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Correlated blinking via time dependent energy transfer in single CdSe quantum dot-dye nanoassemblies
Chemical Physics Letters 572 (2013) 90–95
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Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Correlated blinking via time dependent energy transfer in single CdSe quantum dot-dye nanoassemblies Frank Gerlach 1, Daniela Täuber, Christian von Borczyskowski ⇑ Chemnitz University of Technology, Institute of Physics, Optical Spectroscopy and Molecular Physics, 09107 Chemnitz, Germany
a r t i c l e
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Article history: Received 11 February 2013 In final form 13 April 2013 Available online 19 April 2013
a b s t r a c t Optical confocal spectroscopy on self-assembled single nanoassemblies from CdSe/ZnS quantum dots (QD) and perylene diimide dye molecules demonstrates efficient Förster resonance energy transfer (FRET). Intramolecular dynamics of the flexible dye molecule change the FRET efficiency in course of the detection period of several minutes. This can be followed by correlated observations of luminescence intensities and related spectral shifts of both constituents. Contrary to several experiments on similar assemblies, the FRET efficiencies are by almost one order of magnitude larger in the non-polar liquid solvent TEHOS as compared e.g. to toluene. Experimental and theoretically expected efficiencies are in close agreement with each other. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Energy transfer between molecular moieties after photoexcitation is a fascinating and important phenomenon studied in detail during the last decades for a large variety of systems ranging from antenna pigments of photosynthetic units [1] to semiconductor based nanoassemblies [2]. Among this manifold of materials the combination of organic dye molecules and semiconductor nanocrystals is of special interest since this class is a base for organic/ inorganic photovoltaic devices [3] and biologically relevant nanosensors [4]. Several groups have shown that such hetero-nanoassemblies self-assemble under suitable conditions, that is in the presence of functionalized dyes which can be selectively attached to the surface of e.g. colloidal CdSe semiconductor nanocrystals [5–8]. Such assemblies can be formed either by directed synthesis involving coating with polymers [9,10] or in a dynamic equilibrium [11–13] in liquid solutions. The energy transfer efficiency depends on the energetic and geometric constraints of the constituents of such assemblies [5,10,14]. Additionally, these constraints are subject to changes during the observation time, thus modifying the transfer efficiency. This will be the subject of the present investigation in terms of a case study presently not aiming at statistical relevance. Single molecule detection is an excellent tool to evaluate details of static or dynamic fluctuations of (Förster) resonance energy transfer (FRET), with respect to FRET mechanism, efficiencies and ⇑ Corresponding author. Fax: +49 371 531 3060. 1
E-mail address: [email protected] (C. von Borczyskowski). Present address: Fibotec Fiberoptics GmbH, 98617 Meiningen, Germany.
0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.04.034
competing processes [13,15]. It could be shown, that FRET depends as predicted on the distance between the constituents [10], the relative orientation [16] and the spectral overlap [9,16]. In case of self-assembled assemblies these three variables will depend on observation time as soon as the respective assembly is subject to time dependent fluctuations of the geometry or relative energies. Such long-time dependent phenomena have not yet been studied on the single assembly level, though they are important e.g. with respect to FRET processes in photosynthetic entities. We have already recently realised a suitable model system composed of CdSe/ZnS quantum dots (QD) and functionalized perylene diimide (PDI) dye molecules [13]. The functionalization with pyridyl groups (see Scheme 1) enables attachment of the dye molecules to the QD surface [5,13]. Corresponding assemblies show FRET depending on the specific geometry [16]. However, recent experiments on these systems showed only very small FRET efficiencies since a parallel NON-FRET process has masked the energy transfer [12]. In the present contribution we have chosen the non-polar solvent tetrakis(2-ethylhexoxy)silane (TEHOS) to study subtle effects on FRET such as the structural flexibility of dye molecules, which we investigated recently in quite a detail in toluene, PMMA and on SiO2 [14,17,18]. In contrast to these previous studies, we find that NON-FRET quenching of the photoluminescence (PL) of the CdSe/ZnS energy donor is almost completely suppressed in TEHOS. We will report on a single assembly level for a few selected cases how FRET efficiencies correlate with geometric variables as deduced from the related spectral properties. Since statistics are too low to draw general conclusions this study is intended to demonstrate the capabilities of single molecule detection so far not yet reached.
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Scheme 1. DTPP (di-terpyridyl-perylenediimide). For steric reasons, the molecule can only be attached via 1 or 2 pyridyls of the outermost phenyl rings [16]. The phenoxyl bay groups are very flexible [14,18] and induce a time dependent extention of the perylene chromophoric backbone. The optical transition dipole moment is along the long perylene axis.
2. Experimental Silicon substrates with 100 nm thick thermal oxide (CrysTec, Berlin) were cleaned thoroughly by alternate rinsing with acetone and ethanol (both technical grade), followed by sonication at 50 °C in acetone and in ethanol (both Merck spectroscopic grade) for 15 min each, and finally sonicated at 80 °C for 1 h in piranha solution (1:1 H2O2:H2SO4). In between these steps and at the end, the substrates were rinsed with de-ionised water (Milli-Q) and dried in a flow of nitrogen. Samples were prepared by dipcoating (KSV instruments) the substrates into solutions of liquid 0.3% tetrakis(2-ethylhexoxy)silane (TEHOS) in hexane with highly diluted (TOPO capped) CdSe/ZnS (Evident Technologies) and di-terpyridyl-perylenediimide (DTPP) resulting in 10 nm thick films of TEHOS with nanomolar concentrated CdSe/ZnS and DTPP. DTPP (Scheme 1) has been synthesized by Lang et al. [19]. After sufficient time to allow for evaporation of hexane, optical spectra were investigated with a home-built laser scanning confocal microscope with a numerical aperture of 0.9. The setup has been described in detail elsewhere [20]. Excitation occurs via the 488 nm line of an Ar-ion laser. Samples were laser scanned to detect luminescent spots, the size of which is determined by the focus of the laser. At selected spots time series of luminescence spectra were taken with an integration time of 1 s. We obtain the luminescence intensities via integration of the spectra across the relevant spectral range of the respective constituent. A typical sample contains about 10 localised QDs while the mayority of DTPP undergoes fast diffusion processes. Somewhat less than 10% of the luminescent spots show spectra of both constituents. This is in rough agreement with previous studies [13]. Two typical examples (A and B) from five different samples have been chosen for a detailed spectral analysis. Due to intense optical excitation in the confocal microscope, QDs and molecules easily bleach under ambient conditions and only a few could be studied over a long enough time interval to record spectra. We have not yet implemented investigations of liquid film samples under non-ambient conditions. For this reason we have only investigated a few assemblies. 3. Results and discussion All experiments have been performed in thin films of the nonpolar liquid TEHOS dip-coated onto properly cleaned SiO2 surfaces.
