Lifting the Spectral Crosstalk in Multifluorophore ... - ACS Publications

Article pubs.acs.org/JPCC

Cite This: J. Phys. Chem. C 2017, 121, 26226-26232

Lifting the Spectral Crosstalk in Multifluorophore Assemblies Pavel Moroz,†,‡ William P. Klein,∥ Kiernan Akers,⊥ Abigail Vore,§ Natalia Kholmicheva,†,‡ Natalia Razgoniaeva,†,‡ Dmitriy Khon,⊥ Sebastián A. Díaz,∥ Igor L. Medintz,∥ and Mikhail Zamkov*,†,‡ †

The Center for Photochemical Sciences, ‡Department of Physics, and §Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, United States ∥ Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States ⊥ Department of Chemistry and Biochemistry, St. Mary’s University, San Antonio, Texas 78228, United States S Supporting Information *

ABSTRACT: A general strategy for measuring the energy-transfer efficiencies in multifluorophore assemblies is demonstrated. The present method is based on spectral shaping of the excitation light with molecular solutions representing donor and acceptor fluorophores, which causes a suppressed excitation of the respective donor and acceptor molecules in the sample. The changes in the acceptor emission resulting from spectral shaping of the excitation light are then used to determine the energytransfer efficiencies (Ex) associated with all participating donor−acceptor pairs. Here, the technique is demonstrated through energy-transfer (ET) measurements in a 4-fluorophore construct featuring a DNA supported assembly of three donor/donor-relay (Cy3, Cy3.5, and Cy5) and one acceptor (Cy5.5) molecules. The resulting Ex were validated using the standard photoluminescence (PL) quenching approach as well as measurements of partial 2- and 3-dye assemblies. The present work highlights general benefits of the spectrally shaped excitation approach to measuring donor−acceptor energetics, including the ability to resolve the spectral cross talk between multiple fluorophores and to exclude charge transfer contributions into donor PL quenching.

M

compromised by the spectral overlap between donor and acceptor dyes. Indeed, the spectral crosstalk obscures the individual contributions from participating energy-transfer partners into the emission of a signaling fluorophore (energy acceptor), making it difficult to compare the observed ET efficiencies with the Förster rate equation. A careful selection of excitation/emission filters, often aided by the polarization-gated detection or a multiple-channel photomultiplier analysis, is required for studying systems containing multiple fluorophores.33 Despite these efforts, the experimental uncertainties associated with energy-transfer measurements in multidye systems are often quite confounding.34,35 This is further complicated by competing processes of donor emission quenching due to the charge transfer to an acceptor,36 which cannot be distinguished from the energy-transfer contribution and therefore inflates the experimental ET efficiency. Here, we demonstrate a universal spectroscopic strategy for direct and accurate measurements of energy-transfer efficiencies in multifluorophore assemblies. The present method relies on

easurements of the energy-transfer efficiency on the nanoscale offer a powerful tool for estimating intermolecular distances with subnanometer precision.1−4 By mapping the resonant energy-transfer efficiency between donor and acceptor fluorophores to a known Förster radius, it is often possible to visualize the morphology of chemical hyperstructures5−7 or to monitor complex biological processes, such as DNA folding, protein conformation, and metabolic pathways.8−18 Going beyond binary donor−acceptor label pairs, multifluorophore constructs interacting via a FRET mechanism are often required for visualizing complex biochemical processes or for labeling specific sites of large macromolecules.19−25 FRET-based multifluorophore structures are also being explored for a host of applications in lightharvesting, sensing, encryption, and computing.26−32 The resulting efficiencies associated with contributing energytransfer channels are usually determined from the changes in the donor emission intensity or PL lifetime (Ex = 1 − τdonor−acceptor/τdonor‑only) induced by the proximity of the acceptor moiety (quenching) and are subsequently converted into the respective donor−acceptor distances. The effectiveness of the multicolor labeling approach to imaging complex biological or chemical processes is often © 2017 American Chemical Society

Received: September 21, 2017 Revised: November 4, 2017 Published: November 7, 2017 26226

