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Photoswitchable emission color change in nanodots containing conjugated polymer and photochrome Daigeun Kim, and Taek Seung Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12277 • Publication Date (Web): 23 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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ACS Applied Materials & Interfaces

Photoswitchable emission color change in nanodots containing conjugated polymer and photochrome

Daigeun Kim, Taek Seung Lee*

Organic and Optoelectronic Materials Laboratory, Department of Organic Materials Engineering, Chungnam National University, Daejeon 34134, Korea

Keywords: photoswichable fluorescence color, conjugated polymer, photochrome, nanoparticle, zebrafish imaging

ABSTRACT

A simple approach for the preparation of conjugated polymer (CP)-based fluorescent nanodots containing photochrome (dithienylethene, DTE) is reported. The CP in the nanodots was designed to exhibit dual emissions of blue and green. The photochromic, fluorescent, composite nanodots (PNDs) were able to tune the emission color from green to blue using selective energy

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transfer from the CP to DTE under ultraviolet (UV) irradiation. The UV irradiation-induced ringclosure of the DTE within the PNDs provided a spectral overlap between the green-emission of the CP and the absorption of DTE, leading to quenching of the green emission and, concomitantly, maintaining of the blue emission. The photoswitchable fluorescent PNDs with high on-off green fluorescence contrast was successfully applied in a living zebrafish imaging. Our design strategy provided a versatile tool for constructing a special photomodulated colorchangeable nanostructure in bioimaging.

INTRODUCTION Fluorescent nanostructured materials have attracted great interest in various fields of optoelectronics, such as biosensing, in vivo investigations of cells and protein, and in vivo imaging.1-4 Fluorescent nanomaterials include materials derived from silica and organically modified silica,5,6 hydrophobic and hydrophilic organic polymers,7,8 quantum dots,9,10 carbonaceous nanomaterials,11,12 upconversion materials,13,14 dye-doped metal particles,15 and conjugated polymers.16-18 Such nanoparticles are much less cytotoxic and do not suffer from nonspecific binding by cellular biomolecules, because fluorescent nanoparticles are basically inert and, thereby hardly interact with cellular proteins, resulting in little effect on their optical properties.19,20 In particular, the fluorescent nanomaterials that are based on conjugated polymers (CPs) have attracted considerable attention for biological applications such as biosensing and imaging because they have the combined advantages of nanostructure and a conjugated backbone.21-24 Such composite nanodots integrated with CP (CPdots) are easily prepared by miniemulsion and


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nanoprecipitation techniques and present interesting features such as a high absorption coefficient, high brightness, good photostability, low cytotoxicity, large absorption cross-section, and easy surface modification for bioconjugation compared with organic dyes and inorganic semiconducting nanodots.25-27 These advantages of the CPdots provide critical progress in photoswitchable imaging that needs spatial control of fluorescence, in which the photoswitchable CPdots switch on and off in response to light input in the presence of a photochromic dye.28-30 Photochromic molecules like dithienylethene (DTE) have attracted much interest for potential applications31 in optical switches, memories, and 3-dimensional data storage.32,33 The photochromic behavior of DTE is related to a reversible photo-induced isomerization between a colorless, open-ring form and a planar, colored, closed-ring form.34 The photophysical properties of DTE in triplet state was also investigated in terms of photochromic behavior.35,36 The DTE photochromes have been successfully incorporated into polymer matrices for the development of rapid and stable switching properties.37 Their applications are related to fluorescence photoswitching including cell labeling,38,39 in vivo imaging,40,41 and imaging in living vertebrates.42 Herein, we demonstrate photochromic, fluorescent, composite nanodots (PNDs) containing CPs and DTE that show reversible blue-and-green fluorescence photoswitching in response to UV light (365 nm). Usually, on-and-off photoswitching is common in photochrome-containing composite nanoparticle systems; thus we turned to an unusual nanostructure that changes its emission color color using a CP containing benzothiadiazole (BT) and phenylene groups in the polymer backbone. Because of the presence of electron-accepting BT units linked with electrondonating phenylene groups, the polymer showed distinctive dual emission colors (blue and green). Upon UV irradiation onto PNDs containing CP and DTE, one of the two emission colors


