Controlling Photoswitching via pcFRET in Conjugated Polymer

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C: Physical Processes in Nanomaterials and Nanostructures

Controlling Photoswitching via pcFRET in Conjugated Polymer Nanoparticles Xinzi Zhang, Aiko Kurimoto, Natia L Frank, and Elizabeth J. Harbron J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b06900 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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The Journal of Physical Chemistry

Controlling Photoswitching via pcFRET in Conjugated Polymer Nanoparticles

Xinzi Zhang,† Aiko Kurimoto,‡ Natia L. Frank,‡ and Elizabeth J. Harbron*† † ‡

Department of Chemistry, The College of William and Mary, Williamsburg, VA 23187-8795

Department of Chemistry, University of Victoria, Victoria, British Columbia, V8W 3V6, Canada

*To whom correspondence should be addressed. E-mail: [email protected]

Abstract Nanoparticles prepared from conjugated polymers (CPNs or Pdots) are powerful fluorophores for sensing and imaging applications, including those that require fluorescence photoswitching. We and others have developed CPNs doped with photochromic dyes that act as acceptors in only one of their two forms via fluorescence resonance energy transfer (pcFRET). We recently developed visible-light-responsive CPNs doped with a reverse photochromic spirooxazine dye that has a thermally stable merocyanine form. Off-to-on fluorescence switching occurs when the merocyanine is switched to its non-quenching spirooxazine form. The on-state fluorescence intensity is more than 100 times greater when photoswitching occurs via FRET from the CPNs than when the dyes are excited directly. Temporal control of fluorescence intensity is achieved using a single-color input of variable intensity for fluorescence excitation (read-only mode) and photoswitching (read-write mode). Here we present photokinetic measurements and simulations that establish the origin of the observed fluorescence behavior. We find that highly efficient CPNto-merocyanine energy transfer sensitizes the photochromic reaction, which has a quantum yield of 0.03 in the absence of FRET. Simulated fluorescence trajectories reveal that the low

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photochromic quantum yield is the necessary condition for single-color intensity control in this system. From the simulations, we determine that the product of the photochromic quantum yield and the energy transfer efficiency ( * E) must be no more than 0.05 to produce analogous fluorescence behavior. These findings suggest that the CPNs could be used as an amplifier to drive other low-quantum-yield photoreactions to completion with intensity control.

Introduction Light is an appealing stimulus for molecular transformations because it can be delivered to a sample with high spatiotemporal resolution and control of exposure energy and intensity. In conjunction with light stimulation, photoresponsive molecules can be used to produce molecular motors,1–3 alter material properties,4,5 unmask reactive moieties,6,7 and activate fluorescence in displays8 and super-resolution imaging experiments.9–12 Light functions as an input in all of these applications and as an output in the form of fluorescence in some of them. Many super-resolution microscopy techniques, for example, require fluorescent probes that can be stochastically switched between non-emissive and emissive states using a light signal.10 Likewise, light is used to control the molecular form of photochromic sensors, which may then exhibit analyte-dependent output fluorescence properties.13 Imaging and sensing applications such as these have prompted increased interest in fluorophores with photoswitchable emission properties.14–18 One approach to creating molecular systems that are both highly fluorescent and strongly light-responsive is to combine a fluorophore with a photochromic moiety.19 In response to a light signal, photochromic molecules undergo a reversible transformation between two molecular forms with discrete molecular properties.20 Photoinduced changes in absorption spectra or redox potentials alter the photochrome’s ability to quench the emission of a proximate fluorophore by


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energy or electron transfer mechanisms, respectively.21 The photochromic transformation thus results in the activation or deactivation of fluorescence, a phenomenon that has been exploited in super-resolution microscopy17 and molecular logic gates,22,23 among other applications. We recently reported24 that visible light reversibly photoswitches fluorescence intensity in conjugated polymer nanoparticles (CPNs or Pdots) of the polymer poly[9,9-dioctylfluorenyl2,7-diyl)-co-1,4-benzo-{2,1′-3}-thiadiazole)] (PFBT) doped with spiro[azahomoadamantanephenanthrene-oxazine] (APESO), a negative photochromic dye25 that is non-fluorescent. Due to their exceptional fluorescence brightness, high photostability, aqueous compatibility, and low cytotoxicity,26 CPNs are compelling fluorophores for photoswitching and other imaging and sensing applications.27–29 Dopant APESO’s thermally stable form is a colored merocyanine (MC, Figure

Figure 1. (A) Chemical structures of MC and SO forms of APESO dye and conjugated polymer PFBT. (B) Spectral overlap of CPN fluorescence spectrum (green) with MC absorbance in the form of a difference spectrum obtained from spectra of doped CPNs before and after irradiation (purple). (C) Scheme for reversible photoswitching of CPN fluorescence under single-color control.

