Tetraphenylethene end-capped [1,2,5]thiadiazolo[3,4-c]pyridine with aggregation-induced emission and large two-photon absorption cross-sections

Abstract

Two new donor–acceptor–donor-type two-photon absorption (2PA) dyes (TPEPT1 and TPEPT2) have been synthesized by Suzuki coupling reaction with tetraphenylethene (TPE) as a donor and [1,2,5]thiadiazolo[3,4-c]pyridine (PT) as an acceptor. The 2PA cross sections (σ) values of these dyes measured by the open aperture Z-scan technique are up to 6030 and 2360 GM, respectively. Both of them exhibit good aggregation-induced emission (AIE) properties with fluorescent quantum efficiencies of 7.25% and 5.53% in the red channel. Finally, TPEPT1 with higher brightness was used to perform a cell-imaging proposal.

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Introduction

Since the concept of the two-photon absorption (2PA) process was first proposed by Göppert-Mayer in 1931, there has been strong demand for efficient 2PA dyes.¹ Materials with large two-photon absorption cross sections have many potential applications, including optical power limiting materials,² three-dimensional optical data storage³ and bioimaging.⁴ Due to the researchers' efforts, a series of novel organic materials with large 2PA cross sections have been investigated. The molecular designs include donor–π-bridge–donor (D–π–D)-type, donor–π-bridge–acceptor (D–π–A)-type and donor–acceptor–donor (D–A–D)-type architectures,⁵ and the study shows that the 2PA cross sections of a compound is related to the degree of the intramolecular charge transfer (ICT) and effective π-conjugate length.⁶

Although the 2PA cross sections (σ) values of the materials can be improved by a special molecular design strategy,⁷ most of 2PA dyes are hydrophobic, and their fluorescence is often quenched on aggregation, although they may show high fluorescence efficiency in solutions. This phenomenon is called aggregation-caused quenching (ACQ) effect, which greatly limits their applications, such as bioimaging. Recently, a novel phenomenon of aggregation-induced emission (AIE) is discovered that the AIE luminogens are nonemissive in good solvents and become highly emissive in aggregated state.⁸ Today, some famous organic molecules with AIE effect were discovered, such as TPE⁹ and silole¹⁰ etc. Because of the AIE effect, the hydrophobic 2PA materials with large π system can be fabricated in nanosystem and used in biosensing and bioimaging. The materials possessed 2PA and AIE properties and exhibited high brightness fluorescence in far-red/near-infrared (FR/NIR) region are highly desirable, because they have shown some advantages in bioimaging applications, such as deep penetration depth and avoiding auto-fluorescence.¹¹

Tetraphenylethene (TPE) is a classical luminogen with intriguing AIE characteristic, which has been widely used in optic- and electro-active materials due to its electron-donating property and propeller-like molecular structure.¹² [1,2,5]Thiadiazolo[3,4-c]pyridine (PT) is an excellent acceptor, which has a higher electron affinity than benzothiadiazole (BTD),¹³ thus using PT as an acceptor is conductive to promote ICT process and shift the spectral response reached to the NIR region. In this work, we have designed and synthesized two new D–A–D-type 2PA dyes (TPEPT1 and TPEPT2) by introducing TPE as periphery to the PT core (Scheme 1). These materials show different 2PA properties and unique AIE effects in aggregated state. Furthermore, we exhibited the solid fluorescence and cell imaging of them.

Scheme 1 Molecular structures of TPEPT1 and TPEPT2.

Experimental

General methods and materials

Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under dry N₂ immediately prior to use, and N,N-dimethyl formamide (DMF) was refluxed with calcium hydride and distilled before use. Compound 1 was prepared according to previous published procedures.¹³ All reagents were of high commercial quality and used as received without further purification.

