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The Competition of Charge and Energy Transfer Processes in DonorAcceptor Fluorescence Pairs: Calibrating the Spectroscopic Ruler. Pavel Moroz, Zhicheng Jin, Yuya Sugiyama, D'Andree Lara, Natalia Razgoniaeva, Mingrui Yang, Natalia Kholmicheva, Dmitriy Khon, Hedi Mattoussi, and Mikhail Zamkov ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01451 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018
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The Competition of Charge and Energy Transfer Processes in Donor-Acceptor Fluorescence Pairs: Calibrating the Spectroscopic Ruler.
Pavel Moroz1,2, Zhicheng Jin3, Yuya Sugiyama3, D’Andree Lara4, Natalia Razgoniaeva1,2, Mingrui Yang1,2, Natalia Kholmicheva1,2, Dmitriy Khon4, Hedi Mattoussi3, Mikhail Zamkov1,2
The Center for Photochemical Sciences1 and Department of Physics and Astronomy2, Bowling Green State University, Bowling Green, Ohio 43403 USA. Department of Chemistry and Biochemistry3, Florida State University, Tallahassee, Florida 32303 USA. Department of Chemistry and Biochemistry4, St. Mary’s University, San Antonio, Texas 78228 USA.
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[email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required) Abstract. Sensing strategies utilizing Fӧrster resonance energy transfer (FRET) are widely used for probing biological phenomena. FRET sensitivity to the donor-acceptor distance makes it ideal for measuring the concentration of a known analyte or determining the spatial separation between
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fluorescent labels in a macromolecular assembly. The difficulty lies in extracting the FRET efficiency from the acceptor-induced quenching of the donor emission, which may contain a significant non-FRET contribution. Here, we demonstrate a general spectroscopic approach for differentiating between charge transfer (CT) and energy transfer (ET) processes in donor-acceptor assemblies and apply the developed method for unravelling the FRET/non-FRET contributions in cyanine dye-semiconductor quantum dot (QD) constructs. The present method relies on correlating the amplitude of the acceptor emission to specific changes in the donor excitation profile in order to extract ET-only transfer efficiencies. Quenching of the donor emission is then utilized to determine the non-ET component, tentatively attributed to the charge transfer. We observe that the latter accounts for 50-99% of donor emission quenching in QD-Cy5 and QD-Cy7 systems, stressing the importance of determining the non-FRET efficiency in spectroscopic ruler and other FRET-based sensing applications.
KEYWORDS: energy transfer, nanocrystals, FRET, crosstalk, bleed through.
Measurements of the energy transfer (ET) interactions in live cells and tissues represent one of the most effective biosensing strategies.1-5 In many cases, the structure and dynamics of the cellular environment can be visualized through the interaction between fluorescent labels anchored to specific sites of the investigated molecule/analyte.6-8 These constructs are designed to exhibit changes in Förster resonance energy transfer (FRET) efficiency in response to a specific biological stimulus. The sensing mechanism is usually enabled by correlating such FRET efficiency changes with the donor-acceptor distance within the Förster dipole-dipole approximation, which could be employed either to measure the concentration of a particular analyte that docks at a known distance to a host,9 or to determine the spatial separation between fluorescent labels in targeted macromolecules.10-13 The strong sensitivity of FRET to small distance variations has found widespread applications in the fields of biology and biochemistry,14-
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where the dynamics of conformational changes in biomolecules at a subnanometer length scale leads
to important insights into a variety of processes, including DNA folding,18 protein conformation,19-22 ligand-receptor interaction,23,24 and metabolic pathways.25,26 Experimental measurements of the FRET efficiency are usually performed by analyzing changes in the donor emission caused by docking of the acceptor moiety (Eq. SE1).27-29 The resulting changes in the intensity (or the lifetime) of the donor photoluminescence (PL) can be employed for estimating the portion of the photoinduced energy that is nonradiatively transferred to the proximal acceptor(s). The intrinsic error associated with such measurements comes from non-FRET contributions to donor PL quenching that are related to the acceptor-induced dissociation of donor excitons; these can manifest in a few mechanisms, including charge transfer (CT) and exciton annihilation.30-33 Consequently, the measured total efficiency of the donor PL quenching, ETOT, in general, may include contributions from non-FRET processes. This is particularly problematic for systems featuring a significant driving force for photoinduced charge transfer interactions. Here, estimating the donor-acceptor separation distance, using energy transfer measurements become subject to a sizable error, as non-FRET processes do not obey the law predicted by the dipole-dipole formalism. For instance, a number of recent works have indicated that charge transfer contribution to donor PL quenching could be considerable in case of energy acceptors containing iron,34 ruthenium complex,35 metal ions or organic conductors.36-38 For instance, Lian group has reported36 that FRET was responsible for only 16% of the energy transfer from CdS nanocrystals (NCs) to adsorbed Rhodamine B dye molecules with the remaining 84% being attributed to electron transfer interactions. Similar results were reported for methylene blue adsorbed on CdSe QDs (∼6% FRET; ∼94% charge transfer).39 Quinones and dopamines, which do not fluoresce, were likewise shown to engage in charge (electron and hole) transfer interactions with QD donors.40 Notably, the probability of charge transfer was found to be dependent on the number of acceptor
