Single-Molecule Fluorescence of a Phthalocyanine in PAMAM

J. Phys. Chem. C 2010, 114, 19035–19043

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Single-Molecule Fluorescence of a Phthalocyanine in PAMAM Dendrimers Reveals Intensity-Lifetime Fluctuations from Quenching Dynamics Pedro M. R. Paulo* and Sı´lvia M. B. Costa Centro de Quı´mica Estrutural, Instituto Superior Tećnico, Complexo Interdisciplinar, AV. RoVisco Pais 1, 1049-001 Lisboa, Portugal ReceiVed: August 6, 2010; ReVised Manuscript ReceiVed: September 23, 2010

We report on single-molecule fluorescence of an aluminum phthalocyanine dye associated with PAMAM dendrimers at a glass surface. The fluorescence decays of the phthalocyanine-dendrimer complex deposited from a solution at pH 3 are monoexponential and with lifetimes closely distributed around its natural fluorescence lifetime. However, in the absence of buffer (pH 6), the distribution of fluorescence lifetimes is broadened toward shorter decay times. Fluorescence quenching is attributed to electron transfer between excitedstate phthalocyanine and the dendrimer’s tertiary amines, which are deprotonated at higher pH and therefore can act as efficient electron donors. Single-molecule fluorescence measurements give direct access to static heterogeneity as revealed by the broad lifetime distributions for the quenched systems. However, it also gives evidence of dynamic heterogeneity from the fluorescence time traces. In some traces, it was possible to identify clear intensity changes that are accompanied by lifetime changes and to define discrete intensity-lifetime levels. This behavior was attributed to phthalocyanine-dendrimer conformational dynamics that occur on the millisecond-to-second time scale affecting the efficiency of fluorescence quenching by electron transfer. Faster dynamics are not distinguished as discrete levels but give rise to single-molecule multiexponential fluorescence decays. This rich behavior is not accessible in ensemble measurements, whereby only an average lifetime distribution can be obtained. 1. Introduction Phthalocyanines are planar macrocyclic chromophores constituted by four isoindole units which form a 18 π-electron aromatic system.1,2 They are synthetic analogues to porphyrins, but usually phthalocyanines have more intense S0-S1 transitions (Q-bands) and better fluorescence quantum yields, which makes them promising molecules for fluorescence microscopy.3 Nevertheless, most of the single-molecule studies on phthalocyanines involve scanning tunneling microscopy (STM).4-7 The conducting properties of phthalocyanines have been investigated by STM to evaluate their potential as single-molecule junctions for molecular electronics.8,9 Phthalocyanines are also interesting molecules for photodynamic therapy and solar-energy conversion. For the latter application, phthalocyanines have been incorporated in blends with semiconductor polymers or acceptor molecules, such as perylene diimide, anthraquinone, or fullerene derivatives, to build organic solar cells.10-14 They have also been deposited at nanostructured titanium dioxide to make dye sensitized solar cells.15-17 The overall efficiencies of dye solar cells are still low when compared to conventional devices, but, in some examples, significant incident photon-to-current conversion efficiencies were achieved with phthalocyanines. Although many factors influence the efficiency of dye solar cells, the primary step is always a photoinduced electron-transfer process involving the dye. In this sense, it is important to understand the photophysics and photochemistry of phthalocyanines in nanostructured systems. Single-molecule spectroscopy is a privileged technique for this purpose, because it gives direct information about the individual behavior of fluorescent molecules in heterogeneous media.18-21 However, to the best of our * Corresponding author. E-mail: [email protected].

knowledge, there are no previous accounts in the literature on single-molecule fluorescence spectroscopy of phthalocyanines. Here, we report on single-molecule fluorescence of a phthalocyanine dye in PAMAM dendrimers immobilized directly at a glass surface or in a polymer film. Dendrimers are artificial polymers with a highly branched and regular structure.22,23 They are built from a core unit by successively adding concentric layers of monomers. Each layer is usually called a generation. The dendrimer mass increases exponentially from generation to generation because of branching out of the dendrimer structure. Eventually, a limit generation is reached because of molecular crowding of dendrimer branches within its finite volume. Dendrimers with flexible branches, such as the poly(amido)amine (PAMAM) dendrimers used here, have a globular shape and a size of a few nanometers.24 PAMAM dendrimers have been extensively used as building blocks in nanostructured films for many different functionalities. For instance, they have been employed to build layer-by-layer films because of their polyelectrolyte behavior.25-30 The ability of PAMAM dendrimers to encapsulate small photo- or electroactive molecules allows to disperse such molecules in a controlled way within the film and to avoid complications from self-aggregation. An alternative approach consists of functionalizing photo- or electroactive units at the multiple terminal groups of the dendrimers in order to benefit from possible cooperative effects that improve their optical or redox properties. PAMAM dendrimers have also been used as molecular spacers for metal nanoparticles in films or as nanoreactors for the synthesis of dendrimer encapsulated metal nanoparticles.31-34 Furthermore, PAMAM dendrimers have been conjugated with other typical building blocks in nanostructured systems, such as carbon nanotubes, DNA, or proteins.35-37 Some of the