DOI: 10.1021/acs.jpcc.7b09396 J. Phys. Chem. C 2017, 121, 26226−26232

Article

The Journal of Physical Chemistry C the spectral shaping of the continuous wave excitation light with solutions of donor molecules, which leads to the suppressed excitation of the respective donor species in the sample. The resulting changes in the acceptor emission are then used to determine absolute energy-transfer efficiencies, ED→A, for all participating donor−acceptor pairs. The present technique represents an extension of the recently reported Sample Transmitted Excitation Photoluminescence (STEP) spectroscopy37,38 to the case of multiple fluorophores, which enables ET efficiency measurements in systems with a significant spectral crosstalk component. The present method also benefits from the ability to distinguish between charge and energy-transfer processes that often coexist in donor−acceptor assemblies and allows for accurate measurements of ED→A without employing excitation/detection filter sets, pulsed light sources, or photon counting techniques. As a model system for measuring the energy-transfer efficiencies in a multifluorophore assembly, we employ a 4dye construct (Cy3-Cy3.5-Cy5-Cy5.5) supported on a double helix DNA backbone (see Figure 1a and Table ST3). The fluorophores were chosen to promote a one-directional energy transfer, Cy3 → Cy3.5 → Cy5 → Cy5.5, with any donor molecule, potentially contributing to the emission of the Cy5.5 acceptor (see Figure 2). The design resembles a double crossover alternating even (DAE)-based structure39 with two parallel template strands held together by a series of ∼1 helical turn staples shown in Figure 1a and Table ST3. However, single crossovers were utilized to hold the two parallel template strands together. The single crossovers also allowed for fluorophore labeling at the end of the DNA strands. The de novo sequences were purchased from Integrated DNA Technologies (Coralville, IA) except for the Cy3.5- and Cy5.5-functionalized strands, which were purchased from Operon Biotechnologies, Inc. (Huntsville, AL). The sequences are listed in Table ST3 and were selected such that each binding portion had no more than three complementary bases with nondesired binding domains. The fluorophores were placed at the 5′ ends of the DNA sequences using three carbon spacers. All sequences were synthesized using phosphoramidite chemistry. The DNA constructs were formed in 2.5X PBS (phosphate buffered saline, pH 7.4) by adding all of the strands found on Table ST3, either the fluorophore-labeled or the equivalent unlabeled strand. The mixture was annealed by heating to 94 °C and then decreasing the temperature by 1 °C per minute to 4 °C in a PCR thermocycler. The constructs were characterized by absorbance, fluorescence spectroscopy, and agarose gels. The gels (an example gel is available in the Supporting Information) reported 65 ± 3% fully or perfectly formed structures with that number increasing to 85 ± 2% if we consider partially formed structures. The STEP spectroscopy technique for measuring the cascaded ET efficiencies in the Cy3-Cy3.5-Cy5-Cy5.5 system is based on the assumption that the number of photons emitted by the acceptor molecule (Cy5.5), NFL A , can be expressed as a linear combination of the number of excited states Ni in all donor fluorophores (i = 1...LD) participating in the energy transfer:

Figure 1. (a) Schematic representation of the DAE-based structure with two parallel template strands and a series of ∼1 helical turn staples supporting the Cy3, Cy3.5, Cy5, Cy5.5 fluorophores. The cascaded downhill energy-transfer direction, Cy3 → Cy3.5 → Cy5 → Cy5.5, is indicated by arrows. The associated energy-transfer coefficients (Ei→Cy5.5, i = Cy3, Cy3.5, Cy5) were measured using the STEP spectroscopy that employs shaping of the excitation light with optical filters comprising solutions of each of the four fluorophores. The corresponding changes in the Cy5.5 (acceptor) emission are then used to calculate Ei→Cy5.5. (b) Schematic illustration of the experimental setup. The light from the broad band excitation source is picked by a reference photodiode (PD1) for integrating the photon flux emitted by the halogen lamp during each measurement. The second photodiode (PD2) samples the excitation beam after it passes through the filter solution. The PD2 count is used to calculate the OD of filter solutions in situ. The fluorescence of the acceptor dye, NFL A , is detected using a spectrometer and normalized on the basis of the corresponding level of the photon count received by PD1.