¹H and ¹³C NMR spectra were recorded on a Bruker AM-400 spectrometer using chloroform-d (CDCl₃) as solvent and tetramethylsilane (TMS, δ = 0) as internal reference. Mass spectra were recorded on ESI mass spectrometer and Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometer. A Varian-Cary 500 spectrophotometer was used to measure the UV-Vis spectra. The fluorescence spectra were taken on a Varian-Cary fluorescence spectrophotometer. Quanta-w F-3029 integrating Sphere was used to measure the quantum yield of solid powders. The SEM micrographs were obtained on a JEOL JSM-6360 scanning electron microscope (SEM). An ALV-5000 laser light scattering spectrometer (DLS) was used to measure the size distribution of the nanoparticles. 2PA cross sections of the compounds were measured by femtosecond open-aperture Z-scan technique according to previously described method.¹⁴ The TPF was achieved, at a wavelength of 800 nm, by femtosecond pulses with different intensities. The repetition rate of the laser pulses was 250 KHz, with the 80 fs pulse duration. The Olympus FV1000 confocal laser scanning microscope (CLSM) was used for cell imaging experiments. The cyclic voltammograms of TPEPT1 and TPEPT2 were obtained using a Versastat II electrochemical work station.

2,2′-([1,2,5]Thiadiazolo[3,4-c]pyridine-4,7-diylbis(4,1-phenyle-ne))diacetonitrile (3). In a 50 mL two-necked round-bottom flask, compound 1 (246.8 mg, 0.5 mmol) and Pd(PPh₃)₄ (30.0 mg, 0.026 mmol) were dissolved in 20 mL THF under N₂ atmosphere, and then added 5 mL 2 M potassium carbonate aqueous solution to the mixture. After stirring for 1 h at 60 °C, compound 2 (364.7 mg, 1.5 mmol) dissolved in 10 mL THF was dropwise added into the mixture and stirred at 80 °C for another 10 h. Upon cooling, the mixture was poured into saturated brine and extracted with DCM (50 mL × 3). The combined organic layer was dried over anhydrous MgSO₄ and concentrated using a rotary evaporator. The residue was purified by column chromatography on silica (petroleum ether/ethyl acetate = 8/1, v/v) to afford 64.4 mg product as a yellow solid (yield: 35%).¹H NMR (CDCl₃, 400 MHz, TMS), δ: 8.84 (s, 1H), 8.68 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.2 Hz, 2H), 7.57 (dd, J = 8.0, 5.7 Hz, 4H), 3.89 (s, 2H), 3.88 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz, TMS), δ: 156.64, 151.71, 149.65, 142.69, 136.57, 134.36, 132.25, 130.65, 129.93, 128.55, 128.25, 126.48, 117.52, 23.65, 23.55. HRMS (ESI) (m/z): [(M + H)⁺] calcd for C₂₁H₁₄N₅S, 368.0968, found: 368.0970.

N-Phenyl-4-(1,2,2-triphenylvinyl)aniline (5). In a 100 mL two-necked round-bottom flask, palladium acetate (50.0 mg, 0.22 mmol), tri-tert-butylphosphine solution (1.56 mL, 0.66 mmol) and compound 4 (411.3 mg, 1 mmol) were dissolved in 20 mL toluene under N₂ atmosphere and stirred at room temperature for 30 min. Then added aniline (111.7 mg, 1.2 mmol) and sodium tert-butoxide (134.5 mg, 1.4 mmol) to the mixture and heated to 120 °C for another 20 h. Upon cooling, the mixture was filtered, and the solvent was removed under vacuum. The residue was poured into saturated brine and extracted with DCM (20 mL × 3). The combined organic layer was dried over anhydrous MgSO₄ and concentrated using a rotary evaporator. The residue was purified by column chromatography on silica (petroleum ether/dichloromethane = 10/1, v/v) to afford 270.8 mg product as a solid (yield: 64%). ¹H NMR (CDCl₃, 400 MHz, TMS) δ: 7.22 (d, J = 7.5 Hz, 2H), 7.14–7.04 (m, 13H), 7.01 (d, J = 7.6 Hz, 4H), 6.90 (dd, J = 7.9, 4.9 Hz, 3H), 6.79 (d, J = 8.6 Hz, 2H), 5.64 (s, 1H).