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The dynamic self-assembly of CdSe/ZnS and DTPP in TEHOS results both in assemblies and single constituents, similar to the formation process observed in toluene [11,13]. Thus, luminescent spots observed by confocal microscopy show three types of luminescence spectra related either to CdSe/ZnS, DTPP or both spectra of the two constituents. Figure 1 shows typical luminescence spectra of CdSe/ZnS (left) and DTPP (right) as a function of observation time of several minutes. Spectral positions and intensities vary in time. A time integrated spectrum is shown at the top of each time trace. The fluctuating luminescence intensities are given at the side. CdSe/ZnS shows strong intensity fluctuations known as blinking or luminescence intermittency [21], while blinking is almost negligible for DTPP [14], though moderate intensity fluctuations in combination with spectral fluctuations (spectral diffusion) are quite obvious [19]. The luminescence of CdS/ZnS and DTPP are bleached after 180 and 77 s, respectively. Bleaching of the QD is in most cases preceded by a strong blue shift of the photoluminescence (PL) which is tentatively assigned to photooxidation of the CdSe core [22]. Only a few of the localised luminescent QD spots show spectra of both constituents. Two typical examples (A and B) are shown in Figure 2. In general, the time dependent spectral features are quite similar to those shown already in Figure 1, besides the enhanced blinking of DTPP. At (almost) every blinking event of the QD, the DTPP fluorescence (Fl) intensity is switched ‘off’ when the QD is switched ‘off’ and it is ‘on’ when the QD is ‘on’. A quantitative inspection reveals that the respective luminescence intensities are strongly correlated, as can be directly seen, when plotting the corresponding intensities on top of each other at the bottom of Figure 2. In both cases A and B, the strong blinking of QDs (Figure 1, left) is imposed on DTPP, which now also blinks very frequently. Assembly A (Figure 2, left) shows that the DTPP fluorescence is reduced from a relatively high Fl intensity (about 6 kcts) to a much lower one (about 1 kcts, the background being close to 100 cts) as soon as the QD photoluminescence turns ‘off’ according to a blinking process. When DTPP is photobleached or detached from the QD (at 77 s) the QD photoluminescence increases on average by about a factor of 3. There are only three occasions for which intensity correlation fails, namely at 56, 66, and 72 s, where DTPP fluorescence decreases while the QD photoluminescence increases. This can be assigned to one of the rare blinking events of DTPP. The overall interpretation is that the PL of the QD shows intrinsic blinking and is additionally partly quenched due to FRET from the QD to DTPP. Fluorescence of DTPP, on the other hand, is predominantly caused via FRET. We do not observe a detectable difference in blinking dynamics of isolated QDs and QDs in assemblies. Therefore all the QD blinking features are transferred uniquely to DTPP. Photoexcitation at 488 nm is not selective with respect to the two constituents. Therefore it is at first glance surprising that we observe only a relatively low DTPP fluorescence when the QD is in an ‘off’-state. (In the B-type assembly to be discussed later in detail the Fl intensity in the ‘off’-state of the QD is about 2.5 times stronger than in the A-type assembly.) This can be explained by the orientation of DTPP with respect to the exciting laser beam. The transition dipole moment of DTPP is nearly parallel to the long axis of the chromophoric perylene backbone [14] (see Scheme 1). Reduced direct excitation is achieved when this long axis is orientated parallel to the incident laser beam, which corresponds to case b) in Scheme 2. We reported recently [14] for similar perylene diimides on SiO2 surfaces that the Fl intensity varies in time by up to a factor of 5 without any change in the Fl spectra. We suggested that this is due to fluctuations in the molecular orientation. Later on we could directly show by determination of the three-dimensional dipole orientation [26] that diimide molecules re-orientate by up to 40° with respect to the surface within timescales of seconds. FRET from the (nearly isotropically absorbing) QD to DTPP abandons this
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Figure 1. Typical time dependent spectra of CdSe/ZnS (left) and DTPP (right) with 1 s integration time. The corresponding integrated spectra over the total observation time are shown on top of each spectral trace. The integrated luminescence intensity is depicted on the right side of each time trace.
Figure 2. Top: Spectral time traces of 2 exemplary QD–dye assemblies A (left) and B (right). Each time trace shows the spectral signature of the QD ( 560 nm) and DTTP ( 615 nm). In case A, time ranges of fluctuating (f) and stable (s) intensities (see Figure 3) are indicated. Bottom: Intensity time trace of the 2 constituents of the assembly A (left) and B (right).
Scheme 2. Two different orientations of the DTPP dye on the QD. For steric reasons DTPP can be attached via the terpyridyl groups at only one end of the dye. DTPP is plotted along the optically active transition dipole moment. Laser excitation is perpendicular to the substrate.
selection rule only with respect to absorption while emission of DTPP remains still reduced in case of an unfavourable dipole orientation but is nevertheless visible. The situation is obviously different for assembly B as will be discussed later on. Alternatively to FRET, the intensity correlation might be caused by photoinduced electron transfer from the QD to DTPP [5,15] or vice versa. In that case both constituents become charged and at least the luminescence of DTPP will be completely quenched, while a charged QD will have a similar PL spectrum (but with reduced PL intensity) as the uncharged one but shifted by about 20 meV [21]. The only small spectral shift rules out that FRET from DTPP occurs to a charged QD. The key to rule out charge transfer processes is the manifestation of the intrinsic nature of QD blinking. If charge
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Figure 3. Top: Correlation of normalised DTPP and QD luminescence intensities of assemblies A and B for selected observation time ranges related to the respective time traces in Figure 2. Intensities have been normalised to the respective maximum of each time trace for each constituent. Bottom: Luminescence intensity ratios as a function of observation time. Data for assembly A are shown on the left and assembly B on the right. s and f indicate time ranges of stable and fluctuating intensities, respectively. For correlation with wavelength see Figure 2 top left.
