Energy Transfer Pathways in a Quantum Dot-Based Concentric FRET

Article pubs.acs.org/JPCC

Energy Transfer Pathways in a Quantum Dot-Based Concentric FRET Configuration Miao Wu,† Melissa Massey,† Eleonora Petryayeva, and W. Russ Algar* Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada S Supporting Information *

ABSTRACT: The unique optical properties of semiconductor quantum dots (QDs) are highly advantageous for biological imaging and analysis, particularly when combined with Förster resonance energy transfer (FRET). A recent innovation in this area has been concentric FRET (cFRET), wherein QDs are assembled with multiple copies of two different types of fluorescent label. Although multifunctional biological probes have been developed utilizing cFRET, a detailed photophysical analysis of cFRET has not been undertaken, and energy transfer in these probes has been understood only qualitatively. Here, we characterize a prototypical QD-(A555)M-(A647)N cFRET configuration through photoluminescence (PL) intensity, decay, and photobleaching measurements. This cFRET configuration combines a central, green-emitting QD with Alexa Fluor 555 (A555) and Alexa Fluor 647 (A647) dyes that are assembled to QDs through peptide linkers, where M and N are the numbers of A555 and A647 per QD. Following initial photoexcitation of the QD, the energy transfer pathways are QD-to-A555 and QD-to-A647, which compete with one another, and A555-to-A647, which occurs subsequent to QD-to-A555 energy transfer. A rate analysis, calibrated to the conventional QD(A555)M and QD-(A647)N FRET systems, accurately predicts quenching efficiencies and permits a first approximation of dye/ QD PL intensity ratios in the cFRET configurations. CdSe/CdS/ZnS QDs and CdSeS/ZnS QDs of different sizes but similar emission characteristics are used for these experiments, and they demonstrate the general applicability of the analysis. The interplay between the three FRET pathways and nonideal behavior within this system is discussed with directions for future research. Overall, this study provides a framework and predictive power for the rational design and optimization of novel cFRET probes and biosensors for biological applications.

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INTRODUCTION Colloidal semiconductor nanocrystals, or “quantum dots” (QDs), are of great interest for biological imaging and analysis.1−3 These materials are well-known for their bright, spectrally narrow photoluminescence (PL), which is also resistant to photobleaching.4,5 As recent reviews attest, QDs have been widely utilized as labels for multicolor fluorescence measurements and imaging, single-particle tracking, and superresolution imaging, with applications spanning in vitro assays, cellular imaging, and in vivo imaging.1,6−8 The chemistry associated with these materials is also well developed: methods for the synthesis of CdSe/ZnS and related core/shell nanocrystals are established,9,10 several QD materials are available commercially, a variety of ligand and polymer coatings can be used to transfer QDs into aqueous media,11−13 and numerous methods for bioconjugation have been reported.11,14,15 The cumulative optical properties of QDs, which additionally include larger one-photon and two-photon absorption coefficients, spectrally broad absorption profiles, good quantum yields, and precise wavelength-tuning of PL through size and composition, cannot be matched by other fluorescent materials.4,5 © 2015 American Chemical Society

Adding to the utility of QDs is their ability to participate in Förster resonance energy transfer (FRET),16 which is one of the most versatile techniques for fluorescence-based measurements of biological molecules and systems.17 QDs are most frequently used as FRET donors paired with fluorescent dyes, dark quenchers, or fluorescent proteins as acceptors.18,19 Less frequently, QDs are used as acceptors for lanthanide, bioluminescent, or chemiluminescent donors.7,16 The advantages of QDs as donors include the flexibility to efficiently excite the QD while minimizing direct excitation of the acceptor, the ability to optimize the spectral overlap between the QD and acceptor without introducing problematic crosstalk between their emission, and the ability to further optimize FRET efficiency by arraying multiple acceptors per QD.16 Experimental and theoretical studies have shown that energy transfer between QDs and the aforementioned acceptors is consistent with the Förster mechanism.20−25 FRET and related mechanisms of energy transfer have been harnessed to develop Received: September 3, 2015 Revised: October 16, 2015 Published: November 4, 2015 26183

DOI: 10.1021/acs.jpcc.5b08612 J. Phys. Chem. C 2015, 119, 26183−26195

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The Journal of Physical Chemistry C

QD-A647 FRET systems, despite some nonideal behavior. This study provides a quantitative framework for the rational design, characterization, and optimization of cFRET probes and sensors for biological applications, and suggests directions for further research.

a wide array of QD-based probes and sensors that actively respond to biological targets through modulation of QD and acceptor PL.16,18,19 These developments have been greatly facilitated by a good understanding of FRET and its applicability with QDs. We have been developing “concentric FRET” (cFRET) configurations in which a QD is paired with two different fluorescent dye acceptors, where multiple copies of each dye are positioned on the surface of a sphere that is concentric with the QD, as illustrated in Figure 1. A successful prototype cFRET

