Energy Transfer between Quantum Dots and Conjugated Dye

Article

Energy Transfer Between Quantum Dots and Conjugated Dye Molecules Gary Beane, Klaus Boldt, Nicholas Kirkwood, and Paul Mulvaney J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/jp502033d • Publication Date (Web): 08 Jul 2014 Downloaded from http://pubs.acs.org on July 17, 2014

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The Journal of Physical Chemistry

Energy Transfer Between Quantum Dots and Conjugated Dye Molecules Gary Beane, Klaus Boldt, Nicholas Kirkwood, and Paul Mulvaney∗ School of Chemistry and Bio21 Institute, University of Melbourne, Parkville VIC, 3010, Australia E-mail: [email protected]

Abstract Energy transfer from quantum dots (QDs) to variable length, dye-labeled peptides is reported. We find that existing models used to calculate the efficiency of energy transfer from steady state measurements are insufficient for nanoparticle - dye interactions. To accurately measure the distance dependence as a function of separation, the effects of multiple valencies as well as variations in the luminescence quantum yield of the acceptor dye with separation both need to be taken into consideration. Using Poisson statistics, we account for the distribution of QD:dye ratios. Nevertheless, we find that the actual dependence of the energy transfer efficiency as a function of QD-dye separation obeys an R−n dependence with n = 6.1 ± 0.1 as predicted by Förster Resonance Energy Transfer theory (FRET).

KEYWORDS: energy transfer, quantum dots, FRET, CdSe

Introduction Understanding the photophysics behind the phenomenon of excitation energy transfer has been an area of intense research for over 85 years. 1–5 Much of this research has focused around understanding how the transfer of energy from an excited ‘donor’ fluorophore to a nearby ground state ∗ To

whom correspondence should be addressed

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‘acceptor’ fluorophore depends on distance. Following the pioneering work of Cario et al. and Perrin et al., 2,6 Theodor Förster formulated a quantitative model to explain the relationship between energy transfer efficiency and the spatial separation of the fluorophores based on dipole - dipole coupling. 3 Förster’s theory was subsequently substantiated by Stryer and co-workers, who used rigid polyproline peptides to vary the separation of an α -napthyl donor at the C terminus and a dansyl acceptor at the N terminus. 4 Förster’s theory has become the dominant model now used to describe energy transfer between organic fluorophores, where it is commonly known as Förster Resonance Energy Transfer (FRET). 7 The ubiquitous use of FRET theory has arisen as, although it makes several key assumptions, it fits experimental observations surprisingly well and requires only a few experimental parameters to be measured. Indeed, in many biological situations FRET provides a very useful ‘spectroscopic ruler’ 4 relating easily measurable macroscopic quantities to the nano-scale separation between donors and acceptors.

While Förster originally formulated his theory for the case of conjugated fluorescent molecules of low molecular weight, there have been many new fluorophores discovered which are of large molecular weight such as conducting polymers, green fluorescent proteins and semiconductor nanocrystals or quantum dots.

In particular, there have been numerous studies of energy transfer from semiconductor nanocrystals (QDs) to acceptor dyes. These systems are very attractive for biological labelling studies because QDs have large absorption coefficients; their emission can be tuned by controlling the size of the nanocrystal and they exhibit enhanced photostability, which facilitates long, time-dependent fluorescence studies. However, there are several key challenges to using QDs for FRET studies. These are (i) whether QDs can be considered as point dipoles, (ii) whether conjugated dye molecules are free to rotate and (iii) how to account for multiple dyes being adsorbed to a single QD donor. Nevertheless, despite these concerns, FRET has been applied to describe energy transfer between QD donors and dye molecule acceptors. 8–16 Literature reports about the applicability of using


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FRET to describe systems involving QD donors and dye acceptors vary, with many workers in the area arguing that QDs are well described as point dipoles, 8–12 without the need for either higher multipolar interactions or the calculation of the full electronic interaction. Pons et al. 5 found that the quenching of a CdSe/ZnS core shell structure by dye labeled peptide sequences was consistent with the standard FRET theory, in which the QD is treated as a point dipole.

