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Size- and distance-dependent excitation energy transfer in fluorophore conjugated block copolymer – gold nanoparticle systems
Polymer 59 (2015) 243e251
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Size- and distance-dependent excitation energy transfer in fluorophore conjugated block copolymer e gold nanoparticle systems Rakesh Banerjee 1, Chiranjit Maiti 1, Sujan Dutta, Dibakar Dhara* Department of Chemistry, Indian Institute of Technology Kharagpur, West Bengal 721302, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 September 2014 Received in revised form 9 December 2014 Accepted 3 January 2015 Available online 9 January 2015
In the present work, we have demonstrated size- and distance-dependent excitation energy transfer (EET) phenomenon from polymer-bound fluorophores to gold nanoparticles (GNPs) conjugated to the same polymer. Anthracene labeled two block copolymers of controlled block length were synthesized by reversible-addition fragmentation chain transfer (RAFT) polymerization technique using two-arm dithioester chain transfer agent. The block copolymers were grafted onto the gold nanoparticle (GNP) surfaces by taking advantage of the high affinity of the dithioester end-groups located at the two ends of the polymer chains for the gold surface. GNPs of two different sizes, 20 nm and 55 nm, were used for the present study. The bare and polymer-bound GNPs were characterized by UVevis spectroscopy, transmission electron microscopy and dynamic light scattering measurements. The EET process was monitored through steady-state and time-resolved fluorescence spectroscopic study. Thus we have successfully synthesized fluorophore labeled well-defined triblock copolymers with tailored architectures and subsequently anchored them with GNPs of different sized that enabled us to control the energy transfer process between GNPs and fluorescent polymer. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Nanomaterial energy transfer Fluorescence RAFT polymerization
1. Introduction There has been a growing research interest in gold nanoparticles (GNPs) owing to their unique properties and potential applications in several areas like biomedical materials, optics, and electronics [1]. Thiol chemistry has widely been exploited to modify the surface of AuNPs with synthetic polymers [2e4] and biomacromolecules [5e7]. The affinity of the diothioester end-groups for the gold surfaces have been utilized to link well-defined polymers (synthesized via RAFT polymerization method [8]) to gold surfaces [9e13], as demonstrated by McCormick and T. P. Davis et al. Eeisenberg et al. incorporated GNPs into the central of vesicle wall and core of micelle derived from block copolymers of PS-b-PAA [14,15]. Excitation energy transfer (EET) from a donor molecule to an acceptor molecule is a very well-known natural process, the most common example being the highly efficient light harvesting system involved in photosynthesis. The EET process involves a nonradiative energy transfer from an electronically excited state of a
* Corresponding author. Tel.: þ91 3222 282326; fax: þ91 3222 282252. E-mail addresses: [email protected], [email protected] (D. Dhara). 1 Both the authors have contributed equally to this work. http://dx.doi.org/10.1016/j.polymer.2015.01.004 0032-3861/© 2015 Elsevier Ltd. All rights reserved.
donor molecule (D*) to the ground state of an acceptor molecule (A) [16]. During the EET process, the total energy of the donor (D) e acceptor (A) system is conserved, and hence, often referred as resonance energy transfer (RET). Experimentally, RET is mostly detected either via decrease/increase in the fluorescence intensity of the donor/acceptor, depending on whichever is fluorescent. Thus, this technique is also popularly known as fluorescence resonance energy transfer (FRET) [16]. €rster [17,18] proposed an elegant theory for the rate of In 1948, Fo EET, that predicted that the rate of EET followed a distance dependence of 1/R6 e type, where R is the center-to-center distance €rster theory also predicts that the efficiency of between D and A. Fo ET strongly depends on R and the relative orientation of the participating D and A molecules. This dependence of FRET rate on the distance between the donor and the acceptor has been utilized to develop sensors for various biologically relevant analytes like CO2 [19], glucose [20,21], and metal ions [22,23]. Moreover, on attaching a suitable DA pair (generally dye molecules) to a macromolecule, valuable information on the configuration and conformations of the macromolecule can be obtained from the rate €rster theory works better when small dye molecules of FRET [16]. Fo are used in a DA pair, except for small separation distances. However, use of small dye molecules both as donors and acceptors,
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limits the range of distances that can be probed by EET to less than 10 nm [16], which is indeed small when conformations and dynamics of long and complex polymer molecules are being investigated. In recent years, surface energy transfer (SET) between dye molecule and metal nanoparticles has gained interest because this technique double the range of separations than FRET which would help to understand the large scale conformational dynamics of complex biomolecules in macroscopic detail [24e29]. Persson-Lang [30], Silbey [31], and Gersten-Nitzen [32] have already demonstrated the mechanism of dye quenching at a metal (e.g., gold, silver) surface and the separation of donor and acceptor is d4 dependence. Therefore, development of new polymer-GNP systems
would not only open up possibilities of new applications, also provide newer systems to study as well as controlling the EET process. In this work, new polymer-GNP systems were designed and prepared. We studied the EET process using polymer-bound anthracene as donor (D) and polymer-bound GNPs of different sizes as acceptor (A). Size and distance-dependent EET process from anthracene to GNPs was demonstrated by the help of steady state and time resolved fluorescence spectroscopic studies. 2. Experimental section 2.1. Materials t-Butyl acrylate, styrene, 9-anthracene methanol, 4dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), and gold(III) chloride trihydrate (HAuCl4$3H2O) were purchased from SigmaeAldrich and used without further purification. a,a0 Xylyl-bis(dithiobenzoate) (XBDTB) was synthesized according to the literature reported procedure [33,34]. Trisodium salt of citrate, and trifluoroacetic acid (TFA) were purchased from Sisco Research Laboratories Pvt. Ltd, India, and used as received. 2,20 - Azobisisobutyronitrile (AIBN, SigmaeAldrich) was recrystallized twice from methanol before use. MilliQ water was used in all the experiments. 2.2. Synthesis of anthracene conjugated polystyrene-blockPoly(acrylic acid)-block-polystyrene (P1, Scheme 1a) Synthesis of P1 was consists of the following steps. 2.2.1. Synthesis of Poly(t-Butyl Acrylate) Macro-CTA a,a0 -Xylyl-bis(dithiobenzoate) (XBDTB) (0.16 g, 0.39 mmol) and AIBN (0.012 g, 0.073 mmol) were added to a 50 mL round bottomed flask. t-Butyl acrylate (5.0 g, 39.06 mmol) was then added to it, followed by addition of 4.0 mL of dry DMF. The reaction vessel was then degassed, purged with nitrogen and then placed in an oil bath at 70 C. The polymerization was continued for 6 h, and the product was recovered by precipitation from ice cold MeOH/H2O (2:1). Mn (1H NMR) ¼ 5600, Mn (GPC) ¼ 7100, PDI ¼ 1.3. 2.2.2. Synthesis of polystyrene-block-Poly(t-butyl acrylate)-blockpolystyrene Poly(t-butyl acrylate) macro-CTA (1.0 g, 0.180 mmol) and AIBN (7.3 mg, 0.047 mmol) were added to a 30 ml tube. Styrene (3.7 g, 36.0 mmol) was then added followed by addition of 3.0 ml of dry DMF. The reaction vessel was then degassed thrice, purged with nitrogen and allowed to warm to room temperature. The reaction mixture was then placed in an oil bath at 70 C with vigorous stirring for 16 h. Thereafter, the polymerization was quenched, product diluted with minimal amount of THF and precipitated from ice-cold hexane. The polymer was isolated by filtration and washed several times with hexane and then dried under high vacuum. Poly(t-butyl acrylate)-block-polystyrene was obtained as pale pink solid (54% yield). Mn (1H NMR) ¼ 9800, Mn (GPC) ¼ 11,800, PDI ¼ 1.45.
Scheme 1. Synthetic strategy of (a) anthracene containing polystyrene-block-poly(acrylic acid)-block-polystyrene (P1) e and (b) anthracene containing poly(acrylic acid)block-polystyrene-block- poly(acrylic acid) (P2).