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EXPERIMENTAL SECTION Additional experimental details can be found in the Supporting Information (SI). Materials. CdSeS/ZnS QDs (QD525a) were obtained from Cytodiagnostics (Burlington, ON, Canada), and CdSe/CdS/ ZnS QDs (QD520b) were synthesized using established methods.29,30 Both QD materials were coated with hydrophilic glutathione (GSH) ligands31 and self-assembled with dyelabeled, polyhistidine-terminated peptides (Bio-Synthesis, Inc., Lewisville, TX) as described previously.32,33 Peptide sequences can be found in the SI (Table S1). The peptides were labeled at their distal terminus (a cysteine residue) with maleimide derivatives of A555 and A647 fluorescent dyes (Invitrogen, Carlsbad, CA) and purified using published protocols.34 The model in Figure 1 was generated using UCSF Chimera35 for αhelical peptides and QDs with an average diameter intermediate to the QD520b and QD525a. Samples of QD-peptide-dye conjugates were prepared by mixing GSH-coated QDs (typically 10−20 pmol) with M equivalents of an A555-labeled peptide and N equivalents of an A647-labeled peptide in borate buffered saline (50 mM, pH 8.5, 50 mM NaCl) to a final volume of 100 μL (typically 0.1−0.2 μM final concentration of QD conjugate). The mixtures were let stand in the dark at room temperature for 1−2 h for selfassembly. The conjugation of QDs with polyhistidine peptides has been well characterized and is known to proceed nearly quantitatively at these concentrations.32,33 The maximum loading of the QDs is addressed in the Discussion section and SI. Fluorescence Measurements. Fluorescence spectra were measured with an Infinite M1000 Pro multifunction plate reader (Tecan Ltd., Morrisville, NC). This instrument is equipped with a xenon flash lamp, excitation and emission monochromators, and a photomultiplier tube detector. Spectra were recorded in 2 nm steps with 5 nm monochromator bandwidths. The excitation wavelength for interrogating cFRET was 400 nm. This wavelength efficiently excited the QDs with negligible direct excitation of A555 and A647. An excitation wavelength of either 530 or 545 nm was used to excite A555 directly while minimizing excitation of the QD. An excitation wavelength of 610 nm was used to directly excite A647. Samples were measured in black, nonbinding 96-well microtiter plates (#3650; Corning, Corning, NY). Time-resolved PL measurements were made with a streak camera system. QD samples in a quartz cuvette were excited with laser pulses at 420 nm. Laser pulses were generated with an optical parametric generator (EKSPLA, Vilnius, Lithuania) pumped by a pulsed laser at 355 nm with a 10 Hz repetition rate and 35 ps pulse duration (EKSPLA). Time-resolved spectra were recorded with a combination of a spectrograph (Princeton Instruments, Trenton, NJ, USA) and streak camera (C7700; Hamamatsu Photonics, Hamamatsu, SZK, Japan). Measurements were made in photon counting mode between 430−790 nm with a temporal range of 50 ns. Photobleaching experiments were done on an IX83 inverted epifluorescence microscope (Olympus, Richmond Hill, ON, Canada) with MetaFluor software (Molecular Devices,

Figure 1. Simplified schematic of the prototypical QD-(A555)M(A647)N cFRET configuration. The right-hand side of the figure shows the assembly of the cFRET configuration, including the hydrophilic glutathione (GSH) ligand coating, and A555- and A647-labeled peptides self-assembled through polyhistidine tags. The left-hand side of the figure depicts the three energy transfer rates, k, in the cFRET configuration when the QD is photoexcited.

system combines a green-emitting QD donor with (i) a yellowemitting dye, such as Alexa Fluor 555 (A555), and (ii) a redemitting dye, such as Alexa Fluor 647 (A647). The QD is initially photoexcited and can subsequently transfer its energy to either the A555 or the A647, where the A555 is the better of the two acceptors, but energy transfer to the A647 is not negligible. Following energy transfer from the QD to the A555, the A555 can transfer energy to the A647. The A555 and A647 dyes are selected primarily because of their spectral properties, and spectrally analogous dyes have been successfully substituted (e.g., Cy3 for A555).26 In application, these dyes have been conjugated to green-emitting QDs through biological macromolecules such as synthetic peptides or oligonucleotides, where the selection of the biological molecule has enabled measurement of the activity of two different proteases,27 proprotease activation,27 and the concentration and activity of a protease.26 A cFRET imaging method was also recently developed.28 In each case, energy transfer has been understood only qualitatively and analytical applications have relied on empirical calibration. To further expand the scope and utility of cFRET, it is necessary to have a good physical understanding of the system. Here, we present a detailed photophysical characterization of a prototypical, peptide-based QD-A555-A647 cFRET configuration at the ensemble level, including PL intensity, lifetime, and photobleaching measurements with two different QD materials. Our results reveal competitive and sequential energy transfer within the system, and show that energy transfer efficiencies and PL ratios in the cFRET system can be largely predicted from the corresponding nonconcentric QD-A555 and 26184


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The Journal of Physical Chemistry C Sunnyvale, CA). Samples of QD-peptide-dye conjugates (5 μL, 0.15−0.2 μM) were placed in a 2 × 2 set of wells in a clearbottom 1536-well plate (#782 101; Grenier Bio-One, Monroe, NC). These wells were then covered with optical microplate sealing tape (Corning #6575). 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), an Orca-Flash 4.0 V2 sCMOS camera (C11440; Hamamatsu Photonics), and a motorized emission filter wheel (Olympus). The filter wheel housed 520/40 (center line/ bandwidth in nm), 565/30, and 665 long-pass filters (Chroma Technology Corp., Bellow Falls, VT) for sequential measurement of QD, A555, and A647 emission, respectively. The excitation filter was always 405/20 and paired with a 470 nm cutoff dichroic mirror (Chroma). Samples were continuously illuminated and imaged with all three filter channels at 1 min intervals for 30 min, and at 2 min intervals for an additional 60 min. The total illumination power exiting the objective lens was ∼27 mW. Data was analyzed in ImageJ software (National Institutes of Health, Bethesda, MD).36 Analysis. Peak PL emission intensities, Ip, were calculated from emission spectra by averaging the measured intensities within ±4 nm of the peak emission wavelength. The peak emission wavelengths were approximately 518 nm for QD520b, 526 nm for QD525a, 566 nm for A555, and 668 nm for A647. Total emission intensities, I = αIp, were calculated by multiplying the peak emission intensity by a coefficient, α, that related peak area and peak height (see SI for details). This coefficient was determined from the isolated emission spectra of each QD and dye. The Ip values were corrected for crosstalk between the emission spectra of the QDs, A555, and A647, as described in the SI. FRET efficiencies were calculated from PL spectra according to eq 1, where Ip(D) is the unquenched peak PL intensity of the donor, and Ip(DA) is the donor peak PL intensity in the presence of acceptor. When multiple FRET pathways were available to a donor (e.g., QD donor with both A555 and A647 acceptors coassembled), the efficiency term in eq 1 was instead treated as a quenching efficiency, QD. −1 E = 1 − (Ip(DA)Ip(D) )