4-Bromo-N-phenyl-N-(4-(1,2,2-triphenylvinyl)phenyl)aniline (6). The synthesis method resembles that of compound 5 and the compound 6 was prepared by the reaction of compound 5 (211.6 mg, 0.5 mmol) and 4-bromo-iodobenzene (169.1 mg, 0.6 mmol) to give a yellow solid (147.2 mg, yield: 51%). ¹H NMR (CDCl₃, 400 MHz, TMS) δ: 7.22 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 7.4 Hz, 2H), 7.07–7.00 (m, 11H), 6.96 (dd, J = 14.8, 5.6 Hz, 7H), 6.81 (dd, J = 8.3, 4.6 Hz, 4H), 6.70 (d, J = 8.4 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz, TMS) δ: 147.14, 146.79, 145.47, 143.99, 143.68, 143.47, 140.76, 140.56, 138.58, 132.31, 132.08, 131.37, 129.30, 127.68, 127.64, 127.59, 126.49, 126.41, 126.38, 125.18, 124.44, 123.23, 123.11, 114.76. (EI) (m/z): [M⁺] calcd for C₃₈H₂₈BrN, 577.1405, found: 577.1407.

Dimethyl(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)-phenyl)boronate (7). In a 50 mL Shrek tube, compound 6 (1734.1 mg, 3 mmol) was dissolved in 20 mL anhydrous THF under N₂ atmosphere and cooled to −78 °C. Then n-butyl lithium (2.25 mL, 3.6 mmol) was added slowly into the mixture. After 1 h, trimethyl borate (4.5 mmol, 0.43 mL) was added and slowly warm to room temperature. The reaction stirred for another 2 h and then quenched with dilute hydrochloric acid solution. The mixture was poured into the saturated salt brine and extracted with DCM (50 mL × 3). The combined organic layer was dried over anhydrous MgSO₄ and concentrated using a rotary evaporator to afford the product without further purification.

4-(Bis(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)benzaldehyde (10). The synthesis method resembles that of compound 3 and the compound 10 was obtained by the Suzuki coupling between compound 8 (808.4 mg, 2 mmol) and 9 (262.5 mg, 0.5 mmol) to give an orange solid (261.4 mg, yield: 56%). ¹H NMR (CDCl₃, 400 MHz, TMS) δ: 9.85 (s, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.5 Hz, 5H), 7.37 (d, J = 8.3 Hz, 5H), 7.22 (d, J = 8.5 Hz, 5H), 7.16–7.10 (m, 33H).

4,4′-([1,2,5]Thiadiazolo[3,4-c]pyridine-4,7-diyl)bis(N-phenyl-N-(4-(1,2,2-triphenylvinyl)phenyl)aniline) (TPEPT1). The synthesis method resembles that of compound 3 and the compound 10 was obtained by the Suzuki coupling between compound 1 (62.2 mg, 0.25 mmol) and 7 (571.3 mg, 1 mmol) to give a red solid (172.5 mg, yield: 61%). ¹H NMR (CDCl₃, 400 MHz, TMS) δ: 8.68 (s, 1H), 8.44 (d, J = 8.9 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 9.1 Hz, 2H), 7.30–7.27 (m, 2H), 7.13–6.93 (m, 42H), 6.87–6.82 (m, 6H). ¹³C NMR (CDCl₃, 100 MHz, TMS) δ: 156.77, 151.05, 149.78, 149.69, 148.16, 147.14, 146.88, 145.48, 145.17, 144.02, 143.96, 143.73, 143.69, 143.52, 143.46, 142.18, 140.95, 140.81, 140.64, 140.59, 139.33, 138.88, 132.41, 132.39, 131.41, 131.39, 130.75, 130.08, 129.72, 129.40, 129.37, 128.05, 127.73, 127.67, 126.55, 126.40, 125.64, 125.40, 125.04, 124.24, 123.88, 123.78, 123.51, 123.00, 121.90. HRMS (ESI) (m/z): [(M + H)⁺] calcd for C₈₁H₅₇N₅S, 1132.4413, found: 1132.4409.