2.2.3. Hydrolysis of t-butylacrylate groups of polystyrene-b-Poly(tbutylacrylate)-b-polystyrene Polystyrene-b-poly(t-butyl acrylate)-b-polystyrene (0.5 g, 0.048 mmol with respect to t-butyl acrylate) was dissolved in 5 mL dry DCM, stirred for 15 min followed by addition of trifluoroacetic acid (TFA, 1.5 mL). The reaction mixture was stirred for 16 h at room temperature. Then the solvent was removed in rotary evaporator and TFA was completely removed by azeotropic distillation using
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toluene. The polymer was then dissolved in THF, precipitated from ice cold MeOH/H2O (2:1) mixture and washed several times with MeOH. The polymer was isolated by repeated extraction with DCM and the organic portions combined and dried over anhydrous MgSO4. Thereafter, the solvent was removed under high vacuum for 6 h to recover the product as a brown solid. The conversion of poly(t-butylacrylate) to poly(acrylic acid) was confirmed by 1H NMR analysis. 2.2.4. Conjugation of 9-anthracene methanol with polystyrene-bPoly(acrylic acid)-b-polystyrene Polystyrene-b-poly(acrylic acid)-b-polystyrene (0.22 g, 1.34 mmol with respect to the acid group) was dissolved in dry THF and 9-anthracene methanol (0.14 g, 0.67 mmol) was added at once to it, followed by the addition of 4-dimethylaminopyridine (DMAP, 0.008 g, 0.063 mmol). The reaction mixture was stirred for 20 min at 0 C. N,N0 -Dicyclohexylcarbodiimide (DCC, 0.12 g, 0.57 mmol in 2 ml of dry THF) was added very slowly at this temperature for 1 h and the reaction was continued for another 48 h. The reaction mixture was then filtered to remove the produced dicyclohexylurea (DCU) and the polymer product was recovered by precipitation from cold hexane. The residue was further dissolved in THF and again precipitated from hexane/pet-ether mixture to get the product. The incorporation of the anthracene moiety in the polymer backbone was confirmed by NMR, UVevis and fluorescence spectroscopy. 2.3. Synthesis of anthracene conjugated Poly(acrylic acid)-bpolystyrene-b-Poly(acrylic acid) (P2, Scheme 1b) Synthesis of P2 consists of the following steps. 2.3.1. Synthesis of polystyrene Macro-CTA a,a0 -Xylyl-bis(dirhiobenzoate) (XBDTB) (0.136 g, 0.481 mmol) and AIBN (20 mg, 0.12 mmol) were added to a 30 ml tube. Styrene (2.5 g, 24.0 mmol) was then added to it followed by addition of 1.5 ml of dry DMF. The reaction vessel was then degassed thrice with continuous purging of nitrogen and then warmed to room temperature. The reaction mixture was then placed in an oil bath at 70 C with vigorous stirring for 20 h. Polymerization was quenched and diluted with minimal amount of THF and the polymer was precipitated from ice-cold MeOH. The polymer was isolated by filtration and washed several times with MeOH and then dried under high vacuum. Polystyrene macro-CTA was obtained as pink solid (90% yield). Mn (1H NMR) ¼ 4500, Mn (GPC) ¼ 6700, PDI ¼ 1.2. 2.3.2. Synthesis of Poly(t-butyl acrylate)-b-polystyrene-b-Poly(tbutyl acrylate) Polystyrene macro-CTA (0.4 g, 0.089 mmol) and AIBN (3.6 mg, 0.021 mmol) were added to a 30 ml tube. t-Butyl acrylate (0.91 g, 7.2 mmol) was then added to it which was followed by addition of 2.0 ml of dry DMF. The reaction vessel was then degassed thrice, continuously purged with nitrogen and allowed to warm to room temperature. The reaction mixture was then placed in an oil bath at 70 C with vigorous stirring for 12 h and then the polymerization was quenched and diluted with minimal amount of THF. The polymer was precipitated from ice-cold methanol/water (3/1 v/v) mixture, isolated by filtration, and washed several times with methanol and then dried under high vacuum. Poly(t-butyl acrylate)-b-polystyrene-b-poly(t-butyl acrylate) was obtained as pale pink solid (78% yield). Mn (1H NMR) ¼ 10,400; Mn (GPC) ¼ 12,300, PDI ¼ 1.34. Hydrolysis of the t-butyl acrylate of polystyrene-b-poly(t-butyl acrylate)-b-polystyrene and conjugation of 9-anthracene methanol
245
with the above copolymer were carried out following similar procedure as described earlier as in the case of P1. 2.4. Instrumentation and methods The number average molecular weight of the synthesized polymers was determined by the utilization of 1H NMR spectroscopy [35] and gel permeation chromatographic (GPC) technique. In GPC instrument (Viscotek) we have used polystyrene standards and THF as eluent with a flow rate of 1 mL min1. Dynamic light scattering measurements were performed to determine the hydrodynamic diameter of bare GNPs and GNP embedded polymer using Malvern Zetasizer Nano equipment with a 3.0 mW HeeNe laser operated at 633 nm. Analysis was performed at a fixed detector angle of 173 and a constant temperature of 25 C. The absorption and fluorescence spectra were collected using a Shimadzu (model number, UV-2450) spectrophotometer and a Hitachi (model no. F-7000) spectrofluorimeter, respectively. For steady-state experiments, all the samples were excited at 350 nm. For time resolved fluorescence measurements, we have used a time correlated single photon counting (TCSPC) instrument from IBH, U. K. The instrument response function of this setup was ~0.09 ns. The detailed time-resolved fluorescence setup is described by Hazra et al. [36,37] Briefly, the samples were excited at 350 nm using a picosecond laser diode (IBH, U. K. Nanoled), and the signals were collected at the magic angle (54.7 ) using a Hamamatsu micro-channel plate photomultiplier tube (3809U). The data analysis was performed using IBH DAS version 6 decay analysis software. All the long and short wavelength decays were fitted bi-exponentially by considering c2 becomes close to 1, indicating a good fit. 2.5. Fluorescence quantum yield calculation Fluorescence quantum yields of donor molecules (anthracene) attached to the block copolymer have been calculated by using ethanolic solution of anthracene (quantum yield 0.27) [38] as the secondary standard for the fluorescence quantum yield measurement according to the following eq.:
" Фsample ¼ Фstd:
I=A
sample
A=I
# std:
. h2std:
!
h2sample
where F represents quantum yield, A is absorbance at the excitation wavelength, h is the refractive index, and I is the integrated emission intensity calculated from the area under the emission peak. 2.6. Preparation of GNPs GNPs of pre-determined sizes (20 nm and 55 nm) were synthesised by the reported method of Ferns [39]. Briefly 1.25 mL 0.01 M of HAuCl4.3H2O solution was taken in a 250 mL conical flask and it was mixed with 50 mL of milliQ water which was heated just to boil. 1% solution of trisodium citrate salt was then added and the solution was boiled with constant shaking till deep-red color of the GNP solution appeared. The formation of GNPs was confirmed from UV-vis spectroscopy and DLS, TEM experiments. 2.7. Grafting of block copolymers to GNP surfaces 10 mL of GNP solution was placed in an ice bath under vigorous stirring for 30 min. Cooled polymer solution (300 mL, concentration: 1 mg/mL) was added to the GNP solution, followed by stirring
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for 6 h. Then, the GNPs were purified by centrifugation at 20,000 rpm for 30 min at 5 C followed by re-suspension in cooled water. This process of centrifugation and re-suspension was repeated thrice. Final GNP/polymer solutions were used for characterization and fluorescence measurements as soon as possible. 3. Results and discussion In the present work, we have synthesized new polymer-GNP systems. EET process was demonstrated using polymer-bound anthracene as donor (D) and polymer-bound GNPs of different sizes as acceptor (A). Distance-dependent EET process was followed using two block copolymer-coated GNPs in which the distance between the D and the A was varied simply by altering the sequence of the blocks in the block copolymers. Dithioester groups present at the block-copolymer ends were used to conjugate two different-sized GNPs with two anthracene containing block copolymers viz. polystyrene-block-poly(acrylic acid)-block-polystyrene (P1) and poly(acrylic acid)-block-polystyrene-blockpoly(acrylic acid) (P2) synthesized by RAFT polymerization technique. Size and distance-dependent EET process from anthracene to GNPs was monitored by the help of steady state and timeresolved fluorescence spectroscopic studies. 3.1. Anthracene conjugated block copolymers - synthesis and characterization The chain transfer agent (CTA), a,a0 -Xylyl-bis(dirhiobenzoate) (XBDTB) was used for synthesis of the two block copolymers polystyrene-block-poly(acrylic acid)-block-polystyrene and poly(acrylic acid)-block-polystyrene-block-poly(acrylic acid). The CTA and the polymers were characterized by 1H NMR and 13C NMR spectroscopy (Supporting information e Figs. S1 to S9). The compositions of the polymers were determined by 1H NMR spectroscopy. 9-Anthracene methanol was reacted with acrylic acid containing polymers as synthesized above to attach anthracene fluorophores in the polymer backbones. The coupling reactions were carried out in such a way so that some of the acrylic acid groups remain free (for hydrophilicity). Fig. S6 (Supporting information) showed almost quantitative conversion of poly(tbutylacrylate) to poly(acrylic acid). Furthermore, it is clear from the Fig. S7 and Fig. S8 that the ratio of anthracene units to free acrylic acid units is almost same in both the polymer P1 and P2. Presence of significant amount of free hydrophilic acrylic acid groups (which were not functionalized by anthracene) was confirmed from the peak at 177 ppm of the 13C NMR spectra of the block copolymer P1 (Fig. S9, Supporting information). Also from 13C
Table 1 Characterization of the polymers used in this study. Polymer
Monomer Initiator CTA agent
Mna Mnb PDIb (G/mol) (G/mol)
PtBA macro-CTA PS-b-PtBA-b-PS P1 PS macro-CTA PtBA-b-PS-b-PtBA P2
tBA Styrene e Styrene tBA e
5600 9800 e 4500 10,400 e
a b
AIBN AIBN e AIBN AIBN e
XBDTB PtBA macro-CTA e XBDTB PS macro-CTA e
7100 11,800 13,100 6700 12,300 13,900
1.30 1.45 1.41 1.20 1.34 1.51
From 1H NMR. From GPC.
