Accepted Manuscript
Steady-state and time-resolved fluorescence studies on the conjugation of Rose
Bengal to gold nanorods
Ana-Maria Gabudean, Raluca Groza, Dana Maniu, Simion Astilean
PII: S0022-2860(14)00499-2
DOI: http://dx.doi.org/10.1016/j.molstruc.2014.05.015
Reference: MOLSTR 20614
To appear in: Journal of Molecular Structure
Received Date: 15 February 2014
Revised Date: 8 May 2014
Accepted Date: 8 May 2014
Please cite this article as: A-M. Gabudean, R. Groza, D. Maniu, S. Astilean, Steady-state and time-resolved
fluorescence studies on the conjugation of Rose Bengal to gold nanorods, Journal of Molecular Structure (2014),
doi: http://dx.doi.org/10.1016/j.molstruc.2014.05.015
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* Corresponding author Phone number: +40 264 405300(5188) Fax: +40 264 591906 [email protected]
Steady-state and time-resolved fluorescence studies on the conjugation of Rose
Bengal to gold nanorods
Ana-Maria Gabudean, Raluca Groza, Dana Maniu, Simion Astilean*
Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute in Bio-Nano-Sciences and Faculty of Physics, Babes-Bolyai University,
1 M. Kogalniceanu Str., 400084, Cluj-Napoca, Romania
This paper is dedicated to Professor Simion Simon on the occasion of his 65th birthday.
ABSTRACT
This work examines the fluorescence properties of Rose Bengal (RB) molecules conjugated to
cetyltrimethylammonium bromide (CTAB) - coated gold nanorods (GNRs) by performing
steady-state and time-resolved fluorescence measurements. We show that the quantum yield and
fluorescence lifetime can be significantly modified by the electromagnetic coupling of RB to
GNRs but the overall fluorescence signal depends also on the environmental conditions in which
RB molecules reside - in solution or on solid substrate. For example, we have observed the
increase of fluorescence intensity after binding RB to GNRs (RB@GNR) as result of both non-
radiative rate decrease and contribution from the electromagnetic coupling of RB to GNRs. For
RB@GNRs conjugates deposited on solid substrate we can provide evidence for a clear metal-
enhanced fluorescence (MEF) mechanism by observing the decrease of fluorescence lifetime of
RB from an average of 2.1 ± 0.36 ns, when complexed to CTAB solely, to 1.6 ± 0.26 ns, when
conjugated to GNRs, together with the increase of fluorescence intensity. Our findings contribute
to the development and evaluation of novel fluorescent tags based on plasmonic nanoparticles
for biomedical applications.
Keywords: Rose Bengal, gold nanorods, steady-state fluorescence, time-resolved fluorescence
1. Introduction
The interaction of fluorescent molecules with plasmonic nanoparticles (NPs) has been
intensively addressed over the last decade aiming to design new spectral properties and improved
functionalities in medical diagnostics and biotechnology [1–3]. For instance, the coupling of
photosensitizing molecules to plasmonic NPs has received a great deal of attention due to the
attractive perspectives of performing both fluorescence imaging and photodynamic therapy
(PDT) of cancer [4–6]. Specifically, the metallic moiety can provide the enhancement of the
fluorescence signal though the phenomenon known as metal-enhanced fluorescence (MEF) while
the photosensitizer in close vicinity of NP can offer the basis of performing PDT with enhanced
rate of singlet oxygen generation [6]. Additionally, considering photosensitizer-NP conjugates
with plasmon resonance band located in the near-infrared ”biological window”, it is possible to
exploit the photo-to-heat conversion effect called plasmon-assisted hyperthermia together with
PDT for performing a dual anticancer therapy.