10.1021/jp107412p  2010 American Chemical Society Published on Web 10/15/2010

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approaches described before were employed to develop chemical or biochemical sensing films with improved detection efficiency.38-42 We are interested in dendrimers as molecular scaffolds for the organization of chromophore units in nanostructured photoactive systems. We have previously studied the noncovalent interaction of ionic porphyrins with charged PAMAM dendrimers. One of the systems studied showed evidence of porphyrin self-aggregation at the dendrimer’s surface promoting excitonic interactions,42 whereas the other showed strong fluorescence quenching due to electron transfer between the dendrimer and excited-state porphyrin.44 Photoinduced electron transfer in phthalocyanine complexes with protein cytochrome c was also investigated by some of us by using ensemble fluorescence and transient absorption techniques.45,46 The work presented here addresses analogue systems comprising an anionic phthalocyanine and positively charged PAMAM dendrimers. Fluorescence quenching due to photoinduced electron transfer is also observed in phthalocyanine-dendrimer complexes, and a statistical distribution of quenching rates is inferred from the multiexponential fluorescence decay kinetics. Single-molecule fluorescence allows to gain further insight on the distribution of quenching rates in these systems, although with some caution because of changes in the molecular environment from immobilization of the phthalocyanine-dendrimer complex at a glass surface or in a polymer film. In particular, single-molecule fluorescence measurements revealed both static and dynamic heterogeneities in these systems that otherwise are averaged out in ensemble measurements. 2. Experimental Section Materials. Tetrasulfonated aluminum phthalocyanine was purchased from Porphyrin Products and used as received (99% purity). PAMAM dendrimers of generations 4-7 were supplied by Aldrich as methanolic solutions with concentrations of 5-10 wt %. The methanol was evaporated under vacuum for 48 h, and the viscous dendrimer oil was reconstituted with water to prepare stock solutions. Millipore water (18 MΩ cm) was used in the preparation of all solutions. In the absence of buffer, the Millipore water has a pH of 5.6 units. Poly (vinyl alcohol) with MW 89,000-98,000 was purchased from Aldrich and used as received. The pH 3 buffer was prepared from citric acid (Aldrich, 99+%) and disodium hydrogen phosphate (BDH, 97+%). Glass coverslips for microscopy from Menzel-Glase¨r were thoroughly cleaned with detergent and solvents, then irradiated in a UV/Ozone chamber for more than 1 h, and subjected to plasma cleaning to obtain hydrophilic surfaces. Aqueous solutions used in ensemble fluorescence measurements were prepared with a phthalocyanine concentration of 1 µM and dendrimer concentration of 10 µM. For FCS measurements, both concentrations were decreased down to 1 nM of phthalocyanine and 1 µM of dendrimer. Finally, the solutions used for single-molecule deposition were prepared with a concentration 10 fold lower than those used for FCS. Samples with PVA were prepared from solutions with a polymer concentration around 1 wt %. Single-molecule samples were prepared by spin coating diluted phthalocyanine-dendrimer solutions on glass substrates. Methods. Single-molecule and ensemble time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements were performed in a MicroTime 200 setup from PicoQuant GmbH. This setup has been described in detail before.47 In this work, we used a pulsed diode laser at 639 nm as excitation source (LDH-635-b, PicoQuant) which was operated at a repetition rate of 20 MHz. The laser beam is coupled

Paulo and Costa to an Olympus IX71 inverted microscope through a dichroic beamsplitter (650DRLP, Omega). The detected light is further selected by using a filter with a transmission window between 668 and 723 nm (695AF55, Omega). Confocal detection is achieved with a tube lens and a 50 µm pinhole. Illumination and collection of emitted light are done through a water immersion objective 60× magnification with N.A. of 1.2 (UPLSAPO 60XW, Olympus). Single-photon avalanche diodes (SPCM-AQR-13, Perkin-Elmer) are used for detection. The instrument response function has a fwhm around 500 ps, and the time increment of fluorescence decays is 37 ps/channel. Single-molecule fluorescence decays were acquired with excitation powers in the range of 0.14-1.4 kW/cm2, which corresponds to excitation rates of 105-106 s-1 for the phthalocyanine. Mono- or biexponential decay functions were fitted to the fluorescence decays by using iterative reconvolution with maximum-likelihood estimators. This method proved to be more accurate than least-squares fitting for assessing lifetimes from fluorescence decays with low number of counts.48 The quality of the fits was evaluated with the usual criteria for the χ2 parameter and weighted residuals. The average lifetime of biexponential decays was calculated as the intensity averaged lifetime 〈τ〉 ) ∑iaiτi2/∑iaiτi. Hermite integration was used to evaluate the integral term in the distributed kinetics model (see Results and Discussion Section). 3. Results and Discussion Ensemble Measurements in Solution. The phthalocyanine (Pc) studied here exists in the monomeric form in aqueous solution for the range of concentrations used in this work (below 1 µM). It is negatively charged because of its four sulfonic substituent groups. On the other hand, the PAMAM dendrimers are positively charged because of protonation of the terminal primary amines in water at neutral pH (or below). These two species associate in aqueous solution to form a phthalocyanine-dendrimer (Pc-G4) complex. The absorption and emission spectra of Pc practically do not change their shape upon association with the G4 dendrimer (Figure S1 in the Supporting Information).49 However, the emission intensity drops to about 58% in the Pc-G4 complex (in the absence of buffer, i.e., at pH 6). The fluorescence decay also changes from monoexponential with a lifetime of 4.93 ( 0.02 ns to biexponential with an average lifetime of 2.89 ( 0.02 ns in the Pc-G4 complex (respectively, curves a and b in Figure 1). The quenching effect of the dendrimer on the emission of phthalocyanine is attributed to electron transfer from the tertiary amines of the dendrimer to excited-state phthalocyanine. Tertiary amines are known to be efficient electron donors. By using the redox potential of triethylamine as a model compound for the dendrimer’s tertiary amines, we estimate from Rehm-Weller expression a favorable value of ∆G0 ) -0.22 eV for electron transfer to excited-state aluminum phthalocyanine in aqueous medium.50 To confirm this hypothesis, the fluorescence spectrum and decay of Pc-G4 were measured at pH 3 in aqueous solution. At low pH, the tertiary amines in the dendrimer are protonated, and therefore, they should not act as electron donors. Indeed, we recover an emission intensity and a monoexponential decay for the Pc-G4 complex at pH3 (curve c in Figure 1), which are comparable to those of free Pc. The association between the phthalocyanine and the dendrimer at pH 3 was confirmed by FCS, as it is described further ahead. The fluorescence lifetime of Pc-G4 is slightly longer (5.51 ( 0.02 ns vs 4.93 ( 0.02 ns for free Pc), but this could be related to differences of the local refraction index in the dendrimer

Single-Molecule Fluorescence Phthalocyanine in PAMAM

J. Phys. Chem. C, Vol. 114, No. 44, 2010 19037 SCHEME 1

Figure 1. Ensemble fluorescence decays of tetrasulfonated aluminum phthalocyanine (Pc) in water (curve a) and of its complex with generation 4 PAMAM dendrimer (Pc-G4) in aqueous solution without buffer (curve b) and with pH 3 buffer (curve c). The excitation wavelength was 639 nm, and emission was collected in the interval 668-723 nm. The inset shows the fluorescence lifetime distribution fitted to curve b with eq 1 and, for comparison, the fluorescence lifetimes obtained from monoexponential fits to curves a and c (see text for further details).