There are, however, several problems with their analysis; specifically, that they use the method of Yu et al. 17 to calculate the QD concentration, which has been shown by Jasieniak and co-workers to underestimate the extinction coefficient resulting in a lower real concentration. 18 This means that the QD to dye ratios are underestimated. It is also of note that the shortest QD to dye separation in the study by Pons and co-workers, already has a center-to-center separation of 25 Å. This is significant as the assumption of a point dipole is expected to be more accurate the further the dye is from the QD surface. Several studies have already concluded that the point dipole approximation breaks down when describing energy transfer between QDs and dyes when the dye molecules are near the particle surface. 19–24 Indeed Blaudeck and co-workers 20 have suggested that photoexcited electrons in a QD can tunnel into the local environment, out to 5 Å for a 3.5 nm diameter CdSe QD - which raises the possibility of additional luminescence quenching due to electron transfer from QDs to adsorbed dye molecules. A further complication is that there may be multiple dye molecule acceptors for every QD donor, a situation that does not normally arise for dye - dye energy transfer, and which makes the analysis more complex, as well as raising the issue of interactions between adsorbed dye molecules.

A further consideration that must be addressed for any energy transfer systems involving QDdye systems, is the rigidity of the linker used. Several groups that have studied the effects of using biopolymer and DNA spacers in dye-dye FRET systems have found that significant corrections must be introduced to account for (i) the steric restriction of labels 25 and (ii) for the variability in the separation caused by the inherent flexibility of the linker. 26–28 The effect of linker rigidity has also



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been investigated by Boeneman et al. who found that, especially for biotin-streptaviding type linkers, the assumption of a single, well-defined donor-acceptor separation is invalid. 29 Given the inherent variability in these QD-linker-dye systems, it is worth reflecting on the original choice of linker used to validate FRET theory experimentally. Haugland and Stryer used what they assumed to be a rigid molecule in their early proof of FRET, a polyproline helix 4 (See Figure 1). The rigidity of this molecule arises due to the formation of a characteric helix, known as the polyproline type II helix (PPII) (see Figure 2). This is reported to have a rigid, rod-like structure in ethanol, as evidenced by the well-defined circular dichroism of solutions containing this molecule. 30 However, recent work by Schuler et al. 31 has called into question the rigidity of this molecule, and showed through Monte Carlo simulations that a ‘worm-like-chain’ (WLC) model provides a better description of this molecule. Moreover, they showed that for polyproline lengths much larger than about 20 prolyl residues, it is necessary to explicitly included the chain flexibility when calculating energy transfer efficiencies accurately. Notwithstanding these considerations, polyproline remains the obvious choice as a rigid linker so long as the number of prolyl residues remains low. 26

What is still missing in the literature and is clearly needed, is a systematic experimental validation of FRET theory for energy transfer between QD donors and dye molecule acceptors for a range of QD:dye ratios and for a wide range of clearly defined, donor-acceptor separations. We report herein an approach based on the use of polyproline spacers. We find that there exist significant deviations from the standard FRET model unless several important corrections are made, including: (i) accounting for the statistical distribution of dyes, (ii) ensuring the dye is separated by sufficient distance from the donor to enable the point dipole model to be applied (iii) accounting for the changes in the intrinsic dye QY, due to the change in environment when bound to the QD (iv) proving the relative rigidity of the linker used. We propose a revised model based on the experimental data, which accounts for the distance dependence of energy transfer from a QD to a dye acceptor when using peptide spacers.


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Solid-phase Synthesis Protocol using Automated Peptide Synthesiser Polyproline peptides consisting of 0, 4, 8, 12, 16 and 20 proline residues with an additional glycine residue at the N terminus and a Boc protected Dap residue at the C terminus were synthesised using a CEM Liberty microwave peptide synthesiser. The scale for synthesis was typically 0.1 mmol. Boc−Dap functionalised Wang resin (0.3 g) was added to the reaction vessel of the peptide synthesiser and the peptide sequence programmed into the computer. The deprotection solution used was 20% piperidine with 0.2 M hydroxybenzotriazole in DMF. That the final resin still had an Fmoc protected N terminus was confirmed by a negative result for the TNBS test (no free amines present). The Dap residue was left Boc protected until the final terminal Fmoc−glycine was added.

