Calibration of Dynamic Molecular Rulers
Based on Plasmon Coupling between
Bjo 1rn M. Reinhard,†,‡Merek Siu,§Harish Agarwal,†A. Paul Alivisatos,|,⊥and
Physics Department, Biophysics Graduate Program, and Chemistry Department,
UniVersity of California, Berkeley, California 94720, and Physical Biosciences
DiVision and Materials Sciences DiVision, Lawrence Berkeley National Laboratory,
Berkeley, California 94720
Received August 12, 2005; Revised Manuscript Received September 9, 2005
Pairs of noble metal nanoparticles can be used to measure distances via the distance dependence of their plasmon coupling. These “plasmon
rulers” offer exceptional photostability and brightness; however, the advantages and limitations of this approach remain to be explored. Here
we report detailed plasmon peak versus separation calibration curves for 42- and 87-nm-diameter particle pairs, determine their measurement
errors, and describe experimental procedures to improve their performance in biology, nanotechnology, and materials sciences.
The characterization of nanometer-sized machines, such as
artificial1and biological motors,2and of transient interactions
between individual macromolecules requires stable and
precise tools to measure absolute distances and distance
changes. However, continuous monitoring of distances is
challenging because of the small size of the systems of
interest (tens of nanometers) and the extremely broad range
of time scales. In cell differentiation and tissue growth, for
example, relevant time scales range from nanoseconds to
hours or longer. Molecular rulers based on single dye pair
fluorescence resonance energy transfer (FRET)3have been
the tool of choice for single-molecule measurements of
distance changes.4-7However, like all fluorescence-based
methods, FRET is constrained by the properties of organic
dyes: a short lifetime (<180 s) when continuously il-
luminated and blinking due to trapping in dark states.8,9
Conventional FRET measurements are also limited to a
distance range of <10 nm,10complicating the investigation
of the structural dynamics of large multicomponent systems
such as the ribosome, the DNA loops formed during
transcriptional regulation by distant enhancers,11and the
folding/unfolding of large proteins or RNAs.
In cases where probes with a diameter of 30-40 nm can
be tolerated, the limitations of FRET can be overcome with
a dynamic molecular ruler based on the distance-dependent
plasmon coupling of two noble metal nanoparticles.12A
particle plasmon refers to the collective oscillation of the
free electrons within a metal nanoparticle.13Noble metal
nanoparticles are efficient light scatterers at their plasmon
resonance frequency. The plasmon resonance frequency
depends on the size14-17and shape of the particles,15-19the
dielectric constant of the metal,13,17,20and the surrounding
medium.16,20-23When two nanoparticles are brought into
proximity (within ∼2.5 times the particle diameter)24their
plasmons couple in a distance-dependent manner.25As the
interparticle distance decreases, the coupled plasmon reso-
nance wavelength red-shifts. The distance dependence of the
plasmon coupling has been investigated at fixed interparticle
distances with different nanostructures such as spherical,26
cylindrical,27,28and elliptical nanoparticles,29trigonal prisms,28
and opposing tip-to-tip Au triangle (bowtie) nanostructures.30
In these studies, nanostructures were fabricated with top-
down fabrication techniques such as electron beam lithog-
raphy and thus had fixed interparticle spacing.
In contrast to the wealth of distance versus plasmon
coupling data available for fixed geometries in optically
homogeneous environments, there is currently very little
information available for dynamic geometries in optically
anisotropic environments. For the plasmon ruler to be a
versatile and robust tool in biology and materials science, it
is essential to obtain a wavelength versus distance relation-
ship that is valid under typical experimental conditions:
illumination by unpolarized light of functionalized particle
dimers randomly oriented in space. Ruler calibration can be
done experimentally (by measuring the resonance wavelength
* Corresponding author. E-mail: firstname.lastname@example.org.
†Physics Department, University of California, Berkeley.
‡Physical Biosciences Division, Lawrence Berkeley National Laboratory.
§Biophysics Graduate Program, University of California, Berkeley.
|Chemistry Department, University of California, Berkeley.
⊥Materials Sciences Division, Lawrence Berkeley National Laboratory.
Vol. 5, No. 11
10.1021/nl051592s CCC: $30.25
Published on Web 09/29/2005
© 2005 American Chemical Society
87-nm plasmon rulers is less steep, allowing the measurement
of larger distances and distance changes, albeit with a reduced
resolution. The accuracy of absolute distance measurements
is limited principally by size and shape heterogeneity of the
nanoparticles and the possibility of multiple tether formation
The accuracy of plasmon rulers should improve more than
4-fold for the 42-nm particles and more than 10-fold for the
87-nm particles by employing a simple enrichment and
validation procedure (Figure 6) that exploits a fundamental
advantage of single-molecule approaches: ruler assembly can
be monitored step by step, and at each stage, the nascent
ruler can be compared to a predefined standard such as a
simulated spectrum or empirically obtained standard spec-
trum. This procedure involves, per field of view, the
acquisition of two color images, some simple image process-
ing, and the subsequent acquisition of scattering spectra
(Figure 6). Given our present setup and frame rates, and
assuming the use of custom software and a commercial three-
axis positioning system or a slitless spectrometer, we estimate
that this procedure should identify 1-3 well-behaved plas-
mon rulers per field of view per minute. This approach is
conceptually identical to the one that has been refined over
the past decade for single-molecule fluorescence experiments
in which only FRET pairs fulfilling certain stringent criteria
(e.g., anticorrelation of donor and acceptor emissions and
single-step bleaching) are subject to further analysis.
To take full advantage of the long distance range of the
plasmon ruler, synthetic strategies have to be developed to
lower the surface density of tethering biopolymers and to
improve the size and shape homogeneity of the nanoparticles.
Both of these issues do not present fundamental limitations
of the technique. Already, 10-nm gold nanoparticles can be
functionalized routinely with a single DNA molecule,37and
new promising synthetic approaches to better size and shape
control, like the polymer-mediated polyol process, are being
Acknowledgment. We acknowledge financial support
from the Deutsche Forschungsgemeinschaft (B.M.R.), a
Howard Hughes predoctoral fellowship (M.S.), and a NSF
Graduate Research Fellowship (H.A.). This work was
supported in part by the Director, Office of Energy Research,
Office of Science, Division of Materials Sciences, of the U.S.
Department of Energy under contract no. DE-AC02-
05CH11231 and by the NIH National Center for Research
Resources through the University of California, Los Angeles,
subaward agreement no. 0980 G FD623 through the U.S.
Department of Energy. We are also grateful to Bruce Draine
and Michael Mishchenko for making their DDA and T-
matrix code publicly available and to Phillip Geissler for
his generous allowance of computational time on his cluster.
We thank Aleksandra Radenovic for taking the TEM images
and Hari Shroff for helpful discussions.
Supporting Information Available: Materials and meth-
ods, determination of the DNA coverage of Neutravidin gold
nanoparticle conjugates, and Figure S1. This material is
available free of charge via the Internet at http://pubs.acs.org.
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