1) that converts to a colorless spirooxazine (SO) upon visible irradiation and reverts to MC in the dark. The combination of PFBT CPNs with the MC form of APESO was designed to exhibit photoswitchable fluorescence resonance energy transfer (FRET), also known as photochromic FRET (pcFRET).30 The MC form of the dye is expected to act as a FRET acceptor for the CPNs, as evidenced by the strong spectral overlap between the donor CPN fluorescence spectrum and the acceptor MC absorption spectrum (Figure 1). The SO form of APESO absorbs only in the UV and



is not expected to have any electronic interaction with the PFBT chromophores. Thus, CPN fluorescence is quenched by FRET when the dyes are in their MC form and unquenched when the dyes are switched to the SO form. The APESO-doped CPNs possess many desirable features for photochromic fluorophores: compatibility with an entirely aqueous environment, off-to-on fluorescence switching, and photoswitching induced by visible rather than ultraviolet light. However, this system also exhibits unusual features that deviate from those of the ideal photochrome-fluorophore system. Notably, off-to-on fluorescence switching occurs with a dramatically greater on-state fluorescence intensity when the MC dyes are excited via FRET from the CPNs than when excited directly. Consequently, the highest fluorescence intensity is achieved when exc = switch = 455 nm, which generates the CPN excited state required for both CPN fluorescence and FRET to MC acceptors (Figure 1). This scenario contrasts with the preferred use of distinct colors (exc ≠ switch) for these operations, which enables the fluorescence and photoswitching operations to be controlled independently. In spite of the single-color input conditions employed here, we were able to achieve temporal control of emission intensity by using different light intensities (Iexc ≠ Iswitch). We previously attributed the higher on-state fluorescence intensity observed upon CPNmediated excitation of MC dyes to the light harvesting capability of the CPNs.24 This effect originates in the CPNs’ exceptional absorption characteristics (extinction coefficients 107-108 M1

cm-1)31 and intrinsic exciton diffusion processes that efficiently deliver excited state energy to low

energy acceptors.32 These features enable CPNs to be unusually efficient FRET donors to doped acceptors and provide a qualitative explanation for our previous results. In our system, however, the mechanistic details that enable temporal control of fluorescence by varying the intensity of a single color of irradiation remains unknown.


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Here we present photokinetic measurements and simulations that explain how FRET excitation of MC dyes yields higher fluorescence intensities than direct excitation and how a single-color input of variable irradiation intensity can be used to control fluorescence intensity. Our ultimate goal is to elucidate the molecular and photophysical features of the doped CPNs that enable these properties. We find that the combination of a low-quantum-yield photochromic reaction with a highly efficient transfer of energy from the CPNs to MC dyes enables greatly enhanced fluorescence photoactivation in this system. CPN composition is also crucial, with an excess of MC dye dopants serving as a buffer population to prevent inadvertent fluorescence activation. An additional goal of this work is to determine how generalizable these findings might be to other dye-CPN combinations that could be useful in applications beyond fluorescence intensity modulation. Through simulation of fluorescence trajectories, we demonstrate that specific values of the photochromic quantum yield and energy transfer efficiency are required for intensity-controlled fluorescence activation. The experimentally-determined parameters for APESO-doped CPNs fall fortuitously in the “Goldilocks” region in which fluorescence intensity can be controlled under experimentally feasible irradiation conditions. These results suggest that the CPNs could be used as an amplifier to drive other low-quantum-yield photoreactions to completion, and the simulations provide guidelines for the design of future systems for this purpose.

Experimental Methods Materials. All chemicals were obtained from Acros or Sigma-Aldrich and used as received unless otherwise specified. Poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,1′-3}thiadiazole)] (PFBT) with an average molecular weight of 138,000 and polydispersity 3.1 was



obtained from American Dye Source (Quebec, Canada) and used as received. Granular poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate) (PVB-VA-VA) was obtained from Sigma-Aldrich. Spiro[azahomoadamantane-phenanthrene-oxazine] (APESO) was synthesized as described previously.33 The extinction coefficient of APESO was measured in a mixture of 25% water and 75% DMSO due to insolubility in pure water. Nanoparticle Preparation. CPNs were prepared by a literature procedure.34 Separate stock solutions of PFBT (1 mg/mL) and optically transparent PVB-VA-VA (1 mg/mL) in anhydrous tetrahydrofuran (THF) were stirred overnight under argon. The addition of PVB-VAVA was previously shown to reduce photobleaching of MC dyes.24 A precursor solution was prepared by combining portions of the PFBT and PVB-VA-VA solutions with additional THF to a final concentration of 0.04 mg/mL PFBT and 0.008 mg/mL PVB-VA-VA. This precursor solution was filtered through a 0.7 m filter to remove any aggregates and then sonicated for 30 s to ensure homogeneity. A 1 mL portion of the precursor solution was injected into 8 mL of sonicating ultrapure water, which was then sonicated for an additional 2 min. For APESO-doped CPNs, a freshly-prepared stock solution of APESO in THF was added to the aqueous suspension of CPNs while sonicating, and sonication was continued for an additional 15 s. The volume of APESO stock solution added was adjusted for each preparation so that the concentration of APESO in CPNs would be 8 wt. % relative to PFBT. The THF was removed by a high vacuum pump, and the aqueous suspension of APESO-doped CPNs was filtered through a 0.22 m filter. Undoped control CPNs were prepared by the same method with omission of the dye doping step. Nanoparticle size distributions were measured in aqueous suspension by dynamic light scattering using a Nicomp N3000 Submicron Particle Sizer (Particle Sizing Systems).