Reverse Phase HPLC purification of peptides The first step in the peptide purification was cleavage of the peptides from the resin with cleavage solution (97.5 % TFA with water), which simultaneously deprotected the Boc protected side chain on the Dap residue. The peptide was isolated from the resin by passing the peptide-resin solution through a syringe with a porous teflon frit, into 20 mL of diethyl ether chilled in a dry ice / acetone bath. The solutions were then made up to 50 mL with additional diethyl ether and left in the freezer overnight. After leaving to chill overnight, the samples were centrifuged at 4400 rpm for 5 minutes, and the diethyl ether removed. The peptides were further dried with a nitrogen stream, and then redispersed in a minimum amount of 20 % acetonitrile/water. HPLC separation of polyproline peptides was carried out using Phenomenex Jupiter C4 column (300 Å), 15 × 4.6 mm, 5 µ m, using a 10 - 95 % B (acetonitrile) and a flow rate of 0.5 mL/min. Peptides samples were prepared for injection by adding 20 µ L formic acid, 80 µ L acetonitrile and 400 µ L milliQ water. Fractions were checked for purity by Electrospray ionisation time-of-flight mass spectrometry and pooled to produce a purified peptide product. Peptide solutions were then lyophilized.



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Dye labelling and purification Excess dye (>10 molar excess) was added to ≈ 2 µ mol of peptide prepared to ≈10 µ g/µ L in dry DMF. Excess 0.4 M DIPEA / DMF (dry) was added to the solutions until they attained a grainy texture. The reaction mixtures were covered and vortexed to thoroughly mix the peptide and dye label. The solutions were then allowed to sit for 16 hours with occasional mixing. The final dye-labeled peptides were purified via HPLC with a 1% acetonitrile gradient over 20 minutes with a Phenomenex Jupiter C4 column (300 Å). Electrospray ionisation time-of-flight mass spectrometry confirmed the purity of the resultant dye-labeled peptides, which are denoted G(Dap)-ROX, GPn (Dap)ROX where n = 0, 4, 8, 12, 16 and 20.

Sample preparation The diameter of CdSe/CdS/ZnS nanoparticle solutions was determined to be 5.8 nm via TEM (see SI). Samples for fluorescence measurements were all prepared in ethanol (HPLC grade, Sigma-Aldrich) by ligand exchange with aminopentanol. For steady state experiments sample concentrations of 200 nM with a 1:1 ratio of QDs and dyes were used, unless stated otherwise. The absorbance of the samples at the excitation wavelength was adjusted to be < 0.1. The samples were not degassed but were left to equilibrate for five minutes. The quantum yield of the QDs was measured with respect to a rhodamine 6G (Rh6G) standard under a N2 atmosphere and is shown in Table 1. For photoluminescence measurements (PL) the excitation wavelength was 400 nm and the quantum yield of Rh6G at this wavelength taken to be 0.95. 33

QDs (D = 5.8 ± 0.7 nm) 5 - ROX Rh6G

λabs,max 525 nm 568 nm 529 nm

λPL,max ε (400 nm) 542 nm 8.2 ×105 M−1 cm−1 604 nm 1363 M−1 cm−1 553 nm 5031 M−1 cm−1

τ (ns) QY 5.2 ± 0.1 0.49 ± 0.02 4.3 ± 0.1 0.80 ± 0.04 0.95 33

Table 1: General photophysical properties of the different chemical species used. All data are for species in ethanol. All measured extinction coefficients have errors less than 5 %. 8 ACS Paragon Plus Environment

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Instrumentation UV-Visible absorbance, excitation and photoluminescence (PL) spectra were collected using a Thermo-Electron Varioskan multimode plate reader, with excitation at 400 nm for PL spectra. Time resolved emission measurements were carried out in solution using a confocal microscope in epi-illumination configuration, with an oil-immersion objective (Olympus, PlanApo NA 1.4) and a 470 nm, 10 MHz repetition rate (PicoQuant, LDH-P-C-405). A 550 nm shortpass filter was used to remove emission originating from the dye, and the light was focused onto an avalanche photo-diode (Perkin-Elmer, SPCM-AQR-15). For the measurement of the PL decay times of the individual particles, the photon-counting card (PicoQuant, TimeHarp 200) was set to time-tagged time-resolved mode (TTTR), and the decays were then generated by time-correlated single photon counting (TCSPC). All steady state and time-resolved samples were prepared in triplicate.