(1)

Figure 2. Absorption profiles of the four fluorophores participating in the energy transfer along the DNA backbone (Cy3, Cy3.5, Cy5 → Cy5.5). The corresponding extinction coefficients are 150 000, 150 000, 250 000, and 190 000 M−1 cm−1, respectively. The emission of the acceptor molecule (Cy5.5) as well as the spectral profile of the excitation light (nWL) are also plotted.

where the i = LD + 1 term represents the contribution of the acceptor molecule into its own emission, and Ni are the

numbers of photons absorbed by the ith fluorophore. For detailed derivations, see the Supporting Information. In the

NAFL = QYA ×

LD + 1

∑ i=1

EiNi

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ratio of photons absorbed by these species can be expressed using relative rather than absolute values. Indeed, because the excitation spectral profile nWL(λ) entering eq 3 is the same for (j) both j and k molecules, the N(j) i /Nk ratio can be calculated using a scaled excitation beam profile rather than the actual photon density. Our simulations show that the ensuing accuracy of measurements is only weakly dependent on which k “denominator” fluorophore is chosen for a particular j filter. To indicate that a certain k “denominator” fluorophore is chosen for a particular j, we define a k(j) function (j = 1...LD + 1) that specifies the denominator, N(j) k , for each of LD + 1 equations. In the next step, we exclude the unknown QYA constant by dividing the first LD equations in the system (eq 5) by the last one (j = L D + 1). This strategy allows obtaining L D independent equations containing LD unknown variables, Ei:

case of the Cy3-Cy3.5-Cy5-Cy5.5 system, there are three donor/relay molecules: Cy3, Cy3.5, and Cy5, so LD = 3. QYA is the acceptor emission quantum yield in the donor−acceptor assembly, which could be different from the “nominal” QYA of this molecule (Cy5.5) in solution due to DNA conjugation.40 Ei are the energy-transfer efficiencies given by the fractions of photons absorbed by the ith fluorophore that ultimately result in the formation of an exciton on the acceptor. These include both the direct energy transfer to an acceptor as well as the mediated, cascade ET process, which involves an energy relay through other dyes. The last Ei coefficient in the sum of eq 1 (i = LD + 1) corresponds to the acceptor’s contribution into its emission and is therefore equal to unity:Ei = LD + 1 ≡ 1. Equation 1 contains LD + 1 unknown variables: QYA, Ei (i = 1...LD). The application of LD + 1 excitation filters composed of solutions of each fluorophore in the assembly allows shaping of the excitation light in such a way that it preferentially suppresses the excitation of the corresponding fluorophore in the Cy3-Cy3.5-Cy5-Cy5.5 sample. This results in LD + 1 independent equations: NAFL(j) = QYA ×

LD + 1

∑

EiNi(j) ,

j = 1 .. L D + 1

bj = Aij Ei ,

where NFL A (j) are the numbers of emitted acceptor photons resulting from the application of jth fluorophore as an excitation filter. N(j) i are the numbers of photons absorbed by the ith fluorophore in the case when the excitation light is transmitted through a “filter” solution of the jth molecule, given by Ni(j) =

∫ nWL(λ) × 10−Abs

filter j (λ)

sample

× [1 − 10−Absi

(λ)

] dλ (3)

where nWL(λ)is the spectral photon density of the excitation light, Absfilter (λ) is the absorbance of the jth filter solution, and j Abssample (λ) is the absorbance of the ith fluorophore in the i sample, obtained by fitting the absorbance profile of the investigated system with a weighted sum of donor and acceptor absorbance profiles (see Figure 2): LD + 1

Abs(sample) =

∑

Ci(λ) × Absisample(λ)

i=1

(4)

The sequence of LD + 1 filters to be applied in the path of the excitation light is arbitrary. For simplicity, one can apply the filters in the order of increasing absorbance wavelength. In the case of the current Cy3-Cy3.5-Cy5-Cy5.5 assembly, individual Cy3, Cy3.5, Cy5, and Cy5.5 excitation filters were applied sequentially. Once the order of filters is selected, the system of equations (eq 3) is converted from absolute photon numbers to relative ratios by dividing each equation in the system by N(j) k representing the number of photons absorbed by a donor k, such that k ≠ j. The resulting system is now given as NAFL(j) Nk(j)