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Spectroscopy and Photochemistry. Absorption and fluorescence measurements were made with Varian Cary 50 and Varian Eclipse instruments, respectively. All fluorescence experiments used an excitation wavelength of 455 nm. CPNs were studied in aerated aqueous suspension in semi-micro cuvettes (10 mm x 4 mm interior dimensions). Small sample volumes (150 L) were used to maximize consistency of the visible irradiation intensity throughout the sample. Visible irradiation (455 nm and 590 nm) was provided by a 4-Wavelength High-Power LED Source (ThorLabs, DC4100) with square wave input from a function generator. Irradiation was delivered to the top of the sample cuvette in the absorbance and fluorescence instruments by a liquid light guide (ThorLabs, LLG0538). Photon flux values for 455 nm and 590 nm irradiation were determined by chemical actinometry35 using Aberchrome 670.36

Results and Discussion CPN Preparation, Characterization, and Operation. APESO-doped CPNs for fluorescence photoswitching were prepared by a reprecipitation method in which THF solutions of the CPN components are injected into water. Following THF removal and filtration, the resulting aqueous suspension of doped CPNs is clear and slightly tinted. The size distribution of the doped CPNs was measured in aqueous suspension by dynamic light scattering and is shown in Figure S1. APESO-doped CPNs had an average diameter of 13.6 ± 0.8 nm. The absorption spectrum of the doped CPNs is dominated by the PFBT conjugated polymer chromophores, which exhibit a broad visible absorption around 465 nm (Figure 2). Dyes in the asprepared CPN suspension are predominantly in the MC form, which can be seen in the absorption spectrum as a small peak above 540 nm. MC’s absorption spectrum with max = 570 nm can be seen more clearly in a difference spectrum that removes the polymer absorption (Figure 1). As



described in detail in the SI,

absorbance

Figure 2. Absorption spectra of APESO-doped CPNs in aqueous suspension with irradiation wavelengths marked ( = 455, 590 nm). Inset: Fluorescence spectra of APESO-doped (red) and undoped (black) CPNs.

and

sizing data can be used to estimate that the average comprised

CPN

is

of

4.5

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polymer chains and 110 dye molecules. These MC dyes are effective quenchers of the CPN fluorescence, with as-prepared CPNs exhibiting only trace fluorescence when compared to undoped control CPNs (Figure 2 inset). To determine how the APESO-doped CPNs function with single-color control of the fluorescence intensity, we studied off-to-on fluorescence switching under three different sets of conditions. Scheme 1 depicts the three pathways, all of which begin with the fluorescence “off” state exhibited by the doped CPNs in their as-prepared form. Pathways 1 and 2 are designed for reversible off-to-on fluorescence photoswitching. Pathway 1 conditions directly excite MC to its singlet excited state (MC*), which is required for the photochromic conversion to SO. We previously attempted to study this pathway using switch = 530 nm but found that the results were distorted by CPNs’ small absorption at this wavelength. Here, pathway 1 employs switch = 590 nm, which is to the red of the maximum absorption of MC in the CPN environment (max = 570 nm, Figures 1 and 2) but is not absorbed by the CPNs. Pathway 2 conditions Scheme 1. Pathways for reversible (1 and 2) and irreversible (3) photoactivation of CPN fluorescence.

directly excite the CPNs, which are expected to


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generate MC* via FRET. The 455 nm irradiation used for pathway 2 falls in the main CPN absorption band (Figure 2) and is poorly absorbed by MC. Indeed, 99.95% of 455 nm light is absorbed by the CPNs based on the extinction coefficients of the components at 455 nm. LED irradiation in both pathways 1 and 2 is in the form of a square wave of 5 s duration, which is sufficient to induce the photochromic reaction to a measurable extent without significant photobleaching. Comparison of these pathways will enable us to separate the photophysical roles of the CPNs and MC dyes in the performance of the system. Pathway 3 is an alternate means of off-to-on fluorescence activation that relies on irreversible photobleaching of MC by high intensity 455 nm irradiation. This pathway enables study of CPNs with a low concentration of MC dyes, a regime that is not accessible by pathways 1 and 2. Figure 3 shows the doped CPNs’ peak fluorescence intensity as a function of time under pathway 1 and 2 conditions. Direct excitation of MC dyes with orange light induces a scant 10% increase in CPN fluorescence intensity. In contrast, blue light of slightly lower flux and identical duration successfully activates CPN fluorescence, yielding an 1100% increase in peak fluorescence intensity. On-state fluorescence intensity is more than 100 times greater upon CPNmediated excitation of MC than direct excitation. The fluorescence intensity recovers nearly to its original off-state after irradiation is ceased, demonstrating the reversibility of fluorescence photoswitching by pathway 2. For all experiments, 455 nm light is used to excite CPN fluorescence. The stability of the fluorescence signal prior to the higher intensity irradiation Figure 3. Fluorescence of APESO-doped CPNs in aqueous suspension at 535 nm before, during, and after LED irradiation at 455 nm (photon flux = 7.9 x 10-5 M s-1) and 590 nm (flux = 8.6 x 10-5 M s-1). The shaded area indicates the 5 s irradiation period.