Results We have constructed a series of analogues to the original α -napthyl-polyproline acceptor systems, which also meet some specific, additional requirements. Foremost amongst these is that the peptidedye structures must be capable of binding directly to the surface of the QD, a condition that is met by incorporating a primary amine into the peptide sequence (by means of a glycine residue at the N-terminus of the peptide). Moreover, we require an orthogonal approach to deprotection, as we ultimately want one primary amine to be labeled with dye, and another amine to bind to the surface of the QD. This is achieved using a combination of tert-butoxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc) protection on the (Nβ ) amine side-chain of the 2,3-diaminopropionic acid (Dap) residue and the N-terminus (Nα ) respectively. It is desirable that the donor can be excited at a particular wavelength at which the acceptor does not absorb any of the incident light - this is indeed the case for the present system, as we see in Figure 3b that at 400 nm the extinction coefficient of the QD (8.2 × 105 M−1 cm−1 ) is 600 times higher than that of the dye (1363 M−1 cm−1 ). We also see from this same figure that the QD PL and the dye 9 ACS Paragon Plus Environment


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PL have significant spectral separation, which enables the emission from just the donor or acceptor to be measured, respectively. The structure shown in the upper right-hand corner of Figure 3bb is a general structure for the dye acceptor used throughout this study, 5-carboxy-X-rhodamine (5-ROX) where the substituent R represents the different peptides, which this acceptor labels. A concise summary of some of the key photophysical characteristics of the dye acceptor (5-ROX) and the QD donor are presented in Table 1. If we look at the photoluminescence spectrum of the QD donor in Figure 4a in the absence, and in the presence of 1 stoichiometric equivalent of the shortest, dye labeled spacer G(Dap), we see that the PL of the QD donor is significantly quenched. We observe that in the absence of the QD, the dye PL at the same concentration is much lower than the dye emission in the presence of the QD i.e. the dye emission is enhanced. From these data alone, we can say there is energy transfer from the QD donor to the dye acceptor. To confirm this, one can also monitor the excitation spectrum of the two fluorophores.

In Figure 4a, the PL is measured for excitation at 530nm. We see that the PL at 630 nm is much lower for a solution containing the dye than does a solution containing an equimolar concentration of QD and dye. As the QD has negligible PL at this wavelength, we must infer that the emission from the dye in the presence of the QD originates primarily from the absorption of light by the QD - thus there must be energy transfer.

An important complication in these systems is that dielectric environment around the dye changes as it binds to the QD surface. This causes a drastic reduction in the intrinsic QY of the dye. 34 This effect can be seen by examining the emission of the dye at 630 nm when excited at a wavelength at which only the dye absorbs (575 nm) (Figure 4b). We also note that the emission from the dye-only sample in Figure 4b is significantly larger than for the QD-dye conjugate, when excited at 575 nm. Overall, we see from the excitation spectra that while there is significant energy transfer to the dye, the overall emission intensity is substantially lower due to the environmental quenching effect. This additional quenching must be accounted for in calculating energy transfer efficiencies.


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The final proof that we have energy transfer is provided by measurement of the QD lifetime after the addition of the dye, which results in strong reductions in PL lifetimes, as seen in Figure 5a.

