Self-Quenching, Dimerization, and Homo-FRET in Hetero-FRET

Article pubs.acs.org/JPCC

Self-Quenching, Dimerization, and Homo-FRET in Hetero-FRET Assemblies with Quantum Dot Donors and Multiple Dye Acceptors Erin M. Conroy, Jia Jun Li, Hyungki Kim, and W. Russ Algar* Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada S Supporting Information *

ABSTRACT: The combination of semiconductor quantum dots (QDs) and Förster resonance energy transfer (FRET) is a powerful tool for bioanalysis and imaging. Through FRET, the dye is able to borrow brightness from the QD, and the FRET efficiency can be tuned through the assembly of multiple acceptor dyes per QD. In principle, the fluorescence intensity from acceptor dyes assembled to a QD donor should always exceed that from the dyes alone, but we observed anomalously low acceptor dye fluorescence intensities in FRET systems with a QD donor and multiple Alexa Fluor 610 (A610) or Alexa Fluor 633 (A633) acceptors. In contrast, fluorescence from Alexa Fluor 555 (A555) or Alexa Fluor 647 (A647) acceptors was well-behaved and agreed with theoretical expectations. The difference between these systems was studied using a combination of UV−visible absorption and fluorescence intensity, lifetime, and anisotropy measurements. Anomalous fluorescence from A610 and A633 arose from the formation of nonfluorescent, H-type dimers of these dyes. The monomer−dimer equilibrium was shifted strongly in favor of the dimer as a result of the locally high concentration of dyes assembled to the QD. Both the lower number of monomeric dyes per QD and the introduction of a competitive energy transfer pathway from the QD to dimeric dyes contributed to the low dye fluorescence. Another consequence of the close proximity between the dyes was homo-FRET, which was particularly evident with A555 and A647 acceptors. Homo-FRET did not appear to lead to significant quenching of dye fluorescence, although there was some evidence of low-efficiency energy transfer to dyes that may act as modest energy sinks. The results of this study help inform the rational design of optimized QD−FRET probes for biological applications.

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emission.5 A third advantage arises from the ability of the QD to serve as a scaffold for the assembly of multiple acceptor dyes, which permits increases in the FRET efficiency through the introduction of multiple, approximately equivalent energy transfer pathways.5,8 The QD PL is progressively quenched as more acceptors are assembled, and ideally, FRET-sensitized acceptor dye emission progressively increases. Although typically referred to simply as FRET, the energy transfer between the QD and dye acceptors is more precisely referred to as hetero-FRET because it occurs between two different materials. Hetero-FRET is typically analyzed in terms of QD donor properties such as quenching of its PL emission intensity and decreases in its PL lifetime.8 The emission of QD− conjugated fluorescent dye acceptors is far less analyzed, despite the important role of acceptor emission in many applications of QDs and FRET.3,8−10 An unavoidable consequence of increasing the efficiency of hetero-FRET by assembling multiple dye acceptors per QD donor is that these multiple dye acceptors are brought in close proximity to one another. Given the nanometer-scale size of the QD, the proximity between dyes is also on the order of nanometers and may facilitate dye−dye interactions. Such dye−

INTRODUCTION Semiconductor quantum dot (QD) nanocrystals exhibit bright, spectrally narrow photoluminescence (PL) that can be tuned as a function of nanocrystal size and composition.1−4 These materials also possess large one- and two-photon absorption cross sections, have good quantum yields, and are resistant to photobleaching.2,5 Given these properties, QDs are of interest for a wide range of biological imaging and sensing applications, as summarized in several reviews.1,3,4,6,7 Within these applications, QDs are widely utilized as part of Förster resonance energy transfer (FRET) probes and sensors.8−10 The photophysics of energy transfer with QDs have been found to be consistent with the Förster mechanism,11−16 and this fundamental understanding has facilitated the development of conventional QD−FRET probes with a single donor−acceptor pair, as well as more complex QD−FRET probes with multiple donor−acceptor pairs.10,17 Although QDs can function as acceptors for select donor materials such as luminescent lanthanide complexes and bioluminescent or chemiluminescent substrates,18−20 QDs most commonly function as donors for fluorescent dye and protein acceptors.8−10 Two advantages of QDs as FRET donors are the flexibility to excite the QD at a wavelength that minimizes direct excitation of the acceptor dye, and the ability to optimize the spectral overlap between QD emission and acceptor dye absorption without introducing problematic crosstalk between their © XXXX American Chemical Society

Received: June 10, 2016 Revised: July 9, 2016

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DOI: 10.1021/acs.jpcc.6b05886 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

critically important for the rational design and optimization of QD−FRET probes for biological applications.

dye interactions are often associated with self-quenching of dye fluorescence, the main symptom of which is minimally increasing or even decreasing dye fluorescence intensity as the number of dye molecules increases.21,22 Self-quenching is known to occur with some multiply labeled protein−dye conjugates,21,22 where the proteins, like QDs, act as nanoscale scaffolds to colocalize multiple dye molecules. Commonly cited mechanisms of self-quenching include FRET between the likedye molecules,22−25 referred to as either homo-FRET or energy migration FRET (emFRET),26−29 and the formation of nonfluorescent dimers or larger dye aggregates.30−37 Compared to hetero-FRET, homo-FRET and dimer formation between dye molecules in configurations with a QD donor and multiple dye acceptors has been largely overlooked. Here, we report a spectroscopic investigation of the selfquenching of fluorescent dyes when multiple copies of that dye are conjugated to a QD through peptide linkers. These model systems are illustrated in Figure 1, where the designed