demonstrates that the weak excitation light does not photoswitch fluorescence to its on-state. This result supports the idea that the fluorescence output can be controlled by the intensity of a singlecolor input. The fluorescence photoswitching data in Figure 3 reinforce our previous observations that CPN-mediated excitation of MC (pathway 2) is far more effective for fluorescence photoactivation than direct excitation (pathway 1). This result is sustained at all irradiation photon fluxes studied, as shown in Figure 4A. The extent of fluorescence photoactivation depends on photon flux for both pathways 1 and 2, but direct excitation of MC never yields more than a 10% increase in fluorescence intensity. The fraction of dyes converted from the quenching MC form to the nonquenching SO form, determined from absorbance measurements, shows an analogous dependence on photon flux (Figure 4B). While CPN-mediated excitation of MC switches >85% of dyes from MC to SO at the highest photon fluxes, direct excitation never converts more than 10% of the dye population to SO. The Figure 4 data suggest that isomeric composition of APESO determines the fluorescence intensity in these photoswitching experiments. That is, the extent of fluorescence photoactivation depends on the irradiation wavelength only because the MC to SO conversion depends on irradiation wavelength in this system. While either pathway 1 or 2 conditions could theoretically produce the same fluorescence output, only CPN-mediated excitation of MC

Figure 4. Ratio of CPN fluorescence intensity at 535 nm after 5 s irradiation to that before irradiation (A) and fraction of APESO dye dopants in the MC form after 5 s irradiation (B) as a function of LED photon flux.


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(pathway 2) induces the photochromic conversion to the extent required for significant fluorescence activation. Thus, we must focus on the photochromic conversion of MC to SO in order to understand which photophysical features of the system are responsible for the successful fluorescence photoswitching by pathway 2.

Photokinetics. Kinetic studies of photochromic reactions have been used to establish mechanisms, characterize transient species, and determine rate constants and photochemical quantum yields.37–39 Jares-Erijman and Jovin developed photokinetic analyses specifically for pcFRET systems,30,40,41 including photoswitchable quantum dots with multiple photochromic acceptors.42 Here, we will use the tools of photokinetics to quantify the photophysical parameters that govern the MCSO conversion

and,

hence,

the

fluorescence

photoswitching of the doped CPNs. Figure 5 depicts the key photophysical pathways for the CPNs (left side) and APESO dyes (right side) and the interaction between the two manifolds. The CPNs are a highly heterogeneous system, with a single particle comprised

Figure 5. Top: Key photophysical and photochemical pathways in APESO-doped CPNs, including excitation (exc), fluorescence (fl), internal conversion (ic), fluorescence resonance energy transfer (FRET), photochromic switching (switch), thermal switching (), and photobleaching (bleach). The molecular species are conjugated polymer nanoparticles (CPN), merocyanine (MC) and spirooxazine (SO) forms of APESO, and unknown photoproducts (PP). Excited singlet states are denoted with an asterisk, and SO* is omitted for clarity. Bottom: Representative kinetic trace of the MC absorbance at 570 nm during pathway 2 irradiation (shaded region) and dark thermal recovery (lined region).

of many fluorescent donor sites and MC acceptors. The simplified scheme highlights the key processes for a single donor-acceptor pair, and the actual concentrations of CPNs and dyes will



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factor into the quantitative analysis below. Once generated by direct or CPN-mediated excitation, MC* can decay by undergoing photoswitching to SO, internal conversion, or destructive photobleaching to form unspecified photoproducts (PP). Because the photochromic conversion is our focus, the following discussion neglects the photobleaching pathway. Experimentally, this exclusion is justified by limiting irradiation periods to 5 s under moderate photon fluxes. A representative kinetic trace of the MC absorbance during the photochromic reaction and dark recovery at room temperature is shown at the bottom of Figure 5. The time-dependent concentration of MC under irradiation is governed by one photochemical process (MC SO) and one thermal process (SOMC). Following Micheau,39 the differential rate law is given by Eq. 1, −𝑑[𝑀𝐶] 𝑑𝑡

= (𝑘𝑀𝐶𝑆𝑂 + 𝑘𝑆𝑂𝑀𝐶 )[𝑀𝐶] − 𝑘𝑆𝑂𝑀𝐶 [𝑀𝐶]0

(1)

where kMCSO and kSOMC are the rate constants for the forward photochemical and reverse thermal reactions, respectively. In pathway 1, the direct excitation of MC by orange light occurs without any participation by the CPNs, and kMCSO,1 is given by Eq. 2, 𝑘𝑀𝐶𝑆𝑂,1 = 𝐼0 𝐹590 𝜀𝑀𝐶,590 𝑙Φ𝑀𝐶𝑆𝑂

(2)

where I0 is the incident photon flux, F590 is the photokinetic factor at 590 nm (F = [1 - 10Abs590

]/Abs590), MC,590 is the extinction coefficient of MC in water at 590 nm, l is the path length,

and MCSO is the quantum yield of the photochromic reaction. The poor conversion of MC to SO by pathway 1 (Figure 4B) suggests that MCSO is low; determining its value in the CPN environment is one goal of the photokinetic study. Under pathway 2 conditions, direct excitation of MC is negligible, and it is the ability of the CPNs to absorb and to generate MC* via FRET that is important. The pathway 2 rate constant, kMCSO,2, is given by Eq. 3, 𝑘𝑀𝐶𝑆𝑂,2 = 𝐼0 𝐹455 𝜀𝐶𝑃𝑁,455 𝑙Φ𝑀𝐶𝑆𝑂 𝐸𝑜𝑏𝑠

