Design and Synthesis of AIE-Based Small-Molecule and Nanofibrous Film for Fluorescent Sensing Application

Fluorescent sensors that respond to environmental conditions (temperature, pressure, and pH) have attracted widespread attention in recent years. Generally, traditional solid-state fluorescent materials tend to suffer from aggregation-induced quenching (ACQ) and difficulty of film forming, limiting their extensive applications. Therefore, researchers are focusing more and more attention on fluorescent sensors with aggregation-induced emission (AIE) effects. Herein, the article reports an AIE molecule (TPEBZMZ) containing tetraphenylethylene (TPE) and benzimidazole fragments. The fluorescence properties of TPEBZMZ in solution and aggregation states have been investigated, and the luminescence performance and aggregation structures of solid-state TPEBZMZ after force and acid treatments have been explored. The results show obvious AIE and fluorescent sensing properties of TPEBZMZ, presenting force- and acid-induced discolorations. Moreover, the TPEBZMZ-based fluorescent nanofibrous film is fabricated by electrospinning the solution of TPEBZMZ blended with polylactic acid (PLA), which shows a good nanofiber film structure and exhibits reversible acid-induced discoloration property, even with only 0.5 wt% TPEBZMZ. This work provides a simple strategy to achieve stimulus-responsive fluorescent film.


INTRODUCTION
Fluorescent films composed of π-conjugated materials-based fluorescent sensors and membranous materials are promising sensor technology due to their real-time detection, easy fabrication, noninvasiveness detection, and mechanical stability and flexibility (Guan et al., 2015;Miao et al., 2016). In recent years, they have been widely used in detecting residual pesticides (Peng et al., 2021), enzymes (Zhao et al., 2017a;Zhao et al., 2017b), pollutants (Lee et al., 2019;Zhao et al., 2020b;Sun et al., 2021), explosives (Zhou et al., 2014;Sun et al., 2015;Li et al., 2018;Hao et al., 2021), metal ions (Chen et al., 2020), gases (Moscoso et al., 2020), and temperature and humidity (Jiang et al., 2020;Li et al., 2020), which greatly expanded the applications of fluorescence sensors. Generally, high selectivity and sensitivity are the key characteristics of a successful membranous fluorescent sensor (Sun et al., 2015). Selectivity is mainly related to the specific interaction between fluorescent probe and environmental stimulus or detection objects (Zhou et al., 2017), showing differentiated luminescence characteristics, while sensitivity is primarily determined by fluorescence intensity (Song et al., 2015). Traditional fluorescent materials usually present weak fluorescence in the solid state but high fluorescence in solution due to the widely known aggregationcaused quenching (ACQ) effect. ACQ greatly limits the application of traditional fluorescence materials (Hong et al., 2011). Aggregation-induced emission (AIE), proposed by Tang's research team in 2001, can be used as a perfect solution to the ACQ issue (Luo et al., 2001). AIE behavior is just the opposite of the ACQ fluorescence behavior of traditional fluorescent compounds: AIE luminogen (AIEgen) emits weak fluorescence in solution, whereas it emits strong fluorescence when aggregated or in a solid state (Luo et al., 2001). The AIE phenomenon is related to intermolecular rotation, restricting these rotations in solid state, inducing the fluorescent emission of the corresponding compound (Qin et al., 2012;Liu et al., 2019;Zhao et al., 2020a). Therefore, AIEgen-based films will show specifically emissive characteristics. Thus, AIEgen-based fluorescent films will provide an effective strategy for the development of fluorescent film sensors.
Electrospinning is an effective method to obtain polymerbased nanofibrous films, compared with casting films, showing a larger specific surface area and higher porosity (Xue et al., 2019). Thus, fluorescent films fabricated by electrospinning can improve the sensitivity and response rate (Zhao et al., 2017b). Therefore, much attention has been devoted to nanofibrous fluorescent films to explore their specific sensing properties. For example, Li et al. (2019) have developed an epoxy resin system functionalized with the typical AIE tetraphenylethylene (TPE) groups, forming nanofibrous films by electrospinning, and finally obtained temperature-sensitive fluorescent films probes. Yang et al. (Yang et al., 2018) have reported specific composite nanofibrous films by electrospinning of AIE small molecules (oME-TPA) and polyvinyl alcohol (PVA) and further studied its piezochromic properties related to sensitivities. Zhao et al. (2017a) have grafted TPE derivatives and phloxine B onto electrospun nanofiber film, inducing the static quenching of phloxine B through protamine adsorption and combining them with the aggregation of TPE-based groups, promoting fluorescence emission of nanofiber, which realized effective monitoring of different concentrations of heparin. Therefore, the design and development of novel AIE molecules containing nanofiber fluorescent films will benefit the realization of efficient fluorescent sensing applications.
Herein, we report a novel compound (TPEBZMZ) containing the classic AIE group tetraphenylethylene (TPE) and benzimidazole unit. The TPE group renders the TPEBZMZ molecule AIE property, and the structure of the benzimidazole unit will offer the potential of acid-induced discoloration. The relationship between the structure, aggregate, and luminescence properties of TPEBZMZ in different states and force and acid treatments has been systematically studied. Specifically, TPEBZMZ shows an obvious AIE phenomenon and fluorescent sensing properties of force-and acid-induced discolorations. More importantly, we prepared TPEBZMZbased fluorescent nanofibrous film by electrospinning the solution of TPEBZMZ blended with polylactic acid (PLA), and the nanofibrous film displays a good reversible acid-induced discoloration property. Significantly, this work provides a simple strategy to prepare fluorescent sensing film.