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EXPERIMENTAL SECTION Materials. CdSeS/ZnS QDs in toluene were obtained from Cytodiagnostics (Burlington, ON, Canada) and CdSe/CdS/ ZnS QDs were synthesized using standard hot solvent methods.38,39 These hydrophobic QDs were transferred to aqueous solution through ligand exchange with glutathione as described previously.40 Alexa Fluor 555 C2 maleimide, Alexa Fluor 610-X succinimidyl ester, Alexa Fluor 633 C5 maleimide, and Alexa Fluor 647 C2 maleimide were from Thermo-Fisher Scientific (Burlington, ON, Canada). Peptides were from BioSynthesis, Inc. (Lewisville, TX). These peptides were labeled with the reactive fluorescent dye derivatives, and the labeled peptides were purified by affinity chromatography and desalting. The peptides are denoted as Pep(dye). Additional details and peptide sequences can be found in the Supporting Information. All other reagents were from Sigma-Aldrich (Oakville, ON, Canada) and used as received unless otherwise noted. QD−Peptide Conjugates. Individual samples of QD− [Pep(dye)]n conjugates were prepared by mixing 8−20 pmol of QDs with the desired equivalents of dye-labeled peptide. A polyhistidine sequence in the peptide bound to the surface of the QDs with nanomolar affinity.41,42 This assembly has been shown to be effective in aqueous buffer, and in aqueous buffer with a high percentage of DMSO cosolvent.43 Solutions were diluted to 80−110 μL using borate buffer (50 mM, pH 8.5, 10 mM NaCl) and stood at room temperature for 30−60 min prior to measurements. The final concentrations of conjugates were typically between 0.1 and 0.2 μM. Batch preparation of like conjugates for N measurements was done similarly by scaling quantities by a factor of N. Control samples of n × Pep(dye) were always measured in parallel, in part as a guard against trivial reasons for decreases in fluorescence intensity (e.g., inner filter effects). Absorption and Fluorescence Measurements. Absorption spectra, and fluorescence emission and excitation spectra, were measured with an Infinite M1000 Pro plate reader (Tecan US Inc., Morrisville, NC). Excitation light was from a xenon flash lamp with a double-monochromator for wavelength selection. Excitation and emission bandwidths were ∼5 nm and spectra were collected with a 2 nm step size unless otherwise noted. Fluorescence emission anisotropy was also measured with the Infinite M1000 Pro plate reader using a fluorescence polarization module. For A555, excitation was at 530 nm (∼5 nm bandwidth) and emission was measured at 570 nm (∼5 nm bandwidth). For A647, A633, and A610, excitation was at 590 nm (∼5 nm bandwidth) and emission at 670, 650, or 630 nm, respectively (∼20 nm bandwidth). Time-Resolved Fluorescence. Emission lifetimes were measured using a picosecond laser and streak camera system. Excitation pulses were generated using an EKSPLA PL2241 Nd:YAG cavity dumped laser with a 355 nm wavelength, 35 ps pulses, and 25 mJ output energy. The laser output was tuned between 600 and 630 nm using a picosecond optical parametric generator (EKSPLA Model PG401). Lifetime data were collected using an Acton spectrometer (Princeton Instruments, NJ) and a Hamamatsu streak camera set to a 10 or 20 ns time window. Photobleaching. Photobleaching was done on an IX83 inverted epifluorescence microscope (Olympus, Richmond Hill,

Figure 1. Schematic of a multivalent QD−[Pep(dye)]n conjugate that depicts four photophysical processes that can be observed with these materials: (A) hetero-FRET from the QD donor to the fluorescent dye acceptor; (B) homo-FRET between fluorescent dyes; (C) interactions between dyes to form nonfluorescent dimers; (D) hetero-FRET from the QD donor to the nonfluorescent dimers.

interaction is hetero-FRET from the QD donor to the dye acceptor. However, self-quenching leads to anomalous heteroFRET-sensitized emission from dyes in QD conjugates that, despite the strong light absorption and antennae role of the QD, is less bright than directly excited emission from equivalent amounts of dye in bulk solution. The mechanisms responsible for this anomalous emission are probed through measurement of UV−visible absorption and fluorescence intensity, lifetime, and anisotropy with a variety of QD− peptide−dye conjugates. The conjugates are selected from seven colors of CdSe/CdS/ZnS and alloyed CdSeS/ZnS QDs, and a group of fluorescent dyes that include Alexa Fluor 555 (A555), Alexa Fluor 610 (A610), Alexa Fluor 633 (A633), and Alexa Fluor 647 (A647). A primary mechanism of selfquenching is found to be the formation of nonfluorescent dimers, aided by the locally high concentration of dye molecules at the interface of the QD. Evidence for homoFRET between dye molecules was also observed independent of nonfluorescent dimer formation, and additional inadvertent energy transfer pathways can be inferred in configurations with nonfluorescent dimers. A general understanding of potential self-quenching mechanisms and inadvertent energy transfer pathways in QD donor-based hetero-FRET assemblies is B


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The Journal of Physical Chemistry C ON, Canada) with MetaFluor software (Molecular Devices, Sunnyvale, CA). QD−[Pep(A647)]16 conjugates (80 μL, 0.1 μM) were placed in a well of a clear-bottom 386-well plate (Nunc, Thermo-Fisher Scientific, Mississauga, ON, Canada). A 4.0× magnification (NA = 0.16) objective lens was used for illumination and imaging of the wells. The microscope was equipped with an X-Cite 120XL metal−halide light source (Excelitas Technologies, Mississauga, ON, Canada). The excitation filter was 405/20 and paired with a 510 nm cutoff dichroic mirror (Chroma Technology Corp., Bellows Falls, VT). The illumination power exiting the objective lens was ∼56 mW. The emission from the samples was measured in real time using a Green Wave fiber-coupled CCD spectrometer (StellarNet Inc., Tampa, FL). A long-pass filter with a 530 nm cutoff wavelength was used to block excitation light from reaching the spectrometer. Illumination was interrupted and anisotropy measurements were made at several intervals (approximately 1, 2, 5, 7, 10, 15, 20, and 30 min). Enzymatic Hydrolysis. QD−[Pep(A647)]16 conjugates (0.2 μM) were prepared, and 40 μL was transferred to a microtiter plate well. A solution of trypsin (2 nM, 40 μL) in borate buffer (100 mM, pH 9.2) was added to the same well and the fluorescence anisotropy of the acceptor dye was measured at 1 min intervals for 90 min. Control samples with only dye-labeled peptide and with the addition of only buffer (rather than trypsin solution) were measured in parallel. Data Analysis. In FRET experiments, relative PL intensities were calculated by normalizing the QD and dye peak PL intensities to the PL intensity of the QD donor alone (i.e., without any assembled dye-labeled peptide). With QD− [Pep(dye)]n samples, the dye PL intensity measured from the emission peak was corrected for the difference in area between its emission spectrum and that of the QD, such that the relative PL intensities reflect the total number of photons emitted. FRET efficiencies were calculated from quenching of the peak QD PL intensity according to eq 1, where IQD,n is the measured peak QD PL intensity with an average of n Pep(dye) assembled per QD, and IQD,0 is the intensity when n = 0. Additional details regarding data analysis can be found in the Supporting Information. E = 1 − (IQD, n)IQD,0−1

Figure 2. Normalized absorption and emission spectra for the (A) QDs and (B) fluorescent dyes utilized in this study. The dye absorption and emission spectra are normalized to their respective maxima. The QD emission spectra are normalized to their maxima, whereas the absorption spectra are normalized to the first exciton peaks. See Table 1 for a summary of photophysical properties.