transfer processes caused by DTPP were involved, the QD blinking would be considerably influenced. This is not observed neither in this nor in other but similar experiments [13,22,25]. If charge transfer would apply, the Fl of the dye will increase when the QD is intrinsically ‘off’ and can thus not act as a charge donor. Contrary to this, the Fl of the dye is strongly decreased when the QD does not emit. An additional support that FRET is the reason of the intensity correlation stems from the observation that the QD PL intensity increases when DTPP is bleached. This proofs that QD PL reduction of the ‘on’ state prevails only in the presence of the DTPP acceptor. The B-type assembly (Figure 2, right) shows also a strong correlation of luminescence intensities. However, in this case direct excitation of DTPP is, as compared to the A-type assembly, more clearly visible when the QD is ‘off’. This implies a more favourable orientation of DTPP with respect to laser excitation as is e.g. shown in Scheme 2a. In case B, both constituents are photodegraded after 27 s within the same range of time. In case that the degrading is due to a permanent charge transfer event, this would indeed result in a complete quenching of Fl and considerable reduction of the PL of the QD. Permanent charge separation, however, is very unlikely to be realised in the non-polar solvent TEHOS. To obtain a more quantitative description of FRET we have plotted the luminescence intensity ratio R = IDTPP/IQD in Figure 3 (bottom) for A and B as a function of observation time. In both cases the correlation clearly depends on time. While R changes by more than a factor of 3 in case of A (till DTPP is bleached), the time dependence is less pronounced for B. More details become visible
when plotting (normalised) intensity ratios as a function of selected observation time ranges (Figure 3, top). In case of A, we have selected two characteristic time intervals since the inspection of spectral properties (Figure 2, top left) indicates a discrete jump in wavelength of DTPP at about 27 s. A correlation of (normalised) intensities is quite linear during the first 30 s as described by the increment in Figure 3 (top left). During that time (Figure 2, left) the PL of the QD continuously red shifts, while the Fl of DTPP experiences a blue shift. This will change the spectral overlap at least due to the red shift of the QD (since we do not know, whether the absorption of DTPP shifts in the same manner as the Fl does). As we and others have shown, a spectral shift of DTPP is accompanied by a change of the molecular conformation [14,16]. A closer inspection of the Fl intensity of DTPP in Figure 2 (bottom left) reveals that there is a small remaining intensity when the QD is ‘off’, as can be also seen in Figure 3 (top left) from the remaining IDTPP abscissa value for IQD = 0. The remaining Fl intensity (when the QD is ‘off’) becomes lower in the second time interval (IDTPP = 0 for IQ = 0). This change coincides with the spectral jump of DTPP at 27 s (Figure 2 (top left)), which indicates a change in DTPP geometry [18]. In the second time period (30.5– 80 s) the correlation is less obvious, since the absolute DTPP Fl intensity decreases, which indicates a somewhat reduced FRET efficiency. Pronounced fluctuations of the intensity correlation shown in Figure 3 bottom are observed for times longer than 55 s. In this range, we find several short periods during which, as discussed before, an opposite correlation is observed, probably due to DTPP
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blinking. There seems to be still a correlation at times longer than 80 s (see Figure 3, top left) which, however, is probably due to remaining QD PL in the long wavelength detection channel of DTPP. In this time interval the Fl of DTPP is nearly bleached or alternatively, DTPP is no longer attached to the QD. For assembly B, we observe a strong correlation over the total observation time. Apart from the first 3–5 s, there are almost no spectral shifts both for DTPP and QD. As can be seen in Figure 3 (top right), there seems to be a slightly smaller FRET efficiency in the interval (15.5–30 s) compared to the interval (0.5–15 s), followed by a pronounced increase in the interval (30.5–40 s). During this last short time interval FRET is very effective, but direct excitation of DTPP becomes negligible, since IDTPP = 0 for IQD = 0. From this we conclude that DTPP (or the assembly itself) has changed to an orientation unfavourable for direct excitation. The accompanied spectral jump observed at 26 s indicates a change in the intrinsic molecular geometry. This excludes a reorientation of the assembly since this will not result in a spectral jump. The two earlier time intervals in Figure 3 (top right) show considerable Fl of DTPP, even when the QD is ‘off’, however, the absolute value decreases with observation time. The decrease of Fl of DTPP is, in particular, very pronounced after t = 26 s, where luminescence of both components becomes very weak. At t > 40 s both constituents are ‘off’. In the following we will discuss the observed FRET processes more quantitatively in the framework of Förster resonance energy transfer [23]. Though it is not immediately obvious that FRET in a QD–dye assembly can be described by a dipole–dipole approximation, several experiments have shown that this simplification holds within experimental accuracy [2,5,10]. The energy transfer rate kFRET (r) as a function of the distance r between the centres of QD and DTPP, corresponds to
kFRET ðrÞ ¼
6 Ro ; sD r 1
ð1Þ
with the Förster Radius Ro (including the spectral overlap integral J and the relative orientation of the transition dipole moments) and the intrinsic donor lifetime sD in the absence of PL quenching processes. Since it is known that PL blinking of QDs is related to a fluctuation of sD which will result in an effective sQD[13,17], kFRET will depend on the effective sQD. The PL intensity IQD is to a good approxr r imation proportional tokQD sQD (with the radiative rate kQD ) [23]. The FRET efficiency EFRET corresponds to
EFRET ¼
kFRET kQ þ kFRET þ s1 D
ð2Þ
where kQ describes the intrinsic PL quenching including the NONFRET quenching as induced by merely attaching the acceptor [11– r 1 1 13]. With ðs1 D þ kQ Þ ¼ sQD and sQD ¼ kQD =I QD , the efficiency EFRET will depend approximately linearly on IQD for kFRET ðs1 D þ kQ Þ. For both assemblies A and B we clearly observe a linear dependence of the DTPP fluorescence intensity on IQD. The FRET efficiency EFRET for a DTPP–QD assembly can in principle be also calculated from the fluorescence intensities IDTPP of the acceptor DTPP according to
EFRET ¼
eDTPP ðIDTPPQD IDTPP Þ ; IDTPP eQD
ð3Þ
where IDTPPQD is the DTPP Fl intensity in the assembly. This equation relies via e on the isotropic orientation of the participating entities. However, as we have shown, an isotropic orientation does not apply in the present situation of only one single assembly. Therefore we cannot use this approach to determine absolute values of EFRET. An alternative access to the FRET efficiency is related to quenching of the QD donor PL intensity IQD according to [23]
EFRET ¼ 1
IQDDTTP : IQD
ð4Þ