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(1)

−1 ρ = IdyeIQD

Figure 2. (A) Spectral overlap functions (J(λ), top) and normalized absorption and emission spectra (bottom) for QD520b, QD525a, A555, and A647. The scale bar for the spectral overlap functions represents 3 × 10−11 mol−1 cm5. (B) Representative TEM images of the QD520b and the QD525a. The scale bars are 20 nm.

(2)

PL decay curves, I(t), were fit with a biexponential function, eq 3, where I is the PL intensity, t is time, C is a constant, Ai is an amplitude, and τi is a lifetime. Amplitude-weighted average lifetimes were calculated according to eq 4 and are the most appropriate for the analysis of FRET efficiencies.37

A1τ1 + A 2 τ2 A1 + A 2

along with the corresponding spectra for A555 and A647, and spectral overlap functions. Several properties of the QDs, dyes, and possible FRET pairs are summarized in Table 1. The most important difference was that the average diameter of the CdSeS/ZnS QDs was ∼ 1 nm larger than the CdSe/CdS/ZnS QDs. Representative transmission electron micrographs of the QDs are shown in Figure 2B. For brevity, the binary-core CdSe/CdS/ZnS QDs are denoted as QD520b, and the alloyedcore CdSeS/ZnS QDs are denoted as QD525a. Nearly identical Förster distances were calculated for the QD520b and QD525a with both the A555 and A647 acceptors. QD-to-Dye Energy Transfer in the cFRET Configuration. To study the QD-(A555)M-(A647)N cFRET system, samples were prepared with 36 different (M, N) permutations, where M and N were the average number of A555- and A647-

−1

I(t ) = A1e−tτ1 + A 2 e−tτ2 + C

τave =

(5)

RESULTS QD and Dye Properties. In the prototypical QD-A555A647 cFRET configuration, the QD serves as an initial point of photoexcitation and donor for FRET to the A555 and A647, and concomitantly as a scaffold to colocalize the A555 and A647, thereby creating the proximity for FRET between these dyes. To investigate the possible role of QD size in cFRET, and to confirm the generality of the results in this study, QD-A555A647 cFRET configurations were assembled around CdSe/ CdS/ZnS QDs and alloyed CdSeS/ZnS QDs with similar emission profiles. The normalized absorption and emission spectra of these two QD materials are shown in Figure 2A,

Dye/QD PL ratios were calculated according to eq 2, where Idye is the total PL intensity of the dye, and IQD is the total PL intensity of the QD, in the presence of one another.

−1

−1 E = 1 − (τave(DA)τave(D) )

(3)

(4)

FRET efficiencies were calculated from lifetime data according to eq 5, where τ ave(D) is the unquenched amplitude-weighted average lifetime of the donor, and τave(DA) is the amplitude-weighted average lifetime of the donor in the presence of acceptor.37 26185


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The Journal of Physical Chemistry C Table 1. Photophysical Properties of QDs, Dyes, and FRET Pairs A555 Acceptor

A647 Acceptor

Donor

Dia.a (nm)

εpeakb (M−1 cm−1)

PLmax (nm)

fwhmc (nm)

QYd (%)

τavee (ns)

Jf (cm6 mol−1)

R0g (nm)

Jf (cm6 mol−1)

R0g (nm)

QD520b QD525a A555 A647

3.6 ± 1.4 4.6 ± 0.5

57 800 210 000 155 000 270 000

518 526 566 670

37 30

45 45 10 33

24.3 13.8 0.34 0.86

5.7 × 10−10 6.8 × 10−10

6.0 6.2

6.7 × 10−11 6.5 × 10−11 8.1 × 10−10

4.2 4.2 5.0

a Estimated from TEM micrographs. bApproximate molar absorption coefficient at the first exciton peak for the QDs or the absorbance maximum for the dyes. cEmission spectrum full-width-at-half-maximum. dQuantum yield values were measured for the QDs and are reported by the manufacturer for A555 and A647. eAmplitude-weighted average lifetime calculated from eq 4. fSpectral overlap integral. gFörster distance, calculated assuming an orientation factor of κ2 = 2/3.

Figure 3. Emission spectra for different (M, N) cFRET configurations assembled around (A) QD520b and (B) QD525a. The arrows indicate the trends in PL intensity with increasing amounts of A647 per QD.