LD + 1

= QY ×

∑

Ei × (Ni(j)/Nk(j)),

(6)

where the matrix Aij and the vector bj are given in eqs SE6 and SE7. Notably, the division of the first LD equations in the system (eq 5) allows transitioning from absolute numbers of emitted and absorbed photons (in the left-hand side of eq 5) to their relative ratios. The values of the acceptor emission corresponding to the application of LD excitation filters, NFL A (j = 1...LD), become normalized by the value of the acceptor emission resulting from the application of the acceptor-based FL excitation filter: NFL A (j = 1...LD)/NA (j = LD + 1). Experimental measurements of the acceptor emission associated with the numerator and the denominator of this fraction are recorded simply by changing the excitation filter, which does not alter the detection solid angle or its efficiency. Consequently, the normalization procedure of eq 6 (see also eq SE3) effectively cancels out experimental detection constants allowing the use of the raw emission counts (collected by a spectrometer or a single channel detector) instead of absolute photon numbers in eq 5. According to eq 4, there are four types of input parameters that are needed to obtain the donor−acceptor energy-transfer efficiencies, Ei. These include (i) integrated acceptor emission counts, NFL A (j), resulting from the application of donor and acceptor excitation filters (j = 1...LD + 1), (ii) the spectral profile of the excitation light, nWL(λ), (iii) optical absorptions of donor and acceptor filter solutions, Abssample (λ), and (iv) the i absorption of the Cy3-Cy3.5-Cy5-Cy5.5 DNA construct (Figure 3a). The latter is fitted with a linear combination of individual dye absorption profiles using eq 4. The fitting coefficients are given in Table ST2. Prior to determining the energy-transfer efficiencies in the 4dye construct (Cy3-Cy3.5-Cy5-Cy5.5), a baseline measurement was performed using a solution of Cy5.5 molecules. Because the target contains only the acceptor molecule in this case, all donor contributions determined using the four-filter (Cy3, Cy3.5, Cy5, and Cy5.5) STEP measurements should result in zero ET coefficients, Ei = 0. To perform such a baseline run, a formamide solution of the Cy5.5 fluorophore was excited using the broad-band light source featuring a known spectral density, nWL(λ) (Figure 2). The resulting Cy5.5 emission was collected in a 90° geometry to avoid direct detection of the excitation light. The filter cuvette was then placed in the path of the excitation beam and was sequentially refilled with solutions of the four filter dyes. The resulting emission counts, NFL A (j) (j = 1−4) were recorded and used to calculate 16 elements of the 4 × 4 Aij matrix (eq 6). According to Table 1, the measured

(2)

i=1

i , j = 1 ... L D

j = 1 .. L D + 1

i=1

(5)

Notably, the numbers of photons absorbed by the ith fluorophore in eq 5 are now normalized by the kth molecule (j) absorbed photon count: N(j) i /Nk . Because both j and k molecules occupy the same excitation volume in the sample (assuming a homogeneous distribution of donor species), the 26228


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Figure 4. Summary of the energy-transfer efficiency measurements for a DNA-supported assembly of four dyes (Cy3, Cy3.5, Cy5, Cy5.5). See also Table 1. The E (Cy3.5 → Cy5) value was obtained using a partial construct containing Cy3.5 and Cy5 dyes on a DNA backbone.