nanoenvironment as compared to aqueous medium. The absorption and emission spectra of Pc-G4 show only a slight red shift at pH 3 (Figure S1 in the Supporting Information), which suggests that the electronic structure of the complex is not very different from that of free Pc. The fluorescence decay of Pc-G4 in aqueous solution without buffer (curve b, Figure 1) deviates clearly from the monoexponential decay of Pc in water (or that of Pc-G4 at pH 3). This behavior can be explained by a statistical distribution of phthalocyanine-dendrimer conformations in solution with different electron-transfer efficiencies. There are 62 tertiary amines in a PAMAM dendrimer G4 (generation 4). Each is a potential electron donor for excited phthalocyanine; however, because of the exponential distance dependence of electron-transfer rates, only the closest amines are efficient in quenching phthalocyanine emission. We performed molecular-dynamics simulations of PAMAM G4 dendrimer and calculated the average distance distribution of its tertiary amines to every primary amine at the dendrimer surface over a simulation trajectory 1 ns long. (The terminal primary amines are positively charged and therefore are potential sites for phthalocyanine association with the dendrimer.) The number of tertiary amines within a distance of 10 Å from the dendrimer surface varies between 2 and 16 depending on the position at the surface (Figure S2 in the Supporting Information). Furthermore, the distance of the closest amine can also fluctuate up to 5 Å, which can strongly affect the electron-transfer rate and thus its quenching efficiency. This situation is depicted in scheme 1; as the phthalocyanine-dendrimer conformation changes in solution (see double arrow), the number and distance of tertiary amines close to the phthalocyanine varies, and the system can evolve from a conformation with high electron-transfer rate (strong quenching) to another with low electron-transfer rate (weak quenching). In ensemble measurements, there is a statistical distribution of phthalocyanine-dendrimer conformations with different electrontransfer efficiencies, and this gives rise to a distribution of fluorescence lifetimes which causes the fluorescence decay to deviate from monoexponential behavior. Although some of these conformational changes may occur in the time scale of excited-

state decay (i.e., within a few nanoseconds), we assumed that the average distribution of conformations is not changing with time after each excitation event, and we fitted the fluorescence decay of Pc-G4 in solution without buffer (curve b, Figure 1) with a distributed kinetics model,51

D(t) ∝ e-t/τ0

∫-∞+∞ p(x) e-k (x)×t dx q

(1)

where τ0 is the natural fluorescence lifetime of phthalocyanine and p(x) is a distribution function that weights the contribution of the quenching rate constant kq(x). It was assumed that p(x) ) exp(- x2) and kq(x) ) kq,0 exp(γx); that is, the quenching rate depends exponentially on a variable x that is normally distributed, and γ is a parameter related to the spread of this distribution. Equation 1 gives a good fit with only two adjustable parameters, kq,0 and γ, by setting τ0 to the unquenched fluorescence lifetime of phthalocyanine (as opposed to a biexponential fit that requires three adjustable parameters). The distribution p(x) obtained is represented in the inset of Figure 1 (see curve b) as a function of the respective decay time τ(x) ) [τ0-1 + kq(x)]-1. The distribution is quite symmetrical when represented in this way, and it can be approximated by a Gaussian distribution of lifetimes with an average value of 2.55 ns and a spread of (1.00 ns. The variable x in the distributed kinetics model could be related to a set of coordinates in the conformational space of the phthalocyanine-dendrimer complex, upon which the donor-acceptor distance can be mapped. PAMAM dendrimers have a flexible branching structure, and the position of the tertiary amines (donors) is not fixed in a rigid frame. Therefore, the distance of the tertiary amines to the dendrimer surface, where the phthalocyanine (acceptor) is associated, varies for different locations at the dendrimer surface and also for different conformations of the dendrimer. This was previously shown by molecular-dynamics simulations of an analogue system composed of a negatively charged PAMAM dendrimer and a cationic porphyrin.52 Significant changes in the distribution of donor-acceptor distances were observed in those simulations due to the dynamics of dendrimer branches inducing conformational changes in the porphyrin-dendrimer complex. Hereafter, we give some estimates of the electron-transfer rate between a phthalocyanine and a single tertiary amine in a model dendrimer nanoenvironment to illustrate the strong dependence on donor-acceptor distance and support the distribution of

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Figure 2. Estimates of the electron-transfer rate (A) and fluorescence lifetime (B) for a system consisting of a phthalocyanine (acceptor) and a tertiary amine (donor) in a dendrimer model environment with different degrees of hydration: curve a corresponds to a solvated dendrimer with 44% volume of water inside the dendrimer; curves b-d correspond to the same dendrimer surrounded by a shell of water with thicknesses of 9, 3, and 0 Å, respectively (and surrounded by air on the outside); curve e corresponds to a dendrimer “dry” environment with a water volume fraction of only 11% inside the dendrimer and surrounded by air on the outside (see text for further details).

lifetimes, as it is experimentally observed. For this purpose, the Marcus theory of electron transfer in the limit of hightemperature was employed:53,54

kET )

[

1 2π 2 (∆G0 + λ) V exp p 4λkBT √4πλkBT

]

(2)

For the electronic coupling term, we considered the usual exponential distance dependence, V2 ) V02 exp[-β(R - R0)], with a coupling value of V02 ) 10 cm-1 at contact distance R0 and a damping constant of β ) 1 Å-1.55 The reorganization energy of the medium λ was calculated with a three-phase model to account for the specific dielectric properties of the dendrimer nanoenvironment, where electron transfer occurs, and the surrounding aqueous medium in solution. This model was developed for micellar media, and it has been adapted for dendrimer systems, yielding results comparable to those from more rigorous estimates from molecular-dynamics simulations.52 The values of λ obtained for the phthalocyanine-dendrimer system vary from 0.85 to 1.1 eV for the distance range between R0 and R0 + 4 Å. The same three-phase model was used to calculate the solvation energies and Coulomb interaction of the ion species involved in electron transfer, which contribute to the value of ∆G0.50 Further details of the model can be found in the Supporting Information (Figure S3). The values of kET obtained are shown in Figure 2A (see curve a). These vary by three orders of magnitude, from about 1010 s-1 to 107 s-1, in the distance range between R0 and R0 + 4 Å. This corresponds to changes in the fluorescence lifetime τ ) [τ0-1 + kET(R)]-1 from hundreds of picoseconds to the unquenched lifetime of phthalocyanine (about 5 ns) over the same donor-acceptor distance range (see curve a in Figure 2B). Therefore, even a small conformational change in the dendrimer can change significantly the quenched lifetime of phthalocyanine in the complex, if the donor-acceptor distance is affected by only a few angstroms. The curves b-e in Figure 2 correspond to different hydration degrees of the dendrimer nanoenvironment,

Figure 3. Diffusion coefficient of tetrasulfonated aluminum phthalocyanine (Pc) in water and of its complexes with PAMAM dendrimers of generations 4-7 (Pc-Gn, n ) 4-7) in aqueous solution without buffer (green circles) and with pH 3 buffer (red triangles). The error bars correspond to a relative uncertainty of 20%. The hydrodynamic radius assumed for the complexes were estimated from the radius of gyration given for these dendrimers in ref 56 by using the approximation to an homogeneous sphere: G4, 23.2 Å; G5, 28.3 Å; G6, 36.5 Å; and G7, 42.0 Å. The solid curve shows the estimated diffusion coefficient from Stokes-Einstein equation for the range of hydrodynamic radius represented here. The inset shows the autocorrelation curves obtained for the phthalocyanine-dendrimer systems in aqueous solution without buffer.