Electrospinning Process of PLA-TPEBZMZ and PLA Nanofibrous Films
The electrospinning process was performed on an E05 electrospinning apparatus (supplied by Lepton Technology Co., Ltd., Foshan, China), using a 10 wt% PLA/(DMF:DCM 1:2) solution with or without 0.05 wt% TPEBZMZ as a spinning solution. The nanofibers were collected by a rotating metal cylinder (100 r/min) covered with release paper, with an immobile distance of 20 cm away from the needle tip of the spinneret (0.7 mm inner diameter). The spinning voltage and fluid flow rate were set to 12 kV and 2 ml/h, respectively. The obtained nanofibrous films were dried in a vacuum oven at 70°C for another 12 h to remove the residual solvent completely.

Synthesis and Characterization
The synthetic route of TPEBZMZ is shown in Scheme 1. The intermediate molecule P 4 A was acquired via Suzuki coupling from P 3 Br and 4-formylphenylboronic acid. The final product TPEBZMZ was obtained by Knoevenagel reaction with P 4 A and 2-(cyanomethyl) benzimidazole. As shown in Supplementary  Figures 1-4, TPEBZMZ was characterized using 1 H NMR and 13 C NMR spectroscopies, mass spectrometry, and Fourier transform infrared spectroscopy, confirming that the target compound TPEBZMZ has been successfully synthesized. The thermal stability of TPEBZMZ was investigated by thermal gravity analysis (TGA) under a nitrogen atmosphere, and the result is presented in Supplementary Figure 5. TPEBZMZ showed good thermal stability with 5% weight loss at a temperature (T d ) of 366°C.

AIE Property of TPEBZMZ
Due to the state-of-the-art AIE functional unit, TPE, we proposed that the TPEBZMZ molecule can exhibit the AIE effect, which was firstly examined by the photoluminescence (PL) fluorescence emission spectra of TPEBZMZ/THF/H 2 O solutions with different water content. The resulting spectra, the plot of PL intensity vs. water fraction, and the fluorescence photo images of specific water fraction are shown in Figure 1A. The fluorescence emission of the TPEBZMZ/THF solution in the presence of only the good solvent THF is very weak, and the fluorescence of the solution is almost invisible to the naked eye. When the water content is lower than 70%, the fluorescence spectra show few changes. In contrast, the fluorescence intensity increases sharply, when the water content reaches 80%, and the fluorescence intensity is about 36 times compared to 0% water content. The fluorescence intensity begins to decrease after the water content is further increased to 90%, within a sharp drop of fluorescence intensity at 98% of water content. We speculate that the reason for the change in fluorescence intensity is because TPEBZMZ aggregation started with 80% of water content, and the size of aggregates increased gradually when the water fraction increased to 98%, leading to a remarkable light scattering phenomenon (Thomas et al., 2007). Thus, the PL fluorescence experiment preliminarily confirmed the AIE property of TPEBZMZ.
Optical absorption properties of TPEBZMZ/THF/H 2 O solutions with different water content were investigated using UV-vis absorption spectroscopy. As shown in Figure 1B, the solution absorption spectra exhibit similar characteristics under 0-70% water content, while the obvious decreases of absorption intensities were demonstrated in >80% water content solutions. Moreover, the spectra of >80% water content show the obvious tailing phenomenon in absorption curves, especially presented in the 98% water content solution, which can be attributed to the Mie scattering effect of nano aggregates (Zhang et al., 2015). The tendency of absorption spectra is consistent with the results of fluorescence emission spectra of TPEBZMZ/THF/H 2 O mixture solution with different water content. These results further SCHEME 1 | Synthetic route and chemical structure of TPEBZMZ.
Frontiers in Chemistry | www.frontiersin.org August 2021 | Volume 9 | Article 727631 confirmed that TPEBZMZ shows the AIE effect, which will benefit its further fluorescent sensor application.