Figure 4. (A) Contour plots of the QD PL quenching efficiency as a function of (M, N) for the QD520b and QD525a cFRET configurations. (B) Correlation between the QD PL quenching efficiencies predicted from eq 6 and those measured experimentally from changes in the QD emission intensity. The solid circles are data points from panel A; the open circles are data points from an analogous experiment (see Figure S4 for plots similar to panel A).

labeled peptides assembled per QD. Figure 3 shows emission spectra excited at 400 nm for each (M, N) permutation, grouped according to the number of A555 per QD. Direct excitation of the A555 and A647 was negligible at this excitation wavelength. With both the QD520b and QD525a, the amount of FRET-sensitized A555 PL increased with more A555-labeled peptides per QD, and decreased with more A647-labeled peptides per QD. Conversely, the amount of FRET-sensitized A647 PL increased with increasing numbers of both A555- and A647-labeled peptides per QD. The magnitude of the incremental increase in the intensity of the FRET-sensitized dye emission from A555 and A647 tended to decrease as more of each dye was assembled per QD. These qualitative trends were in agreement with expectations based on the energy transfer pathways depicted in Figure 1. The QD quenching efficiency (eq 1) in the QD-(A555)M(A647)N cFRET configurations is plotted as a function of M and N in Figure 4A. These efficiencies were determined from

the data in Figure 3 and represent the sum of the QD-to-A555 and QD-to-A647 FRET efficiencies. From first-principles, the quenching efficiency, QQD, was expected to follow eq 6, where kQD = (τave(QD))−1, τave(QD) is the unquenched amplitudeweighted average lifetime of the QD, kQD‑A555 is the average rate of energy transfer per A555 acceptor per QD, and kQD‑A647 is the average rate of energy transfer per A647 acceptor per QD. Similar equations have been derived for other QD systems with multiple FRET pathways.38 To test this model, FRET efficiencies were calculated for the (M, 0) and (0, N) data sets (eq 1), and values of kQD−1kQD‑555 and kQD−1kQD‑A647 were subsequently determined by fitting the efficiencies for these two data sets with eq 6. For convenience, the kQD−1kQD‑dye terms were treated as a single variable during fitting (see right-hand side of eq 6). The values of kQD−1kQD‑dye were then used to predict the quenching efficiencies for (M, N) samples with nonzero values of M and N. Figure 4B plots the QD quenching efficiencies predicted in this manner versus the measured QD 26186


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Figure 5. (A) Time-dependent emission spectra for different (M, N) cFRET configurations around QD520b, where M = 0, 4, 8, and 12 A555 per QD, and N = 0, 6, 12, and 18 A647 per QD. The amplitude-weighted average lifetimes for the QD520b are listed for each configuration in units of nanoseconds. (B) QD PL decay profiles for the (M, 0) and (0, N) configurations in panel A. (C) Correlation between the QD PL quenching efficiencies predicted from eq 6 and those measured experimentally from changes in the average QD lifetime.

Table 2. Calculated Energy Transfer Rates and Estimated Donor−Acceptor Distances QD

Measurement

kQD‑A555 (ns−1)a

rQD‑A555 (nm)b

kQD‑A647 (ns−1)a

rQD‑A647 (nm)b

QD520b

Intensity Lifetime Intensity

0.017 ± 0.002 0.016 ± 0.001 0.023 ± 0.002

6.9 7.0 7.5

0.0029 ± 0.0003 0.0028 ± 0.0003 0.0061 ± 0.0003

6.5 6.6 6.3

QD525a a

Rate of energy transfer between the QD donor and one of the indicated dye acceptors. bAverage calculated distance from the center of the QD to the indicated acceptor. Refer to the Discussion section for the uncertainty in r.

(M, 0) and (0, N) data sets were then fit with eq 6, as done previously for the PL intensity data, to calculate kQD−1kQD‑A555 and kQD−1kQD‑A647. These values were again used to predict the QD quenching efficiency, QQD (eq 6), for nonzero (M, N) samples. A shown in Figure 5C, a plot of the predicted quenching efficiency versus the measured quenching efficiency had a slope of approximately unity (Figure 5C includes data derived from Figure 5A and a replicate experiment for which the data is not shown). This result was consistent with the intensity measurements, and the correlation further suggested that eq 6 accounted for the quenching of QD PL in the cFRET configurations. The calculated rates of energy transfer from the QD to the A555 and A647 are summarized in Table 2 and were calculated from the intensity and lifetime data in Figure 3 and Figure 5, respectively. Note that a faster rate of energy transfer does not necessarily imply greater efficiency because of the difference in lifetime between the two QD materials. The center-to-center QD-dye separation distances derived from these energy transfer rates are also tabulated. A555-to-A647 Energy Transfer in the cFRET Configuration. In parallel with the analysis of QD quenching, the A555 quenching efficiency (eq 1) was also determined as a function of N, using the appropriate (M, 0) sample as a reference state. The results, shown in Figure 6A(i) for the same data set as Figure 3, revealed that the measured A555 quenching efficiency was approximately independent of M.

quenching efficiencies. A line of best fit through the data points for the QD520b and QD525a systems had a slope close to unity, indicating a good correlation between theory and experiment. This result suggested that eq 6 was a valid model for quenching of QD PL in the cFRET configuration. Q QD = =

Mk QD ‐ A555 + Nk QD ‐ A647 k QD + Mk QD ‐ A555 + Nk QD ‐ A647 −1 −1 Mk QD k QD ‐ A555 + Nk QD k QD ‐ A647

−1 −1 1 + Mk QD k QD ‐ A555 + Nk QD k QD ‐ A647

(6)

To complement the above analysis, time-resolved PL measurements were made on the QD520b cFRET system using a streak camera system that also provided wavelength resolution. The time-dependent PL emission spectra are shown in Figure 5A for selected (M, N) permutations, prepared separately from the samples for intensity measurements (Figure 3). QD520b PL decay profiles for the (M, 0) and (0, N) samples are shown in Figure 5B, and had biexponential character with short and long lifetime components. These lifetime components are tabulated in the SI (Tables S2−S4) with all of the decay curves (Figure S6), and the amplitudeweighted average lifetimes are given in Figure 5A. Consistent with the PL intensity results, the QD520b lifetime was shortened to a greater degree with the (M, 0) samples than with the (0, N) samples. The FRET efficiencies (eq 5) from the 26187