Figure 3. (a) The absorption profile of the Cy3-Cy3.5-Cy5-Cy5.5 sample (blue ○) fitted with a linear combination of individual dye absorption profiles based on eq 4. Fitting coefficients are given in Table ST2. (b) Illustration of the donor contribution into the acceptor emission window (emission cross talk). The contribution of each dye in the Cy3-Cy5.5 assembly into the detection window (λ ≈ 760−850 nm) is given as a ratio ( f) in the top right corner of the figure as well as in Table ST2.

molecules (Cy3, Cy3.5, and Cy5), whose PL profiles extended into the Cy5.5 detection widow (λ ≈ 760−850 nm). To account for an emission bleed-through, the relative donor contributions into the detection spectral range were determined by fitting the emission profile of the Cy3-Cy3.5-Cy5-Cy5.5 sample with individual emission profiles of the four fluorophores (see Figure 3b). Subsequently, the contribution of each dye into the emission detection range was integrated. According to Figure 3b and Table ST2, Cy5 can contribute up to 22% into the sample emission in the 760−850 range, while Cy3.5 accounts for 4% of the PL counts in the same spectral window. The resulting contributions, f i, were subtracted from the determined energy-transfer efficiencies, giving rise to corrected coefficients, Ei = Ei* − f i (Table 1). The obtained efficiencies were compared to the benchmark values obtained by applying the methodology previously exploited in the literature.27,31 The 4-dye construct was characterized through the end-to-end transfer efficiency (Eee) and the anywhere-toend transfer efficiency (Eae). The Eee is a measure of the excitons reaching the Cy5.5 emitter that were introduced by the initial donor (Cy3) and transported by the relay dyes. The value is calculated by eq 7.

Table 1. Energy-Transfer Efficiency Measurements in DNASupported Assemblies of (Column 2) Four Dyes (Cy3, Cy3.5, Cy5, and Cy5.5), (Column 3) Three Dyes (Cy3.5, Cy5, and Cy5.5), and (Column 4) Two Dyes (Cy5 and Cy5.5)a 1

2

3

4

energy-transfer coefficients

Cy5.5-only (no-donor baseline)

Cy3-...Cy5.5 assembly

Cy3.5-...Cy5.5 assembly

Cy5Cy5.5 assembly

E (Cy3→Cy5.5) E (Cy3.5→Cy5.5) E (Cy5→Cy5.5)

0.021 −0.01 0.014

0.31 0.40 0.42

0.43 0.40

0.41

a

The baseline measurements performed on a solution of Cy5.5 dyes in formamide are summarized in column 1.

efficiency coefficients, averaged over three independent runs, were fairly close to zero with a combined standard deviation of 1.4%. This value provides an estimate of the experimental uncertainty associated with STEP measurements in the present 4-dye system. The DNA supported 4-dye construct was investigated next. The sample was dissolved in 2.5× PBS buffer and placed in a cuvette for a 90° photoluminescence detection. The filter cuvette, placed in the path of the excitation beam, was sequentially refilled with Cy3, Cy3.5, Cy5, and Cy5.5 fluorophore solutions at an approximate optical density of 0.5. The four corresponding values of the sample emission, NFL A (j), were recorded for each case. The intensity of the excitation beam was measured before and after the excitation filter using a 10:90 beamsplitter and a single-channel photodiode (see Figure 1b) to enable in situ measurements of the filter OD, which was subsequently used to calculate the spectral density of the excitation light, nWL(λ) × 10−OD(λ). Ultimately, the measured emission counts, NFL A (j), along with the expected numbers of photons absorbed by the sample for each type of the excitation filter, N(j) i (please see Figure 4), were used to determine coefficients of the Aij matrix in eq 6. Solving the system of linear eqs (eq 6) resulted in a set of Ei* efficiencies that prior to accounting for the emission bleed through (discussed below) were found to be E*Cy3→Cy5.5 = 0.32, E*Cy3.5→Cy5.5 = 0.44, and E*Cy5→Cy5.5 = 0.64. The measured emission count, NFL A (j), can potentially be inflated by the PL contributions from the three donor