and these will be discussed in the section about single-molecule fluorescence of immobilized phthalocyanine-dendrimer systems at glass surfaces. FCS Measurements in Solution. We performed fluorescence correlation spectroscopy of the phthalocyanine-dendrimer systems in solution to verify the association between these two species at concentrations close to those used for spin coating on glass substrates. Besides PAMAM dendrimers of generation 4, also generations 5-7 were used to establish a series of dendrimer sizes and test the accuracy of our FCS measurements. The FCS curves obtained for free Pc and for the complexes Pc-Gn (n ) 4-7) can be well fitted with the well known expression for 3D Brownian diffusion in the range of microseconds-milliseconds (inset of Figure 3). From these fits, the diffusion coefficient of the emitting objects were retrieved and plotted against the hydrodynamic radius known from the literature (see Figure 3). For the dendrimer sizes, the radius of gyration determined by small-angle X-ray scattering in ref 56 were converted to hydrodynamic radius by using the approximation to an homogeneous sphere RH ) 5/3RG.57 A perfect match was obtained between the diffusion coefficient determined from our FCS measurements and those calculated with Stokes-Einstein equation by assuming diffusing objects with the size corresponding to that of the several dendrimer generations measured here. It should be noticed that the dendrimers are not fluorescent when excited at 639 nm, and thus, the emission comes from the phthalocyanine associated in the Pc-Gn complex, which means that these two species form a tight binding complex in solution, as expected. This holds true for a nanomolar concentration of phthalocyanine, as long as the concentration of dendrimer exceeds largely that of phthalocyanine. If both phthalocyanine and dendrimer concentrations are decreased to the nanomolar range, the association is lost, and the diffusion coefficient obtained from FCS measurements is that of free Pc. For this reason, the dendrimer concentration was always maintained in the micromolar range


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Figure 4. Histograms of fluorescence lifetimes obtained from single-molecule fluorescence decays of tetrasulfonated aluminum phthalocyanine complex with PAMAM G4 dendrimer (Pc-G4) deposited directly at a glass surface from a solution with pH 3 buffer (A) and without buffer (B). Histograms for the same phthalocyanine in a PVA film without dendrimer (C) and with G4 dendrimer (D). The dashed curve in part D shows the fluorescence lifetime distribution fitted with eq 1 from the ensemble fluorescence decay of Pc-G4 in aqueous solution (cf. inset of Figure 1). The number of single-molecule traces contributing to each histogram shown here is (A) 113, (B) 106, (C) 112, and (D) 113. The error bars in parts A and C were estimated from data simulation by considering a Gaussian distribution of lifetimes and a sample size of 100 points. It is assumed that the uncertainty in the data of parts B and D is within the same order of magnitude.

for immobilization of the Pc-G4 complex at glass surfaces by spin coating. Finally, the increase of ionic strength in the solution at pH 3 does not seem to dissociate the Pc-Gn complex in the same concentration range, although the diffusion coefficient measured for the Pc-G7 is slightly lower than expected. Single-Molecule Fluorescence of Pc-G4. First, we present the results for the systems immobilized directly on glass. The Pc-G4 complex was deposited from both solutions without buffer and with pH 3 buffer. In both samples, over 100 singlemolecule fluorescence time traces were measured (some examples are shown in Figures S4 and S5 in the Supporting Information). From each trace, it is possible to build a singlephoton counting histogram, which corresponds to the fluorescence decay of that particular single molecule. The singlemolecule fluorescence decays were then fitted individually with either a monoexponential or a biexponential function. The lifetimes retrieved from these fits (or the average lifetimes in the case of biexponential fits) are represented in Figure 4A,B, as normalized histograms, for Pc-G4 deposited from a solution with pH 3 buffer (dep. from pH3) and without buffer (no buffer), respectively. The single-molecule fluorescence decays obtained for Pc-G4 deposited from pH 3 solution are mostly monoexponential (less than 5% are biexponential), and the distribution of lifetimes from this sample is single-peaked with an average lifetime of 5.5 ns (Figure 4A). This value agrees well with the fluorescence lifetime measured in solution for Pc-G4 at pH3 (cf. curve c in Figure 1). The peak in Figure 4A is well described by a Gaussian function with a spread of (0.8 ns. The dispersion of singlemolecule fluorescence lifetimes in a polymer environment are probably related to differences in the local refraction index.58 For instance, we also measured the fluorescence lifetimes of single phthalocyanine molecules in a PVA film without any dendrimer (Figure 4C). The distribution of fluorescence lifetimes is again well described by a Gaussian distribution centered at 5.5 ns, but here, the spread is only (0.6 ns. This probably suggests that the PVA film is more homogeneous in terms of local index of refraction than the dendrimer nanoenvironment. The situation changes significantly for the sample of Pc-G4 deposited from a solution without buffer. Although the distribu-

tion of fluorescence lifetimes also peaks at 5.5 ns, there is a broadening toward shorter lifetimes, with a plateau around 5% (Figure 4B). Furthermore, about 26% of the single-molecule fluorescence decays are biexponential, but this will be discussed below. This clearly shows that phthalocyanine emission is also quenched in the Pc-G4 complex at the glass surface, as it happened in solution without buffer. However, the histogram of lifetimes in Figure 4B does not compare well with the lifetime distribution fitted from the ensemble fluorescence decay in solution (cf. inset of Figure 1) because it shows a large subpopulation of phthalocyanine molecules which are not quenched (peak around 5.5 ns). A simple explanation for the unquenched population would be that the Pc-G4 complex dissociates when deposited at the glass surface. This seems unlikely because a control sample of Pc deposited directly on glass (without any dendrimer) gives a lifetime distribution that is centered at 4.1 ns with a spread of (0.9 ns (results not shown). The broad distribution of lifetimes for Pc deposited directly on glass also suggests that the glass surface is more heterogeneous than the dendrimer or PVA environments. This gives evidence that the peak at 5.5 ns in the lifetime distribution of Pc-G4 sample without buffer is not dissociated Pc on glass and that, alternatively, it should be attributed to an unquenched subpopulation of Pc-G4 complex which is favored upon deposition at the glass surface. For this subpopulation, the efficiency of the electron-transfer reaction, responsible for fluorescence quenching of phthalocyanine in Pc-G4 complex, is drastically reduced at the glass surface in comparison to the aqueous environment. This seems plausible considering that the electron-transfer efficiency might depend significantly on the polarity of the surrounding environment. In the context of the Marcus theory (eq 2), the dependence of electron-transfer rate on polarity comes from the reorganization energy term λ, the solvation energy, and Coulomb interaction terms that contribute to the free energy ∆G0 of the electron-transfer reaction.50 We did some model calculations by assuming different degrees of hydration in the dendrimer environment of Pc-G4 at the glass surface to estimate how local polarity can influence the electron-transfer rate in these systems. Briefly, it was