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of CP (green) could be quenched by the photochromic action of the DTE inside the PNDs, exhibiting blue emission. UV irradiation onto PNDs was supposed to provide ring-closure of DTE, leading to switching-off of the PNDs’ green emission because the green energy of the CP was absorbed by DTE while maintaining blue emission. To the best of our knowledge, this is the first report on the preparation of multiemissive (blue and green) fluorescent PNDs that display emission color variability depending on UV irradiation, and this material should have subsequent applications in fluorescence imaging and switching in particle-based imaging agents. In this study, zebrafish (Danio rerio) were used as an in vivo model to evaluate the feasibility of reversible fluorescence switching by the internalized PNDs. Zebrafish have unique advantageous features over other vertebrate model systems such as mouse, rat, or human.43,44 The zebrafishes allow direct visual detection of pathological embryonic death as well as the investigation of realtime transport and effects of nanoparticles in vivo because of their transparent embryos. Therefore, zebrafish embryos provide a potential opportunity to investigate the effects of nanoparticles on intact cellular systems. Despite the variety of uses of zebrafish in the biomedical field, only a limited number of studies have used fluorescent probes based on nanomaterials for zebrafish imaging.42 Switching by emission color-changeable fluorescence is preferable to on-off switching, because of the greater sensitivity to an increase or decrease in fluorescence intensity from a dark background or initially strong signal, respectively. The fabrication of PNDs in this study is easier and simpler via simple mixing and reprecipitation, compared to previous methods that needed chemical reaction between fluorescent molecules (or conjugated polymer) and photochromes or specific encapsulation. In addition, the photoswitchable PNDs we prepared featured fluorescence color change from green to blue because of use of dual-emissive conjugated polymer unlike previous turn-off method.


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EXPERIMENTAL SECTION Materials and Instrumentation. 1,4-Benzenediboronic acid, tetrakis(triphenylphosphine)palladium(0), and hydroquinone were purchased from Sigma-Aldrich and used without further purification.

A

photochromic

dye,

1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)-3,3,4,4,5,5-

hexafluoro-1-cyclopentene (DTE) was purchased from TCI and used without further purification. 1H NMR spectra were obtained on a Bruker DRX-300 spectrometer (Korea Basic Science Institute). Elemental analysis (EA) was performed with a CE Instruments EA-1110 elemental analyzer. FT-IR spectra were recorded on a Tensor 27 FT-IR spectrometer (Bruker). The UV-Vis absorption spectra were recorded on a Perkin Elmer Lambda 35 spectrometer. Fluorescence spectra were taken using a Varian Cary Eclipse spectrophotometer equipped with a xenon lamp excitation source. Synthesis of CPs. 1,4-Dibromo-2,5-bis(octyloxy)benzene (0.30 g, 0.61 mmol), 4,7-dibromo2,1,3-benzothiadiazole (0.02 g, 0.067 mmol), and 1,4-benzenediboronic acid (0.13 g, 0.81 mmol) were added to a 100 mL round-bottomed flask containing dry toluene (18 mL) and 2 M aqueous sodium carbonate solution (8 mL) under nitrogen. After the addition of tetrakis(triphenylphosphine)palladium(0) (5 mol%) as a catalyst, the mixture was heated to 90 °C and stirred for 40 h. The reaction mixture was poured into acetone and the precipitate was washed with methanol and water. The polymer was obtained after drying in a vacuum oven (yield 0.21 g, 40%). 1H NMR (400 MHz, CDCl3, δ): 8.1–6.8 (m), 4.1–3.8 (m), 3.3–2.8 (t), 2.3-1.8 (d) ppm. 13C NMR (CDCl3, δ): 147.6, 135.4, 128.4, 112.3, 31.9, 24.3, 14.1 ppm. FT-IR (KBr, cm−1): 2939 (C– H), 1618 (C=C), 1151 (aryl C–N), Anal. Calcd. for C26.79H38.19N0.26O1.74S0.13 C, 81.27%; H, 9.72%; N, 0.92%; S, 1.05%. Found: C, 84.47%; H, 10.87%; N, 0.81%; S, 1.05%.