[𝐶𝑃𝑁] [𝑀𝐶]


(3)

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where F455 is the photokinetic factor at 455 nm, CPN,455 is the extinction coefficient of the CPNs at 455 nm, and the concentration ratio [CPN]/[MC] accounts for the molecular composition of the system. In a simple single-donor/single-acceptor FRET system, Eobs would be the FRET efficiency as defined by Förster theory.43 For the APESO-doped CPNs, the efficiency with which CPN* generates MC* involves both FRET and the exciton diffusion processes within the CPN that relay donor excitation to acceptor dyes. Known as amplified FRET, this phenomenon has been explored extensively in CPNs doped with non-photochromic dyes by McNeill and coworkers,32,34,44 who have demonstrated that fluorescence quenching involving both FRET and exciton diffusion can be 2 to more than 4 times more efficient than FRET alone.32 Amplified FRET has also been observed in other types of nanoparticles in which dye-to-dye exciton diffusion occurs.45 Most of the variables in Eqs. 2 and 3 are either known (MC, CPN, l), can be calculated from absorbance (F590, F455, [CPN]/[MC]), or measured by actinometry (I0). Measurement of kMCSO,1 and kMCSO,2 as a function of I0 will enable determination of the key unknown parameters MCSO and Eobs. Kinetic traces analogous to the one shown in Figure 5 were measured for APESO-doped CPNs under pathway 1 and 2 conditions at a range of photon fluxes. Experimental values of kSOMC were extracted from the thermal recovery data using the Guggenheim method for cases in which the concentration at the photostationary state (t) is unknown.46 Values of the photochemical rate constant kMCSO were determined by stepwise numerical simulation. Kinetic traces and their corresponding numerical simulations are shown in Figure 6A for representative pathway 1 and 2 experiments at similar photon fluxes. The extracted values of kMCSO are shown as a function of input photon flux in Figure 6B, from which we will show that MCSO is low and Eobs is high. CPN-mediated excitation clearly yields much larger values of kMCSO than direct excitation. Over the range of photon fluxes studied, 13 ACS Paragon Plus Environment


both curves exhibit good linearity and are fit to lines forced through the origin since kMCSO is equal to zero in the dark. The ratio of the slopes of these lines is equal to 9, meaning that kMCSO,2 is nearly an order of magnitude greater than kMCSO,1 at any given photon flux. In other words, the photochromic reaction is accelerated when run through the CPNs via pathway 2 as compared to direct excitation by pathway 1. Eq. 2 shows that MC,590 and MCSO are key parameters in setting the small values of kMCSO,1.

Figure 6. (A) Representative kinetic traces of MC absorbance at 570 nm during pathway 1 (orange, photon flux 7.7 x 10-5 M s-1) and 2 (blue, photon flux 7.9 x 10-5 M s-1) irradiation, indicated by grey shading. The solid lines are simulated trajectories from which kMCSO values were extracted. (B) kMCSO vs. input photon flux for pathway 1 direct excitation and pathway 2 CPN-mediated excitation.

The value of MCSO can be extracted from the slope of the pathway 1 data in Figure 6B according to Eq. 2, yielding MCSO = 0.03. Given that the value of MC,590 (2 x 104 M-1cm-1) is typical for a small molecule dye, the low value of MCSO in the CPN environment appears to be responsible for the extremely sluggish photochromic reaction observed upon direct excitation. Quantum yields for the photochromic conversion of MC to SO are generally much lower than those for the SO to MC conversion. The mechanism of the photoinduced ring closure of MC has been studied by ultrafast spectroscopy.47,48 Bifurcation through an unknown and short-lived excited state intermediate occurs, such that a majority of the photoproduct funnels back to the open MC form, leading to low quantum yields for generation of the closed SO form (1-2 % typically). The value for MCSO determined here is thus consistent with known properties of merocyanines. For the observed acceleration in kMCSO to occur, the low value of MCSO must be offset in pathway 2 (Eq. 3) by the parameters that describe the excitation of the CPNs and the transfer of 14 ACS Paragon Plus Environment

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energy from CPN* to MC to generate MC*. CPNs are known for their exceptional extinction coefficient values,31 and CPN,455 is 500 times greater than MC,590. However, the CPNs’ enviable absorbance properties will yield accelerated photochromic switching only if Eobs, which encompasses both exciton diffusion and energy transfer efficiency, is also high. From the ratio of the slopes in Figure 6B, we can calculate the value of Eobs by Eq. 4, 𝑠𝑙𝑜𝑝𝑒

𝐹

𝐴𝑏𝑠

𝐸𝑜𝑏𝑠 = 𝑠𝑙𝑜𝑝𝑒2 ∙ 𝐹590 ∙ 𝐴𝑏𝑠590 1

455

(4)