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assumed that the volume fraction of water inside a PAMAM G4 dendrimer is 44% from the swelling effect observed in molecular-dynamic simulations with explicit solvent.59 The electron acceptor (phthalocyanine) and the electron donor (tertiary amine) are considered to be inside the dendrimer, which is represented as a spherical region with dielectric properties that are calculated as the volume fraction average between those of poly(amido)amines and of bulk water. Outside the dendrimer spherical region, we considered a shell of water with thicknesses of 9, 3, and 0 Å (i.e., the dendrimer surrounded by air), as examples of dendrimer environments with successively lower degrees of hydration. Finally, we also assumed a dendrimer “dry” environment with a water volume fraction of only 11%. For these four cases, the reorganization and solvation energies and the Coulomb interaction terms were calculated (see Figure S3 in the Supporting Information), and the Marcus theory was used to estimate the electron-transfer rates for several short distances between a single donor-acceptor pair in the dendrimer model environment with different degrees of hydration (see curves b-e in Figure 2A). It is clear from this simple model that, as the degree of hydration decreases, electron transfer becomes less efficient (it follows from curve b to e). Eventually, for “dry” conditions, fluorescence quenching no longer occurs, or it only occurs at very short distances (see curve e in Figure 2B). Thus, the subpopulation of unquenched lifetimes in the Pc-G4 sample without buffer can be explained by low polarity regions (less hydrated) of the dendrimer environment at the glass surface. These seem to coexist with other more polar regions (more hydrated), where electron transfer can efficiently quench fluorescence emission, which accounts for the broadening of the lifetime distribution toward short decay times. This interpretation seems to be supported by similar measurements done for Pc-G4 without buffer immobilized in a PVA film (Figure 4D). PVA films are known to provide a polar environment, in particular because they can retain small water domains.60,61 Indeed, the unquenched subpopulation of Pc-G4 decreases significantly in the PVA film, and the distribution of lifetimes now peaks at shorter lifetimes (around 1-2 ns). The fluorescence quenching by electron transfer of phthalocyanine in the Pc-G4 complex is more efficient in a PVA film because of its higher polarity than directly on a glass surface. (We notice again that the fluorescence lifetimes of phthalocyanine are not quenched by PVA in the absence of G4 dendrimer, which precludes any direct effect from PVA itself, see Figure 4C.) For comparison purposes, the histogram of lifetimes of Pc-G4 in PVA film is overlapped with the lifetime distribution fitted to the ensemble fluorescence decay of Pc-G4 in solution without buffer (dashed curve in Figure 4D). It is not straightforward to compare these two situations. The Pc-G4 system in solution probably experiences a more homogeneous environment, but conformational dynamics of the phthalocyaninedendrimer complex might contribute with dynamic disorder effects to the ensemble fluorescence decay. On the other hand, the immobilization of Pc-G4 in PVA film could restrain the accessible phthalocyanine-dendrimer conformations, and local heterogeneities of polarity or polarizability in the film might also contribute to broaden the fluorescence lifetime distribution. Nevertheless, the two lifetime distributions shown in Figure 4D are close to each other and suggest similar electron-transfer efficiencies of Pc-G4 in aqueous solution and PVA film. Besides polarity differences due to the relative hydration of dendrimer environments, other factors might influence the efficiency of electron transfer in Pc-G4 at a glass surface. It is known that the interaction of dendrimers with surfaces or

Paulo and Costa interfaces can significantly distort their conformation.62,63 PAMAM dendrimers in solution are approximately spherical (or slightly prolate); but upon deposition at a surface with favorable interactions, they adopt an oblate ellipsoid shape to maximize surface interactions (at low surface coverage). This modification in the dendrimer shape could affect donor-acceptor distances in the Pc-G4 complex when deposited at a glass surface or the dielectric response from the dendrimer environment to electron transfer and thus change the electron-transfer efficiency. These factors could also contribute to explain the differences between the lifetime distributions of Pc-G4 deposited directly on glass and those in PVA film or in solution without buffer. As previously mentioned, another interesting feature of Pc-G4 deposited on glass from a solution without buffer is that about 26% of the single-molecule fluorescence decays are biexponential (by contrast to the other systems with less than 5% biexponential decays). Multiexponential fluorescence decays of single molecules have been previously reported, in some cases also related to quenching by electron transfer used to probe conformational dynamics of biomolecules.64,65 In our dendrimer systems, it was possible to decompose some of the intensity time traces with an overall biexponential fluorescence decay into subintervals with monoexponential decays. In the example shown in Figure 5A, the molecule begins emitting with an intensity level around 1.4 counts/ms (section a), and after 11 s, it decreases to about 0.9 counts/ms (section b). During a short interval between 26.5 and 28.5 s, it goes through a nonemissive period, and then, it continues to emit at 0.9 counts/ms (section c), until at around 40 s, it returns to an intensity level of 1.4 counts/ms (section d). The fluorescence decay of the total trace is biexponential with lifetimes of 4.6 ns (77%) and 1.4 ns (23%). However, the fluorescence decays of each section a-d are monoexponential (lower part Figure 5A). Furthermore, sections a and d, both with an intensity level around 1.4 counts/ms, show the same fluorescence lifetime of 4.9 ns, and similarly, sections b and c also show the same lifetime of 3.1 ns. It is clear in this example that the change in emission intensity is correlated with a change in fluorescence lifetime, which is reversible and is large enough to give an overall biexponential decay. Another example is shown in Figure 5B. Here, the time trace is divided according to three intensity thresholds: sections a, b, and c for the intensity ranges 0.7-1.4, 1.4-2.8, and 2.8-5.3 counts/ms, respectively. The fluorescence decay for each section is monoexponential, and the respective lifetime becomes longer as the intensity threshold increases, from 2.1 ns in section a to 3.7 ns in section b and 4.7 ns in section c. Also here, there are changes in emission intensity that can be correlated with changes in fluorescence lifetime, although, in this case, it is possible to define three discrete intensity-lifetime levels. Other examples are shown in the Supporting Information (Figure S6). It is not always possible to identify discrete intensity-lifetime levels. Sometimes, the intensity fluctuations occur on a time scale faster than the binning time, or the change in intensity is not large enough to be separated into discrete levels. However, when it is possible to identify discrete intensity-lifetime levels, in general, the higher intensity levels correspond to longer fluorescence lifetimes. We attribute these intensity-lifetime fluctuations of single-molecule fluorescence to changes in the phthalocyanine-dendrimer conformation in the Pc-G4 complex at glass surface that affect the efficiency of fluorescence quenching, therefore causing a simultaneous change in emission intensity and fluorescence lifetime.66 Such conformational changes could be a reorganization of the dendrimer branches that change the distance or the number of tertiary amines close