Mechanochromism Property of TPEBZMZ
To evaluate the stimulus-response characteristic of TPEBZMZ, we first tested the fluorescence performance of the original TPEBZMZ (TPEBZMZ-o) powder. As shown in  Figure 6) indicate that TPEBZMZ displays good reversible mechanochromism properties. Solid-state TPEBZMZ changes from yellow-green to orangeyellow after being mechanically stimulated. We speculate that the reason is the change in the aggregation state of the TPEBZMZ molecules due to the action of external mechanical forces (Ma et al., 2016;Ma et al., 2017). Accordingly, the aggregate structures of the TPEBZMZ powder with different treatments were investigated by powder X-ray diffraction (PXRD) measurements. As shown in Figure 3A, a large number of sharp diffraction peaks appeared in the PXRD curve of TPEBZMZ-o, suggesting a relatively regular molecular arrangement and good crystallinity presented in the initial state of TPEBZMZ. While the ground sample TPEBZMZ-g shows weak and even disappeared diffraction peaks, it indicates a relatively disordered and amorphous state exhibited in the TPEBZMZ-g sample. Moreover, the diffraction peaks are partially restored while TPEBZMZ-g is annealed, and the major peaks of the fumed TPEBZMZ-f are also restored and similar to those of TPEBZMZ-o. These results demonstrated the varied aggregated structures of TPEBZMZ with different treatments, which is consistent with its mechanochromism property.
Aggregate structures of TPEBZMZ with different treatments were further characterized by differential scanning calorimetry (DSC) measurements. As shown in Figure 3B, in the first heating curves, TPEBZMZ-o shows an obvious melting peak at 298°C (enthalpy of 25.80 J/g), while TPEBZMZ-g displays a weak melting peak at 290°C with an enthalpy of 4.63 J/g, suggesting  that the mainly amorphous state is presented in the TPEBZMZ-g sample. TPEBZMZ-f and TPEBZMZ-a also have obvious crystal melting peaks near 290°C, but the melting enthalpies (23.77 J/g and 25.90 J/g, respectively) are slightly lower than or almost the same as TPEBZMZ-o, indicating that a certain rearrangement of amorphous structure of the TPEBZMZ sample appeared under annealing or DCM fumigation treatments.