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Figure 6. (A) Characterization of the A555-to-A647 FRET efficiency in the QD520b and QD525a cFRET (M, N) configurations: (i) measured A555 quenching efficiency for cFRET configurations; (ii) predicted quenching of the A555 emission intensity due to the competition for energy transfer from the QD between A555 and A647; (iii) calculated A555-to-A647 energy transfer efficiency for cFRET configurations; and (iv) A555-toA647 energy transfer efficiency measured via direct excitation of the A555 (bypassing FRET from the QD). In panel (iv), the gray shaded region represents the range of efficiencies calculated in the corresponding panel (iii). The data points for M = 1.4 for QD525a at 400 nm excitation were excluded because of poor signal-to-background ratios. (B) Representative A555 PL decay profiles for different (M, N) cFRET configurations for QD520b. The data was from Figure 5A. (C) Summary of changes in the amplitude-weighted average A555 PL lifetime as a function of N, for different values of M.

For a given value of N, the apparent quenching efficiency of M A555 per QD varied with an absolute standard deviation of ±3% or less for both the QD520b and QD525a cFRET configurations. Conversely, the quenching of A555 PL was strongly dependent on N, the number of A647 per QD. There were two anticipated mechanisms of quenching of the A555 PL in the cFRET configuration. The first source of quenching was the competition between A555 and A647 for direct energy transfer from the QD. Although a poorer acceptor than A555, the A647 nonetheless received some energy directly from the QD. The analysis of QD PL quenching efficiencies for (M, N) configurations based on fitting of (M, 0) and (0, N) data was extended to predicting the competitive FRET quenching of the A555 intensity, QA555‑C, with increasing A647 per QD. Eq 7 applied in this case, where the terms are defined analogously to eq 6. The numerator in the second term is the efficiency of QD-to-A555 FRET that would be predicted to be observed in the presence of A647, and the denominator in the second term (bracketed) is what would be predicted to be observed for the same number of A555 without A647 present.

The predicted A555 quenching efficiencies associated with the competition for energy transfer from the QD donor are plotted in Figure 6A(ii). When these predicted values were subtracted from the measured quenching efficiencies, the residual quenching was attributed to energy transfer between A555 and A647, where the A555 was initially sensitized by energy transfer from the QD. The resulting efficiencies are plotted in Figure 6A(iii). To verify this analysis, A555-to-A647 energy transfer was interrogated with excitation at 530 nm, largely bypassing energy transfer from the QD. These energy transfer efficiencies, measured from quenching of the A555 PL intensity for each appropriate (M, 0) reference state, are plotted in Figure 6A(iv). There was good agreement between the calculated A555-to-A647 efficiency values for cFRET and those measured for direct excitation of the A555. Interestingly, the directly excited A555-to-A647 FRET efficiency also appeared to be approximately independent of M. To further analyze the cFRET A555-to-A647 energy transfer with the QD520b, the decay of the A555 PL in the timedependent PL emission spectra in Figure 5A was evaluated. Representative decay profiles are shown in Figure 6B. Similar to the QD520b, the A555 PL decayed with biexponential character. The amplitude-weighted average lifetimes were longer than the 0.35 ns native lifetime of the dye-labeled peptide, which exhibited a monoexponential decay rather than a biexponential one. Notably, the average A555 PL lifetime decreased with more A555 per QD520b, in parallel with shortening of the QD520b PL lifetime, further indicating that

⎡ −1 Mk QD k QD ‐ A555 Q A555 ‐ C = 1 − ⎢ −1 ⎢ 1 + Mk −1 k QD QD ‐ A555 + Nk QDk QD ‐ A647 ⎣ ⎞−1⎤ ⎛ Mk −1 k QD QD ‐ A555 ⎟⎟ ⎥ ⎜ ×⎜ −1 ⎝ 1 + Mk QDk QD ‐ A555 ⎠ ⎥⎦

(7) 26188


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competitive QD-to-A647 energy transfer. In parallel, the A647/ QD PL ratio increased with more A555 per QD and more A647 per QD, as a result of more efficient QD-to-A647 and A555-to-A647 energy transfer. Quantitatively, the QD PL ratios were expected to be a function of the dye and QD quantum yields, and the efficiencies of the relevant energy transfer pathways. In the case of the (M, 0) configurations, the theoretical expression for the A555/ QD PL ratios was given by eq 8, which simplifies to a linear function of the number of A555 acceptors per QD, where Φ is a quantum yield. An analogous expression applied for the A647/QD PL ratio for the (0, N) configurations.

energy transfer from the QD520b was an important determinant of the A555 lifetime. For a fixed value of M, increases in the value of N had only a small effect on the A555 lifetime. The general trend was a decrease in the observed A555 lifetime with increasing N, as shown in Figure 6C; however, the changes were small and difficult to resolve within the precision of the experiment. PL Ratios in the cFRET Configuration. In bioanalytical applications of cFRET, it has not been the energy transfer efficiencies that are measured, but rather the A555/QD and A647/QD PL intensity ratios. The PL ratios calculated for the (M, N) samples in Figure 3 are shown in Figure 7 (crosssectional views of the PL ratio plots can be found in the SI). Similar trends were observed between the QD520b and QD525a configurations. The A555/QD PL ratio increased with more A555 per QD, as a result of more efficient QD-toA555 energy transfer, and decreased with more A647 per QD, as a result of more efficient A555-to-A647 energy transfer and