The dianionic xanthene dye Rose Bengal (RB) is known as potent photosensitizer (singlet
oxygen quantum yield of nearly 76% under 532 nm light irradiation [7]) but as poor emitter in
water (quantum yield of 0.02). Due to their photodynamic activity, RB molecules are applicable
in the treatment of skin diseases like psoriasis and atopic dermatitis [8], inhibition of oral cancer
DNA polymerases [9] or inactivation of various biological species such as vaccinia virus or
Escherichia coli [10]. Other exploitation of RB molecules include photo-catalysis [11], photo-
activation of the fabrication of three-dimensional cross-linked bovine serum albumin
microstructures [12] and staining for the diagnosis of eye disease in ophthalmology [13].
Most recently, the detection and therapy of oral cancer by using RB coupled with gold
nanorods (GNRs) has been demonstrated [14,15]. GNRs represent a particular class of
anisotropic gold NPs with unique optical properties generated by two modes of localized surface
plasmon resonance (LSPR), i.e. transversal and longitudinal modes, and the intense
electromagnetic field concentrated at their ends and corners [16,17]. GNRs have been
demonstrated as versatile plasmonic NPs in photothermal cancer therapy [18,19], detection of
biomarkers [20] or imaging contrast agents [21].
As result of chemical synthesis, GNRs exhibit a positively charged bilayer of
cetyltrimethylammonium bromide (CTAB) surfactant which enables adsorption and transport of
negatively charged photosensitizing drugs as RB molecules. Although both the investigation of
fluorescence properties of RB molecules in relation to their photodynamic activity and the
demonstration of RB@GNRs conjugates formation [22] have been reported, the investigation of
fluorescence properties of RB in interaction with GNRs in solution and solid film have not been
addressed from the perspective of extending both therapeutic and fluorescence properties. In this
work, we studied the fluorescence properties of free RB and RB in RB@GNRs conjugates both
in solution and deposited onto solid substrate through steady-state and time-resolved
fluorescence measurements. Specifically, time-resolved fluorescence spectroscopy can provide
useful information about the dynamic of molecular excitation and local interaction that is not
accessible from steady-state fluorescence data. This technique allows for example discrimination
of fluorophore when placed in heterogeneous environments and the recorded fluorescence
spectra look similarly. As the resonant excitation of LSPR promotes the enhancement of RB
fluorescence intensity, time-resolved fluorescence measurements are indispensable to elucidate
the enhancement mechanism. Moreover, we performed fluorescence lifetime imaging (FLIM)
studies on RB@GNRs conjugates deposited onto solid substrate to reveal spatial and temporal
response of RB molecules in experimental conditions close to biological samples.
2. Experimental Details
2.1. Reagents
Tetrachloroauric acid (HAuCl4•3H2O), cetyltrimethylammonium bromide (CTAB),
ascorbic acid and Rose Bengal (RB) were purchased from Aldrich. Sodium borohydride (NaBH4,
99%) and silver nitrate (AgNO3) were obtained from Merck. All reagents were used as received.
Ultrapure water (resistivity 18.2 MΩ) was used as solvent in all the experiments.
2.2. Samples preparation
CTAB-coated GNRs were synthesized using the seed-mediated growth approach detailed
in a previous publication [23]. For the preparation of RB@GNRs conjugates, we incubated for
several hours a solution of as-prepared GNRs (concentration of 0.57 × 10−9 M) with RB
molecules (the final concentration of RB in the solution was 10−6 M) to allow the attachment of
negatively charged RB to positively charged CTAB-stabilized GNRs. The interaction was
monitored during incubation by absorption spectroscopy. As reference samples for fluorescence
measurements we used 10-6 M RB in water and 10-2 M CTAB aqueous solutions, respectively.