NMR spectra it is clear that the degree of functionalization of the PAA block with anthracene groups is approximately 50%. Gel permeation chromatography (GPC) was also used to analyze the molecular weights and polydispersity of the precursor macro-CTAs and the block copolymers synthesized from them (Fig. 1). The monodispersed nature of the chromatograms and the shifting of the peaks to lower retention volumes in case of the block copolymers, confirmed the formation of reasonably pure and high molecular weight block copolymer The molecular weight data of all the polymers are presented in Table 1. All the polymers synthesized in this study, are expected to contain dithioester end-groups, which could be used to bind to the GNPs surface. 3.2. Synthesis and characterization of GNPs and polymer-coated GNPs Citrate-stabilized GNPs dispersed in water were synthesized using the citrate method [39]. Two different citrate to HAuCl4 ratios were used to synthesize GNPs of two different sizes. The polymercoated GNPs were prepared by treating the synthesized block copolymers with the citrate stabilized GNPs in aqueous solution for a long duration. After the coating process was completed, the polymer-coated particles were isolated by centrifugation, followed by washing and re-suspension in water. This process of centrifugation-washing-re-suspension was repeated thrice to ensure complete removal of any unbound block polymers from the re-suspended polymer-coated GNPs. Eisenberg et al. [14,15]. had described that GNPs can be successfully incorporated in the core of the self-assembled of PS-b-PAA, where GNPs were stabilized mostly into the hydrophobic region produced by the selfassembled nanostructures. The affinity of the diothioester endgroups for the gold surface was shown to be much higher than that of the carboxylate groups. This is the reason why citrate capped GNPs were grafted by P1 and P2 by centrifugation and redispersion method. Furthermore, to avoid any confusion, we have
Fig. 1. GPC chromatograms of anthracene conjugated block copolymers along with their precursor polymers - (a) P1 and (b) P2.
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used almost same block length of poly(acrylic acid) in both the polymer P1 and P2. The re-suspended solution did not show any observable precipitation even after 15 days (Fig. S10, Supporting information), although the characterization and fluorescence measurements were carried out soon after the preparation of the polymer-coated GNP solutions. Fig. 2 (top) shows typical transmission electron microscopy (TEM) images of GNPs synthesized by the citrate method and (bottom) the corresponding polymercoated GNPs. The average particle sizes of the nanoparticles are presented in Table 2. It should be noted that the polymer-coated GNPs were observed somewhat aggregated on the carbon-coated TEM grids during sample preparation. In Fig. 3, typical UVevisible spectra of citrate-stabilized GNPs and the corresponding polymer coated GNPs in aqueous solution are presented. Citrate-stabilized GNPs exhibited localized surface plasmon resonance (LSPR) band with maxima at approximately 522 nm and 527 nm for the two nanoparticles, which is typical of spherical GNPs of approximately 20 nm and 55 nm diameter respectively [1,39]. After being coated with the block copolymer, the 20 nm GNPs exhibited a slight red-shift of the lmax (3 nm for P1 and 5 nm for P2) indicating a small increase in the effective size. However, for the 55 nm GNPs, there was no shift in lmax, which isnot unexpected; as the GNPs were already large enough coating of polymer did not affect its absorption properties significantly. The size of GNPs and the polymer-coated GNPs were characterized by DLS to determine their hydrodynamic sizes. Fig. 4 shows their DLS data, along with that of the P1-coated GNPs. The size distribution of P2-coated GNPs was more or less similar to that of P1 and not shown here. The hydrodynamic sizes obtained from DLS matched closely with those obtained from TEM and UVevis spectroscopic studies. Analysis of the DLS results indicated that the two assynthesized citrate-stabilized nanoparticles had diameters of 19 nm and 50 nm, which were increased to 29 nm and 58 nm respectively on coating with P1.
247
Table 2 Preparation and characterization of prepared GNPs. Entry Amount of 0.01 M HAuCl4.3H2O (mL)
Amount of 1% trisodium salt of citrate (mL)
UV-vis Size of GNPs from UV-vis lmax (nm) (nm)
Size of GNPs by TEM (nm)
1 2
875 300
522 527
18e22 50e60
1.25 1.25
20 55
3.3. Steady-state fluorescence study of the GNPs and polymercoated GNPs It is well-known in literature that if a fluorophore is brought in close proximity to metal nanoparticles like gold, silver, the excited state energy of the fluorophore (D) is easily transferred to the metal nanoparticles (A). In the present work, we essentially have two block copolymeric systems as shown in Scheme 2a. In case of P1, the anthracene molecules are attached to the middle block and are placed away from the GNPs that are linked with the dithioester groups present at the two ends (Scheme 2b) of the P1. In case of P2, the anthracene groups are situated on the two sides and hence closer to the GNP bonded chain ends. Fig. 5 shows the steady-state fluorescence spectra of the polymer-bound anthracene fluorophores (lex ¼ 350 nm) for the two GNPs e polymer conjugate systems. The spectra for the free polymers (not conjugated with GNPs) are also included in Fig. 5. The fluorescence emission spectra of the anthracene molecules were clearly observed in the free polymers P1 and P2. The following observations could be made from the spectral data - (i) for P1, where the anthracene units are in the middle of the polymer chains and hence far from the GNPs, the quenching of anthracene fluorescence increased with the size of the nanoparticles, (ii) for P2, where the anthracene units are at the two sides of the chains and in
Fig. 2. Typical transmission electron microscopy (TEM) images of the two sets of GNPs synthesized by the citrate method (a and b) and the corresponding polymer-coated GNPs (c and d respectively).
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Fig. 3. UVevis absorbance plot of the two citrate stabilized GNPs as prepared in water (bare GNPs) and the polymer-coated GNPs; (a) and (b) corresponds to the same GNPs as shown in Fig. 2a and b respectively.
Fig. 4. DLS histograms for the two GNPs (bottom) and for the same GNPs coated with P1 (top), x-axis represent hydrodynamic size.
close proximity to the GNPs, both GNPs caused significant quenching to the anthracene fluorescence. For P2, as the fluorophores are placed near the GNPs, easy energy transfer occurred from the excited anthracene molecules to the GNPs. However, for P1, the anthracene moieties being far from GNPs, the 55 nm ones have the largest energy accepting capability whereas the 20 nm GNPs are probably too small to influence anthracene molecules from a distance and as a result, practically no energy transfer took place. This size-dependent energy accepting capability of the GNPs is reported in literature and has been attributed to an increase in non radiative decay rate with increasing the size of GNPs [1,40]. The binding between the dithioester groups and the GNPs were found to be very strong. We tried to disrupt this binding process and release the anthracene units by adding several compounds that
Scheme 2. Schematic presentation of the location of the fluorophores (anthracene, donors) and the GNPs (acceptors) in the polymer-coated GNP systems. The GNPs could bind to more than one polymer; for simplicity, one polymer chain conjugated to the GNPs is shown.
