ACUNA ET AL.VOL. 6
’ NO. 4
March 22, 2012
C2012 American Chemical Society
Distance Dependence of Single-
Fluorophore Quenching by Gold
Guillermo P. Acuna,†,*Martina Bucher,‡Ingo H. Stein,‡Christian Steinhauer,‡Anton Kuzyk,§
Phil Holzmeister,†Robert Schreiber,^Alexander Moroz,)
Friedrich C. Simmel,§and Philip Tinnefeld†,*
Fernando D. Stefani,#Tim Liedl,^
†Physical and Theoretical Chemistry?NanoBioScience, TU Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany,‡Angewandte
Physik?Biophysik, Ludwig-Maximilians-Universität, Amalienstrasse 54, 80799 Munich, Germany,§Physik Department, Technische Universität München,
Am Coulombwall 4a, 85748 Garching, Germany,^Fakultät für Physik and Center for Nanoscience, Ludwig Maximilians Universität, Geschwister-Scholl-Platz,
Naturales, Universidad de Buenos Aires, Ciudad Universitaria Pab. 1, 1428 Buenos Aires, Argentina
complex metallic nanostructures can be
created by positioning metallic nanoparti-
cles or nanorods using DNA as scaffolding
material.1?4This scheme has recently taken
technique5to arrange metallic nanoparticles
(MNPs) into complex geometries.6?10The
DNA origami technique additionally offers
the possibility to place various moieties
such as fluorescent dyes on a rigid DNA
scaffold with subnanometer accuracy.11,12
Here, we have combined these unique abil-
ities and revisited the distance-dependent
single gold nanoparticle.
The interaction between metallic nano-
particles and fluorophores has recently
gained considerable attention.13,14On one
hand, the fluorescentdye is ananoreporter
of its immediate dielectric environment.
On the other hand, metallic nanostruc-
tures can enhance fluorescence with great
potential for diagnostic and material
science applications.15Besides theoretical
studies,16,17experiments have so far been
enabled by attaching MNPs to cantilever
tips in a microscope?AFM setup18,19or by
attaching dye-labeled DNA sequences to
the MNPs.20?22Using double-stranded
DNA as a spacer, the distance control is
limited because of DNA's non-negligible
flexibility23and because the DNA strands
have to be oriented vertically on the nano-
particle's surface. Additionally, it is difficult
rophores are incorporated into the DNA
etallic nanostructures offer new
means for light manipulation at
the nanometer scale owing to
to control the stoichiometry of dyes per
We build 2D rectangular DNA origami
and use them as a molecular breadboard
where a single fluorophore12and a gold
nanoparticle7are incorporated with nano-
meter precision at designated positions
within the DNA origami. While the fluo-
*Address correspondence to
Received for review December 23, 2011
and accepted March 22, 2012.
We study the distance-dependent quenching of fluorescence due to a metallic nanoparticle in
proximity of a fluorophore. In our single-molecule measurements, we achieve excellent control over
structure and stoichiometry by using self-assembled DNA structures (DNA origami) as a breadboard
where both the fluorophore and the 10 nm metallic nanoparticle are positioned with nanometer
precision. The single-molecule spectroscopy method employed here reports on the co-localization of
particle and dye, while fluorescence lifetime imaging is used to directly obtain the correlation of
by exact calculations that include dipole?dipole orientation and distances. Fitting with a more
practical model for nanosurface energy transfer yields 10.4 nm as the characteristic distance of 50%
energy transfer. The use of DNA nanotechnology together with minimal sample usage by attaching
experiments exploiting dye?nanoparticle interactions.