2.3. Experimental measurements
The experimental results of this study were obtained by using the following techniques:
(i) absorption spectroscopy, which was performed on a Double-beam Jasco V-670 UV-Vis/NIR
spectrophotometer with 1 nm spectral resolution, equipped with a deuterium lamp (190 to 350
nm) and a halogen lamp (330 to 2700 nm), (ii) steady-state fluorescence spectroscopy by using a
Jasco LP-6500 spectrofluorimeter; we have selected the wavelength of 523 nm for excitation and
bandwidths of 3 nm in both excitation and emission; (iii) time-resolved fluorescence
measurements by employing the MicroTime200 time-resolved confocal fluorescence microscope
system, from PicoQuant equipped with a picosecond diode laser head operating at 510 nm and
40 MHz and a 60x/NA=1.2 water immersion objective; the signal collected through the objective
was spatially and spectrally filtered by a 50 µm pinhole and a FF01-519LP (Semrock, USA)
emission filter, respectively, before being focused on a PDM Single Photon Avalanche Diode
(SPAD) from MPD; the detector signals were processed by the PicoHarp 300 Time-Correlated
Single Photon Counting (TCSPC) data acquisition unit; fluorescence lifetime decays in solution
were obtained at room temperature after dropping the sample on microscope cover glass while
FLIM imaging was performed on samples spin-coated on microscope cover glasses; the
fluorescence lifetimes were obtained through reconvolution of the experimental decay curves
with the instrument response function (IRF) measured by collecting the back scattered light from
the laser; the goodness of the fit was judged by the chi-square values and by inspection of the
residuals; time and spectral information from selected points in the FLIM images were
simultaneously obtained by using a SR-163 spectrograph equipped with a Newton 970 EMCCD
camera from Andor Technology coupled to an exit port of the main optical unit of
MicroTime200 through a 50 µm optical fiber; a 50/50 beamsplitter was used to split the signal
from the analyzed point towards the spectrograph and TCSPC unit of the MicroTime200 system;
the integration time used for the acquisition of the fluorescence spectra was 5 s.
3. Results and discussion
When dealing with fluorescence properties of molecules in solution it is essential to
limit their concentration in order to avoid intermolecular interactions and interference with so
called inner filter effects [24]. Specifically, the attenuation of the excitation light or the re-
absorption of the emitted light when may distort and alter the real fluorescent spectra of the high
concentration samples. Additionally, when exceeding certain concentration the intermolecular
interaction cause the formation of molecular dimmers or small aggregates which further
influence the spectroscopic signal. Consequently, we first performed some preliminary
experiments to calibrate the optimal concentration of RB in solution (data not shown here). Our
findings showed that the inner filter effect and formation of dimmers start to become appreciable
only at concentrations higher than 10-6 M. Therefore to ensure reliable spectroscopic data from
free RB in solution and RB@ GNRs conjugates we work here with RB concentration of 10-6 M.
3.1. Fluorescence of RB@GNRs conjugates in solution
The GNRs present two well-defined surface plasmon resonance bands at 520 and 736
nm, originating from the coherent oscillations of electrons perpendicular and parallel to the
longitudinal axis of GNRs, respectively, as shown in Fig. 1.
Fig. 1. UV-Vis spectra of free RB (blue curve), as-prepared GNRs (black curve) and RB@GNRs
conjugates (red curve). Concentration of RB is 10-6 M, while the concentration of GNRs is 0.57 ×
10−9 M.
The red spectrum in Fig. 1 provides reliable evidence for RB conjugation to GNRs
surface. Indeed, 2 nm red-shifting of the longitudinal plasmon band reveals an increase of local
refractive index which can be explained by accumulation of RB on CTAB-coated GNRs.
Additionally the weak shoulder band which emerges from the envelope of transversal plasmon
band at about 563 nm is consistent with the electronic absorption of RB molecules after binding
to GNPs (see Fig. 1, blue curve). The fluorescence spectrum of RB@GNRs conjugates together
with the spectra of free RB and RB-CTAB as reference samples is presented in Fig. 2. All
spectra were recorded from solutions of identical RB concentration (10-6 M) under excitation at
523 nm wavelength in resonance with the transversal plasmon band of GNRs.
Fig. 2. Steady-state fluorescence spectra of free RB, RB-CTAB and RB@GNRs conjugates in
solution. The final concentration of RB in all samples was 10-6 M. The concentration of CTAB in
RB-CTAB sample was 4×10-6 M. Excitation wavelength at 523 nm.
