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 simion.astilean@phys.ubbcluj.ro

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