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molecules coupled to a donor moiety and therefore difficult to predict for systems with a poorly defined morphology. Additionally, it has been reported that ferrocene may34,41 or may not42 quench the PL of semiconductor nanocrystals. Similar inconsistencies exist in proving whether nanocrystals engage in purely FRET or charge transfer interactions with proximal chromium43 and osmium metal-complexes44 as well as with C60.45 Taken all together, the fundamental competition between FRET and non-FRET processes when considering donor-acceptor pair of dyes and their bioconjugates is still not well understood for the majority of explored systems. Experimentally, the task of differentiating between FRET and non-FRET (e.g., CT) processes in ETbased sensors is rather challenging. Some reported methodologies for identifying CT interactions have been based on obtaining evidence of excited state filling (bleaching of absorbing transitions) in both donor and acceptor counterparts.46 For instance, transient absorption (TA) spectroscopy has been used to track changes in the oxidized or reduced forms of either donor or acceptor moieties, enabling the differentiation between ET and CT contributions.37,47-49 However, definitive evidence for the charge transfer can be challenging to obtain when the acceptor has weak or absent transient absorption features. Here, we report on a general spectroscopic strategy for distinguishing between charge and energy transfer processes in donor-acceptor systems and apply the developed method for measuring the corresponding FRET and non-FRET efficiencies in cyanine dye-semiconductor quantum dot (QD) assemblies. The choice of cyanine fluorophores (Cy5, Cy7) was motivated by the popularity of these molecules in FRET-based sensing schemes where these dyes participate as energy-transfer partners with biological species or other fluorescent molecules.50 To differentiate between the energy and charge transfer
processes,
we
employed
a
recently
developed
Sample
Transmitted
Excitation
Photoluminescence (STEP) spectroscopy approach.51 This method is based on recording changes in the acceptor emission caused by the spectral modulation of the excitation light and is used to determine the
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percentage of donor excitons that were funneled to the acceptor moiety exclusively via the energy transfer mechanism (including any energy transfer processes, such as FRET, Dexter, or non-dipolar ET, which lead to the acceptor excitation without changing its total charge). By using a combination of STEP and donor PL quenching measurements, we observe that in the case of a CdSe/ZnS core/shell QDCy5 system, exhibiting a significant donor-acceptor spectral overlap, up to 50% of donor emission is quenched due to non-FRET processes (such as the QD→Cy5 charge transfer). Meanwhile, in the case of a low-overlap QD-Cy7 construct (where ET is mostly suppressed), the observed 50-60% reduction in the donor PL lifetime was almost entirely attributed to non-FRET processes. Overall, the present experiments indicate that even non-metallic complexes, such as cyanine fluorophores, can engage in non-FRET charge-transfer interactions with the nanocrystal donor, and the relative contribution of these processes into donor emission quenching should be accounted for (using Eq. 3) when used with spectroscopic ruler and other sensing applications.
RESULTS AND DISCUSSION Figure 1 shows the relative positions of excited state energies in QD-Cy5 and QD-Cy7 conjugated systems. The energetics of cyanine dyes have been previously researched in connection with the potential use of these molecules in organic photovoltaic devices.52,53 Based on those studies, we tentatively place HOMO and LUMO levels of a Cy5 dye (see Figs. 1 and SF3) to be energetically above the respective levels of CdSe/ZnS NCs (λexciton = 570 nm). According to Fig. SF3, the CdSe valence band (VB) lies approximately 0.4 eV lower than the Cy5 HOMO level, supporting a possible photoinduced hole transfer from CdSe to Cy5. In the case of a Cy7 dye, both HOMO and LUMO energy levels are straddled by those of CdSe/ZnS NCs favoring the photoinduced transfer of the two charge types in QD to an organic counterpart.
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Figure 1. (a). An energy diagram showing relative positions of excited state levels in QD-Cy5 and QDCy7 conjugated systems. A more detailed description is given in Fig. SF3. The HOMO energies of Cy5 and Cy7 dyes are located ≈ 0.4 eV and ≈ 0.8 eV above the valence band edge of CdSe NCs, respectively, indicating the possibility of a photoinduced QD→CyX hole transfer. (b). Schematics of the STEP approach. An excitation filter with a known optical density is placed in front of the broad band excitation light in order to reduce the number of photons absorbed by a donor, thereby lowering the energy transfer rate. The resulting changes in the acceptor emission intensity are then used to calculate the energy transfer efficiency, ED→A, which represents the percentage of photons absorbed by the donor moiety that are funneled to the acceptor solely via the energy transfer mechanism.
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The details of the STEP spectroscopy for measuring the energy transfer efficiency in donor-acceptor assemblies have been described in recent literature (see Refs. 51,54,55). The method is based on the assumption that the number of photons emitted by an acceptor fluorophore, N APL , depends linearly on the number of excited acceptor (A) and donor (D) molecules, NA and ND, respectively:
N APL = QYA ( N A + E D→ A N D )
(1)
where E D → A is the quantum efficiency for the D→A energy transfer (including any ET process, such as FRET, Dexter, non-dipolar ET, which results in the excitation of A without changing its charge), and
QYA is the emission quantum yield of the fluorophore A in the presence of the fluorophore D (as measured in the donor-acceptor assembly). To determine E D → A , a donor-acceptor sample is excited using a broad-band light source and the emission intensity of the acceptor dye, N APL , is recorded. The excitation light is then spectrally shaped using donor-like or acceptor–like colloidal solutions (Fig. 1b) designed to suppress the excitation of donor or acceptor species in the investigated sample (ND