Table 1. Selected Optical Properties of the QDs and Dyes Used in This Study

(1)

With direct excitation of dye fluorescence, PL intensity data were normalized to the intensity for the n × Pep(dye) samples with the highest value of n.

QD

λfirst (nm)a

εfirst (M−1 cm−1)b

QD525 QD530a QD540a QD550 QD575 QD600 QD650 dye

500 506 518 526 554 584 632 (nm)e

57 800 210 000 210 000 71 600 108 000 232 000 870 000 (M−1 cm−1)f

A555 A610 A633 A647

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RESULTS Materials and Optical Properties. Figure 2A shows the normalized absorption and emission spectra for various CdSe/ CdS/ZnS QDs (QD525, QD550, QD575, QD600, QD650) and alloyed CdSeS/ZnS QDs (QD530a, QD540a), and Figure 2B shows the normalized absorption and emission spectra for fluorescent dyes that were assembled to these QDs through peptide linkers. These dyes included A555, A610, A633, and A647. Of the Alexa Fluor dyes, only the structures of A610 and A647 are openly available. A610 is a sulfonated and halogenated dye with a core rhodamine structure, and A647 is a sulfonated derivative of Cyanine 5 (Supporting Information, Figure S1). It is anticipated that Alexa Fluor 633 is also a rhodamine derivative, and that A555 is a sulfonated derivative of Cyanine 3.

λAbs

552 610 630 650

εmax

155 000 144 000 159 000 270 000

λPL (nm)c

λPL

Φd

524 528 540 548 574 602 648 (nm)c

0.54 0.46 0.48 0.31 0.40 0.53 0.41 Φd

566 630 652 670

0.18 0.53 0.59 0.33

Wavelength of the first exciton peak. bApproximate molar absorption coefficient at the first exciton peak. cWavelength of peak PL emission. d Approximate PL quantum yield. eWavelength of the absorption peak. f Peak molar absorption coefficient. a

Near-Ideal FRET-Sensitized Dye Emission. The brightness of an isolated fluorescent dye is determined by its absorption coefficient and its quantum yield. If that dye is paired as an acceptor with a FRET donor, such as a QD, then the intensity of the dye emission is expected to increase because it retains its direct excitation and gains an additional excitation pathway through FRET from the QD. Equation 2 is an approximation of the acceptor dye emission intensity, IA, where ΦA is the quantum yield of the dye acceptor, εA and εQD are the C


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Figure 3. (A) Model data from eqs 2 and 3 for the total dye acceptor PL intensity. The plots also show the individual contributions from direct excitation and FRET-sensitization from a QD donor. FRET is calculated for r/R0 = 1.25. (B) QD525−[Pep(A555)]n conjugates: (i) representative PL spectra of QD525−[Pep(A555)]n conjugates and parallel n × Pep(A555) control samples; (ii) changes in QD PL intensity (top, main), FRET efficiency (top, inset), and A555 PL intensity (bottom) as a function of excitation wavelength. The estimated εQD525/εA555 ratios were ca. 100, 64, and 11 at 300, 350, and 450 nm, respectively. (C) QD575−[Pep(A610)]n conjugates: (i) representative PL spectra of QD575−[Pep(A610)]n conjugates and parallel n × Pep(A610) control samples; (ii) changes in QD PL intensity (top, main), FRET efficiency (top, inset), and A610 PL intensity (bottom) as a function of excitation wavelength. The estimated εQD575/εA610 ratios were ca. 48 and 99 at 350 and 450 nm, respectively. (D) QD600−[Pep(A633)]n conjugates: (i) representative PL spectra of QD600−[Pep(A633)]n conjugates and parallel n × Pep(A633) control samples (the insets show close-ups of the dye PL); (ii) changes in QD PL intensity (top, main), FRET efficiency (top, inset), and A633 PL intensity (bottom) as a function of excitation wavelength. The estimated εQD600/εA633 ratios were ca. 54, 300, and >104 at 300, 400, and 450 nm, respectively. The legend applies to all panels labeled (ii), where a color/symbol combination denotes the excitation wavelength, and the solid and open symbols represent QD−[Pep(dye)]n conjugates and n × Pep(dye) control samples, respectively.

donor−acceptor distance of r/R0 = 1.25. The curves for εQD/εA = 100 clearly show how a large εQD and nontrivial E can increase the observed brightness of a dye. In all cases, the dye PL from a QD−[Pep(dye)]n conjugate with FRET is predicted to be brighter than an equivalent amount of Pep(dye) without QDs and FRET. Figure 3B shows representative PL emission spectra and relative QD525 and A555 emission intensities for QD525[Pep(A555)]n conjugates at various excitation wavelengths. As expected, the relative QD PL intensity (normalized to n = 0) and the corresponding ensemble FRET efficiency were independent of the excitation wavelength. In contrast, the difference between the A555 PL intensities for QD525− [Pep(A555)]n conjugates and corresponding n × Pep(A555) samples varied with excitation wavelength, which was the effect of varying εQD/εA. For excitation at 300, 350, and 450 nm, the experimental trends were similar to the ideal trends in Figure 3A. The A555 emission for the QD525−[Pep(A555)]n conjugates increased in parallel with n × Pep(A555) samples.

molar absorption coefficients of the acceptor dye and QD donor, respectively, n is the ensemble averaged number of acceptors per QD donor, E is the ensemble FRET efficiency, and C is a proportionality constant that accounts for instrument parameters, the optical path length, and the concentration of QD donors. The ensemble FRET efficiency, E, is defined by eq 3, where r is the average distance between the QD donor and acceptor dye, and R0 is the Förster distance for the QD−dye FRET pair. IA = C ΦA (nεA + εQDE(n))

(2)

E(n) = nR 0 6(r 6 + nR 0 6)−1

(3)