Discussion The results presented here clearly confirm earlier works, which have demonstrated that energy transfer from QDs to dyes is very efficient. However, to confirm that the distance dependence obeys the FÂrster theory, we need to show that the energy transfer efficiency obeys Eq. (1), with γ = 6. 1 1 + (R/R0 )γ

(1)

9 ln (10)κ 2 φD J 128π 5 ε0 NA n40

(2)

E= In dye-dye systems, R0 is given by Eq. (2):

R60 =

where κ is the orientation factor, φD is the quantum yield of the donor, J is the spectral overlap integral for the donor - acceptor system and n0 is the refractive index of the solvent. An assumption in Eq. (1) is that the donor and acceptor fluorophore are in a one-to-one ratio. The energy transfer efficiency when there are n, independent acceptors per QD at a fixed distance R, is given by Eq. (3):

E(n) =

n n + (R/R0 )6

(3)

Up until this point we have approached the construction of the QD-dye system in a manner that is analogous to that of Stryer and Haugland’s system; however we must now consider one way in which the present system is quite dissimilar. While a molecular system can be constructed so that the donor and acceptor bind in a stoichiometric ratio, this is not easily achieved for quenching of



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QDs by molecular acceptors such as dyes. Because there are multiple conjugation groups attached to each QD surface, addition of the dyes results in a statistical distribution of dyes bound to the QDs. If the dyes are labile enough that they can exchange with different quantum dots and if the number of possible dye-binding sites is large (typically larger than 20 sites for a mean ratio of 1), 35 then the dye distribution will obey Poisson statistics. With these assumptions in place, the observed quenching efficiency Eobs by: ∞

Eobs =

∑ E(n)P(n)

(4)

n=0

where Eobs is the observed, ensemble-averaged energy transfer efficiency, E(n) the energy transfer efficiency for QD donors with n dye acceptors and P(n) is the probability of a QD having n acceptors which is given by the Poisson equation:

P(n) =

e−λ λ n n!

(5)

where λ = dye : QD is the mean number of dyes bound to each QD. Combining Eq. (3) and Eq. (5), the observed energy transfer efficiency is: ∞

Eobs =

e−λ λ n n ∑ γ n! n=0 n + (R/R0 )

(6)

where γ is the exponent to be found. There are 2 ways to determine the values of Eobs in each experiment. The first is by measuring the reduction in quantum yield or lifetime of the donor in the presence of the bound acceptor dye. The efficiency of energy transfer is:

Eobs =

kD→A kD→A + kR + kNR

(7)

where kD→A is the rate of energy transfer, kR the rate of radiative decay and kNR the rate of quenching by all other mechanisms. We can express Eq. (7) in terms of either the fluorescence


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intensity or the lifetime of the donor-acceptor complexes (Iobs and τobs ) and the donor fluorophore by itself (ID and τD ):

Eobs =

Iobs τobs kD→A = 1− = 1− kD→A + kD τD ID

(8)

where τobs = (kD→A + kD )−1 and kD = kR + kNR .

While Eq. (8) allows us to attain a direct value of the energy transfer efficiency from the lifetime quenching of the donor fluorophore (τobs ), simply monitoring the steady state quenching of the donor fluorophore (Iobs ) provides only a measure of the total amount of quenching, not the amount due to energy transfer. Hence, we prefer to use the change in lifetime to estimate the rates of energy transfer. The second way to measure the energy transfer efficiency is from the increase in the dye emission in the presence of the donor, from analysis of the excitation spectra.

It is well known that the excitation spectrum and extinction spectrum for a particular chemical species are coincident provided the absorbance is low. 36 From this equivalence, it follows that in donor-acceptor systems in which 100% of the excitation energy passes from donor to acceptor, the sum of the donor and acceptor extinction spectra is the same as the sum of their excitation spectra, provided both are normalised to unity at the acceptor peak (thus both are unitless). Simply put, if the profile of the donor-acceptor excitation spectrum is the same as that of the extinction spectrum, then energy transfer is unity. The reason that we choose to normalise to the dye acceptor peak is that the magnitude of this peak in the excitation spectra is independent of the degree of energy transfer, and serves as a reference for how much donor character, and thus energy transfer, is present in the donor-acceptor complex. For systems in which energy transfer is less than unity, the efficiency of energy transfer is provided from the normalised extinction spectrum of the donor-acceptor complex,



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A, which is equal to the sum of the acceptor and donor excitation spectra such that