KEYWORDS: single-molecule fluorescence.gold nanoparticles.DNA
self-assembly.fluorescence quenching.DNA origami
ACUNA ET AL. VOL. 6
’ NO. 4
origami sheets during a one-pot annealing procedure,
the nanoparticles with 10 nm diameter are attached to
the assembled and dye-labeled sheets after their im-
mobilization on a microscope coverslip. This in situ
synthesis step directly yields a 1:1stoichiometry of dye
to nanoparticle and minimizes sample usage. Immobi-
lization by incorporated biotin?DNA strands also en-
ables a defined orientation of the DNA origami on the
coverslip with respect to the circularly polarized ex-
citation light plane. In our experiment, we place the
single fluorophores in a plane 13.2 nm below the
light plane. For this plane the field enhancement due
to the MNP can be neglected, excluding nanoparticle-
We employ two-color single-molecule spectroscopy
to evaluate reaction yields and to use origami without
MNP as internal standards. Fluorescence lifetime ima-
ging microscopy then yields the fluorescence intensi-
ties and the fluorescence lifetimes from individual dye
molecules that are attached at four dye?nanoparticle
are analyzed and compared to exact calculations fol-
lowing Chew17and Ruppin26for the radiative and
nonradiative rate changes, respectively. We find ex-
cellent agreement with the calculations.
To the best of our knowledge, this work constitutes
the first study of the distance-dependent interaction
between a metallic nanoparticle and a fluorescent
molecule using immobilized DNA origami. The single-
molecule approach directly enables us to map the
distribution ofproperties in potentially heterogeneous
populations. This combination of DNA nanotechnol-
ogy, metallic nanoparticles, and single-molecule spec-
troscopy is readily applicable for bottom-up nano-
photonic systems with increasing complexity.
RESULTS AND DISCUSSION
The studied DNA origami is sketched in Figure 1a.
The DNA origami rectangle (100 ? 70 nm) is folded
from one m13mp18-derived scaffold strand and 226
staple strands.5In the hybridization process, the
three capturing strands (17 bp ∼5.8 nm) for nano-
particle binding, the three biotin-modified strands
(not shown), and the fluorophore-modified staple
strand (ATTO647N, red with an intrinsic quantum yield
origami rectangle contains one ATTO647N dye, and
the red and pink dots in Figure 1a indicate the posi-
tions of this dye in the different samples. We use three
capturing strands to increase the rigidity of the inter-
action between DNA origami and nanoparticle and to
reduce the conformational freedom. Gold nanoparti-
cles are functionalized with single-stranded DNA se-
quences via thiol bonds (see Materials and Methods
for experimental details). These DNA sequences are
complementary to the capturing strands on the origa-
the sole difference being the position of the fluoro-
phore while keeping constant the position of the
capturing strands. Fordistance estimation weconsider
pairs and two adjacent helices, respectively.5,12By
(17 base pairs, ∼5.8 nm) and assuming a 90? angle
between the origami surface and the bound capturing
triple, the four distances between the fluorophore and
the surface of the MNP are d1= 10.1 ( 3.1 nm, d2 =
Figure 1. (a) Sketch of the 2D DNA origami with attached
positions are shown simultaneously (d1?d4), although
every DNA origami sample contains only one red fluoro-
phore. The green dots represent single-stranded DNA la-
beled with Cy3 to visualize the gold nanoparticles. (b) False
color image of a two-color confocal fluorescence scan with
alternating laser excitation indicating the binding of the
MNPs (green, Cy3) to the DNA origami with ATTO647N at
distance d2 (red). Yellow spots indicate co-localization of
lifetime image (FLIM) of the red-excitation channel indicat-
ing quenched (green-blue) and unquenched (orange-red)
ATTO647N dyes. Fluorescence lifetime of unquenched AT-
TO647N is 4.3 ns. Scale bar, 5 μm. (d) Fluorescence decay
(black), instrument response function (red), and fit (blue)
from all photons summed from one exemplary spot.
ACUNA ET AL. VOL. 6
’ NO. 4
Following immobilization on a BSA-biotin-neutravi-
din-coated coverslip, functionalized 10 nm gold nano-
particles are added. Since the nanoparticles bind to
immobilized DNA origami on the glass surface, no
additional purification steps are required, and the
concentration of nanoparticles required is between
100 and 1000 times lower than previously reported.6
tion time of one hour, unbound particles are washed
capturing strands is added to label the MNPs. This
labeling allows determining the binding efficiency of
MNP to DNA origami on the single-molecule level as
well as the nonspecific binding of MNP to the glass
surface in a two-color experiment. Figure 1b shows a
false color image overlaying red fluorescence from the
DNA origami (ATTO647N at distance d2) and green
fluorescence from the MNPs (Cy3). Yellow spots indi-
cate co-localization of MNP and origami structures.