An estimation of EFRET can be obtained using Eq. (4) in case of the type A assembly, since DTPP is photobleached at times longer than 77 s. Taking the maximum PL intensity IQD in that time interval and the maximum of the intensity IQD–DTTP during the time DTPP is not photobleached (Figure 2, bottom left) and using Eq. (4) we obtain EFRET 0.69. This can be compared to the two events at 57 and 72 s, when DTPP is ‘off’ due to blinking, providing IQD as compared to an average IQD–DTPP when DTPP is ‘on’ again. According to Eq. (4) this results in EFRET 0.55. Both values are in reasonable agreement with EFRET 0.85 recently calculated according to the Förster model [16]. The model takes only the distance RDA between the centres of the two point dipoles and R0 into account. RDA depends on the relative orientation of the DTPP dipole and has a minimum (which results in a maximum of EFRET) for a parallel orientation of the DTPP dipole to the QD surface. Due to steric reasons (caused by the pyridyl rings forming the ’bonds’ to the QD) this is the most probable orientation. Some variations of EFRET are expected since IQD depends on the (intrinsic) quenching process. For type B assembly we did not find a situation for which DTPP is ‘off’ (while the QD is ‘on’) and we therefore cannot apply Eq. (4). From a comparison of the increments of the intensity ratios in Figure 3 (top) we conclude that EFRET is somewhat lower for the B as compared to the A assembly. These FRET efficiencies larger than 0.5 are in contradiction to what we have published recently both for DTPP (ensemble and single assemblies) [13–16] and porphyrins (ensemble) [5,11,12,22,24] for which we found FRET efficiencies of less than 0.1. We have related these previous findings of low FRET efficiencies to the presence of competing NON-FRET processes, which open a quenching pathway other than energy transfer by merely attaching a dye molecule [11–13]. All our previous experiments have been performed in toluene or on SiO2 surfaces. As we have discussed recently in quite detail, [11] NON-FRET effects are related to ligand attachment/detachment processes. TEHOS as a non-polar solvent will hardly solvate the polar (TOPO) ligands. This will result in a well shielded ligand shell of the QD suppressing NON-FRET quenching processes. Moreover, polar solvents will stabilize charges at the QD surface, which also reduces the PL lifetime and thus the PL intensity. Finally we will discuss the variations of FRET during the observation time as it is evident from Figure 3. Since there is no inherent systematic spectral change during observation time for DTPP or QD, at least not for the assembly, we can basically assign the decrease of the intensity ratio to a decrease of FRET. There are at least 4 reasons why FRET varies during observation time, namely changes of the: (i) spectral overlap, (ii) donor–acceptor distance, (iii) the relative orientation of transition dipole moments, or (iv) the effective donor lifetime. Figure 2 (top left) clearly shows that a change in spectral overlap (case (i)) is present at least for assembly A during the time range of (0–25 s). Making use of Förster theory we can calculate the increase in spectral overlap to be about 4%, which is not enough to explain the change in the increment in Figure 3 when comparing FRET at earlier and later times. Recently we have investigated single perylene diimide type molecules on SiO2 interfaces and in polymers with respect to spectral diffusion in relation to fluctuations of the bay group orientation [18,20], which results in large spectral shifts of up to Dk = 30 nm. In the present case we use the non-polar solvent TEHOS as a matrix. Though the statistics are lower as compared to the previous investigations, the principle fluorescence band is between 600 and 625 nm accompanied by a spectral diffusion in the range of 5–15 nm. Comparing the spectral diffusion of
F. Gerlach et al. / Chemical Physics Letters 572 (2013) 90–95
non-aggregated DTTP (Figure 1 (right)) with that in aggregates A or B, it can be seen that spectral jumps occur more frequently in the aggregates. According to what we have found previously [20] for DTPP in the polymer PMMA and on SiO2, this might be caused by a less fixed structure of DTPP within an aggregate (see also Ref. [16]). This will in principle also influence EFRET. For assembly A we find in Figure 3A (bottom) that the intensity ratio IDTPP/IQD can be tentatively divided into 2 categories ‘s’ and ‘f’. ‘s’ corresponds to stable and ‘f’ to fluctuating intensity ratios on a 1 s averaging time scale. These intervals are also indicated in Figure 2A. We find on average slightly more spectral jumps in the ‘f’ as compared to the ‘s’ interval, though this at the limit of experimental accuracy. The spectral jumps mask a precise assignment of the FRET efficiency since each DTPP configuration is related to a different Fl intensity [18] and thus modifies the luminescence intensity ratio (Figure 3) possibly without changing the FRET efficiency. 4. Conclusions The present experiments are demonstrative examples how Förster resonance energy transfer (FRET) can be described in detail for single QD–dye assemblies, which show strongly correlated luminescence intensities of the QD and the dye. Though in liquid solution, these assemblies are stable for at least several minutes but do not diffuse in space due to the observed immobility of the QDs which adsorb to the substrate. The FRET efficiency is larger than 0.5 and approaches the theoretically expected values. In the nonpolar solvent TEHOS, NON-FRET PL quenching processes are effectively suppressed [25], contrary to earlier observations in more polar solvents or on surfaces. Due to their intrinsic flexibility, DTPP dye molecules undergo a large variety of conformations on the experimental time scale of seconds to minutes. For this reason, FRET becomes dependent on observation time, which can be followed quite in detail. This clearly demonstrates that single molecule experiments are an unsurpassed tool to investigate subtle conformational changes and the related impact on interchromophoric dynamics. It is very obvious that more statistical relevant experiments are needed to understand these complex processes quantitatively. Envisaged experiments are related to optically polarised excitation and detection [26] to obtain direct information on the (time dependent) molecular orientation. Complementing time resolved experiments on the ns time scale will provide direct information on FRET dynamics.
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Acknowledgements Financial support by the DFG (FOR 877 ‘From local constraints to macroscopic transport’) is gratefully acknowledged. We thank Prof. F. Würthner, Würzburg University, for the gift of DTPP molecules. References [1] M. Chen, R.D. Schliep, Z.-L. Willows, B.A. Cai, B.A. Neilan, H. Scheer, Science 329 (2010) 1318. [2] A.R. Clapp, I.L. Medintz, H. Mattoussi, Chem. Phys. Chem. 7 (2006) 47. [3] I. Gur, N.A. Former, C.P. Chen, A.G. Kanaras, A.P. Alivisatos, Nano Lett. 7 (2007) 409. [4] H. Lu, O. Schops, U. Woggon, C.M. Niemeyer, JACS 130 (2008) 4815. [5] E. Zenkevich, F. Cichos, A. Shulga, E.P. Petrov, T. Blaudeck, C. von Borczyskowski, J. Phys. Chem. B. 109 (2005) 8679. [6] A.M. Funston, J.J. Jasienank, P. Mulvaney, Adv. Mater. 4 (2008) 4280. [7] T. Ren et al., JACS 130 (2008) 17242. [8] I.I. Zenkevich et al., J. Phys. Chem. C. 115 (2011) 21535. [9] I. Potapova, R. Mruk, C. Hübner, R. Zentel, T. Básche, A. Mews, Angew. Chem. 117 (2005) 2490. [10] A.R. Clapp, I.L. Medintz, J.M. Mauro, B.R. Fischer, M.G. Bawendi, H. Mattoussi, JACS 126 (2004) 301. [11] T. Blaudeck, E.J. Zenkevich, M. Abdel-Motaleb, K. Szwaykowska, D. Kowerko, F. Cichos, C. von Borczyskowski, Chem. Phys. Chem. 13 (2012) 959. [12] T. Blaudeck, E.I. Zenkevich, F. Cichos, C. von Borczyskowski, J. Phys. Chem. C. 112 (2008) 20251. [13] D. Kowerko, J. Schuster, N. Amecke, M. Abdel-Mottaleb, R. Dobwara, F. Würthner, C. von Borczyskowski, Phys. Chem. Chem. Phys. 12 (2010) 4112. [14] D. Kowerko, J. Schuster, C. von Borczyskowski, Mol. Phys. 107 (2009) 1911. [15] A. Issac, S. Jin, T. Lian, JACS 130 (2008) 11280. [16] D. Kowerko, S. Krause, N. Amecke, M. Abdel-Mottaleb, J. Schuster, C. von Borczyskowski, Int. J. Mol. Sci. 10 (2009) 5239. [17] D. Kowerko, Dynamic Prozesses in Functionalised Perylene Bisimide Molecules, Semiconductor Nanocrystals and Assemblies, Ph.D. Thesis, TU Chemnitz, 2010. [18] S. Krause, D. Kowerko, R. Börner, C.G. Hübner, C. von Borczyskowski, Chem. Phys. Chem. 12 (2011) 303. [19] E. Lang, F. Würthner, J. Köhler, Chem. Phys. Chem. 6 (2005) 935. [20] S. Krause, P.F. Aramendía, D. Täuber, C. von Borczyskowski, Phys. Chem. Chem. Phys. 13 (2011) 1754. [21] F. Cichos, C. von Borczyskowski, M. Orrit, Curr. Opin. Colloid Interface Sci. 12 (2007) 272. [22] E.I. Zenkevich, A.P. Stupak, D. Kowerko, C. von Borczyskowski, Chem. Phys. 21 (2012) 406. [23] J. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Springer, New York, 2006. [24] E.I. Zenkevich, T. Blaudeck, A. Milekhin, C. von Borczyskowski, Int. J. Spectrosc. 2012 (2012) 1, http://dx.doi.org/10.1155/2012/97179. [25] C. von Borczyskowski, E.I. Zenkevich, Springer Tracts in Nanoscience, in print. [26] R. Börner, D. Kowerko, S. Krause, C. von Borczyskowski, C.G. Hübner, J. Chem. Phys. 137 (2012) 164202.