455

where the photokinetic factors and absorbance values are obtained from absorption spectra. Eobs for this system is 0.93, which means that the CPNs transfer energy to MC acceptor dyes in a nearly lossless fashion. Thus, the high on-state fluorescence intensity observed for the APESO-doped CPNs under pathway 2 conditions is due to the exceptional absorbance properties of the CPNs and the combination of exciton diffusion and energy transfer processes that deliver CPN excitation energy to MC. In effect, the CPNs act as an amplifier to drive an inefficient photochemical reaction nearly to completion. The analysis above assumes a FRET mechanism for the quenching of CPN fluorescence by the MC dyes. While the fluorescence off-state could be produced by FRET, photoinduced electron transfer (PET), or a combination of mechanisms, experimental evidence supports a strong FRET contribution. Figure 7A shows that the CPN fluorescence spectrum is blue-shifted when the dyes are in the MC form. Undoped control CPNs and doped CPNs in which the dyes have been completely photobleached exhibit virtually indistinguishable fluorescence spectra when normalized, but the weak residual fluorescence from doped CPNs in their off-state (Figure 2A inset) is blue-shifted ca. 8 nm relative to the unquenched spectra. To explore this finding, we prepared a series of CPNs with different dye doping levels, measured their spectra, and compared their fluorescence intensities (FDA) relative to the undoped control (FD). As the concentration of 15 ACS Paragon Plus Environment


MC in the CPNs is increased, the fluorescence spectra shift, losing intensity on the red edge and gaining it on the blue. Due to the noisiness of the highly quenched spectra, the effect is most easily quantified on the blue edge, where we determined the wavelength at which the fluorescence intensity is 25% of its maximum value (blue25). As shown in Figure 7B, blue25 is linearly correlated with the fluorescence

intensity quenching:

the

more

quenched the fluorescence (higher FD/FDA), the bluer the spectrum. It has previously been shown that residual fluorescence in conjugated polymer films undergoing FRET to dye dopants is blue-

Figure 7. (A) Normalized fluorescence spectra of undoped CPNs (black) and APESO-doped CPNs as prepared (red) and after dye photobleaching by pathway 3 (blue). (B) Ratio of integrated fluorescence intensity of undoped CPNs (FD) to that of CPNs doped with varying amounts of APESO (FDA) vs. the wavelength at which blue-edge fluorescence intensity is 25% of its value at the max.

shifted relative to undoped controls.49 When the spectral overlap that is required for FRET occurs on the red side of the polymer emission, low energy excitons are able to transfer their energy to dye acceptors more efficiently than high energy excitons that have less spectral resonance with acceptors. The APESO-doped PFBT CPNs have their strongest spectral overlap on the red side of the polymer emission (Figure 1), and the spectral blue shift observed with increased fluorescence quenching is indicative of FRET. The fact that excitation of the CPNs induces the photochromic MCSO reaction also suggests a FRET mechanism. We previously determined that the most thermodynamically favorable PET reaction in this system would produce MC●¯—PFBT●+,24 which would yield quenched fluorescence. However, electrochemical studies suggest that electrochemical switching


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between the MC and SO forms does not occur in the azahomoadamantyl spirooxazines. While PET could contribute to the off-state fluorescence quenching, the spectral shifts and sensitized photochromic reaction are indicative of FRET as the dominant mechanism.

Intensity Control. Having established that pathway 2 conditions (exc = switch = 455 nm) yield accelerated photochromic switching, it is important to determine why fluorescence activation is not observed when exc is applied to the sample during fluorescence measurements. In the pathway 2 fluorescence activation trace shown in Figure 3, for example, no significant fluctuation in fluorescence intensity is observed during the measurement until the higher intensity irradiation is applied. The ability to measure fluorescence without perturbing it (read-only) is a reproducible feature of the APESO-doped CPNs. The Figure 4 data suggested a correlation between the fraction of dyes in the MC form and the fluorescence intensity but did not provide sufficient information to explain this feature. We must more rigorously quantify the relationship between MC concentration and fluorescence intensity to investigate the origins of this intensity control of fluorescence activation. Much of the data needed to correlate MC concentration with fluorescence intensity can be obtained from pathway 2 experiments such as those shown in Figure 3 for fluorescence. However, the brief irradiation periods employed throughout this work were optimized for reversibility and do not fully convert the MC population to SO. To access the lowest MC quencher concentrations and highest on-state fluorescence intensities, we utilize pathway 3, an alternate fluorescence photoactivation pathway that is irreversible (Scheme 1). Blue irradiation at higher flux and longer duration than used for reversible pathway 2 induces irreversible photobleaching of MC, destroying the fluorescence quenching ability of the dyes. Shown in Figure 8A-B, this pathway yields a 17 ACS Paragon Plus Environment


4700% increase in peak fluorescence intensity upon irradiation. The fluorescence intensity remains stable after irradiation is ceased, indicating that the APESO dyes have lost their ability to convert to the quenching MC form. Indeed, the absorbance spectrum of the doped CPNs shows the complete disappearance of MC’s absorption peak following the irradiation period (Figure 8B inset). Combining data from pathway 2 and 3 experiments enables us to correlate CPN fluorescence intensities with the full range of MC quencher concentrations, from all dyes in the MC form (MC fraction XMC = 1) to none (XMC = 0). CPN fluorescence intensity at 535 nm and MC absorbance at 570 nm were

Figure 8. (A) Fluorescence of APESO-doped CPNs in aqueous suspension at 535 nm during dye photobleaching induced by pathway 3 irradiation (455 nm, I0 = 3.5 x 10-4 M s-1, grey shading). (B) APESOdoped CPNs before (red) and after (blue) dye photobleaching. Inset: Partial absorption spectra showing that intial MC peak (red) disappears after photobleaching (blue). (C) Fluorescence quenching vs. fraction of dyes in the MC form (XMC). FDA and FD are the peak fluorescence intensities of the dye-doped and undoped CPNs, respectively.