2,2′-([1,2,5]Thiadiazolo[3,4-c]pyridine-4,7-diylbis(4,1-phenyle-ne))bis(3-(4-(bis(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)-amino)phenyl)acrylonitrile) (TPEPT2). In a 50 mL two-necked round-bottom flask, compound 3 (66.3 mg, 0.18 mmol), compound 10 (504.0 mg, 0.54 mmol) and potassium tert-butoxide (224.4 mg, 2 mmol) were dissolved in 20 mL methanol under N₂ atmosphere and refluxed for 24 h. Upon cooling, the solid precipitate was filtered, washed with cold methanol and purified by column chromatography on silica (petroleum ether/DCM = 1/1, v/v) to afford 126.6 mg product as a red solid (yield: 32%). ¹H NMR (CDCl₃, 400 MHz, TMS) δ: 8.93 (s, 1H), 8.81 (d, J = 7.7 Hz, 2H), 8.16 (d, J = 6.7 Hz, 2H), 7.90 (dd, J = 15.2, 10.0 Hz, 6H), 7.55 (d, J = 7.7 Hz, 8H), 7.38 (d, J = 7.7 Hz, 8H), 7.23 (d, J = 7.6 Hz, 8H), 7.15–7.09 (m, 64H), 6.92 (d, J = 8.1 Hz, 6H), 6.59 (d, J = 8.6 Hz, 6H). ¹³C NMR (THF-d8, 100 MHz, TMS) δ: 156.08, 149.29, 145.36, 143.42, 143.38, 143.32, 142.32, 142.05, 140.80, 140.21, 137.66, 136.10, 131.36, 130.86, 130.80, 130.53, 130.44, 129.84, 129.08, 127.28, 127.16, 127.12, 127.05, 125.95, 125.91, 125.85, 125.27, 125.19, 124.91, 120.77, 120.67. MALDI-TOF: [(M + H)⁺] calcd for C₁₆₃H₁₁₁N₇S, 2198.8700, found: 2198.8692.

Results and discussion

Synthesis

The detailed synthetic routes of TPEPT1 and TPEPT2 were shown in Scheme 2. The compound 3 was obtained by the Suzuki coupling reaction between compound 1 and 2. The compound 5 was synthesized by Buckwald–Hartwig reaction between compound 4 and aniline, followed by the reaction with 4-bromo-iodobenzene, yielding compound 6. The important intermediate 10 was obtained by the Suzuki coupling between compound 8 and 9 according to the published literature.¹⁵ Finally, the target compound TPEPT1 was prepared by Suzuki coupling reaction between compound 1 and 7. TPEPT2 was obtained by a typical Knoevenagel condensation between compound 3 and 10. All of the intermediates and final compounds were purified by column chromatography and confirmed by satisfactory spectroscopic data (shown in ESI†).

Scheme 2 Synthetic routes of TPEPT1 and TPEPT2.

One-photon absorption

Fig. 1 shows the normalized one-photon absorption spectra of TPEPT1 and TPEPT2 in DMF and DMF/water mixtures (90% water in volume) at 1 × 10⁻⁵ M. The maxima (λ_max) absorption of TPEPT1 and TPEPT2 are located at 510 nm and 475 nm in DMF, respectively. It is obviously shown that TPEPT2 is blue-shifted by 35 nm compared to TPEPT1, which can be attributed to the larger steric hindrance of donor moiety of TPEPT2, reducing the planarity of the molecule and affecting the conjugation. It is also noted that the absorption spectra of TPEPT1 and TPEPT2 in DMF/water (90% water in volume) mixtures are all red-shifted and broadened. In addition, the absorbance of TPEPT1 and TPEPT2 decrease with the increase of water content (shown in Fig. S1†), respectively. The phenomena may be caused by the formation of their nano-aggregates.¹⁶

Fig. 1 Normalized one-photon absorption spectra of TPEPT1 and TPEPT2 in DMF and DMF/water mixtures (90% water in volume) at a concentration of 1 × 10⁻⁵ M.