Plots of eq 2 for εQD/εA ratios of 10 and 100 are shown in Figure 3A, and additional plots for 1 and 1000 can be found in the Supporting Information (Figure S2). In these simulations, the FRET efficiency varies from ∼20% for one acceptor per QD up to ∼80% for 15 acceptors per QD, which is typical for many QD−dye hetero-FRET systems and corresponds to an average D


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Figure 4. (A) QD525-[Pep(A610)]n conjugates: (i) representative PL spectra of QD525−[Pep(A610)]n and parallel n × Pep(A610) control samples (insets); (ii) changes in QD PL intensity (main panel), FRET efficiency (inset), and (iii) A610 PL intensity as a function of excitation wavelength. The estimated εQD525/εA610 ratios were ca. 70, 24, and 68 at 300, 350, and 450 nm, respectively. Analogous data for (B) QD530a− [Pep(A610)]n conjugates and n × Pep(A610) control samples. The estimated εQD530a/εA610 ratios were ca. 84, 100, and 174 at 300, 350, and 450 nm, respectively. The legend applies to all panels (ii)−(iii), where a color/symbol combination denotes the excitation wavelength, and the solid and open symbols represent QD−[Pep(A610)]n conjugates and n × Pep(A610) control samples, respectively.

and as with A610, the trend in the A633 PL intensity was nonmonotonic, decreasing for n > 10. In this case, the A633 PL intensity from the QD600−[Pep(A633)]n conjugates intersected and fell below the A633 PL intensity for the corresponding n × Pep(A633) samples. For excitation at 300 and 400 nm, the intersection points were ca. n = 15 and n = 25, respectively. An intersection point was not observed for excitation at 450 nm but could be extrapolated to n > 40. As with A610, the value of n at the intersection point increased as εQD/εA increased. When the A633 was directly excited at 615 nm without excitation of the QD600, large negative deviations in the A633 PL intensity for the QD600−[Pep(A633)]n conjugates versus n × Pep(A633) samples were observed for n ≥ 8. The nonmonotonic trends in A610 and A633 PL intensity with QD−[Pep(dye)]n conjugates were anomalous with respect to eqs 2 and 3, which predict a steady increase in dye PL intensity with increasing n, as was observed with QD− [Pep(A555/A647)]n conjugates. The results with A610 and A633 suggested a QD-induced mechanism of dye selfquenching. The results also suggested that larger values of εQD/εA should partially mitigate the effects of self-quenching by increasing the value of n at which directly excited dye PL (without the QD) exceeds the PL with FRET-sensitization (on the QD). Verification of the Effect of εQD/εA. The value of εQD/εA is controlled by the selection of QD donor, dye acceptor, and excitation wavelength. In Figure 3, the value of εQD/εA was altered by varying the excitation wavelength. Alternatively, εQD/ εA can be changed by varying the QD donor, as shown in Figure 4, where εQD/εA was altered by replacing QD525 with QD530a in QD−[Pep(A610)]n conjugates. The alloyed-CdSeS core QD530a had a 3.6-fold larger εQD (first exciton peak) than their binary CdSe core QD525 counterparts but had a similar emission spectrum. For both the binary and alloyed QDs, the quenching of QD PL and the ensemble FRET efficiency were again independent of excitation wavelength. With binary QD525, the trend in A610 fluorescence intensity with increasing n was anomalous, with the intensity for the QD conjugates leveling off or slightly decreasing at n ≥ 10, and intersecting with directly excited fluorescence from n × Pep(A610) samples at n ∼ 13, 14, and 16 for excitation

Indeed, the best-fit curve for the QD525−[Pep(A555)]n data in Figure 3B had a linear component with a slope equal to that for the linear trend for n × Pep(A555). The only indication of nonideal behavior was a small negative deviation for the QD525−[Pep(A555)]n conjugates relative to the n × Pep(A555) samples at large values of n for excitation at 560 nm, which directly excited the A555 without exciting the QD525. Analogous results were observed with QD550 and alloyed QD530a and QD540a paired with A555 (Supporting Information, Figure S3). Experiments were also done with QD600−[Pep(A647)]n conjugates and n × Pep(A647), and the trends in the A647 PL intensity also agreed with the expectations of eq 2 (Supporting Information, Figure S4). QD−A555 and QD−A647 FRET pairs thus behaved as expected. Anomalous FRET-Sensitized Dye Emission. Figure 3C shows representative PL emission spectra and relative QD575 and A610 emission intensities for QD575−[Pep(A610)]n conjugates at various excitation wavelengths. These data had both similarities and differences when compared to the QD− A555/A647 FRET-pair data. As before, the relative QD PL intensity and ensemble FRET efficiency were independent of excitation wavelength, in agreement with expectations. However, the trends in the A610 PL intensity for QD575− [Pep(A610)]n conjugates were nonmonotonic, increasing and then decreasing for n > 8 in a manner where the intensities converged with those for the n × Pep(A610) samples at larger values of n. For excitation at 350 nm, this convergence was at ca. n = 15, whereas for excitation at 450 nm, where εQD/εA was larger, this convergence was extrapolated to n > 20. When the conjugates were excited at 560 nm, which directly excited the A610 without exciting the QD575, significant negative deviations in the A610 PL intensity for the QD575− [Pep(A610)]n conjugates versus n × Pep(A610) samples were observed at n ≥ 10. Similar results were observed for A610 with QD525 and QD550 samples (vide inf ra and see Supporting Information, Figure S5). Figure 3D shows representative spectra and relative QD600 and A633 emission intensities for QD600−[Pep(A633)]n conjugates and n × Pep(A633) samples at various excitation wavelengths. Again, the relative QD PL intensity and ensemble FRET efficiency were independent of excitation wavelength, E