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Preparation of PNDs. The fabrication of CP-based nanodots containing the DTE was carried out by the reprecipitation method. A suitable amount of DTE was added to a THF solution of CP (0.25, 0.5, and 1 wt% with respect to 1 mL THF), and then the solution was stirred for 1 h. The solution was quickly dispersed in deionized water (10 mL) under tip-sonication. After removal of THF by heating, the PNDs were filtered by a syringe filter (0.45 µm) to obtain PNDs with uniform size. Finally, the PNDs (DTE/CP = 2) were prepared at the concentration of 36 mg/L. The CPdot dispersion remained clear and stable for 2 weeks without aggregation. Photochromic behavior of PNDs. The as-prepared PNDs in aqueous solution (2 mL) were exposed to UV (254 nm) for 3 min to close the ring of DTE in PNDs, and then, the PNDs were re-exposed to visible light (630 nm) for 3 min to recover the initial open-ring structure. Maintenance and fluorescence imaging of zebrafish. Zebrafishes were raised and kept under standard laboratory conditions at 28.5 °C. Zebrafish embryos were obtained from natural spawning. Embryos were staged at specific hours post fertilization (hpf). For fluorescence imaging, 55 hpf zebrafish embryos were incubated with 5 mL of PNDs solution (36 mg/L) for 40 min in egg water (10 mL). After the PNDs treatment, embryos were washed for 30 min with egg water and then exposed with a monochromic 254 nm hand-held UV lamp (310 µW/cm2) for 3 min. The embryos were alive for 30 days after washing of remained PNDs. The fluorescence images were recorded using an Olympus BX53 fluorescence microscope. Fluorescence images of blue (440-470 nm) and green channels (530-560 nm) were recorded for zebrafish before and after UV irradiation.

RESULTS AND DISCUSSION


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For the construction of photoswitchable PNDs, a CP with phenylene and BT groups as a fluorescent polymer was synthesized via the Suzuki coupling reaction (Scheme 1). The chemical structure of the CP was confirmed by NMR, FT-IR, and EA. The polymer was readily soluble in common organic solvents, such as chloroform and THF. The molecular weight of the CP was determined by gel permeation chromatography using THF as an eluent with a polystyrene standard. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of CP were found to be 11700 and 12400, respectively, with a polydispersity index of 1.05. The molar composition (m : n) was observed to be 0.87 : 0.13 as determined by EA. The polymer was composed of a larger amount of the phenylene segment (as an electron donor) and a smaller amount of the BT unit (as an electron acceptor), exhibiting dual emissions (blue and green) in the solid state via intra- and intermolecular energy transfer.45,46 The fluorescence spectrum of CP in PNDs showed both blue (382 nm) and green (538 nm) emissions, originating from the phenylene and BT units, respectively, in the same backbone structure of the CP (Figure 1). Because of non-efficient singlet energy transfer, the dual emission of the CP is possible. The unique phenomenon of dual fluorescence can produce a long-wavelength emission output that would be useful for in vivo imaging, because the large Stokes’ shift of CP prevents unnecessary signals from autofluorescence. DTE was used as a photochromic switching constituent, which is a bisthienylethene derivative and has highly efficient, stable, and reversible photochromic ability with stability between the colorless, open-ring form and the colored, closed-ring isomer with broad absorption around 600 nm (Figure S1).40,47,48 These properties were considered to be suitable for stable photoswitching of the green-to-blue emission change induced by selective energy transfer from the CP to closedring DTE upon exposure to UV light, enabling light-driven emission color control. As such, the