A = χA + Eobs χD

(9)

where χA and χD are the excitation spectra of the donor and acceptor respectively (normalised to the acceptor peak) and Eobs is the efficiency of energy transfer from the donor to the acceptor fluorophore. While Eq. (9) is correct for the case of energy transfer in which the local dielectric environment of the acceptor is constant, in the present system this is not the case. As can be seen in Figure 4b, the QY of the dye changes with the length of the linker, which complicates the use of Eq. (9) and neccessitates its revision. Thus rearranging Eq. (9) and accounting for the reduced QY of the acceptor, our final equation becomes

Eobs =

Aζ − χA χD

(10)

where the term ζ is a correction for the difference in QY of bound dye molecules compared to the free dye in bulk ethanol solution. Eq. (8) and Eq. (10) provide 2 independent measurements of Eobs . The first assumes that the decrease in QD PL lifetime can be attributed to energy transfer alone, while the second method records the fractional increase in acceptor emission due to the QD. We now use both these measurements of Eobs in Eq. (6) to extract the parameter γ .

In Figure 6a we plot fits of the experimental data from the excitation spectra (black circles) and from the quenching of the QD donor (blue squares) to Eq. (6). The best fits yield values of γ = 5.6 and 5.6 for the two data sets respectively. Eq. (6). should also account for the Poisson distribution and in Figure 6b the efficiency of energy transfer is plotted for various dye:QD ratios using the single quencher G(Dap)-ROX; it is again well fit with Eq. (6) with γ = 6.1. These data all strongly indicate that for these spacers, the energy transfer efficiency closely follows FRET theory. It is 14 ACS Paragon Plus Environment

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worth noting that any error in the QD core-shell diameter would result in changes to R0 and not to the value of γ itself (see Figure 7a). Indeed, only errors in parameters that are distant dependent will cause changes in the value of γ . For example, if there are distance dependent deviations in the orientation of the donor and acceptor, through the κ 2 term, these could account for the observed deviation of the γ from 6. We also see from Figure 7b that a 1/R4 dependence of the efficiency of energy transfer on separation is not valid for this system, and fits the experimental data poorly. A 1/R4 dependence has been reported for quenching of dye photoluminescence by small metal particles.

Importantly, it is worth noting that the efficiencies shown in Figure 5 are calculated from the enhancement of the dye PL (which occurs only if there is energy transfer) and the quenching of the PL lifetime of the QD (which may be caused by either electron or energy transfer) respectively. As is immediately apparent from Figure 6, the distance dependence as a function of QD-dye separation is the same for both sets of data, strongly suggesting that electron transfer in the present system is negligible.

What is evident from the data presented here is that, while FRET is likely to be a valid formalism for explaining energy transfer in QD-dye systems, it must be used with care, as there are a number of significant factors to take into consideration. For example, it is essential to allow for the Poisson distribution of dyes bound to the QDs - failure to do so leads to artificially low values for the FRET exponent, and makes the distance dependence look much weaker than it really is. We also found that the intrinsic dye QY is strongly reduced when bound to the QD - this environmental effect can be accounted for by measuring the dye PL at wavelengths to the red of the QD absorption spectrum when the dye is both bound and free in solution. Moreover, we are forced to assume that the value of κ is a constant and does not change with QD-acceptor separation. Finally, we have assumed strictly dipole-dipole coupling between the QD and dye and cannot exclude quadrupole-dipole coupling 37–39



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Conclusions Energy transfer has been well studied for dye-dye systems, and its ubiquitous use in the biological sciences has proven countless times the validity of the mathematical formalism. In contrast the practical application of FRET to QD-dye systems has received far less attention, and there are real differences in these systems that set them apart from the conventional dye-dye systems. As we have shown in this paper, it is imperative that Poisson statistics be used when evaluating energy transfer in QD-dye systems. Moreover, we have also shown that there is an anomalous distance dependence for QD-dye energy transfer with significantly less energy transfer occurring than would be expected. We attribute these deviations to either variance in the value of the orientation factor κ as a function of separation or to some small degree of divergence from the simple dipole-dipole model of energy transfer. We conclude that more work is necessary to fully elucidate the energy transfer efficiency of QD-dye interactions.