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Correlated blinking via time dependent energy transfer in single CdSe quantum dot-dye nanoassemblies Frank Gerlach 1, Daniela Täuber, Christian von Borczyskowski ⇑ Chemnitz University of Technology, Institute of Physics, Optical Spectroscopy and Molecular Physics, 09107 Chemnitz, Germany
a r t i c l e
i n f o
Article history: Received 11 February 2013 In final form 13 April 2013 Available online 19 April 2013
a b s t r a c t Optical confocal spectroscopy on self-assembled single nanoassemblies from CdSe/ZnS quantum dots (QD) and perylene diimide dye molecules demonstrates efficient Förster resonance energy transfer (FRET). Intramolecular dynamics of the flexible dye molecule change the FRET efficiency in course of the detection period of several minutes. This can be followed by correlated observations of luminescence intensities and related spectral shifts of both constituents. Contrary to several experiments on similar assemblies, the FRET efficiencies are by almost one order of magnitude larger in the non-polar liquid solvent TEHOS as compared e.g. to toluene. Experimental and theoretically expected efficiencies are in close agreement with each other. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Energy transfer between molecular moieties after photoexcitation is a fascinating and important phenomenon studied in detail during the last decades for a large variety of systems ranging from antenna pigments of photosynthetic units [1] to semiconductor based nanoassemblies [2]. Among this manifold of materials the combination of organic dye molecules and semiconductor nanocrystals is of special interest since this class is a base for organic/ inorganic photovoltaic devices [3] and biologically relevant nanosensors [4]. Several groups have shown that such hetero-nanoassemblies self-assemble under suitable conditions, that is in the presence of functionalized dyes which can be selectively attached to the surface of e.g. colloidal CdSe semiconductor nanocrystals [5–8]. Such assemblies can be formed either by directed synthesis involving coating with polymers [9,10] or in a dynamic equilibrium [11–13] in liquid solutions. The energy transfer efficiency depends on the energetic and geometric constraints of the constituents of such assemblies [5,10,14]. Additionally, these constraints are subject to changes during the observation time, thus modifying the transfer efficiency. This will be the subject of the present investigation in terms of a case study presently not aiming at statistical relevance. Single molecule detection is an excellent tool to evaluate details of static or dynamic fluctuations of (Förster) resonance energy transfer (FRET), with respect to FRET mechanism, efficiencies and ⇑ Corresponding author. Fax: +49 371 531 3060. 1
E-mail address: [email protected] (C. von Borczyskowski). Present address: Fibotec Fiberoptics GmbH, 98617 Meiningen, Germany.
0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.04.034
competing processes [13,15]. It could be shown, that FRET depends as predicted on the distance between the constituents [10], the relative orientation [16] and the spectral overlap [9,16]. In case of self-assembled assemblies these three variables will depend on observation time as soon as the respective assembly is subject to time dependent fluctuations of the geometry or relative energies. Such long-time dependent phenomena have not yet been studied on the single assembly level, though they are important e.g. with respect to FRET processes in photosynthetic entities. We have already recently realised a suitable model system composed of CdSe/ZnS quantum dots (QD) and functionalized perylene diimide (PDI) dye molecules [13]. The functionalization with pyridyl groups (see Scheme 1) enables attachment of the dye molecules to the QD surface [5,13]. Corresponding assemblies show FRET depending on the specific geometry [16]. However, recent experiments on these systems showed only very small FRET efficiencies since a parallel NON-FRET process has masked the energy transfer [12]. In the present contribution we have chosen the non-polar solvent tetrakis(2-ethylhexoxy)silane (TEHOS) to study subtle effects on FRET such as the structural flexibility of dye molecules, which we investigated recently in quite a detail in toluene, PMMA and on SiO2 [14,17,18]. In contrast to these previous studies, we find that NON-FRET quenching of the photoluminescence (PL) of the CdSe/ZnS energy donor is almost completely suppressed in TEHOS. We will report on a single assembly level for a few selected cases how FRET efficiencies correlate with geometric variables as deduced from the related spectral properties. Since statistics are too low to draw general conclusions this study is intended to demonstrate the capabilities of single molecule detection so far not yet reached.
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Scheme 1. DTPP (di-terpyridyl-perylenediimide). For steric reasons, the molecule can only be attached via 1 or 2 pyridyls of the outermost phenyl rings [16]. The phenoxyl bay groups are very flexible [14,18] and induce a time dependent extention of the perylene chromophoric backbone. The optical transition dipole moment is along the long perylene axis.
2. Experimental Silicon substrates with 100 nm thick thermal oxide (CrysTec, Berlin) were cleaned thoroughly by alternate rinsing with acetone and ethanol (both technical grade), followed by sonication at 50 °C in acetone and in ethanol (both Merck spectroscopic grade) for 15 min each, and finally sonicated at 80 °C for 1 h in piranha solution (1:1 H2O2:H2SO4). In between these steps and at the end, the substrates were rinsed with de-ionised water (Milli-Q) and dried in a flow of nitrogen. Samples were prepared by dipcoating (KSV instruments) the substrates into solutions of liquid 0.3% tetrakis(2-ethylhexoxy)silane (TEHOS) in hexane with highly diluted (TOPO capped) CdSe/ZnS (Evident Technologies) and di-terpyridyl-perylenediimide (DTPP) resulting in 10 nm thick films of TEHOS with nanomolar concentrated CdSe/ZnS and DTPP. DTPP (Scheme 1) has been synthesized by Lang et al. [19]. After sufficient time to allow for evaporation of hexane, optical spectra were investigated with a home-built laser scanning confocal microscope with a numerical aperture of 0.9. The setup has been described in detail elsewhere [20]. Excitation occurs via the 488 nm line of an Ar-ion laser. Samples were laser scanned to detect luminescent spots, the size of which is determined by the focus of the laser. At selected spots time series of luminescence spectra were taken with an integration time of 1 s. We obtain the luminescence intensities via integration of the spectra across the relevant spectral range of the respective constituent. A typical sample contains about 10 localised QDs while the mayority of DTPP undergoes fast diffusion processes. Somewhat less than 10% of the luminescent spots show spectra of both constituents. This is in rough agreement with previous studies [13]. Two typical examples (A and B) from five different samples have been chosen for a detailed spectral analysis. Due to intense optical excitation in the confocal microscope, QDs and molecules easily bleach under ambient conditions and only a few could be studied over a long enough time interval to record spectra. We have not yet implemented investigations of liquid film samples under non-ambient conditions. For this reason we have only investigated a few assemblies. 3. Results and discussion All experiments have been performed in thin films of the nonpolar liquid TEHOS dip-coated onto properly cleaned SiO2 surfaces.