tracked as a function of time during MCSO thermal reversion (XMC > 0.25) and pathway 3 photoactivation (XMC < 0.25). The fluorescence and absorbance datasets were synchronized in time to yield Figure 8C, in which fluorescence intensity is given by 1-FDA/FD and a value of 1 indicates complete quenching (i.e., off). Figure 8C shows that the fluorescence quenching is relatively insensitive to MC concentration in the high XMC regime. Indeed, XMC must decrease to 0.63 before the fluorescence quenching drops below 99% of its initial level. The doping level in these CPNs substantially exceeds what is necessary to generate the fluorescence off-state. This excess population of MC


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quenchers enables intensity control of the fluorescence output because they can undergo the photochromic MCSO conversion without significantly affecting the intensity of the fluorescence off-state. When fluorescence activation is desired, increased irradiation intensities can be applied to access the lower MC concentrations at which fluorescence is activated. The extent of fluorescence quenching is very sensitive to changes in MC population at XMC  0.2, with 90% of the maximum quenching occurring by XMC = 0.23. The dramatic fluorescence quenching observed at these low quencher concentrations is typical for multi-chromophoric assemblies in which a single dye can act as a FRET acceptor for multiple donor chromophores.50 In the APESOdoped CPNs, irradiation conditions can be used to control whether the fluorescence output is in read-only mode (XMC > 0.63) or read-write mode (XMC < 0.23). Molecular composition clearly plays a critical role in the observed fluorescence photoswitching of the APESO-doped CPNs and must be considered along with the key photophysical metrics measured in the previous section.

Simulation. We have shown that pathway 2 fluorescence activation in the APESO-doped CPNs is governed by photophysical parameters (CPN, Eobs, MCSO), CPN composition (initial XMC in the CPNs), and irradiation inputs (flux, duration). With this evidence in mind, the photochromic MCSO conversion in APESO can be viewed as a slow, low-quantum-yield photoreaction that is accelerated and driven nearly to completion when run through the CPN amplifier, which is activated by an intensity switch. An important goal of this work is to determine how generalizable this phenomenon might be to other photochemical reactions. That is, could the CPNs be used to drive other low-quantum-yield photoreactions with intensity control? How low must the quantum yield of the photoreaction be for a system to function as the APESO-doped CPNs do?



The photokinetic analysis and the quenching relationship explored in the previous section (Figure 8C) equip us with the tools to answer these questions. We used the photokinetic model to simulate the change in XMC through time for a series of photon fluxes of pathway 2 blue irradiation (Figure S2). The resulting XMC values can then be translated to fluorescence intensities using an empirical relationship obtained from the data used to construct Figure 8C. The result is simulated time trajectories of fluorescence activation (FDA/FD) for specific fluxes and different combinations of Eobs and MCSO. At a given flux, the time dependence of fluorescence activation can be changed dramatically by altering the product of Eobs and MCSO. Since high energy transfer efficiency is a feature of most dye-doped CPNs with good donor-acceptor spectral overlap, we set Eobs to our experimentally determined value (0.93) and varied only the quantum yield. The trends observed in these simulations can also be produced by fixing the quantum yield and varying Eobs. As shown in Figure 9A for a typical photon flux, varying  leads to enormous differences in the fluorescence activation trajectories. The highest values of  (e.g. 0.12) produce overly sensitive photoswitches that would switch to the fluorescence on-state immediately upon exposure to ambient light. Such systems would be difficult to control due to the small difference between the irradiation times that yield read-only and read-write fluorescence. The lowest values of  (< 0.02) produce systems that would require extended irradiation periods to activate fluorescence. If high  gives activation without control and low  gives control without activation, then we must turn to the intermediate regime in which both fluorescence activation and control can be achieved. The trajectory that matches our experimental  of 0.03 has a slow enough rise to confer control but still activates at short enough times to be experimentally viable, especially if full activation (FDA/FD = 1) is not required. Qualitatively, the Figure 9A simulations match not only our own data but also those of Osakada, et al. who recently studied fluorescence photoswitching in CPNs doped 20 ACS Paragon Plus Environment

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with diarylethene (DE) photochromes.51 CPNs doped with a higher quantum yield DE ( = 0.015) exhibited undesirably rapid fluorescence activation upon light exposure (t½ = 3.1 s), making it challenging to control read-only vs. read-write fluorescence. In contrast, CPNs doped with a lower quantum yield DE ( = 0.0018) activated more slowly (t½ = 60.4 s), enabling temporal control of fluorescence intensity. The results of our simulation can be extrapolated to predict the range of Φ values amenable to intensity control. To determine the combinations of Φ and photon flux that facilitate intensity control, we define the read-only flux as that which results in FDA/FD = 0.1, given a 5 s irradiation time that matches our experimental conditions for the APESO-doped CPNs. The read-write flux is, in turn, defined as that which gives FDA/FD = 0.9 within the same 5 s period (see Figure 9B and caption). The green and white shaded areas mark the fluorescence onstate and off-state, respectively, while the grey shaded area shows the range of flux values that permits controlled photoswitching for each value of Φ. For an imaging light source with a minimum tunable interval of ~5 μW cm-2, the maximum Φ that affords controlled switching is