are known to complex strongly with the GNPs viz. b-cyclodextrin, 3-mercaptopropanoic acid, glutathione, dodecane thiol, butane thiol. However, none of these compounds was able to dislodge the GNPs bound to the dithioesters of our polymers. Only a slight increase in the fluorescence intensity was noticed on addition of dodecane thiol. 3.4. Time-resolved fluorescence study of the GNPs and polymercoated GNPs After investigating the EET process by steady-state fluorescence spectroscopy, more confirmatory study of the EET process was conducted by carrying out TCSPC experiments of the free and the GNP-bound block copolymers containing anthracene groups as fluorophores. Fig. 6 shows the time-resolved fluorescence decay curves. The lifetime data for all the samples are listed in Table 3. From the data presented in Table 3 and from Fig. 6, it can be clearly noted that the average lifetime of the anthracene moieties decreased because of EET to the GNPs. For example, the average lifetime value of the anthracene in P1 decreased from 2.86 ns to 2.38 ns and 1.96 ns in presence of 20 nm and 55 nm GNPs respectively. Similarly, the average lifetime of anthracene in P2 decreased from 2.95 ns to 1.33 ns and 1.15 ns in presence of 20 nm and 55 nm GNPs respectively. The energy transfer between Au nanoparticle and a dye provided a new paradigm to design an optical-based molecular ruler for long distance measurements [41,42]. This ET originates from the interactions of the electromagnetic field of the donor dipole with
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Fig. 5. Steady-state fluorescence emission spectra of polymer-bound anthracene molecules where the polymer chains are either conjugated to GNPs or free in the solution.
the free conduction electrons of the accepting metal and it is established that the separation between donor and acceptor is d4 dependent. In this study, the distance between the donor (anthracene) and the acceptor (GNPs) has been calculated by using
the surface energy transfer (SET) method. The characteristic distance at which a dye (donor) will display equal probabilities for energy transfer and spontaneous emission (d0) is calculated using the Persson model [30,43].
Fig. 6. Time-resolved fluorescence decay profiles of polymer-bound anthracene molecules where the polymer chains are either conjugated to GNPs or free in the solution.
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Table 3 Lifetime data for the anthracene molecules attached to the block copolymer in presence and absence of GNPs. System
t1 (ns)
a1
t2 (ns)
a2
c2
Avg t (ns)
P1 P1 P1 P2 P2 P2 -
1.38 0.19 1.23 1.74 1.15 1.03
0.73 0.29 0.86 0.78 0.90 0.89
6.88 3.28 6.35 7.23 2.93 2.11
0.27 0.71 0.14 0.22 0.10 0.11
1.33 0.93 1.19 1.10 1.34 1.45
2.86 2.38 1.96 2.95 1.33 1.15
GNP (20 nm) GNP (55 nm) GNP (20 nm) GNP (55 nm)
Table 4 Nanometal surface energy transfer (NSET) parameters for the anthracene molecules attached to the block copolymer in presence of GNPs. System
lex (nm)
d0 (Å)
ФET (%)
d (Å)
kNSET (s1)
P1 - GNP (20 nm) P1 - GNP (55 nm) P2 - GNP (20 nm) P2 - GNP (55 nm)
350 350 350 350
55 55 55 55
16 31 55 61
83.25 67.18 52.31 49.18
0.67 1.57 4.14 5.30
108 108 108 108
bonded chain ends. Our strategy was to synthesize well-defined triblock copolymers of ABA and BAB type of architectures, so that the anthracene moieties could be either placed in the middle of the chain (say the B block in ABA) or at the sides (in BAB). This enabled us to precisely control the distance between the donor anthracene groups and the acceptor Au particles present at the chain termini. Besides, the effect of size of the acceptor molecules in the EET process could also be studied by using GNPs of different sizes. In case of the BAB type copolymer, where the anthracene groups were present closer to the GNPs, a significant quenching of anthracene fluorescence was observed. However, in case of the ABA type copolymer, where the anthracene groups were placed at a larger distance from the GNPs, quenching was observed only by the biggest sized (55 nm) GNPs. Hence, our work shows that block copolymers with tailored architectures, synthesized by the RAFT technique, can be utilized to bond to Au particles through the functionalized chain-ends and that eventually enables in better understanding of the controlled EET process. Acknowledgment
d0 ¼
0:225 c3 Фdye
!1=4
u2dye uF kF
Фdye is the quantum efficiency of anthracene, udye the frequency of the donor electronic transition,uF the Fermi frequency, and kF Fermi wave vector of the metal [30]. The d0 value is calculated using Фdye ¼ 0.32, udye ¼ 4.55 1015 s1, uF ¼ 8.4 1015 s1, and kF ¼ 1.2 108 cm-1, and velocity of light (c) ¼ 3 1010 cm s1. The lifetime quenching data is assumed to be more accurate than the steady-state fluorescence intensity quenching data in order to determine the energy transfer efficiency. The equation, ФET ¼ 1
tDA tD
is used to calculate the time-resolved energy transfer efficiencies, where tDA and tD are the lifetime of donor in the presence and absence of acceptor (GNPs), respectively. According to Persson model, the rate of surface energy transfer is given by
kNSET ¼
1 d0 4 tD d
where (d) is the distance between molecular centers of the donor to surface of nanoparticles acceptor. Information on the above mentioned useful spectral parameters are listed in Table 4. Table 4 reflects that anthracene labeled triblock copolymers (P1), in which polystyrene chain act as a spacer, helps in reducing non radiative energy transfer and more efficiently in case of smaller size GNPs. Thus fluorophore labeled block copolymers with tailored architectures are able to control the energy transfer process by appropriate choosing of chain transfer agent in RAFT polymerization technique. 4. Conclusion In the present work, we demonstrated the size- and distancedependence of the EET process using a polymeric system, wherein anthracene moieties attached to the polymer chains transfer energy to the GNPs bound to the two ends of the same chain. The dithioester end-groups introduced at the polymer chainends by virtue of controlled polymerization using dithioester chain transfer agent (RAFT technique) have been exploited to form GNP-
Financial support from SRIC, Indian Institute of Technology Kharagpur (project code NPA with institute approval numbers - IIT/ SRIC/CHY/NPA/2014-15/81) is thankfully acknowledged. The authors acknowledge Prof. N. Sarkar for providing some laboratory facilities. Research fellowships to R. Banerjee and C. Maiti from UGC, New Delhi, and to S. Dutta from CSIR, New Delhi, are also acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.polymer.2015.01.004. References [1] Daniel MC, Astruc D. Chem Rev 2004;104:293e346. [2] Brust M, Walker M, Bethell D, Schiffrin DJ, Whyman R. Chem Soc Chem Commun 1994:801e2. [3] Lowe AB, Sumerlin BS, Donovan MS, McCormick CL. J Am Chem Soc 2002;124: 11562e3. [4] Shan J, Nuopponen M, Jiang H, Viitala T, Kauppinen E, Kontturi K, et al. Macromolecules 2005;38:2918e26. [5] Yuan JJ, Schmid A, Armes SP. Langmuir 2006;22:11022e7. [6] Li Z, Jin R, Mirkin CA, Letsinger RL. Nucl Acids Res 2002;30:1558e62. [7] Alivisatos AP, Johnsson KP, Peng X, Wilson TE, Loweth CJ, Bruchez MPJ, et al. Nature 1996;382:609e11. [8] Fustin CA, Colard C, Filali M, Guillet P, Duwez AS, Meier MAR, et al. Langmuir 2006;22:6690e5. [9] Boyer C, Whittaker MR, Chuah K, Liu J, Davis TP. Langmuir 2010;26:2721e30. [10] Boyer C, Whittaker MR, Luzon M, Davis TP. Macromolecules 2009;42: 6917e26. [11] Sumerlin BS, Lowe AB, Stroud PA, Zhang P, Urban MW, McCormick CL. Langmuir 2003;19:5559e62. [12] McCormick CL, Lowe AB. Acc Chem Res 2004;37:312e25. [13] Smith AE, Xu X, Abell TU, Kirkland SE, Hensarling RM, McCormick CL. Macromolecules 2009;42:2958e64. [14] Mai Y, Eisenberg A. Macromolecules 2011;44:3179e83. [15] Mai Y, Eisenberg A. J Am Chem Soc 2010;132:10078e84. [16] Lakowicz JR. Plenum New York 1983. €rster T. Ann Phys 1948;437:55. [17] Fo €rster T. Sinanoglu O Ed Academic Press New York 1995. [18] Fo [19] Sipior J, Bambot S, Romauld M, Carter GM, Lakowicz JR, Rao G. Anal Biochem 1995;227:309e18. [20] Lakowicz JR, Maliwal B. Anal Chim Acta 1993;271:155e64. [21] Rolinski OJ, Birch DJS, McCartney LJ, Pickup JC. Phys Med Biol 2001;46: N221e6. [22] Birch DJS, Holmes AS, Darbyshire M. Meas Sci Technol 1995;6:243e7. [23] Rolinski OJ, Birch DJS, Holmes AS. J Biomed Opt 1998;3:346e56. [24] Dulkeith E, Ringler M, Klar TA, Feldmann J, Javier AM, Parak WJ. Nano Lett 2005;5:585e9. [25] Yun CS, Javier A, Jennings T, Fisher M, Hira S, Peterson S, et al. J Am Chem Soc 2005;127:3115e9.