Images for the four different DNA origami samples
are acquired with alternating laser excitation for quasi-
simultaneous imaging of both colors.27A non-negligi-
ble fraction of nonspecific binding of MNPs to the
surface visible in Figure 1b does not affect our mea-
surements since the MNP?fluorophore interaction
decays fast with the distance and can be neglected
at a distance of more than one pixel (50 nm).
Besides the intensity, we simultaneously record the
time lapse with respect to the corresponding laser
pulse for each photon.27,28This time-correlated sin-
gle-photon counting allows the extraction of the fluo-
rescence lifetime information for each pixel by fitting a
single exponential to the histogram of photon arrival
times. The resulting fluorescence lifetime image is
shown in Figure 1c. Clearly, two populations of yel-
low-red (long unquenched fluorescence lifetime)
and blue-green spots (short quenched fluorescence
lifetime) are visible. Comparison with Figure 1b indi-
cates that the spots with shorter fluorescence lifetime
represent DNA origami carrying both the red dye and
MNPs is also used in control experiments to ensure
that the dye in the origami structure (ATTO647N) is
solely influenced by the MNP and not by the Cy3
labeling the MNP.
the relative change in fluorescence intensity and life-
MNP bound to the DNA origami. For optimal compar-
ison of origami structures with and without an MNP
bound, thebindingefficiencyisadjusted toabout50%
(53% for the full scan underlying Figure 1b,c) by
adapting the concentration of gold nanoparticles
and the incubation time. The origami structures with-
out nanoparticle then serve as internal control of
intensity and fluorescence lifetime, avoiding artifacts
and broadening related to sample preparation, align-
ment, and focus position, which can occur when
controls are measured separately. Notably, this in situ
synthesis enables a yield of ∼90% by increasing in-
cubation time and MNP concentration.
properties by summing up all photons within one spot
detected by a spot-finding algorithm. Fluorescence
decay histograms constructed from these photons
generally contain more than a thousand photons.
Decays are well described by a single-exponential
decay using a reconvolution algorithm that accounts
for the instrument response function (Figure 1d). For
results are summarized in Figure 2. In addition to the
intensity plot against fluorescence lifetime, a fre-
quency count plot of the intensity and the lifetime is
included on top of the axes.
The similarity of the DNA origami populations with-
out MNP in the different samples (in black in
Figure 2a?d) shows that all measurements are carried
out under comparable and reproducible conditions
with only some variations in the width of the fluores-
cence intensity histograms. In contrast, the population
of DNA origami with MNPs (in red in Figure 2a?d)
shows a clear dependence on the distance to the
MNP's surface. From a to d in Figure 2, as the distance
between the fluorophore and the surface of the MNP
increases, the intensity and the fluorescence lifetime
also increase until both reach the same magnitude as
the population without MNP for the sample with the
largest distance (d4 = 28.6 ( 3.1 nm). A more subtle
finding is that for the shorter distances the intensity is
correlated with the lifetime, indicating that inhomoge-
neous broadening is contributing to the width of the
distributions. This inhomogeneity does not average
out on the time scale of the experiment and might
be caused by the size and shape distribution of the
particles or by incomplete hybridization to all three
capturing strands. These effects leadtoa correlation of
intensity and lifetime within one sample, visible in
Figure 2a and b. We use this inhomogeneity to esti-
mate an upper limit of the error of the particle?dye
distance by converting the lifetime distribution into a
distance distribution (vide infra).
From the results, the relative change influorescence
intensity and lifetime is directly extracted, and the
mean and standard deviations are shown as black
DNA origami leads to a quenching of its fluorescence
intensity and a reduction of its fluorescence lifetime.