J. Phys. Chem. C, Vol. 114, No. 44, 2010 19041

Figure 5. Single-molecule fluorescence time traces of tetrasulfonated aluminum phthalocyanine complex with PAMAM G4 dendrimer (Pc-G4) deposited directly at a glass surface from an aqueous solution without buffer. Part A is an example of a trace that shows two intensity-lifetime levels: sections a, d with a lifetime of 4.9 ns and sections b, c with a lifetime of 3.1 ns. The fluorescence decays of each sections identified are shown under the fluorescence time trace. Part B is an example of a trace in which case three intensity-lifetime levels can be identified: sections a, b, and c for intensities between 0.7-1.4, 1.4-2.8, and 2.8-5.3 counts/ms, respectively. The respective lifetimes are given in the fluorescence decays of each section, as shown under the fluorescence time trace.

to the phthalocyanine. By decreasing the distance to the closest tertiary amine by only a few angstroms, the efficiency of electron transfer to the phthalocyanine can increase substantially, thereby decreasing its fluorescence lifetime (see Figure 2). In previous molecular-dynamics simulations of an analogue porphyrindendrimer system, it was observed that the folding motion of a dendrimer branch over the porphyrin could increase the number of close tertiary amines by approximately 2 fold and cause an equivalentincreaseinelectron-transferrate.52 Theintensity-lifetime fluctuations that are distinguishable as discrete levels in the single-molecule fluorescence traces occur on the millisecondto-second time scale. Conformational dynamics leading to intensity-lifetime changes on faster time scales cannot be distinguished as discrete levels but contribute to a broadening in the intensity distribution of the “on” level and to a multiexponential fluorescence decay. This is observed in some time traces that have a biexponential decays but do not show distinguishable discrete intensity-lifetime levels. In those cases, the biexponential decay probably reflects a more complex lifetime distribution (and not simply a two- or three-“state” system as in the examples of Figure 5). Single-molecule fluorescence gives direct access to conformational dynamics, and several studies have been reported in the literature, particularly in the context of single-pair Fo¨rster energy transfer (FRET).67,68 The same concepts are applicable to single-pair electron transfer, although on a different length scale, because electron transfer typically occurs over a few angstroms, whereas FRET can take place within a few nanometers. However, the phthalocyanine-dendrimer systems studied here are not good model systems, mostly because of the flexible structure of the dendrimer. The potential-energy surface

of the dendrimer’s conformational space is probably very rough with multiple accessible minima giving rise to a wide distribution of dendrimer conformations. The distribution of tertiary amines (donor) within the dendrimer volume changes with its conformation, and so does the distance to the phthalocyanine (acceptor) on the dendrimer’s surface. Furthermore, the noncovalent character of phthalocyanine-dendrimer association could also contribute to widen the spread of donor-acceptor distances in these systems. The conformational heterogeneity of phthalocyanine-dendrimer systems is reflected in the distribution of fluorescence lifetimes, as previously discussed, and also in the diversity of blinking behavior in single-molecule fluorescence traces through its influence on the forward and back electron-transfer rates. (The blinking behavior of single-molecule fluorescence traces in phthalocyanine-dendrimer systems will be addressed elsewhere, in relation to studies on the influence of excitation power.) Nevertheless, it is interesting to notice that, even though phthalocyanine-dendrimer systems are rather heterogeneous (from a conformational point of view), it is possible to identify cases that behave like two- or three-level systems regarding quenching dynamics by electron transfer. This information would not be accessible from ensemble spectroscopic techniques which average over many phthalocyaninedendrimer conformations with diverse electron-transfer efficiencies. 4. Conclusions Single-molecule fluorescence quenching of a phthalocyanine by PAMAM dendrimers revealed both static and dynamic heterogeneity in these systems. Fluorescence quenching is attributed to electron transfer from the dendrimer’s tertiary

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amines to excited-state phthalocyanine. These species form a complex in solution because of electrostatic interaction, as it was confirmed by FCS measurements. The ensemble fluorescence decay of the phthalocyanine-dendrimer complex in solution (without buffer) can be described by a Gaussian distribution of lifetimes shorter than the natural lifetime of free phthalocyanine. This was attributed to a statistical distribution of phthalocyanine-dendrimer conformations with different electron-transfer efficiencies due to slight variations in the distance from the closest dendrimer amines (donor) to the phthalocyanine (acceptor). At low pH, the dendrimer amines become protonated, and fluorescence quenching no longer occurs. The phthalocyanine-dendrimer complex was deposited at a glass surface from a solution without buffer and with pH 3 buffer, and a comparable behavior was found in single-molecule fluorescence measurements. The phthalocyanine-dendrimer system without buffer shows a broad distribution of fluorescence lifetimes that spreads toward the short lifetimes. However, there is still a large subpopulation of unquenched phthalocyanine molecules that is not present in the lifetime distribution obtained in solution. This difference was attributed to low-polarity environments (i.e., less hydrated dendrimer) at the glass surface, where electron transfer is not efficient enough to compete with radiative decay and cause fluorescence quenching. Similar measurements of the phthalocyanine-dendrimer complex in PVA film, which is known to provide a polar environment, showed efficient fluorescence quenching and a lifetime distribution more comparable with that in solution. Single-molecule fluorescence measurements give direct access to static heterogeneity as revealed by the broad lifetime distributions for the quenched systems. However, it also gives evidence of dynamic heterogeneity from the fluorescence time traces. This behavior was attributed to phthalocyanine-dendrimer conformational dynamics that affect the efficiency of fluorescence quenching by electron transfer and that occur on the millisecond-to-second time scale. Faster dynamics are not distinguished as discrete levels but give rise to single-molecule multiexponential decays. This rich behavior is not accessible in ensemble measurements, and only an average lifetime distribution can be obtained. Further studies will address the blinking behavior of the singlemolecule fluorescence traces to infer about back electron-transfer processes in these systems. Acknowledgment. The authors acknowledge Fundaçaõ para a Cieˆncia e a Tecnologia for financial support through the Project REEQ/115/QUI/2005. Supporting Information Available: Absorption and emission spectra of Pc-G4; average distance distribution between the tertiary amines and two selected primary amines at the dendrimer’s surface obtained from molecular-dynamics simulations; description of three-phase model adapted for calculation of the reorganization and solvation energies and the Coulomb interaction terms in the Marcus model of electron transfer; single-molecule fluorescence time traces of Pc-G4 deposited at glass surface. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) de la Torre, G.; Claessens, C. G.; Torres, T. Chem. Commun. 2007, 2000–2015. (2) Kobayashi, N.; Ogata, H.; Nonaka, N.; Luk’yanets, E. A. Chem.sEur. J. 2003, 9, 5123–5134.