As shown, compared to the fluorescence emission of free RB in water solution, located at
566 nm, the emission of RB@GNRs conjugates is red-shifted to 579 nm. This is a bathochromic
shift ascribed to a change of the environment conditions of RB after binding to the double layer
of CTAB. The binding between RB molecules and GNRs it should be mediated by electrostatic
interaction which occurs between positively charged layer of CTAB surfactant present at the
surface of gold surface and dianionic charge of RB molecule [25].
What is more interesting is the enhancement of fluorescence intensity of RB molecules
conjugated to GNRs via CTAB layer relative to fluorescence intensity of free RB molecules in
water (reference sample). Besides, we have noticed that when RB molecules are mixed to CTAB
molecules in water, the fluorescence intensity is also enhanced relative to reference sample (see
black solid and dashed spectra in Figure 2) and for a given concentration of 10-6 M the
fluorescence increases as function of CTAB concentration (10-6 - 10-2 M) to reach a saturation
level. This fluorescence increase effect can be explained by the fact that CTAB molecules
associate spontaneously in water and form cationic colloidal micelles as function of
concentration, which in turn can complex or accommodate the anionic RB molecules inside of
micelles [25]. Actually, such protected RB molecules are less exposed to water polar
environment and therefore exhibit enhanced fluorescence relative to reference sample (see
Figure 2). On the other hand, when RB molecules are bound to GNRs via CTAB layer, the RB
molecules are partially exposed to water which makes to increase the non-radiative deactivation
and explains a moderate fluorescence enhancement despite the interplay of electromagnetic
coupling to GNRs (see Figure 2). It is well known that fluorophores should have their
fluorescence emission totally quenched when situated extremely close or in direct contact to a
metal surface, due to a transfer of energy from the excited state to the metal [26]. However, in
our case the RB molecules are placed at about 4 nm from the surface of GNRs due to CTAB
spacer which make quenching rate less effective. Therefore, in our opinion, it is prone to errors
to compare fluorescence spectra of two complexed forms of RB in term of proper fluorescence
enhancement because of different chemical and electromagnetic environments in which
molecules reside and some uncertainty related to amount of complexed molecules in each
sample. On the contrary, it is meaningful to compare fluorescence lifetime measurements as the
fluorescence lifetime is generally absolute, being independent of the concentration of RB. This is
why we reveal the relative influence of different contributions to excitation and de-excitation
processes in each sample from fluorescence lifetime measurements. In the following in order to
elucidate the proper mechanism of fluorescence intensity enhancement we investigate the
fluorescence lifetime of RB in three different environments in solution: free in water solution,
complexed to CTAB surfactant and conjugated to CTAB-coated GNRs.
The fluorescence lifetime decay of RB@GNRs conjugates recorded in solution is
presented in Fig. 3. For comparison the plot also displays the histograms obtained for 10 -6 M RB
in aqueous solution and 10-2 M solution of CTAB.
Fig. 3. Normalized fluorescence lifetime decays of free RB, RB-CTAB and RB@GNRs
conjugates in solution. Final concentration of RB in the samples: 10-6 M. IRF represents the
instrument response function. Excitation: 510 nm. Laser power: 0.36 µW.