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The dynamic self-assembly of CdSe/ZnS and DTPP in TEHOS results both in assemblies and single constituents, similar to the formation process observed in toluene [11,13]. Thus, luminescent spots observed by confocal microscopy show three types of luminescence spectra related either to CdSe/ZnS, DTPP or both spectra of the two constituents. Figure 1 shows typical luminescence spectra of CdSe/ZnS (left) and DTPP (right) as a function of observation time of several minutes. Spectral positions and intensities vary in time. A time integrated spectrum is shown at the top of each time trace. The fluctuating luminescence intensities are given at the side. CdSe/ZnS shows strong intensity fluctuations known as blinking or luminescence intermittency [21], while blinking is almost negligible for DTPP [14], though moderate intensity fluctuations in combination with spectral fluctuations (spectral diffusion) are quite obvious [19]. The luminescence of CdS/ZnS and DTPP are bleached after 180 and 77 s, respectively. Bleaching of the QD is in most cases preceded by a strong blue shift of the photoluminescence (PL) which is tentatively assigned to photooxidation of the CdSe core [22]. Only a few of the localised luminescent QD spots show spectra of both constituents. Two typical examples (A and B) are shown in Figure 2. In general, the time dependent spectral features are quite similar to those shown already in Figure 1, besides the enhanced blinking of DTPP. At (almost) every blinking event of the QD, the DTPP fluorescence (Fl) intensity is switched ‘off’ when the QD is switched ‘off’ and it is ‘on’ when the QD is ‘on’. A quantitative inspection reveals that the respective luminescence intensities are strongly correlated, as can be directly seen, when plotting the corresponding intensities on top of each other at the bottom of Figure 2. In both cases A and B, the strong blinking of QDs (Figure 1, left) is imposed on DTPP, which now also blinks very frequently. Assembly A (Figure 2, left) shows that the DTPP fluorescence is reduced from a relatively high Fl intensity (about 6 kcts) to a much lower one (about 1 kcts, the background being close to 100 cts) as soon as the QD photoluminescence turns ‘off’ according to a blinking process. When DTPP is photobleached or detached from the QD (at 77 s) the QD photoluminescence increases on average by about a factor of 3. There are only three occasions for which intensity correlation fails, namely at 56, 66, and 72 s, where DTPP fluorescence decreases while the QD photoluminescence increases. This can be assigned to one of the rare blinking events of DTPP. The overall interpretation is that the PL of the QD shows intrinsic blinking and is additionally partly quenched due to FRET from the QD to DTPP. Fluorescence of DTPP, on the other hand, is predominantly caused via FRET. We do not observe a detectable difference in blinking dynamics of isolated QDs and QDs in assemblies. Therefore all the QD blinking features are transferred uniquely to DTPP. Photoexcitation at 488 nm is not selective with respect to the two constituents. Therefore it is at first glance surprising that we observe only a relatively low DTPP fluorescence when the QD is in an ‘off’-state. (In the B-type assembly to be discussed later in detail the Fl intensity in the ‘off’-state of the QD is about 2.5 times stronger than in the A-type assembly.) This can be explained by the orientation of DTPP with respect to the exciting laser beam. The transition dipole moment of DTPP is nearly parallel to the long axis of the chromophoric perylene backbone [14] (see Scheme 1). Reduced direct excitation is achieved when this long axis is orientated parallel to the incident laser beam, which corresponds to case b) in Scheme 2. We reported recently [14] for similar perylene diimides on SiO2 surfaces that the Fl intensity varies in time by up to a factor of 5 without any change in the Fl spectra. We suggested that this is due to fluctuations in the molecular orientation. Later on we could directly show by determination of the three-dimensional dipole orientation [26] that diimide molecules re-orientate by up to 40° with respect to the surface within timescales of seconds. FRET from the (nearly isotropically absorbing) QD to DTPP abandons this
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Figure 1. Typical time dependent spectra of CdSe/ZnS (left) and DTPP (right) with 1 s integration time. The corresponding integrated spectra over the total observation time are shown on top of each spectral trace. The integrated luminescence intensity is depicted on the right side of each time trace.
Figure 2. Top: Spectral time traces of 2 exemplary QD–dye assemblies A (left) and B (right). Each time trace shows the spectral signature of the QD ( 560 nm) and DTTP ( 615 nm). In case A, time ranges of fluctuating (f) and stable (s) intensities (see Figure 3) are indicated. Bottom: Intensity time trace of the 2 constituents of the assembly A (left) and B (right).
Scheme 2. Two different orientations of the DTPP dye on the QD. For steric reasons DTPP can be attached via the terpyridyl groups at only one end of the dye. DTPP is plotted along the optically active transition dipole moment. Laser excitation is perpendicular to the substrate.
selection rule only with respect to absorption while emission of DTPP remains still reduced in case of an unfavourable dipole orientation but is nevertheless visible. The situation is obviously different for assembly B as will be discussed later on. Alternatively to FRET, the intensity correlation might be caused by photoinduced electron transfer from the QD to DTPP [5,15] or vice versa. In that case both constituents become charged and at least the luminescence of DTPP will be completely quenched, while a charged QD will have a similar PL spectrum (but with reduced PL intensity) as the uncharged one but shifted by about 20 meV [21]. The only small spectral shift rules out that FRET from DTPP occurs to a charged QD. The key to rule out charge transfer processes is the manifestation of the intrinsic nature of QD blinking. If charge
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Figure 3. Top: Correlation of normalised DTPP and QD luminescence intensities of assemblies A and B for selected observation time ranges related to the respective time traces in Figure 2. Intensities have been normalised to the respective maximum of each time trace for each constituent. Bottom: Luminescence intensity ratios as a function of observation time. Data for assembly A are shown on the left and assembly B on the right. s and f indicate time ranges of stable and fluctuating intensities, respectively. For correlation with wavelength see Figure 2 top left.