Paulo and Costa (3) Peng, X.; Draney, D. R.; Volcheck, W. M.; Bashford, G. R.; Lamb, D. T.; Grone, D. L.; Zhang, Y.; Johnson, C. M. Proc. SPIE 2006, 6097, E970. (4) Lippel, P. H.; Wilson, R. J.; Miller, M. D.; Wo¨ll, C.; Chiang, S. Phys. ReV. Lett. 1989, 62, 171–174. (5) Watanabe, F.; Mcclelland, G. M.; Heinzelmann, H. Science 1992, 262, 1244–1247. (6) Pan, S.; Zhao, A.; Wang, B.; Yang, J.; Hou, J. AdV. Mater. 2010, 22, 1967–1971. (7) Wang, Y. F.; Kro¨ger, J.; Berndt, R.; Va´zquez, H.; Brandbyge, M.; Paulsson, M. Phys. ReV. Lett. 2010, 104, 176802. (8) Wu, S. W.; Nazin, G. V.; Chen, X.; Qiu, X. H.; Ho, W. Phys. ReV. Lett. 2004, 93, 236802. (9) Mikaelian, G.; Ogawa, N.; Tu, X. W.; Ho, W. J. Chem. Phys. 2006, 124, 131101. (10) Tang, C. W. Appl. Phys. Lett. 1986, 48, 183–185. (11) Kerp, H. R.; Donker, H.; Koehorst, R. B. M.; Schaafsma, T. J.; van Faassen, E. E. Chem. Phys. Lett. 1998, 298, 302–308. (12) Rostalski, J.; Meissner, D. Sol. Energy Mater. Sol. Cells 2000, 63, 37–47. (13) Kim, I.; Haverinen, H. M.; Wang, Z.; Madakuni, S.; Li, J.; Jabbour, G. E. Appl. Phys. Lett. 2009, 95, 023305. (14) Xi, X.; Meng, Q.; Li, F.; Ding, Y.; Ji, J.; Shi, Z.; Li, G. Sol. Energy Mater. Sol. Cells 2010, 94, 623–628. (15) Nazeeruddin, Md. K.; Humphry-Baker, R.; Gra¨tzel, M.; Murrer, B. A. Chem. Commun. 1998, 719–720. (16) Clifford, J. N.; Palomares, E.; Nazeeruddin, Md. K.; Gra¨tzel, M.; Nelson, J.; Li, X.; Long, N. J.; Durrant, J. R. J. Am. Chem. Soc. 2004, 126, 5225–5233. (17) Levitsky, I. A.; Euler, W. B.; Tokranova, N.; Xu, B.; Castracane, J. Appl. Phys. Lett. 2004, 85, 6245–6247. (18) Tamarat, Ph.; Maali, A.; Lounis, B.; Orrit, M. J. Phys. Chem. B 2000, 104, 1–16. (19) Tinnefeld, P.; Sauer, M. Angew. Chem., Int. Ed. 2005, 44, 2642– 2671. (20) Michalet, X.; Weiss, S.; Ja¨ger, M. Chem. ReV. 2006, 106, 1785– 1813. (21) Wo¨ll, D.; Braeken, E.; Deres, A.; De Schryver, F. C.; Uji-i, H.; Hofkens, J. Chem. Soc. ReV. 2009, 38, 313–328. (22) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. ReV. 1999, 99, 1665–1688. (23) Astruc, D.; Boisselier, E.; Ornelas, C. Chem. ReV. 2010, 110, 1857– 1959. (24) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polymer J. 1985, 17, 117–132. (25) Wang, J.; Chen, J.; Jia, X.; Cao, W.; Li, M. Chem. Commun. 2000, 511–512. (26) Wang, J.; Jia, X.; Zhong, H.; Luo, Y.; Zhao, X.; Cao, W.; Li, M. Chem. Mater. 2002, 14, 2854–2858. (27) Khopade, A. J.; Caruso, F. Langmuir 2002, 18, 7669–7676. (28) Khopade, A. J.; Caruso, F. Nano Lett. 2002, 2, 415–418. (29) Khopade, A. J.; Caruso, F. Langmuir 2003, 19, 6219–6225. (30) Moraes, M. L.; Baptista, M. S.; Itri, R.; Zucolotto, V.; Oliveira, O. N., Jr. Mater. Sci. Eng., C 2008, 28, 467–471. (31) Srivastava, S.; Frankamp, B. L.; Rotello, V. M. Chem. Mater. 2005, 17, 487–490. (32) Park, M.-H.; Ofir, Y.; Samanta, B.; Rotello, V. M. AdV. Mater. 2009, 21, 2323–2327. (33) Qiana, L.; Liua, Y.; Songa, Y.; Lia, Z.; Yang, X. Electrochem. Commun. 2005, 7, 1209–1212. (34) Crespilho, F. N.; Zucolotto, V.; Brett, C. M. A.; Oliveira, O. N., Jr.; Nart, F. C. J. Phys. Chem. B 2006, 110, 17478–17483. (35) Siqueira, J. R., Jr.; Gasparotto, L. H. S.; Oliveira, O. N., Jr.; Zucolotto, V. J. Phys. Chem. C 2008, 112, 9050–9055. (36) Ogasawara, S.; Ikeda, A.; Kikuchi, J.-i. Chem. Mater. 2006, 18, 5982–5987. (37) Zhang, H.; Hu, N. J. Phys. Chem. B 2007, 111, 10583–10590. (38) Li, G.; Li, X.; Wan, J.; Zhang, S. Biosens. Bioelectron. 2009, 24, 3281–3287. (39) Siqueira, J. R., Jr.; Abouzarb, M. H.; Poghossianb, A.; Zucolottoa, V.; Oliveira, O. N., Jr.; Scho¨ning, M. J. Biosens. Bioelectron. 2009, 25, 497–501. (40) Siqueira, J. R., Jr.; Werner, C. F.; Ba¨cker, M.; Poghossian, A.; Zucolotto, V.; Oliveira, O. N., Jr.; Scho¨ning, M. J. J. Phys. Chem. C 2009, 113, 14765–14770. (41) Tomita, S.; Sato, K.; Anzai, J.-i. J. Colloid Interface Sci. 2008, 326, 35–40. (42) Singha, P.; Onoderaa, T.; Mizutaa, Y.; Matsumoto, K.; Miura, N.; Toko, K. Sens. Actuators B 2009, 137, 403–409. (43) Paulo, P. M. R.; Gronheid, R.; De Schryver, F. C.; Costa, S. M. B. Macromolecules 2003, 36, 9135–9144. (44) Paulo, P. M. R.; Costa, S. M. B. J. Phys. Chem. B 2005, 109, 13928–13940.