This interaction is strongly distance dependent as
previously reported.29The exact solution for a dipole
interacting with a metallic nanoparticle was already
obtained17,26based on a quasi-static approximation,16
but in both cases there is no analytical representation
ACUNA ET AL. VOL. 6
’ NO. 4
of the distance dependence.30For gold NPs with a
diameter of <2 nm, a distance dependence of 1/d4has
energy transfer (NSET) model developed by Persson
and Lang.33However, for 10 nm Au NPs this model
deviates from the experimental results.22In order to
test our results, we include the theoretical calculation
of the relative change in intensity and lifetime accord-
Since in our experiments the dipole moment of the
fluorophore is randomly oriented and changes during
the measurement, we considered both a tangential
and a radial orientation for the calculations together
with a natural weighted average (considering a 2-fold
degeneracy of the tangential orientation) of the two
orientations (in blue, red, and black, respectively, in
MNP and the fluorophore. The results obtained show
Figure3. (a)Relativechangeinfluorescence intensityand (b) fluorescence lifetimeas afunctionof thedistancebetweenthe
fluorophore and the MNP. The black squares represent the mean values and standard deviations obtained from the single-
NSET calculation (with d0= 8.38 nm), respectively.
Figure 2. Intensity versus fluorescence lifetime plots together with their corresponding frequency count plots. The black
squares and the red circles represent the DNA origami structures without and with metallic nanoparticle, respectively. From
(a) to (d) the distance between the fluorophore and the surface of the metallic nanoparticle is increased in four steps, d1 =
10.1 ( 3.1 nm, d2 = 11.2 ( 3.1 nm, d3 = 14.3 ( 3.1 nm, and d4 = 28.6 ( 3.1 nm.
ACUNA ET AL. VOL. 6
’ NO. 4
an excellent agreement of the exact solution with the
For the NSET model we observe a deviation for the
calculated d0value of 8.38 nm (see Figure 3a). The
d0value corresponds to the R0value in Förster theory
and is the distance at which the energy transfer
efficiency is 50%. The d0value is calculated by the
and spectral properties of the dye and bulk constants
of Au.33Allowing d0to vary, we obtain an excellent fit
with an experimental d0value of 10.4 nm (not shown,
almost identical to the weighted average). Owing to
dependence, this model might be useful for practical
purposes34?36if experimental d0values are accessible,
for example, on the basis of DNA origami based
distance rulers. Here, the combination of metal parti-
cleswithorganic dyesexceedsthe typicalFRETregime
found around 10.4 nm and is dominated by nonradia-
tive rate changes.
In summary, we have performed single-molecule
fluorescence quenching studies of a fluorophore by a
metallic nanoparticle. For the first time, we have used
DNA origami structures as a breadboard, where both
the fluorophore and the MNP are positioned with
nanometer precision. The results obtained show a
significant quenching of the fluorescence intensity
and a reduction of the fluorescence lifetime for a
fluorophore?MNP distance smaller than 15 nm, while
for longer distances the interaction between the MNP
and the fluorophore tends to disappear. The experi-
mental results are in very good agreement with theo-
The combination of the DNA origami assay together
with our surface immobilization technique provides
several advantages: very small sample amounts are
required, and we are able to identify which fluoro-
phores have a nanoparticle in the vicinity. In addition,
the use of DNA origami will enable experiments in
which nanoparticles arranged in specific geometries
interact with fluorescent dyes. The observed distance
dependence also indicates that nanoparticle?dye in-
teractions on DNA origami can be used as an indicator
for dynamic processes occurring on DNA origami. Fluo-
rescence intensity and fluorescence lifetime can, for
example, report on binding events at specific positions
or distance changes related to DNA walkers, spiders, or
assembly lines37,38in a distance range difficult to access
by conventional FRET or super-resolution microscopy.