transfer processes caused by DTPP were involved, the QD blinking would be considerably influenced. This is not observed neither in this nor in other but similar experiments [13,22,25]. If charge transfer would apply, the Fl of the dye will increase when the QD is intrinsically ‘off’ and can thus not act as a charge donor. Contrary to this, the Fl of the dye is strongly decreased when the QD does not emit. An additional support that FRET is the reason of the intensity correlation stems from the observation that the QD PL intensity increases when DTPP is bleached. This proofs that QD PL reduction of the ‘on’ state prevails only in the presence of the DTPP acceptor. The B-type assembly (Figure 2, right) shows also a strong correlation of luminescence intensities. However, in this case direct excitation of DTPP is, as compared to the A-type assembly, more clearly visible when the QD is ‘off’. This implies a more favourable orientation of DTPP with respect to laser excitation as is e.g. shown in Scheme 2a. In case B, both constituents are photodegraded after 27 s within the same range of time. In case that the degrading is due to a permanent charge transfer event, this would indeed result in a complete quenching of Fl and considerable reduction of the PL of the QD. Permanent charge separation, however, is very unlikely to be realised in the non-polar solvent TEHOS. To obtain a more quantitative description of FRET we have plotted the luminescence intensity ratio R = IDTPP/IQD in Figure 3 (bottom) for A and B as a function of observation time. In both cases the correlation clearly depends on time. While R changes by more than a factor of 3 in case of A (till DTPP is bleached), the time dependence is less pronounced for B. More details become visible
when plotting (normalised) intensity ratios as a function of selected observation time ranges (Figure 3, top). In case of A, we have selected two characteristic time intervals since the inspection of spectral properties (Figure 2, top left) indicates a discrete jump in wavelength of DTPP at about 27 s. A correlation of (normalised) intensities is quite linear during the first 30 s as described by the increment in Figure 3 (top left). During that time (Figure 2, left) the PL of the QD continuously red shifts, while the Fl of DTPP experiences a blue shift. This will change the spectral overlap at least due to the red shift of the QD (since we do not know, whether the absorption of DTPP shifts in the same manner as the Fl does). As we and others have shown, a spectral shift of DTPP is accompanied by a change of the molecular conformation [14,16]. A closer inspection of the Fl intensity of DTPP in Figure 2 (bottom left) reveals that there is a small remaining intensity when the QD is ‘off’, as can be also seen in Figure 3 (top left) from the remaining IDTPP abscissa value for IQD = 0. The remaining Fl intensity (when the QD is ‘off’) becomes lower in the second time interval (IDTPP = 0 for IQ = 0). This change coincides with the spectral jump of DTPP at 27 s (Figure 2 (top left)), which indicates a change in DTPP geometry [18]. In the second time period (30.5– 80 s) the correlation is less obvious, since the absolute DTPP Fl intensity decreases, which indicates a somewhat reduced FRET efficiency. Pronounced fluctuations of the intensity correlation shown in Figure 3 bottom are observed for times longer than 55 s. In this range, we find several short periods during which, as discussed before, an opposite correlation is observed, probably due to DTPP
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blinking. There seems to be still a correlation at times longer than 80 s (see Figure 3, top left) which, however, is probably due to remaining QD PL in the long wavelength detection channel of DTPP. In this time interval the Fl of DTPP is nearly bleached or alternatively, DTPP is no longer attached to the QD. For assembly B, we observe a strong correlation over the total observation time. Apart from the first 3–5 s, there are almost no spectral shifts both for DTPP and QD. As can be seen in Figure 3 (top right), there seems to be a slightly smaller FRET efficiency in the interval (15.5–30 s) compared to the interval (0.5–15 s), followed by a pronounced increase in the interval (30.5–40 s). During this last short time interval FRET is very effective, but direct excitation of DTPP becomes negligible, since IDTPP = 0 for IQD = 0. From this we conclude that DTPP (or the assembly itself) has changed to an orientation unfavourable for direct excitation. The accompanied spectral jump observed at 26 s indicates a change in the intrinsic molecular geometry. This excludes a reorientation of the assembly since this will not result in a spectral jump. The two earlier time intervals in Figure 3 (top right) show considerable Fl of DTPP, even when the QD is ‘off’, however, the absolute value decreases with observation time. The decrease of Fl of DTPP is, in particular, very pronounced after t = 26 s, where luminescence of both components becomes very weak. At t > 40 s both constituents are ‘off’. In the following we will discuss the observed FRET processes more quantitatively in the framework of Förster resonance energy transfer [23]. Though it is not immediately obvious that FRET in a QD–dye assembly can be described by a dipole–dipole approximation, several experiments have shown that this simplification holds within experimental accuracy [2,5,10]. The energy transfer rate kFRET (r) as a function of the distance r between the centres of QD and DTPP, corresponds to
kFRET ðrÞ ¼
6 Ro ; sD r 1
ð1Þ
with the Förster Radius Ro (including the spectral overlap integral J and the relative orientation of the transition dipole moments) and the intrinsic donor lifetime sD in the absence of PL quenching processes. Since it is known that PL blinking of QDs is related to a fluctuation of sD which will result in an effective sQD[13,17], kFRET will depend on the effective sQD. The PL intensity IQD is to a good approxr r imation proportional tokQD sQD (with the radiative rate kQD ) [23]. The FRET efficiency EFRET corresponds to
EFRET ¼
kFRET kQ þ kFRET þ s1 D
ð2Þ
where kQ describes the intrinsic PL quenching including the NONFRET quenching as induced by merely attaching the acceptor [11– r 1 1 13]. With ðs1 D þ kQ Þ ¼ sQD and sQD ¼ kQD =I QD , the efficiency EFRET will depend approximately linearly on IQD for kFRET ðs1 D þ kQ Þ. For both assemblies A and B we clearly observe a linear dependence of the DTPP fluorescence intensity on IQD. The FRET efficiency EFRET for a DTPP–QD assembly can in principle be also calculated from the fluorescence intensities IDTPP of the acceptor DTPP according to
EFRET ¼
eDTPP ðIDTPPQD IDTPP Þ ; IDTPP eQD
ð3Þ
where IDTPPQD is the DTPP Fl intensity in the assembly. This equation relies via e on the isotropic orientation of the participating entities. However, as we have shown, an isotropic orientation does not apply in the present situation of only one single assembly. Therefore we cannot use this approach to determine absolute values of EFRET. An alternative access to the FRET efficiency is related to quenching of the QD donor PL intensity IQD according to [23]
EFRET ¼ 1
IQDDTTP : IQD
ð4Þ