Associated Content Supporting Information Available Additional details of the analysis method and ensemble characteristics results can be found in the Supporting Information. This material is available free of charge via the Internet at http: //pubs.acs.org/.

Author Information Notes The authors declare no competing financial interests.


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Acknowledgement K.B. acknowledges the support of the Alexander von Humboldt Foundation through a Feodor Lynen research fellowship. N.K. would like to thank the Melbourne Materials Institute for support through an MMI/CSIRO scholarship. PM thanks the ARC for support under grant DP130102134.

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[25] Hoefling, M.; Lima, N.; Haenni, D.; Seidel, C. A. M.; Schuler, B.; Grubmueller, H. Structural Heterogeneity and Quantitative FRET Efficiency Distributions of Polyprolines through a Hybrid Atomistic Simulation and Monte Carlo Approach. PLOS ONE 2011, 6, e19791. [26] Dolghih, E.; Ortiz, W.; Kim, S.; Krueger, B. P.; Krause, J. L.; Roitberg, A. E. Theoretical Studies of Short Polyproline Systems: Recalibration of a Molecular Ruler. J. Phys. Chem. A 2009, 113, 4639–4646. [27] Doose, S.; Neuweiler, H.; Barsch, H.; Sauer, M. Probing Polyproline Structure and Dynamics by Photoinduced Electron Transfer Provides Evidence for Deviations from a Regular Polyproline Type II Helix. Proc. Natl. Acad. Sci. 2007, 104, 17400–17405. [28] Sahoo, H.; Roccatano, D.; Hennig, A.; Nau, W. M. A 10-A Spectroscopic Ruler Applied to Short Polyprolines. J. Am. Chem. Soc. 2007, 129, 9762–9772. [29] Boeneman, K.; Deschamps, J. R.; Buckhout-White, S.; Prasuhn, D. E.; Blanco-Canosa, J. B.; Dawson, P. E.; Stewart, M. H.; Susumu, K.; Goldman, E. R.; Ancona, M.; Medintz, I. L. Quantum Dot DNA Bioconjugates: Attachment Chemistry Strongly Influences the Resulting Composite Architecture. ACS Nano 2010, 4, 7253–7266. [30] Ronish, E.; Krimm, S. The Calculated Circular Dichroism of Polyproline I1 in the Polarizability Approximation. Biopolymers 1974, 13, 1635–1651. [31] Schuler, B.; Lipman, E. A.; Steinbach, P. J.; Kumke, M.; Eaton, W. A. Polyproline and the "Spectroscopic Ruler" Revisited with Single-Molecule Fluorescence. Proc. Natl. Acad. Sci. 2005, 102, 2754–2759. [32] Boldt, K.; Kirkwood, N.; Beane, G. A.; Mulvaney, P. Synthesis of Highly Luminescent and Photo-Stable, Graded Shell CdSe/Cdx Zn1−x S Nanoparticles by In Situ Alloying. Chem. Mater. 2013, 25, 4731–4738.


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Graphical TOC Entry

Energy transfer from a Quantum dot donor to a dye acceptor is assumed to proceed via Förster Resonance Energy Transfer (FRET) - but does it?


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A

PL (a.u.)

QD peptide1-ROX QD-peptide1-ROX

450

525

600 675 wavelength (nm)

750

B QD peptide1-ROX QD-peptide1-ROX QD-peptide2-ROX QD-peptide3-ROX

PL (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

350

400

450 500 550 wavelength (nm)

600

Figure 4: A) The photoluminescence of 3 ethanolic solutions containing 200 nM green QDs (green trace), 200 nM G(Dap)-ROX (blue trace) and a solution containing 200 nM of both QDs and G(Dap)-ROX (purple trace). The solution containing an equimolar concentration of each of QD and dye shows a marked reduction in the PL of the QD at a wavelength of 530 nm and an enhancement of the PL of the dye at a wavelength of 600 nm. Excitation wavelength is 400 nm. B) The first three excitation spectra correspond to those shown in A) where the PL of the dye at a wavelength of 630 nm is monitored while scanning the excitation wavelength from 350 - 620 nm. It is evident from B) that the origin of the enhanced PL of the dye is from energy transfer from the QD donor to the dye acceptor. Note that the QY of the G(Dap)-ROX in solution is different to the QY when it is bound to the QD, as evident from the decreased PL of the dye at wavelengths at which the QD does not absorb. The traces in grey and orange are GP4 (Dap)-ROX and GP8 (Dap)-ROX respectively and demonstrate that the QY of the dye increases wtih the length of the linker. 24 ACS Paragon Plus Environment