Meantime, we also investigated their optical properties in the different solvents, such as toluene (TOL), dichloromethane (DCM), THF and DMF. As shown in Fig. 2, the absorption bands of the two compounds remain almost identical in the above solvents, and the real absorbance was shown in the Fig. S2.† However, a significant bathochromic shift of emission band and the decrease of fluorescence intensities can be observed with the increase in solvent polarity (Fig. 3). Since the two dyes possess D–A structural units, their solvent effects may be caused by the intramolecular charge transfer (ICT) in the excited state,¹⁷ which is confirmed through the linear dependence of stokes shift (Δν) on solvent polarity parameter (Δf) together with the large slope of the Δν–Δf plot (shown in Fig. S3–S4 and Tables S1–S2†).¹⁸

Fig. 2 Normalized one-photon absorption spectra of (A) TPEPT1 and (B) TPEPT2 in different solvents at concentration of 1 × 10⁻⁵ M.

Fig. 3 (A and B) Emission spectra for TPEPT1 and TPEPT2 in different solvents at the concentration of 1 × 10⁻⁵ M. λ_ex: 510 nm (TPEPT1) and 475 nm (TPEPT2). Inset: the fluorescence emission spectra of TPEPT1 and TPEPT2 in DCM and DMF, and the fluorescence intensity for them are multiplied by a factor of 100. (C and D) Photographs of TPEPT1 and TPEPT2 in different solvents under UV light.

In addition, the electrochemical behaviors of TPEPT1 and TPEPT2 were investigated by cyclic voltammetry using 0.1 M tetra-butylammonium hexafluorophosphate as supporting electrolyte in dichloromethane solution with platinum button working electrodes, a platinum wire counter electrode, and an SCE reference electrode. The SCE reference electrode was calibrated using a ferrocene/ferrocenium (Fc/Fc⁺) redox couple as an external standard. The cyclic voltammograms of TPEPT1 and TPEPT2 are shown in Fig. S5.† It can be obtained from Fig. S5† that the first half-wave potentials (E_ox) of TPEPT1 and TPEPT2 are 1.04 and 1.04 V, respectively. Therefore, the ground state oxidation potential corresponding to the HOMO energy levels are −5.74 and −5.74 eV (vs. vacuum) according to the equations HOMO = −e(E_ox + 4.7) (eV), respectively. The equal HOMO energy levels of TPEPT1 and TPEPT2 indicate that the HOMO energy level mainly depends on the donor moiety. All the parameters of electrochemical properties of TPEPT1 and TPEPT2 are listed in Table S3.† The zero–zero transition energies (E_g) of TPEPT1 and TPEPT2 are 1.88 eV and 2.02 eV, respectively. Calculated from E_HOMO + E_g, the lowest unoccupied molecular orbital (LUMO) energy levels are −3.86 and −3.72 eV, respectively. It is clear that TPEPT2 has weaker electron-withdrawing ability than that of TPEPT1, which may be caused by the introduction of phenylacrylonitrile, replacing thiadiazolo[3,4-c]pyridine unit as the main acceptor and decreasing the electron-withdrawing ability.

AIE properties

Both of TPEPT1 and TPEPT2 are soluble in common organic solvents, such as toluene, THF and DMF, but insoluble in pure water. To investigate the AIE attributes of the compounds, we used the anhydrous DMF as the good solvent and water as the poor solvent to prepare the stable water dispersions of nano-aggregates of TPEPT1 and TPEPT2 by precipitation method.¹⁹ Fig. 4A shows the corresponding emission spectra of TPEPT1 in aqueous solution with different DMF/water ratios at a concentration of 1 × 10⁻⁵ M. It could be seen that TPEPT1 almost had no fluorescence in pure DMF, but fluorescence began to increase with the water added slowly, and when the water content reached to 30%, the fluorescent intensity was boosted to the maximum and the peaks located at 667 nm with a 32-fold increase (Fig. 4B). Apparently, its emission was caused by aggregation-induced effects. To explore its AIE nature, fluorescence quenching was mainly due to the vibration and rotation of the molecular in pure organic solution that offer a non-radiative transition from the excited states. On the contrary, intramolecular rotation was restricted upon aggregation, which blocked the non-radiative decay path, thus making the fluorescence output. However, fluorescence reached maximum at the f_w = 30%, and further increase in water content caused the decrease in PL intensity. This phenomenon is normal among the compounds with AIE properties, which may be caused by the variation in the packing mode of the molecules in aggregates.²⁰ We can observe similar phenomenon of TPEPT2 (Fig. 4C). The photoluminescence intensity (PL) intensity was very weak in the pure DMF, but when the water content reached to 60% in volume, they gave the maximum luminescence and the peaks located at 669 nm with 7.5-fold increase (Fig. 4D), indicating the compound also had AIE activity.