R. Banerjee et al. / Polymer 59 (2015) 243e251 [26] Jennings TL, Schlatterer JC, Singh MP, Greenbaum NL, Strouse GF. Nano Lett 2006;6:1318e24. [27] Sen T, Sadhu S, Patra A. Appl Phys Lett 2007;91:043104e11. [28] Govorov AO, Bryant GW, Zhang W, Skeini T, Lee J, Kotov NA, et al. Nano Lett 2006;6:984e94. [29] Liu GL, Yin Y, Kunchakarra S, Mukherjee B, Gerion D, Jett SD, et al. Nat Nanotechnol 2006;1:47. [30] Persson BNJ, Lang ND. Phys Rev B 1982;26:5409e15. [31] Chance R, Prock A, Silbey R. Adv Chem Phys 1978;60:1. [32] Gersten J, Nitzan A. J Chem Phys 1981;75:1139e52. [33] Le TPT, Moad G, Rizzardo E, Thang SH. PCT Int Appl WO Patent 9801478;1998. [34] Banerjee R, Maiti S, Dhara D. Green Chem 2014;16:1365e73.
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Maiti C, Dey D, Mandal S, Dhara D. J Phys Chem B 2014;118:2274e83. Hazra P, Sarkar N. Chem Phys Lett 2001;342:303e11. Hazra P, Chakrabarty D, Sarkar N. Langmuir 2002;18:7872e9. Dawson WR, Windsor MW. J Phys Chem 1968;72:3251e60. Frens G, Kolloid Z. Nat Phys Sci 1973;241:20e2. Dulkeith E, Morteani AC, Niedereichholz T, Klar TA, Feldmann J, Levi SA, et al. Phys Rev Lett 2002;89:203002e11. [41] Mandal S, Ghatak C, Rao VG, Ghosh S, Sarkar N. J Phys Chem C 2012;116: 5585e97. [42] Sen T, Patra A. J Phys Chem C 2012;116:17307e17. [43] Jennings TL, Singh MP, Strouse GF. J Am Chem Soc 2006;128:5462e7.
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Size- and distance-dependent excitation energy transfer in fluorophore conjugated block copolymer e gold nanoparticle systems Rakesh Banerjee 1, Chiranjit Maiti 1, Sujan Dutta, Dibakar Dhara* Department of Chemistry, Indian Institute of Technology Kharagpur, West Bengal 721302, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 September 2014 Received in revised form 9 December 2014 Accepted 3 January 2015 Available online 9 January 2015
In the present work, we have demonstrated size- and distance-dependent excitation energy transfer (EET) phenomenon from polymer-bound fluorophores to gold nanoparticles (GNPs) conjugated to the same polymer. Anthracene labeled two block copolymers of controlled block length were synthesized by reversible-addition fragmentation chain transfer (RAFT) polymerization technique using two-arm dithioester chain transfer agent. The block copolymers were grafted onto the gold nanoparticle (GNP) surfaces by taking advantage of the high affinity of the dithioester end-groups located at the two ends of the polymer chains for the gold surface. GNPs of two different sizes, 20 nm and 55 nm, were used for the present study. The bare and polymer-bound GNPs were characterized by UVevis spectroscopy, transmission electron microscopy and dynamic light scattering measurements. The EET process was monitored through steady-state and time-resolved fluorescence spectroscopic study. Thus we have successfully synthesized fluorophore labeled well-defined triblock copolymers with tailored architectures and subsequently anchored them with GNPs of different sized that enabled us to control the energy transfer process between GNPs and fluorescent polymer. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Nanomaterial energy transfer Fluorescence RAFT polymerization
1. Introduction There has been a growing research interest in gold nanoparticles (GNPs) owing to their unique properties and potential applications in several areas like biomedical materials, optics, and electronics [1]. Thiol chemistry has widely been exploited to modify the surface of AuNPs with synthetic polymers [2e4] and biomacromolecules [5e7]. The affinity of the diothioester end-groups for the gold surfaces have been utilized to link well-defined polymers (synthesized via RAFT polymerization method [8]) to gold surfaces [9e13], as demonstrated by McCormick and T. P. Davis et al. Eeisenberg et al. incorporated GNPs into the central of vesicle wall and core of micelle derived from block copolymers of PS-b-PAA [14,15]. Excitation energy transfer (EET) from a donor molecule to an acceptor molecule is a very well-known natural process, the most common example being the highly efficient light harvesting system involved in photosynthesis. The EET process involves a nonradiative energy transfer from an electronically excited state of a
* Corresponding author. Tel.: þ91 3222 282326; fax: þ91 3222 282252. E-mail addresses: [email protected], [email protected] (D. Dhara). 1 Both the authors have contributed equally to this work. http://dx.doi.org/10.1016/j.polymer.2015.01.004 0032-3861/© 2015 Elsevier Ltd. All rights reserved.
donor molecule (D*) to the ground state of an acceptor molecule (A) [16]. During the EET process, the total energy of the donor (D) e acceptor (A) system is conserved, and hence, often referred as resonance energy transfer (RET). Experimentally, RET is mostly detected either via decrease/increase in the fluorescence intensity of the donor/acceptor, depending on whichever is fluorescent. Thus, this technique is also popularly known as fluorescence resonance energy transfer (FRET) [16]. €rster [17,18] proposed an elegant theory for the rate of In 1948, Fo EET, that predicted that the rate of EET followed a distance dependence of 1/R6 e type, where R is the center-to-center distance €rster theory also predicts that the efficiency of between D and A. Fo ET strongly depends on R and the relative orientation of the participating D and A molecules. This dependence of FRET rate on the distance between the donor and the acceptor has been utilized to develop sensors for various biologically relevant analytes like CO2 [19], glucose [20,21], and metal ions [22,23]. Moreover, on attaching a suitable DA pair (generally dye molecules) to a macromolecule, valuable information on the configuration and conformations of the macromolecule can be obtained from the rate €rster theory works better when small dye molecules of FRET [16]. Fo are used in a DA pair, except for small separation distances. However, use of small dye molecules both as donors and acceptors,
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limits the range of distances that can be probed by EET to less than 10 nm [16], which is indeed small when conformations and dynamics of long and complex polymer molecules are being investigated. In recent years, surface energy transfer (SET) between dye molecule and metal nanoparticles has gained interest because this technique double the range of separations than FRET which would help to understand the large scale conformational dynamics of complex biomolecules in macroscopic detail [24e29]. Persson-Lang [30], Silbey [31], and Gersten-Nitzen [32] have already demonstrated the mechanism of dye quenching at a metal (e.g., gold, silver) surface and the separation of donor and acceptor is d4 dependence. Therefore, development of new polymer-GNP systems
would not only open up possibilities of new applications, also provide newer systems to study as well as controlling the EET process. In this work, new polymer-GNP systems were designed and prepared. We studied the EET process using polymer-bound anthracene as donor (D) and polymer-bound GNPs of different sizes as acceptor (A). Size and distance-dependent EET process from anthracene to GNPs was demonstrated by the help of steady state and time resolved fluorescence spectroscopic studies. 2. Experimental section 2.1. Materials t-Butyl acrylate, styrene, 9-anthracene methanol, 4dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), and gold(III) chloride trihydrate (HAuCl4$3H2O) were purchased from SigmaeAldrich and used without further purification. a,a0 Xylyl-bis(dithiobenzoate) (XBDTB) was synthesized according to the literature reported procedure [33,34]. Trisodium salt of citrate, and trifluoroacetic acid (TFA) were purchased from Sisco Research Laboratories Pvt. Ltd, India, and used as received. 2,20 - Azobisisobutyronitrile (AIBN, SigmaeAldrich) was recrystallized twice from methanol before use. MilliQ water was used in all the experiments. 2.2. Synthesis of anthracene conjugated polystyrene-blockPoly(acrylic acid)-block-polystyrene (P1, Scheme 1a) Synthesis of P1 was consists of the following steps. 2.2.1. Synthesis of Poly(t-Butyl Acrylate) Macro-CTA a,a0 -Xylyl-bis(dithiobenzoate) (XBDTB) (0.16 g, 0.39 mmol) and AIBN (0.012 g, 0.073 mmol) were added to a 50 mL round bottomed flask. t-Butyl acrylate (5.0 g, 39.06 mmol) was then added to it, followed by addition of 4.0 mL of dry DMF. The reaction vessel was then degassed, purged with nitrogen and then placed in an oil bath at 70 C. The polymerization was continued for 6 h, and the product was recovered by precipitation from ice cold MeOH/H2O (2:1). Mn (1H NMR) ¼ 5600, Mn (GPC) ¼ 7100, PDI ¼ 1.3. 2.2.2. Synthesis of polystyrene-block-Poly(t-butyl acrylate)-blockpolystyrene Poly(t-butyl acrylate) macro-CTA (1.0 g, 0.180 mmol) and AIBN (7.3 mg, 0.047 mmol) were added to a 30 ml tube. Styrene (3.7 g, 36.0 mmol) was then added followed by addition of 3.0 ml of dry DMF. The reaction vessel was then degassed thrice, purged with nitrogen and allowed to warm to room temperature. The reaction mixture was then placed in an oil bath at 70 C with vigorous stirring for 16 h. Thereafter, the polymerization was quenched, product diluted with minimal amount of THF and precipitated from ice-cold hexane. The polymer was isolated by filtration and washed several times with hexane and then dried under high vacuum. Poly(t-butyl acrylate)-block-polystyrene was obtained as pale pink solid (54% yield). Mn (1H NMR) ¼ 9800, Mn (GPC) ¼ 11,800, PDI ¼ 1.45.