An estimation of EFRET can be obtained using Eq. (4) in case of the type A assembly, since DTPP is photobleached at times longer than 77 s. Taking the maximum PL intensity IQD in that time interval and the maximum of the intensity IQD–DTTP during the time DTPP is not photobleached (Figure 2, bottom left) and using Eq. (4) we obtain EFRET 0.69. This can be compared to the two events at 57 and 72 s, when DTPP is ‘off’ due to blinking, providing IQD as compared to an average IQD–DTPP when DTPP is ‘on’ again. According to Eq. (4) this results in EFRET 0.55. Both values are in reasonable agreement with EFRET 0.85 recently calculated according to the Förster model [16]. The model takes only the distance RDA between the centres of the two point dipoles and R0 into account. RDA depends on the relative orientation of the DTPP dipole and has a minimum (which results in a maximum of EFRET) for a parallel orientation of the DTPP dipole to the QD surface. Due to steric reasons (caused by the pyridyl rings forming the ’bonds’ to the QD) this is the most probable orientation. Some variations of EFRET are expected since IQD depends on the (intrinsic) quenching process. For type B assembly we did not find a situation for which DTPP is ‘off’ (while the QD is ‘on’) and we therefore cannot apply Eq. (4). From a comparison of the increments of the intensity ratios in Figure 3 (top) we conclude that EFRET is somewhat lower for the B as compared to the A assembly. These FRET efficiencies larger than 0.5 are in contradiction to what we have published recently both for DTPP (ensemble and single assemblies) [13–16] and porphyrins (ensemble) [5,11,12,22,24] for which we found FRET efficiencies of less than 0.1. We have related these previous findings of low FRET efficiencies to the presence of competing NON-FRET processes, which open a quenching pathway other than energy transfer by merely attaching a dye molecule [11–13]. All our previous experiments have been performed in toluene or on SiO2 surfaces. As we have discussed recently in quite detail, [11] NON-FRET effects are related to ligand attachment/detachment processes. TEHOS as a non-polar solvent will hardly solvate the polar (TOPO) ligands. This will result in a well shielded ligand shell of the QD suppressing NON-FRET quenching processes. Moreover, polar solvents will stabilize charges at the QD surface, which also reduces the PL lifetime and thus the PL intensity. Finally we will discuss the variations of FRET during the observation time as it is evident from Figure 3. Since there is no inherent systematic spectral change during observation time for DTPP or QD, at least not for the assembly, we can basically assign the decrease of the intensity ratio to a decrease of FRET. There are at least 4 reasons why FRET varies during observation time, namely changes of the: (i) spectral overlap, (ii) donor–acceptor distance, (iii) the relative orientation of transition dipole moments, or (iv) the effective donor lifetime. Figure 2 (top left) clearly shows that a change in spectral overlap (case (i)) is present at least for assembly A during the time range of (0–25 s). Making use of Förster theory we can calculate the increase in spectral overlap to be about 4%, which is not enough to explain the change in the increment in Figure 3 when comparing FRET at earlier and later times. Recently we have investigated single perylene diimide type molecules on SiO2 interfaces and in polymers with respect to spectral diffusion in relation to fluctuations of the bay group orientation [18,20], which results in large spectral shifts of up to Dk = 30 nm. In the present case we use the non-polar solvent TEHOS as a matrix. Though the statistics are lower as compared to the previous investigations, the principle fluorescence band is between 600 and 625 nm accompanied by a spectral diffusion in the range of 5–15 nm. Comparing the spectral diffusion of
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non-aggregated DTTP (Figure 1 (right)) with that in aggregates A or B, it can be seen that spectral jumps occur more frequently in the aggregates. According to what we have found previously [20] for DTPP in the polymer PMMA and on SiO2, this might be caused by a less fixed structure of DTPP within an aggregate (see also Ref. [16]). This will in principle also influence EFRET. For assembly A we find in Figure 3A (bottom) that the intensity ratio IDTPP/IQD can be tentatively divided into 2 categories ‘s’ and ‘f’. ‘s’ corresponds to stable and ‘f’ to fluctuating intensity ratios on a 1 s averaging time scale. These intervals are also indicated in Figure 2A. We find on average slightly more spectral jumps in the ‘f’ as compared to the ‘s’ interval, though this at the limit of experimental accuracy. The spectral jumps mask a precise assignment of the FRET efficiency since each DTPP configuration is related to a different Fl intensity [18] and thus modifies the luminescence intensity ratio (Figure 3) possibly without changing the FRET efficiency. 4. Conclusions The present experiments are demonstrative examples how Förster resonance energy transfer (FRET) can be described in detail for single QD–dye assemblies, which show strongly correlated luminescence intensities of the QD and the dye. Though in liquid solution, these assemblies are stable for at least several minutes but do not diffuse in space due to the observed immobility of the QDs which adsorb to the substrate. The FRET efficiency is larger than 0.5 and approaches the theoretically expected values. In the nonpolar solvent TEHOS, NON-FRET PL quenching processes are effectively suppressed [25], contrary to earlier observations in more polar solvents or on surfaces. Due to their intrinsic flexibility, DTPP dye molecules undergo a large variety of conformations on the experimental time scale of seconds to minutes. For this reason, FRET becomes dependent on observation time, which can be followed quite in detail. This clearly demonstrates that single molecule experiments are an unsurpassed tool to investigate subtle conformational changes and the related impact on interchromophoric dynamics. It is very obvious that more statistical relevant experiments are needed to understand these complex processes quantitatively. Envisaged experiments are related to optically polarised excitation and detection [26] to obtain direct information on the (time dependent) molecular orientation. Complementing time resolved experiments on the ns time scale will provide direct information on FRET dynamics.
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Acknowledgements Financial support by the DFG (FOR 877 ‘From local constraints to macroscopic transport’) is gratefully acknowledged. We thank Prof. F. Würthner, Würzburg University, for the gift of DTPP molecules. References [1] M. Chen, R.D. Schliep, Z.-L. Willows, B.A. Cai, B.A. Neilan, H. Scheer, Science 329 (2010) 1318. [2] A.R. Clapp, I.L. Medintz, H. Mattoussi, Chem. Phys. Chem. 7 (2006) 47. [3] I. Gur, N.A. Former, C.P. Chen, A.G. Kanaras, A.P. Alivisatos, Nano Lett. 7 (2007) 409. [4] H. Lu, O. Schops, U. Woggon, C.M. Niemeyer, JACS 130 (2008) 4815. [5] E. Zenkevich, F. Cichos, A. Shulga, E.P. Petrov, T. Blaudeck, C. von Borczyskowski, J. Phys. Chem. B. 109 (2005) 8679. [6] A.M. Funston, J.J. Jasienank, P. Mulvaney, Adv. Mater. 4 (2008) 4280. [7] T. Ren et al., JACS 130 (2008) 17242. [8] I.I. Zenkevich et al., J. Phys. Chem. C. 115 (2011) 21535. [9] I. Potapova, R. Mruk, C. Hübner, R. Zentel, T. Básche, A. Mews, Angew. Chem. 117 (2005) 2490. [10] A.R. Clapp, I.L. Medintz, J.M. Mauro, B.R. Fischer, M.G. Bawendi, H. Mattoussi, JACS 126 (2004) 301. [11] T. Blaudeck, E.J. Zenkevich, M. Abdel-Motaleb, K. Szwaykowska, D. Kowerko, F. Cichos, C. von Borczyskowski, Chem. Phys. Chem. 13 (2012) 959. [12] T. Blaudeck, E.I. Zenkevich, F. Cichos, C. von Borczyskowski, J. Phys. Chem. C. 112 (2008) 20251. [13] D. Kowerko, J. Schuster, N. Amecke, M. Abdel-Mottaleb, R. Dobwara, F. Würthner, C. von Borczyskowski, Phys. Chem. Chem. Phys. 12 (2010) 4112. [14] D. Kowerko, J. Schuster, C. von Borczyskowski, Mol. Phys. 107 (2009) 1911. [15] A. Issac, S. Jin, T. Lian, JACS 130 (2008) 11280. [16] D. Kowerko, S. Krause, N. Amecke, M. Abdel-Mottaleb, J. Schuster, C. von Borczyskowski, Int. J. Mol. Sci. 10 (2009) 5239. [17] D. Kowerko, Dynamic Prozesses in Functionalised Perylene Bisimide Molecules, Semiconductor Nanocrystals and Assemblies, Ph.D. Thesis, TU Chemnitz, 2010. [18] S. Krause, D. Kowerko, R. Börner, C.G. Hübner, C. von Borczyskowski, Chem. Phys. Chem. 12 (2011) 303. [19] E. Lang, F. Würthner, J. Köhler, Chem. Phys. Chem. 6 (2005) 935. [20] S. Krause, P.F. Aramendía, D. Täuber, C. von Borczyskowski, Phys. Chem. Chem. Phys. 13 (2011) 1754. [21] F. Cichos, C. von Borczyskowski, M. Orrit, Curr. Opin. Colloid Interface Sci. 12 (2007) 272. [22] E.I. Zenkevich, A.P. Stupak, D. Kowerko, C. von Borczyskowski, Chem. Phys. 21 (2012) 406. [23] J. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Springer, New York, 2006. [24] E.I. Zenkevich, T. Blaudeck, A. Milekhin, C. von Borczyskowski, Int. J. Spectrosc. 2012 (2012) 1, http://dx.doi.org/10.1155/2012/97179. [25] C. von Borczyskowski, E.I. Zenkevich, Springer Tracts in Nanoscience, in print. [26] R. Börner, D. Kowerko, S. Krause, C. von Borczyskowski, C.G. Hübner, J. Chem. Phys. 137 (2012) 164202.