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A

B 2.5

0

10

QD reference G(Dap)-ROX GP20(Dap)-ROX

QD - ROX (100 %) QD - G(Dap)-ROX QD - GP20(Dap)-ROX ROX reference

2.0

-1

PL (a.u.)

10

PL (a.u.)

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-2

10

1.5

1.0

-3

10

0.5 -4

10

0

50

100

150

0.0 400

200

time (ns)

450

500 550 wavelength (nm)

600

Figure 5: A) The fluorescence lifetime of ethanolic solutions of QDs collected via TCSPC for various donor-acceptor spacings. The waveforms are normalised to the maximum number of counts. The solutions were excited using a 470 nm pulsed diode laser with a FWHM of 50 ps, and the emission collected using a 530/10 nm shortpass filter. B) The excitation spectra of the different QD-dye systems from G(Dap)-ROX to GP20 (Dap)-ROX, where the dye and QD are in equimolar concentrations. The spectra are normalised to the dye peak at 560 nm. The black trace represents the theoretical maximum energy transfer situation where only 1 dye can bind to the donor and there is 100% energy transfer - in this scenario the excitation spectrum would be the same as the sum of the absorbance spectra of the QD donor and the dye acceptor, when normalised to the acceptor peak.



A 1.0

FRET efficiency

0.8

0.6

0.4 efficiency from lifetimes fit to Eq. (6) efficiency from PL fit to Eq. (6)

0.2

0.0 0

40

80

120

distance (Å)

B 1.0 Experimental data fit to Eq. (6) 0.8

FRET Efficiency

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0.6

0.4

0.2

0.0 2

3

4

5 6 7

0.1

2

3

1 [dye] : [QD]

Figure 6: A) Calculated FRET efficiency from either the excitation spectrum data using Eq. (10) (empty black squares) or the fluorescence lifetime measurements using Eq. (8) (empty red circles) to calculate Eobs in Eq. (6). The black and red dashed lines through each data set, represent best fits to the data using Eq. (6). The parameter λ was held constant at 1, with the fitted values for the steady state and time resolved data sets being; R0 = 79 Å, γ = 5.5 for the steady state data and R0 = 78, γ = 5.6). B) Calculated FRET efficiency from Excitation spectra for 4 different stoichiometries 0.1, 0.3, 1 and 3 equivalents of dye per QD (black open circles) and fit to Eq. (6) where R is set to 50.9 Å and λ is the independent variable. Fitting of the experimental data gives a γ value of 6.1 ±0.08. Error estimates for the efficiencies are one standard deviation, while distance estimates assume a 5% variation.


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A 2 95 Å 4

1

ln(1/Eobs - 1)

3

0 2

-1

PL (a.u.)

1

-2 0

-3

63 Å -1

4.0

4.2 ln(R)

4.4

4.6

B 2 1

ln(1/Eobs - 1)

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0 -1 Data !=4

-2

! = 5.6 !=6

-3

4.0

4.2

4.4

4.6

ln(R)

Figure 7: A) The quantity on the left, which is related to the observed efficiency is plotted with respect to the natural logarithm of the donor-acceptor separation. The data (empty circles) are found to be well fit (solid black line) using Eq. (6), which give a value for R0 of 79.0 ± 1.6 and γ = 5.6 ± 0.5 Å respectively. The grey shaded area represents 20% variation from the mean in the value of R, which would change the value of R0 (lower dashed line is 63 Å, upper trace is 96 Å) but would not change the value of γ . B) The data (empty circles) and fit to data (γ = 5.6, solid black line) compared with the case where γ = 6 and γ = 4.


Energy Transfer between Quantum Dots and Conjugated Dye

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