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The Journal of Physical Chemistry C wavelengths of 350, 300, and 450 nm. With alloyed QD530a, the A610 PL intensity again leveled off and slightly decreased at n ≥ 10, but it nonetheless remained higher than the directly excited A610 fluorescence from n × Pep(A610) samples, and no intersection could be reliably extrapolated (n > 20). Analogous contrast was observed between QD550−[Pep(A610)]n and QD540a−[Pep(A610)]n conjugates (Supporting Information, Figure S5). These results confirmed the role of εQD/εA in eq 2 and the ability of large εQD/εA to at least partially mitigate the effects of self-quenching. Mechanism of Self-Quenching. To investigate the mechanism(s) of self-quenching, additional spectroscopic measurements were made on various QD−[Pep(dye)] n conjugates. Working from the hypothesis that self-quenching originated from the formation of nonfluorescent complexes, homo-FRET, or a combination of these two processes, these additional measurements included UV−visible absorption and fluorescence excitation, lifetime, and anisotropy. Ground-State Absorption. The UV−visible absorption spectrum of selected QD−[Pep(dye)]n conjugates was measured and compared to that of n × Pep(dye) samples. As shown in Figure 5A, the absorption spectrum of A555 in QD525−[Pep(A555)]10 conjugates was nearly identical to the spectra for samples of 10 × Pep(A555), and analogous results were obtained with QD530a and QD575 assembled with Pep(A555). Similarly, no change in the absorption spectrum of A647 was observed between QD600−[Pep(A647)]24 conjugates and 24 × Pep(A647) (Supporting Information, Figure S6). These results were consistent with the minimal selfquenching of these dyes in QD−[Pep(dye)]n conjugates. In contrast to the results with A555 and A647, the absorption spectra of A610 and A633 were different between QD− [Pep(dye)]n and n × Pep(dye) samples. With both Pep(A610) and Pep(A633) assembled to QDs, the hypsochromic shoulder of the dye spectrum intensified to the point of becoming the most prominent peak, as shown in Figure 5A with QD525− [Pep(A610)]n and QD600−[Pep(A633)]n. Analogous results for A610 were also obtained with QD530a and QD575 (Supporting Information, Figure S6). The absorption spectra of a series of different n for QD525−[Pep(A610)]n and QD600− [Pep(A633)]n conjugates was measured and, as summarized in Figure 5B, the intensity of the hypsochromic shoulder/peak increased in parallel with n (Supporting Information, Figures S7−S8 for full spectra). This ratio increased much more modestly for only n × Pep(A633) (Supporting Information, Figures S8−S9). These observed changes in the shape of the absorption spectra were characteristic of the formation of dye dimers. As the dimerization of dyes is driven, in part, by low solubility of the dye monomer, the absorption spectrum for QD525−[Pep(A610)]10 was compared between buffer and buffer with 25% v/v DMSO, where DMSO was an effective cosolvent for the dye. The intensity of the hypsochromic shoulder decreased with the added DMSO and the normal absorption peak recovered (Supporting Information, Figure S10). Results similar to those with A610 were obtained for QD600−[Pep(A633)]n conjugates. Figure 5B shows that the shoulder/peak absorbance ratios for A633 stayed relatively constant with 20% v/v DMSO and a series increasing n. The effect of DMSO as a favorable cosolvent was consistent with the formation of A610 and A633 dimers with increasing amounts of Pep(dye) assembled to the QD.

Figure 5. (A) Absorption spectra of QD−[Pep(dye)]n and n × Pep(dye): (i) QD525, A555 and n = 10; (ii) QD525, A610 and n = 10; (iii) QD600, A633 and n = 24. The QD contribution to the absorption spectrum has been subtracted from the main panels but is shown in the insets. (B) Summary of changes in the absorption spectra of Q525−[Pep(A610)]n and QD600−[Pep(A633)]n with increasing n, plotted as the ratio of the relative absorbance for the shoulder versus the original dye peak (566 nm/610 nm for A610, 582 nm/628 nm for A633). Data are shown for A610 and A633 in aqueous buffer, and for A633 in aqueous buffer with 20% v/v DMSO added. Data for n × Pep(A633) can be found in the Supporting Information and have trends similar to that for buffer with DMSO.

Fluorescence Excitation. Given the results of the groundstate absorption measurements, fluorescence excitation spectra were measured for A610 and A633. As shown in Figure 6A, the excitation spectrum for A610 in a QD525−[Pep(A610)]10 conjugate was significantly less intense than the spectrum for a control sample with 10 × Pep(A610). Normalization revealed that the excitation spectra had the same shape regardless of the difference in intensity, suggesting that the A610 dimers were either nonfluorescent or, at most, very weakly fluorescent. The excitation spectrum of QD600−[Pep(A633)]30 conjugates was also measured and compared to that for 30 × Pep(A633). Figure 6B shows that the excitation spectrum of this conjugate had a primary component associated with direct excitation of the A633 and a secondary component associated with FRETsensitization through the QD donor. As with the A610, the A633 component was less intense with the QD600−[Pep(A633)]30 conjugate than with 30 × Pep(A633) but did not vary in shape. The effect of adding DMSO to the buffer was again investigated, and it had the effect of restoring the intensity of the A610/A633 excitation spectrum for QD−[Pep(A610/ F


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Figure 6. PL excitation spectra for QD−[Pep(dye)]n conjugates versus n × Pep(dye) samples without QDs: (A) QD525 with Pep(A610) and n = 10; (B) QD600 with Pep(A633) and n = 30. Both insets show the PL excitation spectra in the main panels normalized to their maxima.

A633)]n conjugates to a level that was only slightly less than the corresponding n × Pep(A610/A633) sample (Supporting Information, Figures S10−S11). These cumulative results were consistent with the absorption spectra results and confirmed the nonfluorescent character of the formed dimers. Time-Resolved Fluorescence. To further investigate selfquenching, time-resolved fluorescence measurements were made on QD525−[Pep(dye)]n samples. As it is known that polyhistidine-terminated peptides self-assemble to QDs according to a Poisson distribution,14 we prepared samples for timeresolved measurements with two values of n. The first value, n = 20, corresponded to the observation of significant selfquenching with A610 and A633. The second value, n = 0.1, corresponded to an ensemble where >99% of the individual QDs were expected to be assembled with either none or only one Pep(dye). With n = 0.1, and with direct excitation of the dye at a wavelength that minimally excited QD PL, the lifetime of the dye assembled to the QD could be determined without influence from significant nearest-neighbor or self-quenching interactions. Figure 7 shows the PL decay curves for QD525− [Pep(dye)]n and n × Pep(dye) samples, where n = 0.1 or 20 and the dyes were A610, A633, and A647. In all cases, the dye PL lifetimes were practically indistinguishable between QD525−[Pep(dye)]0.1, 0.1 × Pep(dye), and 20 × Pep(dye) samples for all three dyes, and only a small decrease (8−15%) was observed for the QD525−[Pep(dye)]20 samples. The minimal change in dye fluorescence lifetime was consistent with self-quenching of A610 and A633 via the formation of nonfluorescent dimers, and the small decrease in lifetime for all three QD520−[Pep(dye)]20 samples is discussed later. Fluorescence Anisotropy. The results presented in the foregoing sections indicated the formation of nonfluorescent dimers but did not address the possibility of homo-FRET. To investigate the possibility of homo-FRET within QD−[Pep(dye)]n conjugates, fluorescence anisotropy measurements were made. To avoid artifacts and unnecessary complications in the analysis, the conjugates were designed and interrogated in a manner that directly excited dye fluorescence and