The fluorescence lifetimes of RB in the three solutions was assessed by reconvolution
technique using the IRF presented in the plot. The typical fluorescence decay of free RB in water
solution is mono-exponential revealing a lifetime of 75 ps, in good agreement with previous
results [27]. On the other hand, the decay of RB-CTAB could only be fitted with a two-
exponential function revealing a fast component of 75 ps (10 %) and a slow component of 680 ps
(90 %) corresponding to an amplitude averaged fluorescence lifetime 620 ps. In the case of RB-
CTAB system, the fast component (set at 75 ps) seems to correspond to the free RB molecules
from solution, while the slow component could be attributed to the RB molecules in interaction
with the micellar CTAB. It is well-known that changes in the quantum yield and lifetime of a
fluorophore are governed by the radiative and the nonradiative processes in the excited state
[26]. RB is considered extremely sensitivity to its local polarity and weakly fluorescence in
highly polar media such as water. However, it can become extremely fluorescent upon inclusion
in nonpolar environments. Since the CTAB provides a significantly less polar environment than
water, an increase in the lifetime will be observed due to decrease of non-radiative desexcitation
rate. The increase of fluorescence lifetime of similar dianionic dyes in micellar solutions has
been previously shown by Aydin et al. [28]. Finally, when conjugated to CTAB-capped GNRs,
RB exhibits 570 ps mono-exponential fluorescence decay. The absence of a fast component in
the recorded decay may indicate that there are no free RB molecules in the solution. It is known
that the emission rates and the distribution of the radiated energy of a fluorophore can be
modified by nearby metallic surfaces. As a result, in the close proximity of metallic surfaces, the
lifetime of fluorophores is decreases significantly, due to the increase of the radiative rate [29].
However, through binding with the CTAB layer, the non-radiative rate of RB decreases
significantly, as shown by the reference measurements performed on RB-CTAB. Consequently,
the 570 ps lifetime of RB@GNRs can be estimated as a combination of decreased non-radiative
rate due to the presence of CTAB layer and increased radiative rate induced by the presence of
metallic surfaces in close proximity. The reduction in the fluorescence lifetime of the
fluorophore despite an increase in the fluorescence intensity is a characteristic feature of the
fluorophore-plasmon coupling effect. We suppose that the origin of the enhancement mechanism
is a combination of the increase of both the radiative emission rate, due to GNRs acting as
optical nanoantennas, and the excitation rate, owing to the resonant excitation of GNRs. More
discussions of the MEF mechanism, as well as a theoretical estimation of the MEF factor are
presented in our previous paper reporting on the dual-modal spectroscopic performance of RB
conjugated to GNRs [30].
3.2. Fluorescence of RB@GNRs conjugates on solid substrate
When RB@GNRs nanoparticles are deposited on solid substrate strong electromagnetic
coupling can occur between tips and lateral surface of GNRs. Due to various locations in which
RB molecules reside, a high heterogeneity in both excitation and relaxation rate is expected. We
therefore analysed the distribution of fluorescence lifetime of RB in RB@GNRs conjugates
through FLIM technique. As reference sample we used a film of CTAB surfactant entrapping RB
molecules, which formed on glass substrate after solvent evaporation. Fig. 4A shows
representative FLIM images recorded from RB@GNRs conjugates and RB-CTAB complex
respectively, as deposited on glass substrate. The corresponding fluorescence lifetime histograms
extracted from FLIM images are represented in Fig. 4B.
Fig. 4. (A) FLIM images of RB-CTAB (left) and RB@GNRs (right) conjugates deposited on
glass surface (Scale bar 3 μm). Both images are represented on identical intensity and lifetime
scales; (B) Lifetime histograms of above FLIM images; (C) Representative fluorescence lifetime
decay curves and (D) Representative steady-state fluorescence spectra collected from the points
marked in the two FLIM images. Excitation: 510 nm. Laser power: 0.28 µW.
As indicated by the colour scale of FLIM images, a drastic decrease of fluorescence
lifetime of RB is noticed in the case of RB@GNRs as compared to the reference sample.
Specifically, the lifetime histograms reveal an average lifetime of 1.6 ± 0.26 ns for RB in
RB@GNRs film relative to 2.1 ± 0.36 ns for the reference sample. It is worth noting that FLIM
image (and corresponding lifetime histogram) indicates a higher number of counts collected from
RB@GNRs sample than from the reference sample, under identical experimental conditions.