IA555 ⎛ ΦA555 ⎞ EQD ‐ A555 ⎟⎟ = ⎜⎜ IQD ⎝ ΦQD ⎠ (1 − EQD ‐ A555) ⎛Φ ⎞ −1 k QD ‐ A555M = ⎜⎜ A555 ⎟⎟k QD ⎝ ΦQD ⎠

(8)

Experimentally, the trends in the A647/QD PL ratio for (0, N) configurations were approximately linear as expected; however, the trends in the A555/QD PL ratio for (M, 0) configurations were curved, and empirically fit by a quadratic function (see SI, Figure S7). Both of these results were consistent with previous studies.39−41 Similar to the analysis of FRET efficiencies, the (M, 0) and (0, N) data in Figure 7A−B was used as a basis for predicting the A555/QD and A647/QD PL ratios for nonzero (M, N) configurations. This process is described in detail in the SI, where eqs 9−10 were ultimately used as semiempirical models to predict PL ratios. The values of kQD−1kQD‑A555 and kQD−1kQD‑A647 were obtained from the (M, 0) and (0, N) data in Figure 4, and kA555−1kA555‑A647 was from fits to the (M, N) data in Figure 6A(iv). It is important to note that ΦA555/ΦQD in eq 9 was treated as a calibrated variable to account for the nonlinear trend in the A555/QD PL ratio with (M, 0) configurations. That is, the values for ΦA555/ΦQD as a function of M were determined from the (M, 0) data, and systematically increased with increasing M. These values were then applied to prediction of the (M, N) PL ratio data. ⎞ ⎛ IA555 ⎛ ΦA555 ⎞ −1 1 ⎟⎟Mk QDk QD ‐ A555⎜ ⎟ = ⎜⎜ −1 IQD ⎝ 1 + NkA555kA555 − A647 ⎠ ⎝ ΦQD ⎠ (9)

⎛ Φ ⎞⎡ IA647 −1 k QD ‐ A647 = ⎜⎜ A647 ⎟⎟⎢Nk QD IQD ⎝ ΦQD ⎠⎢⎣ ⎛ Nk−1 k ⎞⎤ A555 A555 − A647 −1 ⎟⎥ k QD ‐ A555⎜ + Mk QD −1 ⎝ 1 + NkA555kA555 − A647 ⎠⎥⎦

(10)

Figure 7C plots the predicted PL ratios versus the measured PL ratios for both the QD520b and QD525a cFRET configurations. For the A555/QD PL ratio, there was nearunity correlation for the QD525a. The correlation for all of the QD520b data indicated a small overestimate for the predicted values, although the data for less than 14 total peptides per QD had a near-unity correlation. The predicted A647/QD PL ratios were somewhat underestimated for the QD525a and, to a larger degree, for the QD520b. The correlations for A647/QD PL ratios were also more scattered than that for the A555/QD PL ratios. These trends and anomalies are discussed in detail later.

Figure 7. PL ratios for different (M, N) permutations of (A) the QD520b cFRET system, and (B) the QD525a cFRET system. (C) Correlations between the predicted (eqs 9−10) and measured A555/ QD and A647/QD PL ratios for the cFRET configurations in panels A and B. The dashed line for the A555/QD520b PL ratio is a fit through all points with M + N < 14 (open circles) with a slope of 1.02. The slope of the solid line for all data points (filled and open circles) is 1.15. 26189


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The Journal of Physical Chemistry C Photobleaching. To further characterize the prototypical cFRET configuration, the photobleaching behavior of four different samples was investigated: QD, QD-(A555)M, QD(A647)N, and QD-(A555)M-(A647)N, where M = 8 and N = 10 for QD520b, and M = 7 and N = 9.3 for QD525a. Results for the QD520b are shown in Figure 8A. The PL from the

increase in the A555 PL intensity for the latter sample. Similarly, slow photobleaching of the A555 was observed with the QD525a-(A555)7-(A647)9.3 and QD525a-(A555)7 samples. The main difference versus the QD520b was that the QD525a appeared to bleach more significantly, and, as consequence, no increase in QD525a PL intensity was observed with bleaching of the A647. Nonetheless, bleaching of the QD525a remained slower with QD525a-(A555)7 and QD-(A647)9.3 samples, and minimal with the QD525a-(A555)7-(A647)9.3.

■

DISCUSSION QD Materials. The QD520b and QD525a were interesting to compare because they had comparable ensemble emission spectra but distinct physical properties. In addition to their different compositions, the QD520b were (on average) smaller than the QD525a, less monodisperse, and had different morphologies. Despite these differences, relative energy transfer rates, kQD−1kQD‑dye, were comparable between the two QD materials. The relative rate for QD-to-A555 energy transfer was slightly higher with QD520b (0.41 vs 0.34), and relative rates of QD-to-A647 and A555-to-A647 energy transfer were higher with QD525a, but not by a significant amount (0.075 vs 0.069, and 0.094 vs 0.084, respectively). The FRET-estimated QD-toA555 distance was ∼0.5 nm longer for the QD525a than the QD520b, where the effective radius of the QD525a was ca. 0.5 nm larger on average; however, there was no significant difference between the estimated QD-A647 distances between the QD520b and QD525a. The inability to resolve a clear sizeeffect on cFRET could be a result of factors such as uncertainty in the concentration of QDs,42−44 their polydispersity, nonspherical morphology, assumption of a FRET orientation factor (κ2 = 2/3), and normal batch-to-batch variation. Although more accurate absolute distances would be obtained by introducing a correction for the Poisson distribution of assembled peptides,24,45 the difference in distance between the two QD materials would not change significantly. Given the foregoing, we estimate an uncertainty of ∼1 nm in the QD-dye separations. Nonetheless, the QD-dye separations are comparable to what would be expected for a peptide α helix oriented normal to the QD surface with extension of the dye linker (6.1−6.7 nm for QD520b, and 0.5 nm longer for QD525a), and much less than the upper limit set by the contour length of the peptide and extension of the dye linker (>12 nm).46,47 Importantly, none of these possible sources of uncertainty affect the evaluation of energy transfer in the (M, N) cFRET configurations from (M, 0) and (0, N) data because the analysis was self-consistent. The effectively indistinguishable cFRET behavior between the QD520b and QD525a also suggests that cFRET is likely to be supported by various sizes and shapes of QDs, provided that the dimensionality is comparable to relevant Förster distances. Energy Transfer Efficiencies. The results in Figures 4B and 5C demonstrated that energy transfer in the prototypical QD-(A555)M-(A647)N cFRET configurations was largely predictable from measurements of the energy transfer rates for conventional QD-(A555)M and QD-(A647)N FRET configurations. In a conventional FRET system, an increasing number of a given dye acceptor per QD increases both the rate and efficiency of energy transfer. In the cFRET configurations, the overall quenching of the QD PL arose from the sum of the energy transfer rates to both the A555 and A647 acceptors (eq 6); however, the QD-to-dye energy transfer efficiencies were a function of the competition between the two dyes to accept the