⎛ FAD − FA′ QY ⎞ A ⎟⎟ Eee = ⎜⎜ F ⎝ D QYD ⎠

(7)

where QYA and QYD are the fluorescent quantum yields of the final acceptor and initial donor, respectively, F denotes the integrated fluorescence intensity of the emitters, FAD is the emission of the Cy5.5 in the presence of the donor (as well as all intermediate dyes), and FA′ is the fluorescence intensity of the final acceptor with the relay dyes (Cy3.5 and Cy5) but not the initial donor. This consideration separates the Eee from the Eae, which is calculated similarly to Eee with the modification that FA′ is replaced by FA, the direct excitation of Cy5.5 when it is present alone. For the 4-dye rail construct, the Eee was 0.28 and the Eae was 0.54 (Table ST5). The Eee was slightly lower than the STEP reported ECy3‑Cy55 as might be expected as the value requires a second control structure (Cy3.5-Cy5.5) to correct for downstream excitation. The control structure does not take into account the decreased competition for photons that occurs by eliminating the Cy3, which leads to a slight overcorrection. This factor may also be concentration and photon flux-dependent, making it hard to account for. The STEP strategy overcomes the limitation by working only with the original structure. The Eae is higher as it incorporates all of the direct excitation of the relay components, which have higher Ei as seen in Table 1. 26229


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Figure 5. Illustration of the measured ET efficiencies associated with downhill transitions in a Cy3.5-Cy5-Cy5.5 three-dye system obtained using the STEP approach. To unveil intermediate transitions, STEP measurements were performed on three systems: (a) a three-dye assembly (Cy3.5, Cy5, Cy5.5); (b) a two-dye assembly (Cy3.5, Cy5.5); and (c) a two-dye assembly (Cy3.5, Cy5).

To investigate the character of energy flow along the DNA backbone, we measured the contributions of the individual energy-transfer pathways within a Cy3.5-Cy5-Cy5.5 three-dye system. In general, the task of distinguishing these partial terms entering the total Cy3.5 →Cy5.5 ET efficiency is difficult to achieve through steady-state PL quenching measurements due to a substantial spectral crosstalk between the three fluorophores. By employing STEP measurements, such individual contributions can be unraveled more effectively via the “resonant” suppression of targeted donor excitations. In the case of the investigated Cy3.5-Cy5-Cy5.5 assembly, the excitation energy absorbed by the Cy3.5 fluorophore can be transferred to the end acceptor (Cy5.5) through the two possible channels: either directly, Cy3.5 → Cy5.5, or via an intermediate Cy5 relay dye (Cy3.5 → Cy5 → Cy5.5). Along these lines, the STEP approach was employed to determine the energy-transfer efficiencies in partial assemblies of (Cy3.5, Cy5) and (Cy3.5, Cy5.5) fluorophores. The resulting ET coefficients associated with relevant downhill transitions in a Cy3.5-Cy5Cy5.5 three-dye system are given in Table ST4 as well as in Figure 5. Here, the (Cy3.5, Cy5.5) assembly was treated as a three-dye construct despite a missing Cy5 component and was sequentially subjected to the application of three excitation filters: Cy3.5, Cy5, and Cy5.5. The resulting ET efficiency corresponding to a missing donor filter (Cy5) was found to be 0.004%, which is well within the expected uncertainty of 4%. According to Figure 5, the direct ET from Cy3.5 to Cy5.5 contributed 0.31, while the two-step cascade transfer (Cy3.5 → Cy5 → Cy5.5) gave rise to the corresponding hop efficiencies of 0.68 and 0.40. The addition of the relay results in a higher overall transfer, 0.43, an increase of ∼40% from the 0.31 measured without the relay. The interesting detail from the measurements is that the inclusion of the relay is not additive as it competes with the long distance transfer by introducing a competitive pathway. Using the determined rates and a Cy3.5 fluorescence lifetime of 2.0 ns,41 we can determine that the direct E is now only 0.13, down from 0.31. In addition to the 0.27 value estimated from the relay pathway, this is 0.40, within range of the measured 0.43. Overall, the individual contributions seem to agree with the total efficiency observed in this system. In conclusion, we have demonstrated a spectroscopic strategy for measuring the energy-transfer efficiencies in molecular