Figure 7. Normalized PL decay curves and lifetimes for QD525− [Pep(dye)]n conjugates and n × Pep(dye) samples with n = 0.1 and n = 20 for (A) A610, (B) A633, and (C) A647. Two lifetimes, τ, are shown in each panel: the dye lifetime for the QD525−[Pep(dye)]20 conjugate (red label; the uncertainty is from the fit to the decay curve) and the average lifetime that represents the QD525−[Pep(dye)]0.1, 0.1 × Pep(dye), and 20 × Pep(dye) samples (black label; the uncertainty is one standard deviation of the average). The dashed gray line in each panel is the instrument response function. The UV−visible absorption spectra of these samples were also measured to confirm dimerization (A610, A633) or no dimerization (A647).

minimized the opportunity for hetero-FRET between the QD and dye. The first two systems were (i) QD650−[Pep(A555)]n conjugates, interrogated with an excitation wavelength of ∼530 nm, and (ii) QD530a−[Pep(A647)]n and QD525−[Pep(A647)]n conjugates, interrogated with excitation wavelength of ∼590 nm. A555 and A647 were selected for initial measurements because of their apparent resistance to selfquenching via dimerization. With the QD530a/QD525− [Pep(A647)]n conjugates, hetero-FRET was minimized because there was only small spectral overlap between the QD530a/ QD525 emission and A647 absorption, and the excitation wavelength was longer than the first exciton peak in the absorption spectrum of the QD530a/QD525, thus minimizing formation of excited-state QD donors. With the QD650− [Pep(A555)]n conjugates, there was no spectral overlap between the QD650 emission and dye absorption. Although there was overlap between the A555 emission and QD650 absorption, QDs are poor FRET acceptors.44 Figure 8A(i-ii) shows changes in dye fluorescence anisotropy associated with QD525−[Pep(A647)]n , QD530a−[PepG


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Figure 8. (A) Change in the dye fluorescence anisotropy for QD−[Pep(dye)]n and n × Pep(dye) samples with increasing n for (i) A647, (ii) A555, (iii) A610, and (iv) A633 assembled to various QDs. (B) Change in the A647 fluorescence anisotropy for QD530a-[Pep(A647)]16 conjugates and 16 × Pep(A647) control samples as a function of time, with and without trypsin (TRP) added. The trypsin hydrolyzes the peptide. (C) Change in A647 fluorescence anisotropy for QD530a-[Pep(A647)]16 conjugates with progressive photobleaching of the A647, tracked through the A647/QD530a PL ratio.

(A647)]n, and QD650−[Pep(A555)]n as a function of n. Also shown are analogous data for control samples with n × Pep(dye). Total dye fluorescence was also estimated from the anisotropy experiments and was used to confirm self-quenching (or the lack thereof) in the samples (Supporting Information, Figures S12 and S13). For both the A555 and A647, the fluorescence anisotropy of the Pep(dye) samples was approximately constant, whereas the fluorescence anisotropy for the QD−[Pep(dye)]n samples progressively decreased as n increased, which was consistent with homo-FRET driven by the close proximity between the dyes in the QD conjugates. To further confirm the relationship between n and the dye fluorescence anisotropy, additional experiments were done with QD530a−[Pep(A647)]n conjugates. In one experiment, QD530a−[Pep(A647)]16 conjugates and 16 × Pep(A647) were subjected to proteolytic digestion with trypsin, which changed the number of A647 per QD without changing the number of bound peptides (albeit with changes in peptide length). Figure 8B shows the change in the A647 fluorescence anisotropy as a function of time after adding trypsin. The anisotropy remained constant for the Pep(A647) alone, whereas the A647 anisotropy for the QD530a−[Pep(A647)]16 sample progressively increased as trypsin cleaved an A647labeled fragment of the peptide from the QD. This result was also consistent with homo-FRET. In a third experiment, the A647 in QD530a−[Pep(A647)]16 conjugates was gradually photobleached, thus changing the number of A647 per QD without any change in the peptides. Photobleaching was tracked through changes in the A647/QD PL ratio (with excitation at 400 nm to induce a small amount of heteroFRET) and the A647 fluorescence anisotropy was measured at regular intervals, as shown in Figure 8C. The anisotropy decreased approximately linearly with changes in A647/QD PL ratio, whereas the A647 fluorescence anisotropy of QD530a− [Pep(A647)]16 control samples that were not subject to photobleaching remained approximately constant. Overall, these results strongly suggested that homo-FRET occurred in QD−[Pep(A555/A647)]n conjugates, and that the possibility

of homo-FRET in self-quenched systems with A610 and A633 should be evaluated. Figure 8A(iii) shows changes in the directly excited dye fluorescence anisotropy for QD525−[Pep(A610)]n and QD530a−[Pep(A610)]n conjugates. The anisotropy decreased slightly up to n = 6 before increasing back to the anisotropy of the Pep(A610) alone. Figure 8A(iv) shows the results of an analogous experiment with QD530a−[Pep(A633)]n conjugates. Again, there was a slight initial decrease in the anisotropy up until approximately n = 6 or 8, but no further changes in the case of A633. Although not conclusive, these results suggested the potential for homo-FRET between A610/A633 in QD− [Pep(A610/A633)]n conjugates, but with interference from the formation of nonfluorescent dimers.