Scheme 1. Synthetic strategy of (a) anthracene containing polystyrene-block-poly(acrylic acid)-block-polystyrene (P1) e and (b) anthracene containing poly(acrylic acid)block-polystyrene-block- poly(acrylic acid) (P2).
2.2.3. Hydrolysis of t-butylacrylate groups of polystyrene-b-Poly(tbutylacrylate)-b-polystyrene Polystyrene-b-poly(t-butyl acrylate)-b-polystyrene (0.5 g, 0.048 mmol with respect to t-butyl acrylate) was dissolved in 5 mL dry DCM, stirred for 15 min followed by addition of trifluoroacetic acid (TFA, 1.5 mL). The reaction mixture was stirred for 16 h at room temperature. Then the solvent was removed in rotary evaporator and TFA was completely removed by azeotropic distillation using
R. Banerjee et al. / Polymer 59 (2015) 243e251
toluene. The polymer was then dissolved in THF, precipitated from ice cold MeOH/H2O (2:1) mixture and washed several times with MeOH. The polymer was isolated by repeated extraction with DCM and the organic portions combined and dried over anhydrous MgSO4. Thereafter, the solvent was removed under high vacuum for 6 h to recover the product as a brown solid. The conversion of poly(t-butylacrylate) to poly(acrylic acid) was confirmed by 1H NMR analysis. 2.2.4. Conjugation of 9-anthracene methanol with polystyrene-bPoly(acrylic acid)-b-polystyrene Polystyrene-b-poly(acrylic acid)-b-polystyrene (0.22 g, 1.34 mmol with respect to the acid group) was dissolved in dry THF and 9-anthracene methanol (0.14 g, 0.67 mmol) was added at once to it, followed by the addition of 4-dimethylaminopyridine (DMAP, 0.008 g, 0.063 mmol). The reaction mixture was stirred for 20 min at 0 C. N,N0 -Dicyclohexylcarbodiimide (DCC, 0.12 g, 0.57 mmol in 2 ml of dry THF) was added very slowly at this temperature for 1 h and the reaction was continued for another 48 h. The reaction mixture was then filtered to remove the produced dicyclohexylurea (DCU) and the polymer product was recovered by precipitation from cold hexane. The residue was further dissolved in THF and again precipitated from hexane/pet-ether mixture to get the product. The incorporation of the anthracene moiety in the polymer backbone was confirmed by NMR, UVevis and fluorescence spectroscopy. 2.3. Synthesis of anthracene conjugated Poly(acrylic acid)-bpolystyrene-b-Poly(acrylic acid) (P2, Scheme 1b) Synthesis of P2 consists of the following steps. 2.3.1. Synthesis of polystyrene Macro-CTA a,a0 -Xylyl-bis(dirhiobenzoate) (XBDTB) (0.136 g, 0.481 mmol) and AIBN (20 mg, 0.12 mmol) were added to a 30 ml tube. Styrene (2.5 g, 24.0 mmol) was then added to it followed by addition of 1.5 ml of dry DMF. The reaction vessel was then degassed thrice with continuous purging of nitrogen and then warmed to room temperature. The reaction mixture was then placed in an oil bath at 70 C with vigorous stirring for 20 h. Polymerization was quenched and diluted with minimal amount of THF and the polymer was precipitated from ice-cold MeOH. The polymer was isolated by filtration and washed several times with MeOH and then dried under high vacuum. Polystyrene macro-CTA was obtained as pink solid (90% yield). Mn (1H NMR) ¼ 4500, Mn (GPC) ¼ 6700, PDI ¼ 1.2. 2.3.2. Synthesis of Poly(t-butyl acrylate)-b-polystyrene-b-Poly(tbutyl acrylate) Polystyrene macro-CTA (0.4 g, 0.089 mmol) and AIBN (3.6 mg, 0.021 mmol) were added to a 30 ml tube. t-Butyl acrylate (0.91 g, 7.2 mmol) was then added to it which was followed by addition of 2.0 ml of dry DMF. The reaction vessel was then degassed thrice, continuously purged with nitrogen and allowed to warm to room temperature. The reaction mixture was then placed in an oil bath at 70 C with vigorous stirring for 12 h and then the polymerization was quenched and diluted with minimal amount of THF. The polymer was precipitated from ice-cold methanol/water (3/1 v/v) mixture, isolated by filtration, and washed several times with methanol and then dried under high vacuum. Poly(t-butyl acrylate)-b-polystyrene-b-poly(t-butyl acrylate) was obtained as pale pink solid (78% yield). Mn (1H NMR) ¼ 10,400; Mn (GPC) ¼ 12,300, PDI ¼ 1.34. Hydrolysis of the t-butyl acrylate of polystyrene-b-poly(t-butyl acrylate)-b-polystyrene and conjugation of 9-anthracene methanol
245
with the above copolymer were carried out following similar procedure as described earlier as in the case of P1. 2.4. Instrumentation and methods The number average molecular weight of the synthesized polymers was determined by the utilization of 1H NMR spectroscopy [35] and gel permeation chromatographic (GPC) technique. In GPC instrument (Viscotek) we have used polystyrene standards and THF as eluent with a flow rate of 1 mL min1. Dynamic light scattering measurements were performed to determine the hydrodynamic diameter of bare GNPs and GNP embedded polymer using Malvern Zetasizer Nano equipment with a 3.0 mW HeeNe laser operated at 633 nm. Analysis was performed at a fixed detector angle of 173 and a constant temperature of 25 C. The absorption and fluorescence spectra were collected using a Shimadzu (model number, UV-2450) spectrophotometer and a Hitachi (model no. F-7000) spectrofluorimeter, respectively. For steady-state experiments, all the samples were excited at 350 nm. For time resolved fluorescence measurements, we have used a time correlated single photon counting (TCSPC) instrument from IBH, U. K. The instrument response function of this setup was ~0.09 ns. The detailed time-resolved fluorescence setup is described by Hazra et al. [36,37] Briefly, the samples were excited at 350 nm using a picosecond laser diode (IBH, U. K. Nanoled), and the signals were collected at the magic angle (54.7 ) using a Hamamatsu micro-channel plate photomultiplier tube (3809U). The data analysis was performed using IBH DAS version 6 decay analysis software. All the long and short wavelength decays were fitted bi-exponentially by considering c2 becomes close to 1, indicating a good fit. 2.5. Fluorescence quantum yield calculation Fluorescence quantum yields of donor molecules (anthracene) attached to the block copolymer have been calculated by using ethanolic solution of anthracene (quantum yield 0.27) [38] as the secondary standard for the fluorescence quantum yield measurement according to the following eq.:
" Фsample ¼ Фstd:
I=A
sample
A=I
# std:
. h2std:
!