MATERIALS AND METHODS
Theoretical Calculations. The radiative and nonradiative rates
normalized to the free decay were calculated following refs 17,
26, and 39 for a 10 nm gold nanoparticle with a dielectric
constant of ?12.15 þ i1.13. The calculations were performed at
the maximum emission wavelength of ATTO647N, that is,
669 nm. The relative change in radiative and nonradiative rates
and fluorescence lifetime considering the intrinsic quantum
yield of 0.65 for ATTO647N as described in ref 29. Numerical
simulations done with commercial FDTD software (www.cst.
com) showed that the field enhancement due to the MNP is
negligible at the plane where the fluorophores are placed, and
therefore we assumed that the relative change in intensity is
equal to the relative change in quantum yield since there is no
change in the excitation process. Changesof the excitation rate
strongest field enhancement but rather below the particle. The
the quantum yield given by qy = 1? [1/(1 þ (d/d0)4)] where d is
the distance between the dye and the MNP surface. d0=
8.38 nm is calculated from the Persson and Lang model33
considering the quantum yield of the ATTO647N to be 0.65
and the emission peak at 669 nm.
DNA Origami Structure. For the DNA origami structure, we
adopted the design from the original publication.5The proce-
dure followed for creating and folding the DNA structures as
well as the biotin-labeled staple strands are included in ref 12.
Labeling ofDNAorigamistructures withATTO647N atpositions
d1, d2, d3, and d4 was obtained by modifying the staples r7t2f,
r7t4f, r7t6f, and r7t12f at the 30-end (using the same nomen-
clature as in ref 5). In every origami sample, the three capturing
strands were added by modifying the staples r5t2e, r5t4f, and
Functionalization of the Gold Nanoparticles. Ten-nanometer gold
nanoparticles were purchased from BBInternational (www.bbi-
gold.com). The DNA functionalization was performed by GNA
Biosolutions GmbH (www.gna-bio.de) as described in ref 20
with the following DNA sequence containing a thiol modifica-
tion at the 30end: 50-TCT CAA CTC GTA AAA AA-Thiol-S-30.
Binding oftheGoldNanoparticlestotheDNAOrigami Structures. The
hybridization of the nanoparticles with the DNA origami struc-
tures as well as the subsequent measurements was performed
on chambered glass slides (Lab-tek, Nunc). The chambers were
passivated and functionalized with BSA-biotin-neutravidin. The
DNA origami structures (1 nM in PBS) were added and washed
away once the desired surface coverage was reached (after
approximately 5 min). Then the gold nanoparticles were added
(100 pM in PBS with 600 mM NaCl). In order to reach a binding
yield of around 50%, the incubating time was set to 45 min. For
nanoparticle labeling, the DNA sequence 50-TT TTT TAC GAG
TTG AGA-30-Cy3 was finally supplied at a concentration of 1 nM
for 5 min. Prior to measurement, the buffer was exchanged for
PBS with 2 mM Trolox for blinking suppression and the cham-
bers were sealed.
Determination of Intensity and Lifetime. Fluorescence intensity
and lifetime measurements were carried out on a custom-built
confocal setup based on an Olympus IX-71 inverted micro-
scope. We employed alternating laser excitation with indepen-
dent detection.27,28Excitation was carried out by a pulsed
supercontinuum laser (NKT-Photonics) set to 640 and 533 nm.
Theexcitation polarizationwas circularly polarized employing a
quarter-wave plate and a half-wave plate (B. Halle Nachfl. GmbH).
The alternating time period was 1 ms using an acousto-optical
tunable filter. The fluorescent light was separated by appropriate
filters and detected in different APDs as described in ref 40 with a
time resolution of 150 ps. The lifetime values were extracted by
reconvoluting the instrument response function using the Fluofit
software from Picoquant (www.picoquant.com).
ACUNA ET AL.VOL. 6
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Conflict of Interest: The authors declare no competing
Acknowledgment. The authors are grateful to Joachim
Stehr, Fabian Baumann, and Andreas Gietl for fruitful discus-
sions. This work was supported by DFG (TI329/6-1, LI1743/2-1),
the DFG Excellence Cluster Nanosystems Initiative Munich, a
starting grant of the European Research Council (ERC), the
Volkswagen Foundation, and the Center for NanoScience.
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39. A Fortran F77 source code for the CHEW calculation can
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