Figure 8. Photobleaching of the QD, A555, and A647 in various FRET configurations assembled around (A) QD520b and (B) QD525a. The cFRET configuration is QD-(A555)M-(A647)N, where M = 8 and N = 10 for QD520b, and M = 7 and N = 9.3 for QD525a. The legend in panel A also applies to panel B. The QD is directly excited, and all dye emission is FRET-sensitized.

QD520b sample was stable for 30 min before a ∼ 20% decrease, presumably due to photobleaching, was observed over the final 60 min. In contrast, there was no significant decrease in the QD PL intensity over 90 min for the QD520b-(A555)8 sample. With the QD520b-(A647)10 sample, the most notable result was >90% photobleaching of the A647 emission within 90 min. In parallel with photobleaching of the A647, the QD PL intensity increased ∼30% as a result of the loss of QD-toA647 FRET, with a subsequent decrease that was likely due to slow photobleaching of the QD itself. These trends were largely mirrored with the QD520b-(A555)8-(A647)10 cFRET sample. The A647 photobleached at the same rate as it did in the QD520b-(A647)10, with a concomitant ∼20% increase in the QD PL intensity before photobleaching of the QD became significant, albeit at a slower rate than with the QD520b(A647)10 sample. The smaller increase in QD PL with photobleaching of the A647 and the slower rate of QD photobleaching were both attributed to QD-to-A555 energy transfer in the cFRET sample. The loss of A555-to-A647 energy transfer and competitive QD-to-A647 energy transfer was also evident through the ∼100% increase in A555 PL upon photobleaching of the A647. Following this increase, the A555 appeared to bleach at a rate similar to the QD520b-(A555)8 sample. Photobleaching results for the QD525a are shown in Figure 8B. Overall, the observed trends were qualitatively similar to those observed with the QD520b. Rapid photobleaching of A647 PL was again observed with the QD525a-(A647)9.3 and QD525a-(A555)7-(A647)9.3 samples, as was a concomitant 26190


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independence observed in experiments. First, a dependence on M is predicated on the assumption that the total number of peptides per QD determines the average distance between the assembled peptides and dye labels. This assumption is tantamount to the peptides assembling as far from one another as possible. Although a previous study has suggested that such an interfacial distribution of peptides self-assembled to QDs is more likely than a random distribution,27 the possibility of some random character in the distribution cannot be ruled out with the limited data and characterization available in the literature. The A555-to-A647 FRET efficiency for a truly random distribution would have little or no dependence on M.27 Second, the dependence on M may be difficult to resolve within the precision of the experiment. The measured efficiencies were found to agree with one another with an absolute standard deviation of ±3%, which translates into a useful confidence interval of at least ±6%. If the magnitude of the dependence on M is comparable, then the trend could go undetected. Third, there may be a competing process, such as homo-FRET between A555 molecules, that prevents the A555to-A647 FRET efficiency from measurably increasing with increasing M, perhaps by delocalizing the excitation energy of the specific A555 molecule that is initially excited (either directly or by energy transfer from the QD). It is worth noting that QD-assembled, directly excited dye donor-dye acceptor FRET efficiencies were previously observed to be independent of the number of donors in a different system,50 suggesting that the observation is not necessarily unique to the QD-A555-A647 system. PL Ratios. Although the qualitative trends in the PL ratios for (M, N) cFRET configurations agreed with expectations, quantitative prediction of these PL ratios from conventional (M, 0) and (0, N) configurations was less accurate than predictions of energy transfer efficiency. Overall, the PL ratio predictions were closer to the measured values for lower numbers of dye-labeled peptide per QD, for the A555/QD PL ratios versus the A647/QD PL ratios, and for QD525a versus QD520b. Given that the A555/QD PL ratio did not follow the theoretically expected linear trend for the baseline (M, 0) configurations, the good agreement between the predicted and measured A555/QD PL ratios for (M, N) configurations with QD525a and QD520b was quite remarkable, and suggests that eqs 9−10 are a useful first approximation for the PL ratios observed in the cFRET configurations. In that regard, an important consideration is the rapid increase in PL ratio at high FRET efficiencies, which are associated with higher numbers of dye-labeled peptide per QD. The value of the E/(1 − E) term in eq 8 increases from ca. 2 to 4 to 9 for efficiency values of 70%, 80%, and 90%, respectively. Variations or deviations in FRET efficiency on the order of a few percent may thus be a nontrivial contribution to deviations observed in the correlation between the predicted and measured A555/QD PL. A physical possibility for deviations between the predicted and measured A555/QD PL ratios, particularly at higher numbers of acceptor dyes on the QD520b, is that self-assembly of the peptides saturates at ∼13 total peptides per QD; however, gel electrophoresis and spectroscopic data suggest a loading capacity in the range of 24 ± 6 peptides per QD. Moreover, assumption of a maximum loading of 13 peptides per QD (or any higher values) and implementation of a statistical correction for the values of M and N does not significantly improve the correlation for the A555/QD520b PL ratios and A647/QD PL ratio. Any effects associated with