h2sample
where F represents quantum yield, A is absorbance at the excitation wavelength, h is the refractive index, and I is the integrated emission intensity calculated from the area under the emission peak. 2.6. Preparation of GNPs GNPs of pre-determined sizes (20 nm and 55 nm) were synthesised by the reported method of Ferns [39]. Briefly 1.25 mL 0.01 M of HAuCl4.3H2O solution was taken in a 250 mL conical flask and it was mixed with 50 mL of milliQ water which was heated just to boil. 1% solution of trisodium citrate salt was then added and the solution was boiled with constant shaking till deep-red color of the GNP solution appeared. The formation of GNPs was confirmed from UV-vis spectroscopy and DLS, TEM experiments. 2.7. Grafting of block copolymers to GNP surfaces 10 mL of GNP solution was placed in an ice bath under vigorous stirring for 30 min. Cooled polymer solution (300 mL, concentration: 1 mg/mL) was added to the GNP solution, followed by stirring
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R. Banerjee et al. / Polymer 59 (2015) 243e251
for 6 h. Then, the GNPs were purified by centrifugation at 20,000 rpm for 30 min at 5 C followed by re-suspension in cooled water. This process of centrifugation and re-suspension was repeated thrice. Final GNP/polymer solutions were used for characterization and fluorescence measurements as soon as possible. 3. Results and discussion In the present work, we have synthesized new polymer-GNP systems. EET process was demonstrated using polymer-bound anthracene as donor (D) and polymer-bound GNPs of different sizes as acceptor (A). Distance-dependent EET process was followed using two block copolymer-coated GNPs in which the distance between the D and the A was varied simply by altering the sequence of the blocks in the block copolymers. Dithioester groups present at the block-copolymer ends were used to conjugate two different-sized GNPs with two anthracene containing block copolymers viz. polystyrene-block-poly(acrylic acid)-block-polystyrene (P1) and poly(acrylic acid)-block-polystyrene-blockpoly(acrylic acid) (P2) synthesized by RAFT polymerization technique. Size and distance-dependent EET process from anthracene to GNPs was monitored by the help of steady state and timeresolved fluorescence spectroscopic studies. 3.1. Anthracene conjugated block copolymers - synthesis and characterization The chain transfer agent (CTA), a,a0 -Xylyl-bis(dirhiobenzoate) (XBDTB) was used for synthesis of the two block copolymers polystyrene-block-poly(acrylic acid)-block-polystyrene and poly(acrylic acid)-block-polystyrene-block-poly(acrylic acid). The CTA and the polymers were characterized by 1H NMR and 13C NMR spectroscopy (Supporting information e Figs. S1 to S9). The compositions of the polymers were determined by 1H NMR spectroscopy. 9-Anthracene methanol was reacted with acrylic acid containing polymers as synthesized above to attach anthracene fluorophores in the polymer backbones. The coupling reactions were carried out in such a way so that some of the acrylic acid groups remain free (for hydrophilicity). Fig. S6 (Supporting information) showed almost quantitative conversion of poly(tbutylacrylate) to poly(acrylic acid). Furthermore, it is clear from the Fig. S7 and Fig. S8 that the ratio of anthracene units to free acrylic acid units is almost same in both the polymer P1 and P2. Presence of significant amount of free hydrophilic acrylic acid groups (which were not functionalized by anthracene) was confirmed from the peak at 177 ppm of the 13C NMR spectra of the block copolymer P1 (Fig. S9, Supporting information). Also from 13C
Table 1 Characterization of the polymers used in this study. Polymer
Monomer Initiator CTA agent
Mna Mnb PDIb (G/mol) (G/mol)
PtBA macro-CTA PS-b-PtBA-b-PS P1 PS macro-CTA PtBA-b-PS-b-PtBA P2
tBA Styrene e Styrene tBA e
5600 9800 e 4500 10,400 e
a b
AIBN AIBN e AIBN AIBN e
XBDTB PtBA macro-CTA e XBDTB PS macro-CTA e
7100 11,800 13,100 6700 12,300 13,900
1.30 1.45 1.41 1.20 1.34 1.51
From 1H NMR. From GPC.
NMR spectra it is clear that the degree of functionalization of the PAA block with anthracene groups is approximately 50%. Gel permeation chromatography (GPC) was also used to analyze the molecular weights and polydispersity of the precursor macro-CTAs and the block copolymers synthesized from them (Fig. 1). The monodispersed nature of the chromatograms and the shifting of the peaks to lower retention volumes in case of the block copolymers, confirmed the formation of reasonably pure and high molecular weight block copolymer The molecular weight data of all the polymers are presented in Table 1. All the polymers synthesized in this study, are expected to contain dithioester end-groups, which could be used to bind to the GNPs surface. 3.2. Synthesis and characterization of GNPs and polymer-coated GNPs Citrate-stabilized GNPs dispersed in water were synthesized using the citrate method [39]. Two different citrate to HAuCl4 ratios were used to synthesize GNPs of two different sizes. The polymercoated GNPs were prepared by treating the synthesized block copolymers with the citrate stabilized GNPs in aqueous solution for a long duration. After the coating process was completed, the polymer-coated particles were isolated by centrifugation, followed by washing and re-suspension in water. This process of centrifugation-washing-re-suspension was repeated thrice to ensure complete removal of any unbound block polymers from the re-suspended polymer-coated GNPs. Eisenberg et al. [14,15]. had described that GNPs can be successfully incorporated in the core of the self-assembled of PS-b-PAA, where GNPs were stabilized mostly into the hydrophobic region produced by the selfassembled nanostructures. The affinity of the diothioester endgroups for the gold surface was shown to be much higher than that of the carboxylate groups. This is the reason why citrate capped GNPs were grafted by P1 and P2 by centrifugation and redispersion method. Furthermore, to avoid any confusion, we have
Fig. 1. GPC chromatograms of anthracene conjugated block copolymers along with their precursor polymers - (a) P1 and (b) P2.
R. Banerjee et al. / Polymer 59 (2015) 243e251
used almost same block length of poly(acrylic acid) in both the polymer P1 and P2. The re-suspended solution did not show any observable precipitation even after 15 days (Fig. S10, Supporting information), although the characterization and fluorescence measurements were carried out soon after the preparation of the polymer-coated GNP solutions. Fig. 2 (top) shows typical transmission electron microscopy (TEM) images of GNPs synthesized by the citrate method and (bottom) the corresponding polymercoated GNPs. The average particle sizes of the nanoparticles are presented in Table 2. It should be noted that the polymer-coated GNPs were observed somewhat aggregated on the carbon-coated TEM grids during sample preparation. In Fig. 3, typical UVevisible spectra of citrate-stabilized GNPs and the corresponding polymer coated GNPs in aqueous solution are presented. Citrate-stabilized GNPs exhibited localized surface plasmon resonance (LSPR) band with maxima at approximately 522 nm and 527 nm for the two nanoparticles, which is typical of spherical GNPs of approximately 20 nm and 55 nm diameter respectively [1,39]. After being coated with the block copolymer, the 20 nm GNPs exhibited a slight red-shift of the lmax (3 nm for P1 and 5 nm for P2) indicating a small increase in the effective size. However, for the 55 nm GNPs, there was no shift in lmax, which isnot unexpected; as the GNPs were already large enough coating of polymer did not affect its absorption properties significantly. The size of GNPs and the polymer-coated GNPs were characterized by DLS to determine their hydrodynamic sizes. Fig. 4 shows their DLS data, along with that of the P1-coated GNPs. The size distribution of P2-coated GNPs was more or less similar to that of P1 and not shown here. The hydrodynamic sizes obtained from DLS matched closely with those obtained from TEM and UVevis spectroscopic studies. Analysis of the DLS results indicated that the two assynthesized citrate-stabilized nanoparticles had diameters of 19 nm and 50 nm, which were increased to 29 nm and 58 nm respectively on coating with P1.