Figure 9. (A) Simulated time trajectories for fluorescence activation at Eobs = 93% and I0 = 5 x 10-5 M s-1 for the specified values of . FDA and FD are the peak fluorescence intensities of the dye-doped and fully activated CPNs, respectively. (B) Simulated range of irradiation conditions amenable to intensity-controlled pathway 2 photoswitching. The upper series of dots marks the minimum flux that yields the fluorescence on-state (FDA/FD = 0.9), and the lower series marks the maximum flux that maintains the fluorescence off-state (FDA/FD = 0.1). The gray box indicates Φ values conducive to intensity control for 5 s irradiation periods. Photon flux values were converted to irradiance in μW cm-2 to facilitate comparison to other systems.



~0.05. Above this threshold value of Φ, the difference between the read-only and read-write fluxes is too small for controlled switching using an irradiation source such as our LED. The threshold shifts to higher Φ values when shorter irradiation periods are simulated and to lower Φ values with longer irradiation. This finding is consistent with the data of Osakada, et al., who also observed controlled activation of fluorescence but with lower-quantum-yield dyes and longer irradiation periods than employed in our work.51 While our simulations and Osakada’s experimental work focused on varying Φ, it is important to note that it is the product of Φ and Eobs that governs the extent to which fluorescence activation is controlled in such systems. Since Eobs is nearly 100% in our case and likely to be similarly high in other dye-doped CPNs designed for FRET, the upper limit of the product (Φ * Eobs) should be ~0.05, given a 5 s irradiation period. Our measurements, analyses, and simulations are based on CPNs studied in aqueous suspension, but these findings should also be applicable to CPNs dispersed on slides or added to cells when photoswitching is induced under comparable irradiation conditions to those employed here. Osakada’s DE-doped CPNs described above undergo fluorescence activation upon visible light irradiation in both single particle and live cell imaging experiments.51 CPNs in these sample environments continue to show the slower, more controlled fluorescence activation when the lower quantum yield DE dye is used. Cells labeled with photochromic CPNs that undergo visible-lightactivated fluorescence have also been demonstrated by Chiu and coworkers for optical painting and subsequent fluorescence activated cell sorting.52 The present work shows that using lowquantum-yield photoswitches in applications such as these enables intensity control of fluorescence photoswitching.


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Conclusions APESO-doped CPNs exhibit high-contrast fluorescence photoswitching when a single color (455 nm) is used both to excite fluorescence (read-only) and to induce photoswitching (readwrite). Irradiation intensity functions as a switch to toggle between these modes. CPN composition and the photochromic quantum yield together enable this single-color intensity control. The CPNs are doped with an excess population of dyes in the MC form, which quench CPN fluorescence via FRET and act as a buffer against inadvertent fluorescence photoactivation. The quantum yield for the photochromic reaction was determined by photokinetic analysis to be 0.03 in the CPN environment in the absence of FRET. This relatively low value means that the photochromic conversion occurs slowly when MC is excited directly. When MC is excited via FRET from the CPNs, however, the inefficient photoreaction is offset by the CPNs’ high extinction coefficient and the efficient transfer of energy from the CPNs to MC via FRET and exciton diffusion. The photochromic reaction is thus accelerated when run through the CPN amplifier. Simulations demonstrate that the experimentally determined photochromic quantum yield falls fortuitously in a regime in which both full activation of fluorescence and control of read-only vs. read-write mode are possible. To extend this paradigm to other dye-doped CPNs, additional simulations were used to define the range of photochemical quantum yield values over which activation with control is possible, assuming the same doping levels and efficient energy transfer observed here. The upper limit for retention of intensity control is a ~0.05 quantum yield for 5 s irradiation. The same result can be obtained by fixing the quantum yield and varying the energy transfer efficiency, provided that (Φ * Eobs) remains ~0.05. We intend to apply these findings to the design of future CPNs doped with dyes that undergo low-quantum-yield photoreactions.

Supporting Information. Supplemental figures and CPN composition information. 23 ACS Paragon Plus Environment


Acknowledgements We gratefully acknowledge support of this work by NSF (CHE-1464699).

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A

1 - FDA / FD

1 2 100 575 3 bleach 4 0 5 500 550 600 650 700 0 20 40 60 80 Wavelength (nm) Time (s) 6 7 MC per polymer chain 8 0 5 10 15 20 C9 1.0 10 11 0.8 12 0.6 13 14 0.4 15 0.2 16 ACS Paragon Plus Environment 17 0.0 18 0.0 0.2 0.4 0.6 0.8 1.0 Fraction MC (XMC) 19

A

1 2 3 4 5 6 7 8 B9 10 11 12 13 14 15 16 17 18 19 20

The Journal of Physical Chemistry Page 38 of 39

ACS Paragon Plus Environment

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


↑f l uor . i nt ensi t y

MC

CPN on

ex cessi ve ΦMC→ SO opt i mal ΦMC→ SO r eadonl y ΦMC→ SO

CPN of f ACS Paragon Plus Environment

SO

bl ueLED i nt ensi t y→

Controlling Photoswitching via pcFRET in Conjugated Polymer

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