Fig. 4 Emission spectra of (A) TPEPT1 and (C) TPEPT2 in DMF and DMF/water mixtures with different water fractions (f_w). Plots of relative emission intensity (I/I₀) versus the composition of the aqueous mixtures of (B) TPEPT1 and (D) TPEPT2; I₀ = emission intensity in pure DMF solution. Solution concentration: 1 × 10⁻⁵ M; λ_ex: 510 nm (TPEPT1) and 475 nm (TPEPT2). Inset: photographs of (B) TPEPT1 and (D) TPEPT2 in pure DMF (0%) and DMF/water mixtures under 365 nm UV irradiation.

To further confirm the AIE properties, we also continued to study their PL behaviors in the solid states (Fig. 5). The emission spectra of the two dyes are located at 656 nm (TPEPT1) and 671 nm (TPEPT2) with the fluorescence quantum yield (ϕ_f) of 7.25% and 5.53%, respectively. As shown in spectra and inset photos of figures, such materials emit red to NIR fluorescence that may have potential applications in bioimaging.

Fig. 5 (A) Emission spectra of the solid powders of TPEPT1 and TPEPT2. λ_ex: 510 nm (TPEPT1) and 475 nm (TPEPT2) (B) photos of TPEPT1 and TPEPT2 as solid powders under 365 nm UV light illumination.

Two-photon absorption properties

The 2PA cross sections (σ) of the TPEPT1 and TPEPT2 were determined by a previously described method, using femtosecond open-aperture Z-scan technique.²¹ Fig. 6 (A and C) show the open Z-scan data, and the 2PA coefficient was obtained by data fitting. The σ values can be calculated by the formula of σ = hvβ/N₀, where N₀ = N_AC is the number density of the absorption centers, N_A is the Avogadro constant, C represents the solute molar concentration and β is the 2PA coefficient.²² The values of σ for TPEPT1 and TPEPT2, at the wavelength of 800 nm, are 6030 and 2360 GM, respectively. The σ value of TPEPT2 is smaller than that of TPEPT1. When compared to TPEPT1, TPEPT2 has a large donor moiety with much steric hindrance, which reduces the planarity of the molecule and affects the ICT state, as confirmed by the absorption spectrum. It is well known that a greater degree of charge transfer in donor–acceptor is more conductive to the increase of the σ value. Fluorescence spectra in two-photon excitation experiment of the TPEPT1 and TPEPT2 under different laser intensity are shown in Fig. 6 (B and D). The linear dependence of fluorescence intensity on the square of the excitation intensity, as shown in the inset, confirms that two-photon absorption is the main excitation mechanism of the intense fluorescent emission.²³ The two dyes have a higher σ value than that of some published compounds based on TPE, such as 3TPE–BODIPY,²⁴ which may be caused by the higher electron affinity of [1,2,5]thiadiazolo[3,4-c]pyridine, promoting the ICT process and increasing the 2PA cross sections.

Fig. 6 Open-aperture Z-scan trace (A) and fluorescence spectra (B) under different excitation power densities of PTEPT1 in toluene at the concentration of 1 × 10⁻³ M. (C and D) The corresponding spectra of PTEPT2 in the same experimental conditions. ΔT/T represents the laser transmittance and Z represents the distance between the sample and the focus. Concentration: 1 × 10⁻⁵ M. Inset: 2PF intensities versus square of the excitation power density (I_pump²/(GW × cm⁻⁴)).