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DISCUSSION Nonfluorescent Dimer Formation. The ground-state absorption spectra of A610 and A633 associated with QD− [Pep(dye)]n conjugates progressively changed in a manner consistent with the formation of nonfluorescent dimers as n increased. The intensity of the main absorption band decreased while the relative intensity of the hypsochromic shoulder increased and became a defined and more intense peak, which is characteristic of H-dimers or H-aggregates.45 A decrease in the intensity of the fluorescence excitation spectra of these dyes was also observed, but without a concomitant change in spectral shape, confirming that the dimers were effectively nonfluorescent. The efficient formation of aggregates larger than dimers was likely precluded by steric effects associated with conjugation of the dyes to peptides (that were themselves assembled to the QDs), albeit that dimers are also the dominant form of aggregate for most nonbioconjugated dyes in bulk solution. Dimer formation has been reported in the literature for a variety of dyes, including xanthene dyes,33,36 BODIPY dyes,32,34 and cyanine dyes.46,47 The increasing formation of dimers as n increases for QD−[Pep(dye)]n conjugates is analogous to the well-characterized increase in dimer formation as free dye H


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QD to nonfluorescent dimeric dye acceptors. The dimer absorption spectrum maintains spectral overlap with the QD. Reduction of the monomeric acceptor dye fluorescence in hetero-FRET pairs will thus be associated with two processes: (i) a decrease in hetero-FRET efficiency because of the reduced number of monomeric acceptors and (ii) a decrease in heteroFRET due to the competition between the remaining monomeric dyes and the new dimeric dyes to accept energy from the same QD donor. The pathway in (ii) is indicated by the progressive and monotonic increase in QD quenching efficiency as n increases, despite the formation of nonfluorescent dimers. The hyposchromic shift in absorption from monomeric dye to dimeric dye will increase the qualitative spectral overlap with the QD donor and help offset any decrease in the molar absorption coefficient. Equations 2 and 3 can thus be modified to eqs 4 and 5, respectively, where n = m + d and r is taken to be consistent between monomer and dimer.

concentration increases. The assembly of multiple dyes per QD results in a high local concentration of that dye, and the greater extent of dimer formation observed with QD−[Pep(dye)]n conjugates versus n × Pep(dye) control samples indicates that this high local concentration has the effect of shifting the monomer−dimer equilibrium toward the dimer. The ability of a DMSO cosolvent to reduce dimer formation is consistent with greater solubility for the monomer and corresponding shift in the equilibrium toward the monomer. The factors that are likely to affect dimer formation include the structure of the fluorescent dye, its aqueous solubility, and the character of the linkers between the dyes and QD. As an example of the latter, the peptides in this study were relatively flexible and significant dimer formation was observed with Pep(A633) assembled to QDs, and to some extent, with increasing concentration in solution. In contrast, we observed no indications of dimer formation with A633-labeled oligonucleotides in a previous study,48 where dimerization was likely prevented by the electrostatic repulsion and steric hindrance between oligonucleotides. Regarding the structure of the dye, we observed that A555 and A647 (putative sulfonated cyanine dyes) were minimally susceptible to dimerization, whereas A610 and A633 (putative rhodamine derivatives) were very susceptible. We also observed anomalous rhodamine B (RhB) emission in QD− [Pep(RhB)]n conjugates (data not shown) that was consistent with the trends for A610 and A633, and expected given the well-known formation of nonfluorescent RhB dimers.30,36,37,49 Although it may appear that rhodamine-type acceptor dyes should be avoided in favor of cyanine dyes, we do not posit that the latter family of dyes uniformly avoid self-quenching through dimerization. Candidate acceptor dyes should be considered case-by-case. The dimerization of RhB and other xanthene dyes has been attributed to a combination of attractive van der Waals interactions between dye monomers and more favorable interactions of the dimer with solvent and ions (both factors can overcome repulsive electrostatic interactions between monomers).50 Moreover, the tendency of RhB derivatives to dimerize has been greatly reduced by modification with multiple alkyl sulfonate appendages,50 analogous to the appendages on A647. The use of hydrophilic dyes is thus likely to be the best defense against dimerization in multivalent dye assemblies with QDs and other NP scaffolds. Regarding shifts in monomer−dimer equilibria, the Pep(dye) assembled to a QD can be approximated to occupy a spherical volume, Vconjugate, with a radius approximately equal to the sum of the radius of the QD and the contour length of the peptide linker. As a first approximation, the dimer−monomer ratio can be argued to be proportional to the bulk solution-phase dimerization equilibrium constant, K = [D]/[M]2, as d/m2 ≈ K/NAVconjugate, where [D] and [M] are the bulk concentrations of dimer and monomer, d and m are the number of dimers and monomers per QD, and NA is Avogadro’s number. For n = 10 Pep(dye) per QD with [QD] ≈ 10−7 M, and Vconjugate defined by a radius of 10 nm (∼10−21 L), the effective concentration of Pep(dye) is on the order of 10−3 M, with a corresponding bulk concentration (i.e., without QD assembly) of 10−6 M. Although this first approximation does not account for many factors, it serves to illustrate how the assembly of multiple peptides per QD can promote dimerization through a high local concentration. A consequence of the formation of nonfluorescent dimers is that there will be two hetero-FRET pathways with a QD donor: (i) from the QD to a monomeric dye acceptor and (ii) from the

IA = C ΦM (mεM + εQDE(m))

(4)

E(m) = mR 0,M 6(r 6 + mR 0,M 6 + dR 0,D6)−1

(5)

Anisotropy, Homo-FRET, and Energy Migration. The observation of homo-FRET between dyes coassembled to a QD was not surprising given that we have previously observed hetero-FRET between different fluorescent dyes coassembled to a QD.17,48 Decreases in fluorescence anisotropy are typically the only means of detecting homo-FRET because changes in fluorescence intensity and lifetime do not accompany pure homo-FRET.27,51−54 We took care to evaluate homo-FRET between dyes assembled to QDs while minimizing potential interference from hetero-FRET between the QD and dyes and utilized three different experimental formats with A647 to ensure consistency with homo-FRET. It seems clear that there is homo-FRET between Pep(A555) or Pep(A647) conjugated to QDs; however, the fluorescence anisotropy experiments with Pep(A610) and Pep(A633) were less straightforward to interpret. The A610 anisotropy with QD−[Pep(A610)]n conjugates showed a small decrease before increasing and plateauing at the same value as the n × Pep(A610) without QDs. The trend in the A633 fluorescence anisotropy with QD− [Pep(A633)]n conjugates was somewhat different, as the anisotropy started at a level that was higher than Pep(A633) alone and decreased without a subsequent rise, although still convergent with the anisotropy for n × Pep(A633) without QDs. These trends may reflect an onset of homo-FRET disrupted by dimer formation, which leaves too few monomeric dyes to interact efficiently through homo-FRET. The few and isolated monomeric dyes assembled to the QD may thus retain the same anisotropy observed in bulk solution. A caveat to this interpretation is the larger uncertainty in the data points for A633 and A610 versus A555 and A647. Although the anisotropy results with A610/A633 are not as definitive with respect to homo-FRET as with A555/A647, the results are not inconsistent with this possibility. Another result to consider is the small decrease in A610, A633, and A647 lifetime with QD−[Pep(dye)]20 versus QD− [Pep(dye)]0.1 and 0.1 × or 20 × Pep(dye). For A610 and A633, the very similar lifetimes between the latter three samples are consistent with the ground-state formation of nonfluorescent dimers, which is a static quenching mechanism. Dynamic quenching mechanisms would be expected to decrease the dye lifetime, and given the degree of dye quenching observed, the I