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PNDs containing both CP and DTE exhibited blue fluorescence upon exposure to UV light because of energy transfer from the green emission of the CP to the DTE, which allowed for the combination of fluorescence and high-contrast photoswitching. The absorption band of the closed-ring DTE (571 nm) was responsible for the FRET-based fluorescence change, and thus its spectral overlap with an energy donor (CP) was of importance for efficient FRET (Figure 1). In our system, the absorption band of the closed-ring DTE (571 nm) overlapped with the green emission band of the bluish green-emissive CP (538 nm); thus, the PNDs displayed selective quenching of green emission by the energy absorption of the closed-ring DTE under UV illumination. The photoswitchable change in emission color by UV irradiation is illustrated in Scheme 2. Initially, the PNDs showed blue and green emissions from the structural feature of the CP. After irradiation of UV light (254 nm), the green emission (538 nm) of CP was transferred to the closed-ring DTE via FRET, because a new absorption band of DTE was formed (571 nm). As a result, only the blue emission (382 nm) of the CP in PNDs remained. As soon as visible light (630 nm) was irradiated, the initial bluish-green fluorescence of PNDs was restored. CP and DTE-integrated PNDs were prepared via the reprecipitation method, which provided very fine composite nanoparticles that were dispersed stably in the aqueous medium. A scanning electron microscopic (SEM) image showed that the obtained PNDs were spherically-shaped and the size of the PNDs was 210 ± 5 nm, as determined by SEM image analysis by averaging more than 80 particles (Figure 2a). The densely assembled PNDs allowed integration of two species, leading to easy energy transfer for dual emissions via photochromic fluorescence switching to accomplish efficient photoswitchable fluorescence imaging. One of the advantages provided by the physically integrated system was the ease of optimizing the composition of the emitting and photochromic constituents. The effect of composition on the fluorescence photoswitching was


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investigated by varying the weight ratio (r) during the preparation of PNDs (r = [DTE]/[CP] by weight, where r = 0.5, 1, and 2). When the value of r was 0.5 or 1, the concentration of CP was too high to obtain effective fluorescence quenching of the CP under the condition of 3-min UV irradiation time, exhibiting on-off fluorescence contrast of 43% and 50%, respectively (Figure S2). The use of more DTE (r = 2) enabled us to obtain well-defined photochromic behaviors of PNDs, which showed complete quenching of CP in PNDs via efficient energy transfer with an on-off fluorescence contrast of 94% (Figure S3). We speculate that the larger amount of DTE served not only as the energy acceptor but also as spacers to prevent aggregation-induced selfquenching of the CP.49 Thus, PNDs of r = 2 were used in subsequent photoswitching experiments. The fluorescence of the PNDs could be changed from State 1 (bluish green emission) to State 2 (blue emission) by alternate photochromic behavior between the colorless and colored states of DTE, respectively, where the green emission could be quenched through FRET in State 2. Figure 2b demonstrates the changes in fluorescence emission of the PNDs via their State 1/State 2 photoswitching. Initially, the PNDs were observed to emit blue and green emissions without significant self-quenching, in which the green fluorescence was dominant by the naked-eye. The absorption of the PNDs (570 nm) was increased upon 3-min UV irradiation because of the ringclosing of DTE in PNDs and, concomitantly, the green emission of the PNDs decreased. The successful overlap between the green emission of CP and absorption of closed-ring DTE was obtained within the PNDs framework. Because of the energy transfer from CP to DTE within PNDs, the green emission was quenched, whereas the blue emission (382 nm) was maintained and could be observed by the naked eye (inset photograph in Figure 2b).


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The PNDs exhibited reversible photochromic modulation, in which the absorption band of the closed-ring DTE isomer at 573 nm showed repeated increase and decrease under alternating UV irradiation (254 nm) and visible (630 nm) light, respectively, resulting in successful fluorescence photoswitching of PNDs. Figure 3a demonstrates the change in green emission at 542 nm over the UV irradiation time. The PNDs showed complete quenching of the green emission at 542 nm upon UV illumination for 3 min. Subsequently, after visible light (630 nm) irradiated the green-quenched PNDs for more than 2 min, the original green emission was recovered. The saturation of the ring-closure of DTE was faster (

Photoswitchable Emission Color Change in ... - ACS Publications

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