Under the excitation of 80 fs pulse, 800 nm laser, TPEPT1 and TPEPT2 in the DMF/water mixtures emit NIR fluorescence with peaks located at 669 nm and 672 nm (Fig. 7), respectively. Compared with the spectra in Fig. 4 (A and C), the good overlap between the one- and two-photon excitation fluorescence indicates that the emissions result from the same excited state, regardless of the different modes of excitation.²⁵ The inset photos show the two-photon fluorescence (2PF) in pure DMF and DMF/water mixtures under the excitation of 800 nm laser.

Fig. 7 Two-photon fluorescence emission spectra and 2PA emissions images for (A) TPEPT1 and (B) TPEPT2 in the DMF and DMF/water mixtures at a concentration of 1 × 10⁻⁵ M. λ_ex: 800 nm. Inset: 2PF images of TPEPT1 and TPEPT2 in pure DMF (0%) and DMF/water mixtures under the excitation of 80 fs pulse, 800 nm laser.

Nanoparticles fabrication and cell imaging

The sizes distribution of TPEPT1 and TPEPT2 nanoparticles were investigated by dynamic light scattering (DLS), indicating that the mean diameter are approximately 193 nm and 184 nm, respectively. Scanning electron microscopy (SEM) was also performed to study the morphology of the nanoparticles, suggesting that they are in spherical shape with an average size of around 170 nm and 160 nm, respectively, which are slightly smaller than those of DLS due to the shrinking of samples in the vacuum dry state (Fig. 8). Also, we do the SEM measurement of TPEPT1 and TPEPT2 in TOL, THF, DCM and DMF, respectively. The results show that they are unable to form aggregation of nanoparticles in pure organic solvents (shown in the Fig. S6†).

Fig. 8 Particle size distribution and morphology of (A) TPEPT1 in DMF/H₂O (1 : 9 v/v) and (B) TPEPT2 in DMF/H₂O (1 : 9 v/v) mixtures studied by DLS and SEM at the concentration of 1 × 10⁻⁵ M. The scale bar is 500 nm.

We further study potential applications of PT-based 2PF fluorophore in bioimaging. Between the two dyes, TPEPT1 had a higher fluorescence quantum yield than TPEPT2, so we selected TPEPT1 to investigate the applicability in fixed cell imaging. The experiment was studied by CLSM with RAW264.7 fixed cell line as an example. RAW264.7 cell line was incubated for 24 h at 37 °C under 5% CO₂ atmosphere and fixed by 4% paraformaldehyde for 20 min. PBS solution (3 mL × 5) was used to wash out the remained paraformaldehyde on the slide. The TPEPT1 solution (10 μM, 2 mL) was added into culture dish for another 30 min, and then the sample was washed by PBS solution for 5 times and examined under CLSM. As shown in Fig. 9(A–C), the TPEPT1 was aggregated closely around cell nucleus and emitted fluorescence signal in far red/near infrared region (625–725 nm). The Z-scan cell imaging figure further determined that the position of dyes located in the cytoplasm closed to nucleus in spatial position (Fig. 9D).

Fig. 9 Cell images of RAW264.7 fixed cells after 30 min incubation with TPEPT1. (A–C) Fluorescence, bright field, and overlay images of the RAW264.7 fixed cells. (D) The corresponding confocal Z-scan images (λ_ex = 488 nm). Fluorescent signals collection from 625 nm to 725 nm.

Conclusions

In this work, we successfully designed and synthesized two D–A–D-type dyes (PTEPT1/2) with good 2PA and AIE properties. Compared to the published compounds with similar structure, the two dyes have large two-photon absorption cross sections value. Especially for TPEPT1, the σ value is up to 6030 GM. Both of them emit red fluorescence in the solid states with the fluorescence quantum efficiencies of 7.25% and 5.53%, respectively. The imaging experiment reveals that these types of fluorophores can be the potential materials for bioapplication.

Acknowledgements

This work was supported by the National Basic Research 973 Program (2013CB733700 and 2013CB834701), NSFC/China (2116110444, 21172073 and 91233207) and the Science Fund for Creative Research Groups (21421004).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Characterization data: ¹H NMR, ¹³C NMR, MS-TOF. See DOI: 10.1039/c4ra09789k

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