excitation energy of the QD. Decreases in A555 emission with an increasing number of A647 per QD were a result of quenching via the A555-to-A647 energy transfer pathway, as well as a decrease in the sensitization rate because of the competitive QD-to-A647 energy transfer pathway. A correction for this competition was necessary to resolve the A555-to-A647 energy transfer efficiency when the A555 was sensitized via FRET from the QD. With this correction, the A555-to-A647 energy transfer efficiencies measured for QD sensitization were comparable to those measured for direct excitation of the A555. These efficiencies were determined through PL intensity measurements. PL decay measurements were relatively insensitive to A555-to-A647 energy transfer because the subnanosecond intrinsic lifetime of the A555, which decreased with energy transfer to the A647, was much shorter than the observed lifetime of several nanoseconds. The lengthening of the observed A555 lifetime to several nanoseconds is attributed to asynchronous FRET-sensitization by QDs across the ensemble. In general, apparent lengthening of an acceptor lifetime is expected when the intrinsic donor lifetime is much longer than the intrinsic acceptor lifetime.22,48,49 Another aspect of A555-to-A647 energy transfer that warrants discussion is whether the efficiency of this pathway should depend on both the number of A555, M, and the number of A647, N. After photoexcitation, the energy of a single exciton generated in a QD will be dissipated as heat, emitted as a photon, or transferred via FRET to a proximal acceptor (see Figure S1 for a flowchart). Thus, there was only one excited state donor in the system at a given time, whether it was the QD or an A555 molecule, and regardless of how many A555 were assembled per QD. Net rates of energy transfer from A555 to an A647 acceptor, kA555‑A647, were expected to be determined by eq 11, where kA555 = τA555−1 is the intrinsic relaxation rate of the A555 donor, R0,A555‑A647 is the A555-A647 Förster distance, and ri is the distance between the A555 donor and the ith A647 acceptor. N

kA555 − A647 =

∑ kA555(R 0,A555 − A647/ri)6 i=1

(11)

It is clear from the summation in eq 11 that the efficiency of energy transfer depends on N. In addition, the average value of ri may depend on the total number of peptides per QD. Given the finite surface area of a QD, more peptides per QD would be expected to result in closer average proximity between peptides and thus dye labels across the ensemble. This contribution to the rate of energy transfer would depend on both M and N, although the overall rate of energy transfer would be expected to have a stronger dependence on N due to its two putative contributions to eq 11. Although a strong dependence on N was observed in experiments, no clear dependence on M was observed. For the QD520b-sensitized A555-to-A647 FRET efficiency data in Figure 6A(i), the measured quenching of A555 PL was independent of M, such that the correction for the competition between QD-to-A555 and QD-to-A647 energy transfer introduced a small dependence on M by default. However, analogous data for the QD525a, as well as data for direct excitation of A555-to-A647 energy transfer with both QD materials in Figure 6A(iii−iv), exhibited no systematic dependence on M. We speculate that three factors may contribute to the apparent discrepancy between the expected dependence of the A555-to-A647 FRET efficiency on M and the approximate 26191


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transfer from a QD donor to an assembled dye acceptor. The efficiencies of energy transfer from the QD to the A555 and A647 acceptors are given by eqs 13 and 14, respectively, where rQD‑A555 and rQD‑A647 are the average distances from the center of the QD to the assembled A555 and A647 dyes. These equations assume radial symmetry for both acceptors relative to the QD and are ensemble averaged, neglecting the Poisson distribution for the assembly of both acceptors.24 It is apparent from these equations that energy transfer in the cFRET configuration can be tuned through choice of the length of the peptide or other linker for each dye acceptor, and through control of the number of each acceptor. The control of these parameters is a potential means of compensating for the small spectral overlap between the QD and the A647 (or another red dye) in a cFRET configuration.

approaching the maximum peptide loading of the QDs were thus likely secondary, particularly with respect to the A647/QD PL ratio. Another physical possibility for the observed deviations in dye/QD PL ratios is reabsorption (i.e., secondary inner filter effects).51 Reabsorption of QD emission by A555 is consistent with the nonlinear trend in the A555/QD PL ratio, and reabsorption of A555 emission by A647 could lead to underestimated A647/QD PL ratios. Nonetheless, several observations argue against significant reabsorption effects. First, the difference in the QD-to-A555 FRET rate measured through quenching of QD520b PL intensity and through decreases in QD520b PL lifetime was only ∼5%. Quenching that increases faster than decreases in lifetime would be expected if there were significant reabsorption.51 In other work, we have also found that analogous QD625a-A647 FRET pairs yield linear trends in PL ratio even though greater reabsorption effects would be expected since A647 is more strongly absorbing than A555.39,41 In addition, the dye concentrations (and therefore absorbance values) for cFRET measurements with the QD520b and QD525a samples were identical, yet the experimental A555/QD525a PL ratios matched predictions significantly better than the experimental A555/QD520b PL ratios. Lastly, the absorbance values at the QD, A555, and A647 peak emission wavelengths were

Energy Transfer Pathways in a Quantum Dot-Based Concentric FRET

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