomerization of low
concentrations of amyloid precursors. Given this success, the
use of LSPR technology also holds promise as one of the best
detection techniques for the screening of oligomerization-
blocking drugs. Because the molecular causes and mechanisms
of AD are not fully understood, devices that provide insight
into the aggregated states of biological species and their
interactions at native concentrations will help in screening
patients for disease and possibly for studying drug interactions
with target species. This work represents the first steps toward
making this possible.
Acknowledgment. The authors gratefully acknowledge fi-
nancial support from the Nanoscale Science and Engineering
Initiative of the National Science Foundation under NSF Award
EEC-0118025. Any opinions, findings, and conclusions or
recommendations expressed in this material are those of the
authors and do not necessarily reflect those of the National
Science Foundation. W.L.K. gratefully acknowledges support
from NIH. W.L.K. is co-founder of Acumen Pharmaceuticals,
which has the sole license to patent rights owned by North-
western University and the University of Southern California
for use of ADDLs in the development of Alzheimer’s-related
therapeutics and diagnostics.
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Teplow, D. B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 330.
(37) Fernando, S. A.; Wilson, G. S. J. Immunol. Methods 1992, 151, 67.
(38) Fernando, S. A.; Wilson, G. S. J. Immunol. Methods 1992, 151, 47.
A R T I C L E S
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