Single-Molecule Fluorescence Phthalocyanine in PAMAM (45) Laia, C. A. T.; Costa, S. M. B.; Phillips, D.; Beeby, A. J. Phys. Chem. B 2004, 108, 7506–7514. (46) Laia, C. A. T.; Costa, S. M. B.; Ferreira, L. F. V. Biophys. Chem. 2006, 122, 143–155. (47) Togashi, D. M.; Costa, S. M. B.; Sobral, A. J. F. N. Biophys. Chem. 2006, 119, 121–126. (48) Maus, M.; Cotlet, M.; Hofkens, J.; Gensch, T.; De Schryver, F. C. Anal. Chem. 2001, 73, 2078–2086. (49) No changes are observed in the absorption spectra of phthalocyanine upon association with the dendrimer in the region of the Q-band (transition to S1 state); however, in the region of the Soret band (transition to S2 state), a shift and broadening of this band toward longer wavelengths is observed. (50) The Rehm-Weller expression can be written as

(

∆G0 ) (EDox - EAred)B + 1 -

)

1 (S - Sr) - Wr + Wp - hν εB p

where EDox and EAred stand for the oxidation and reduction potential of the donor and acceptor, respectively, in a medium of permittivity εB; Sp,r are the solvation energies of individual reactant and product ions in vacuum; Wr,p are the solvation energies and Coulomb interaction of the ions, and hν is the excited-state energy. The expressions for Sp,r and Wr,p can be found in Tavernier, H. L.; Barzykin, A. V.; Tachiya, M.; Fayer, M. D. J. Phys. Chem. B 1998, 102, 6078–6088. For EDox, we used a value of 0.97 V (vs. SCE) for the oxidation potential of triethylamine in water taken from Okutsu, T.; Ooyama, M.; Hiratsuka, H.; Tsuchiya, J.; Obi, K. J. Phys. Chem. A 2000, 104, 288–292. For EAred, we used a value of-0.5 V (vs. SCE) for the reduction potential of aluminum phthalocyanine in DMSO taken from Ou, Z.; Shen, J.; Kadish, K. M. Inorg. Chem. 2006, 45, 9569–9579. The excitedstate energy of the phthalocyanine is around 1.82 eV. (51) Albery, W. J.; Bartlett, P. N.; Wilde, C. P.; Darwent, J. R. J. Am. Chem. Soc. 1985, 107, 1854–1858. (52) Paulo, P. M. R.; Canongia Lopes, J. N.; Costa, S. M. B. J. Phys. Chem. B 2008, 112, 14779–14792. (53) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 881, 265– 322. (54) Marcus, R. A. J. Chem. Phys. 1984, 81, 4494–4500. (55) The value of V20 ) 10 cm-1 is small enough to justify the nonadiabatic approximation in eq 2 but at the same time large enough to

J. Phys. Chem. C, Vol. 114, No. 44, 2010 19043 quench the excited-state lifetime of the phthalocyanine down to subnanosecond values. The value of β ) 1 Å-1 is typical for electron tunneling through an aliphatic carbon donor-acceptor bridge and therefore is appropriate for the PAMAM dendrimer systems. (56) Prosa, T. J.; Bauer, B. J.; Amis, E. J. Macromolecules 2001, 34, 4897–4906. (57) Goldstein, H.; Poole, C.; Safko, J. Classical Mechanics, 3rd ed.; Addison Wesley: San Francisco, 2000. (58) Valleé, R. A. L.; Van der Auweraer, M.; De Schryver, F. C.; Beljonne, D.; Orrit, M. ChemPhysChem 2005, 6, 81–91. (59) Maiti, P. K.; Çag˘ın, T.; Lin, S.-T.; Goddard, W. A., III. Macromolecules 2005, 38, 979–991. (60) Hou, Y.; Bardo, A. M.; Martinez, C.; Higgins, D. A. J. Phys. Chem. B 2000, 104, 212–219. (61) Clifford, J. N.; Bell, T. D. M.; Tinnefeld, P.; Heilemann, M.; Melnikov, S. M.; Hotta, J.-i.; Sliwa, M.; Dedecker, P.; Sauer, M.; Hofkens, J.; Yeow, E. K. L. J. Phys. Chem. B 2007, 111, 6987–6991. (62) Bliznyuk, V. N.; Rinderspachert, F.; Tsukruk, V. V. Polymer 1998, 39, 5249–5252. (63) Betley, T. A.; Banaszak Holl, M. M.; Orr, B. G.; Swanson, D. R.; Tomalia, D. A.; Baker, J. R., Jr. Langmuir 2001, 17, 2768–2773. (64) Yang, H.; Luo, G.; Karnchanaphanurach, P.; Louie, T.-M.; Rech, I.; Cova, S.; Xun, L.; Xie, X. S. Science 2003, 302, 262–266. (65) Jia, Y.; Sytnik, A.; Li, L.; Vladimirov, S.; Cooperman, B. S.; Hochstrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7932–7936. (66) Besides fluorescence quenching by electron transfer, other factors can change the intensity of the “on” level in single-molecule fluorescence traces (rotational dynamics, spectral diffusion, and so forth). Indeed, we observe intensity fluctuations in traces of unquenched single phthalocyanine molecules that have monoexponential decays. This is probably related to rotational dynamics of phthalocyanine in more flexible environments. This effect is more pronounced here, because the excitation light is not circularly polarized, and in-plane rotation is enough to change emission intensity. Spectral diffusion does not seem plausible, because the emission light is collected through a filter with a broad wavelength window. (67) Rhoades, E.; Gussakovsky, E.; Haran, G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3197–3202. (68) Nettels, D.; Gopich, I. V.; Hoffmann, A.; Schuler, B. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2655–2660.

JP107412P

Single-Molecule Fluorescence of a Phthalocyanine in PAMAM

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