Moreover, we point out that the sensitive spectrograph coupled to the MicroTime200 system is
able to record simultaneously with FLIM images the fluorescence spectra from regions of
interest. As result, the corresponding fluorescence spectra recorded from selected locations
marked with numbers in Fig. 4A show higher fluorescence intensity from RB@GNRs relative to
reference (see Fig. 4D). The decrease of fluorescence lifetime corroborated with the increase of
fluorescence intensity represents a clear demonstration for the operability of MEF mechanism in
the presence of GNRs. The relative large dispersions of fluorescence lifetime in the two
histograms (Fig. 4B) can be related to both chemical and electromagnetic environment of RB
molecules. Additionally, several decay profiles (Fig. 4C) extracted from FLIM images show that
the reference sample exhibits almost monoexponential profiles while RB@GNRs sample exhibit
faster multiexponential profiles which inform about the strong electromagnetic coupling between
fluorophore and GNPs [31]. We can see notable differences between fluorescence performances
of RB when investigated on solid substrate relative to water. While the steady-state fluorescence
spectra exhibit similar shape when solid substrate (Figure 4D) and solution measurements are
compared (Figure 2), the time-resolved measurements show important differences both in shape
of fluorescence decay and fluorescence lifetimes (Figure 4C and Figure 3). In particular there is a
notable increase of fluorescence lifetime of RB in RB@GNRs on solid substrate (1.6 ns) relative
to RB@GNRs in water (0.57 ns). A similar tendency is observed when compared lifetime of RB-
CTAB in two environments (2.1 ns relative to 0.62 ns). These findings are consistent in both
cases with decrease of nonradiative decay rate after decreasing local polarity by water
evaporation and entrapping RB into CTAB matrix. In addition the broad lifetime distribution
measured on solid substrate (Figure 4B) can be explain by the “static” heterogeneity existing
from spot to spot in the deposited film relative to sampling of averaged “kinetic” heterogeneity
in solution. On the other side, the overlap between the plasmon resonance and the absorption and
emission of fluorophore as well as the orientation of the molecular transition moment are
different in solution containing almost not interacting RB@GNRs comparative to substrate
where a more effective interparticle plasmonic coupling occurs. As result, the molecular
photophysical process on solid substrate is different in term of fluorescence lifetime and
emission quantum yield. Our results demonstrate the advantages of using time-resolved
fluorescence and in particular FLIM, in the investigation of interaction between chromophoric
molecules and plasmonic nanoparticles in various chemical and electromagnetic environments.
4. Conclusions
In this paper we have examined the fluorescence of RB molecules conjugated to GNRs in
solution and on solid substrate and we have shown that MEF is operational in both cases. We
have observed the increase of fluorescence intensity in solution as result of non-radiative rate
decrease after binding RB to CTAB, which competes with the electromagnetic coupling of RB to
GNRs. On the other hand, RB@GNRs conjugates deposited on solid substrate exhibit a clear
MEF mechanism revealed by the decrease of fluorescence lifetime and increase of fluorescence
intensity. Time-resolved fluorescence measurements and FLIM imaging are extremely useful
approaches for characterization of the electromagnetic coupling which occurs between
fluorophores and plasmonic nanoparticles. Our findings point toward exciting possibility to
perform fluorescence lifetime-based intra-cellular imaging through FLIM using as local probe
RB@GNRs conjugates. Although the photodynamic action of RB has been intensively explored,
the investigation of RB coupled to plasmonic nanoparticles for applications in cancer therapy
needs to be extended. Work is in progress in our laboratory towards anticancer dual therapy
based on PDT combined with plasmon resonance photothermal therapy.
Acknowledgments
This work was supported by Babes - Boyai University under the project number 34045/2013 –
Grants for young scientists.
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HIGHLIGHTS • Examination of fluorescence performance of Rose Bengal conjugated to gold nanorods • Emission enhancement controlled by chemical and electromagnetic environment • Radiative and non-radiative deactivation revealed by fluorescence lifetime studies • Promising applications of Rose Bengal-gold nanorods in imaging and cancer theraphy