The statistical uncertainties associated with STEP measurements of Ei coefficients were obtained using run-to-run averaging (approximately 3−4 runs for each construct). The resulting standard deviation of 3.2% was comparable to the 1.4% uncertainty observed in the Cy5.5-only baseline run (Table 1, column 1). The experimental Ei coefficients were also compared to those obtained in STEP measurements of partial dye assemblies featuring 3-dye (Cy3.5-Cy5-Cy5.5) and 2-dye (Cy5-Cy5.5) systems, shown in Table 1, rows 4 and 5, respectively. These partial constructs were fabricated using the same fluorophore anchoring geometry as in the case of the investigated Cy3-Cy3.5-Cy5-Cy5.5 target. The Cy5-Cy5.5 target was also investigated using a conventional STEP approach that relies on f-ratio measurements (see Figure SF1 and ref 37 for details). According to Figure SF1b, the Cy5 → Cy5.5 energy-transfer efficiency was 43%, which is close to the value determined using a matrix method (eq 6). On the basis of the average magnitude of observed discrepancies in baseline and partial samples, we expect the total measurement error to be within 4%. We note that the Eee and Eae values can have uncertainties in the 8−10% range for similar systems.41 Finally, the accuracy of STEP measurements resulting from the application of the fluorophore filter set (Cy3, Cy3.5, Cy5, Cy5.5) was compared to that of analogous STEP measurements utilizing 4 notch filters (bandwidth = 40 nm, centered at absorption maxima of the four investigated dyes). To this end, we have simulated the propagation of the 3% uncertainty in the emission intensity (ΔNFL) on the ensuing error in the determination of the three energy-transfer coefficients, Ei, for both the fluorophore-based and the notch-type excitation filter sets. The results are summarized in Figure SF2, which shows that the employment of fluorophore filters leads to overall lower uncertainties. The experimental error of STEP was attributed to two potential sources: (1) the nonlinearity of the relationship between emitted and absorbed photons (eq 1), which is likely to be small under low power excitation regime, and (2) the sample absorbance fitting error of eq 4. Additional sources of error could be associated with the ambiguity in the optical density of the filter solution due to its colloidal instability and incomplete mixing, as well as the uncertainty in the actual spectral shape of the excitation light due to excitation dispersion issues and exciton filter self-fluorescence. 26230


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The Journal of Physical Chemistry C assemblies featuring multiple donor fluorophores. The present method is based on spectral shaping of the excitation light using donor molecule solutions, which suppresses the excitation of respective donor species in the sample. The changes in the acceptor emission corresponding to the inhibited excitation of donor molecules in the sample were used to determine the energy-transfer efficiencies corresponding to all donor molecules participating in the energy transfer. The technique was demonstrated using a 4-dye model system (Cy3, Cy3.5, Cy5, Cy5.5 dyes supported on a DNA backbone) where the observed ET efficiencies were validated on the basis of standard PL measurements as well as measurements of partial 2- and 3dyes assemblies featuring the same conjugation geometries. The experimental error was further assessed using a baseline run utilizing acceptor-only solutions. The demonstrated ability of STEP to lift the spectral cross talk was subsequently employed for resolving the individual contributions associated with interdye energy-transfer channels into the total end-to-end ET efficiency in a Cy3.5-Cy5.5 system. The demonstrated approach for measuring energy-transfer efficiencies in systems with multiple donor molecules represents a potentially useful strategy for determining intermolecular distances in multifluorophore assemblies, such as multiply labeled DNA constructs or systems with several fluorescent proteins. Particular benefits of the present method stem from the two important features: (i) the ability to resolve the spectral cross talk between multiple molecules participating in the energy-transfer reaction, and (ii) the ability to exclude charge transfer pathway of photoluminescence quenching in supported dyes that can potentially inflate the energy-transfer efficiency. Other advantages of the demonstrated STEP spectroscopy include the utilization of steady-state, broadband light sources and the universal scalability across visible and near-IR spectral ranges due to filter free operation.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b09396. Experimental section, additional figures, and details of the calculation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sebastián A. Díaz: 0000-0002-5568-0512 Igor L. Medintz: 0000-0002-8902-4687 Mikhail Zamkov: 0000-0002-8638-2972 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge OBOR “Material Networks” program and NSF Award CBET-1510503 for financial support. K.A. was funded by the Welch foundation grant no. U-0047.

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REFERENCES

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