247
Table 2 Preparation and characterization of prepared GNPs. Entry Amount of 0.01 M HAuCl4.3H2O (mL)
Amount of 1% trisodium salt of citrate (mL)
UV-vis Size of GNPs from UV-vis lmax (nm) (nm)
Size of GNPs by TEM (nm)
1 2
875 300
522 527
18e22 50e60
1.25 1.25
20 55
3.3. Steady-state fluorescence study of the GNPs and polymercoated GNPs It is well-known in literature that if a fluorophore is brought in close proximity to metal nanoparticles like gold, silver, the excited state energy of the fluorophore (D) is easily transferred to the metal nanoparticles (A). In the present work, we essentially have two block copolymeric systems as shown in Scheme 2a. In case of P1, the anthracene molecules are attached to the middle block and are placed away from the GNPs that are linked with the dithioester groups present at the two ends (Scheme 2b) of the P1. In case of P2, the anthracene groups are situated on the two sides and hence closer to the GNP bonded chain ends. Fig. 5 shows the steady-state fluorescence spectra of the polymer-bound anthracene fluorophores (lex ¼ 350 nm) for the two GNPs e polymer conjugate systems. The spectra for the free polymers (not conjugated with GNPs) are also included in Fig. 5. The fluorescence emission spectra of the anthracene molecules were clearly observed in the free polymers P1 and P2. The following observations could be made from the spectral data - (i) for P1, where the anthracene units are in the middle of the polymer chains and hence far from the GNPs, the quenching of anthracene fluorescence increased with the size of the nanoparticles, (ii) for P2, where the anthracene units are at the two sides of the chains and in
Fig. 2. Typical transmission electron microscopy (TEM) images of the two sets of GNPs synthesized by the citrate method (a and b) and the corresponding polymer-coated GNPs (c and d respectively).
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R. Banerjee et al. / Polymer 59 (2015) 243e251
Fig. 3. UVevis absorbance plot of the two citrate stabilized GNPs as prepared in water (bare GNPs) and the polymer-coated GNPs; (a) and (b) corresponds to the same GNPs as shown in Fig. 2a and b respectively.
Fig. 4. DLS histograms for the two GNPs (bottom) and for the same GNPs coated with P1 (top), x-axis represent hydrodynamic size.
close proximity to the GNPs, both GNPs caused significant quenching to the anthracene fluorescence. For P2, as the fluorophores are placed near the GNPs, easy energy transfer occurred from the excited anthracene molecules to the GNPs. However, for P1, the anthracene moieties being far from GNPs, the 55 nm ones have the largest energy accepting capability whereas the 20 nm GNPs are probably too small to influence anthracene molecules from a distance and as a result, practically no energy transfer took place. This size-dependent energy accepting capability of the GNPs is reported in literature and has been attributed to an increase in non radiative decay rate with increasing the size of GNPs [1,40]. The binding between the dithioester groups and the GNPs were found to be very strong. We tried to disrupt this binding process and release the anthracene units by adding several compounds that
Scheme 2. Schematic presentation of the location of the fluorophores (anthracene, donors) and the GNPs (acceptors) in the polymer-coated GNP systems. The GNPs could bind to more than one polymer; for simplicity, one polymer chain conjugated to the GNPs is shown.
are known to complex strongly with the GNPs viz. b-cyclodextrin, 3-mercaptopropanoic acid, glutathione, dodecane thiol, butane thiol. However, none of these compounds was able to dislodge the GNPs bound to the dithioesters of our polymers. Only a slight increase in the fluorescence intensity was noticed on addition of dodecane thiol. 3.4. Time-resolved fluorescence study of the GNPs and polymercoated GNPs After investigating the EET process by steady-state fluorescence spectroscopy, more confirmatory study of the EET process was conducted by carrying out TCSPC experiments of the free and the GNP-bound block copolymers containing anthracene groups as fluorophores. Fig. 6 shows the time-resolved fluorescence decay curves. The lifetime data for all the samples are listed in Table 3. From the data presented in Table 3 and from Fig. 6, it can be clearly noted that the average lifetime of the anthracene moieties decreased because of EET to the GNPs. For example, the average lifetime value of the anthracene in P1 decreased from 2.86 ns to 2.38 ns and 1.96 ns in presence of 20 nm and 55 nm GNPs respectively. Similarly, the average lifetime of anthracene in P2 decreased from 2.95 ns to 1.33 ns and 1.15 ns in presence of 20 nm and 55 nm GNPs respectively. The energy transfer between Au nanoparticle and a dye provided a new paradigm to design an optical-based molecular ruler for long distance measurements [41,42]. This ET originates from the interactions of the electromagnetic field of the donor dipole with
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Fig. 5. Steady-state fluorescence emission spectra of polymer-bound anthracene molecules where the polymer chains are either conjugated to GNPs or free in the solution.
the free conduction electrons of the accepting metal and it is established that the separation between donor and acceptor is d4 dependent. In this study, the distance between the donor (anthracene) and the acceptor (GNPs) has been calculated by using
the surface energy transfer (SET) method. The characteristic distance at which a dye (donor) will display equal probabilities for energy transfer and spontaneous emission (d0) is calculated using the Persson model [30,43].
Fig. 6. Time-resolved fluorescence decay profiles of polymer-bound anthracene molecules where the polymer chains are either conjugated to GNPs or free in the solution.
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Table 3 Lifetime data for the anthracene molecules attached to the block copolymer in presence and absence of GNPs. System
t1 (ns)
a1
t2 (ns)
a2
c2
Avg t (ns)
P1 P1 P1 P2 P2 P2 -
1.38 0.19 1.23 1.74 1.15 1.03
0.73 0.29 0.86 0.78 0.90 0.89
6.88 3.28 6.35 7.23 2.93 2.11
0.27 0.71 0.14 0.22 0.10 0.11
1.33 0.93 1.19 1.10 1.34 1.45
2.86 2.38 1.96 2.95 1.33 1.15
GNP (20 nm) GNP (55 nm) GNP (20 nm) GNP (55 nm)
Table 4 Nanometal surface energy transfer (NSET) parameters for the anthracene molecules attached to the block copolymer in presence of GNPs. System
lex (nm)
d0 (Å)
ФET (%)
d (Å)
kNSET (s1)
P1 - GNP (20 nm) P1 - GNP (55 nm) P2 - GNP (20 nm) P2 - GNP (55 nm)
350 350 350 350
55 55 55 55
16 31 55 61
83.25 67.18 52.31 49.18
0.67 1.57 4.14 5.30
108 108 108 108
bonded chain ends. Our strategy was to synthesize well-defined triblock copolymers of ABA and BAB type of architectures, so that the anthracene moieties could be either placed in the middle of the chain (say the B block in ABA) or at the sides (in BAB). This enabled us to precisely control the distance between the donor anthracene groups and the acceptor Au particles present at the chain termini. Besides, the effect of size of the acceptor molecules in the EET process could also be studied by using GNPs of different sizes. In case of the BAB type copolymer, where the anthracene groups were present closer to the GNPs, a significant quenching of anthracene fluorescence was observed. However, in case of the ABA type copolymer, where the anthracene groups were placed at a larger distance from the GNPs, quenching was observed only by the biggest sized (55 nm) GNPs. Hence, our work shows that block copolymers with tailored architectures, synthesized by the RAFT technique, can be utilized to bond to Au particles through the functionalized chain-ends and that eventually enables in better understanding of the controlled EET process. Acknowledgment
d0 ¼
0:225 c3 Фdye
!1=4
u2dye uF kF
Фdye is the quantum efficiency of anthracene, udye the frequency of the donor electronic transition,uF the Fermi frequency, and kF Fermi wave vector of the metal [30]. The d0 value is calculated using Фdye ¼ 0.32, udye ¼ 4.55 1015 s1, uF ¼ 8.4 1015 s1, and kF ¼ 1.2 108 cm-1, and velocity of light (c) ¼ 3 1010 cm s1. The lifetime quenching data is assumed to be more accurate than the steady-state fluorescence intensity quenching data in order to determine the energy transfer efficiency. The equation, ФET ¼ 1
tDA tD
is used to calculate the time-resolved energy transfer efficiencies, where tDA and tD are the lifetime of donor in the presence and absence of acceptor (GNPs), respectively. According to Persson model, the rate of surface energy transfer is given by
kNSET ¼
1 d0 4 tD d
where (d) is the distance between molecular centers of the donor to surface of nanoparticles acceptor. Information on the above mentioned useful spectral parameters are listed in Table 4. Table 4 reflects that anthracene labeled triblock copolymers (P1), in which polystyrene chain act as a spacer, helps in reducing non radiative energy transfer and more efficiently in case of smaller size GNPs. Thus fluorophore labeled block copolymers with tailored architectures are able to control the energy transfer process by appropriate choosing of chain transfer agent in RAFT polymerization technique. 4. Conclusion In the present work, we demonstrated the size- and distancedependence of the EET process using a polymeric system, wherein anthracene moieties attached to the polymer chains transfer energy to the GNPs bound to the two ends of the same chain. The dithioester end-groups introduced at the polymer chainends by virtue of controlled polymerization using dithioester chain transfer agent (RAFT technique) have been exploited to form GNP-
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