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limited quantities of material. For example, we observed degrees of dimer formation with QD−[Pep(dye)]n conjugates at amounts of Pep(dye) that were orders of magnitude less than what would have been required to see the same degree of dimerization in bulk solution.

decrease in lifetime would be expected to be much larger than observed. The small decrease in lifetime may thus be indicative of nonequivalent homo-FRET pathways, where one or more monomeric dyes act as a modest energy sink by having a faster rate of nonradiative relaxation due to their local environment. In the case of A610 and A633, low-efficiency energy transfer from monomeric dyes to nonfluorescent dimers may also be possible. Implications of Self-Quenching and Homo-FRET. A significant advantage of QDs is their high brightness. In FRET pairs with QD donors, this brightness is, in part, transferred to a fluorescent dye acceptor. This advantage is largely negated by self-quenching processes that reduce the FRET-sensitized dye fluorescence to a level that is less than direct excitation of dye alone. An understanding of self-quenching phenomena associated with multivalent QD−dye conjugates is therefore important for designing effective QD−FRET probes for biological applications. Nonetheless, self-quenching through dimerization can potentially be exploited in certain experimental situations where there is value in probing the dye directly without FRET sensitization by the QD. As we describe in the Supporting Information, it appeared possible to use trends in acceptor dye fluorescence to estimate the maximum loading of QDs with Pep(dye), and it should be possible to track protease-catalyzed hydrolysis of Pep(dye) via the recovery of dye fluorescence with alleviation of self-quenching. Indeed, we showed that hydrolysis could be tracked by anisotropy changes with loss of homo-FRET between non-self-quenching dyes, and there was an indication that anisotropy trends related to homo-FRET could also be used to estimate maximum loading of Pep(dye) on QDs (Supporting Information). HomoFRET, like self-quenching through dimer formation, may thus be a useful spectroscopic means of gaining insight into the structure or environment at the periphery of a QD without relying on measurements of QD PL. However, unlike dimer formation, there does not appear to be a significant drawback to the occurrence of homo-FRET in hetero-FRET assemblies with QD donors. The confirmation of homo-FRET may also have implications for understanding energy transfer in emergent “concentric FRET” (cFRET) probes, which depend on energy transfer between multiple copies of two different fluorescent dyes coassembled around a common QD.17,48 A555 and A647 are common dyes in these probes. Photophysical analysis of energy transfer in these systems has, to date, focused on hetero-FRET between the two types of dye but has not explicitly considered homo-FRET between like dyes, which may have a nontrivial role in determining the overall energy transfer dynamics and efficiencies. Dyes with a tendency to self-quench should also be avoided in cFRET, as our present study was partially motivated by an initial observation that Pep(A610) was ineffective in a cFRET configuration. In addition to the above discussion, the observed selfquenching is another example of how multivalent nanoparticle bioconjugates provide opportunities that are not readily accessible in bulk states. For example, the concept of avidity has been described with nanoparticles with multiple conjugated affinity ligands or their targets, and is characterized by enhanced binding.55−57 Here, in our study, the high local concentration of X achieved through the assembly of QD−Xn conjugates had the effect of shifting a concentration-dependent equilibrium relative to bulk solution. Assembly to a NP may thus enable studies of systems that have relatively low equilibrium constants and

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CONCLUSIONS We have shown that the coassembly of multiple fluorescent dyes to a QD through a flexible linker such as a peptide can lead to the formation of nonfluorescent dimers. The high local concentration of assembled dye in the nanoscale volume surrounding the QD shifts the monomer−dimer equilibrium much further toward the dimer than is observed with an equal amount of dye in bulk solution. These nonfluorescent dimers are the cause of anomalous FRET-sensitized dye emission that can negate the brightness advantage of QD−FRET probes. Energy transfer from the QD donor to the fluorescent monomeric dye acceptors decreases because of both the lower number of monomeric dyes per QD and the competition with nonfluorescent dimers for accepting energy from the QD. Whereas some dyes are very prone to the formation of dimers (e.g., A610, A633), other dyes strongly favor their monomeric states (e.g., A555, A647) and their FRET-sensitized dye emission follows the expected trends with retention of the brightness advantage of QD donors. Although there is evidence for homo-FRET between the monomeric dyes in these latter systems, the homo-FRET pathway does not appear to lead to significant quenching of dye emission. Dyes that exhibit strong self-quenching through dimerization should be avoided in the design of QD−FRET assays and sensors; however, both selfquenching and homo-FRET may prove to be useful spectroscopic tools for studying structure and processes at the QD−bioconjugate interface.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.6b05886. Ligand exchange, peptide sequences and dye labeling, FRET calculations, quantum yield measurements, details of data analysis; characteristics of QD−dye FRET pairs, model data for directly excited and FRET-sensitized dye emission, additional examples of well-behaved QD− [Pep(A555/A647)]n conjugates and anomalous QD− [Pep(A610)]n conjugates, additional absorption and fluorescence excitation spectra, further discussion on fluorescence anisotropy, peptide assembly in DMSO, and prospective methods for determining loading (PDF)

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AUTHOR INFORMATION

Corresponding Author

*W. R. Algar. Tel: 1-604-822-2464. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), and the University of British Columbia for support of this research. J.J.L. is grateful for support from NSERC through the CREATE NanoMat training program. H.K. is grateful for support through an NSERC USRA J


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award. W.R.A. is grateful for a Canada Research Chair (Tier 2) and a Michael Smith Foundation for Health Research Scholar Award. We thank Saeid Kamal at the UBC LASIR facility for assistance with lifetime measurements.

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