Acid-Base Discoloration Property of TPEBZMZ
The protonateable benzimidazole unit presented in the TPEBZMZ molecule renders the variable electron cloud density of TPEBZMZ after being protonated, further, in turn, affecting its fluorescence performance. Thus, TPEBZMZ-o was fumigated by hydrogen chloride gas to obtain the protonated acid-fumigated TPEBZMZ powder (TPEBZMZ-h). As shown in Figure 4A, the TPEBZMZ-h sample displays orange fluorescence with an emission peak at 589 nm, which shows an obvious red shift of 55 nm compared to the TPEBZMZ-o solid. Furthermore, the TPEBZMZ-h solid fumigated NH 3 vapor (TPEBZMZ-n) shows a fluorescence color turning back to yellow-green, and its fluorescence spectrum is almost restored to that of TPEBZMZo. Figure 4B shows the repeated fluorescence spectra of TPEBZMZ by HCl fuming and NH 3 fuming cycles, displaying modest repeatability of acid-base discoloration under three cycles. These results prove that the TPEBZMZ powder exhibits good reversible acid-base discoloration performance.
The proposed reaction mechanism about TPEBZMZ treated with HCl and NH 3 is shown in Figure 5, and the frontier molecular orbital distributions of TPEBZMZ and HClprotonated TPEBZMZ (TPEBZMZ-HCl) were further determined using DFT by Gaussian 09 at the B3LYP/6-311G(d,p) level. The electron density distribution of the lowest unoccupied molecular orbital (LUMO) of TPEBZMZ is localized in the central molecule and tends to be fastened to the cyano unit.  The highest occupied molecular orbital (HOMO) of TPEBZMZ shows a well-proportioned electron density distribution. Moreover, the electron density distributions of the LUMO and HOMO of TPEBZMZ-HCl exhibit a delocalization effect, which tends to localize in the benzimidazole hydrochloride unit and TPE unit, respectively. Correspondingly, TPEBZMZ and TPEBZMZ-HCl exhibit LUMO/HOMO levels of −2.62/−5.68 and −2.86/−5.89 eV, respectively, presenting the bandgaps of 3.06 and 3.03 eV. The red-shift fluorescence spectra of HClprotonated TPEBZMZ could be attributed to the delocalization effect of its electron density distributions, which may explain the acid-base discoloration property of TPEBZMZ.

Fluorescent Sensing Property of TPEBZMZ-Based Nanofibrous Film
Generally, small-molecule fluorescent powders exhibit poor filmforming properties requiring expensive film-forming equipment (e.g., vacuum evaporator), which will limit their practical application (Hong et al., 2011). To overcome this drawback, TPEBZMZ was blended with a PLA dimethylformamide (DMF)/DCM mix solution by electrospinning to obtain the TPEBZMZ-based nanofibrous film (TPEBZMZ-m). The morphology of TPEBZMZ-m and a pure PLA nanofibrous film was tested using scanning electron microscopy (SEM). The corresponding SEM images are shown in Figures 6A-D. TPEBZMZ-m shows an almost similar microstructure compared to that of the pure PLA nanofibrous film, both of which possess filaments with diameters of 500-800 nm.
To evaluate the fluorescent sensing property of TPEBZMZ-m, the acid-induced discoloration experiment was performed. The results of the acid-induced discoloration experiment are shown in Figure 7A. Unlike the pure PLA nanofibrous film (Supplementary Figure 7B), the nascent TPEBZMZ-m film emits significant green fluorescence under ultraviolet light (confirmed by laser confocal microscope, Supplementary  Figures 7C-E), even with only 0.5 wt% TPEBZMZ. After fuming by HCl vapor, the resulting film (TPEBZMZ-m-h) exhibits an obvious yellow fluorescence color. Moreover, when the TPEBZMZ-m-h sample was fumed with a NH 3 vapor (TPEBZMZ-m-n), the fluorescence of the sample became green again. To quantitatively characterize the fluorescence emission wavelength of TPEBZMZ-m after different treatments, we used fluorescence spectroscopy to test the electrospinning nanofibrous film samples after different treatments. As shown in Figure 7A, the TPEBZMZ-m sample shows a fluorescence emission peak of 496 nm, while the TPEBZMZ-m-h sample exhibits a fluorescence emission peak FIGURE 5 | Proposed reaction mechanism for TPEBZMZ treated with HCl and NH 3 , and the frontier molecular orbital distributions of TPEBZMZ and HCl-fumed TPEBZMZ (TPEBZMZ-HCl) determined using DFT by Gaussian 09 at the B3LYP/6-311G(d,p) level.
Frontiers in Chemistry | www.frontiersin.org August 2021 | Volume 9 | Article 727631 6  Frontiers in Chemistry | www.frontiersin.org August 2021 | Volume 9 | Article 727631 7 of 563 nm, with a red shift of 67 nm compared to TPEBZMZ-m. Meanwhile, the TPEBZMZ-m-n sample shows an obvious blueshift fluorescence emission compared to the TPEBZMZ-m-h sample and presenting a fluorescence emission peak at 496 nm, coinciding with the spectroscopy of TPEBZMZ-m. Figure 7B shows the repeated fluorescence spectra of TPEBZMZ-m by HCl fuming and NH 3 fuming cycles, displaying good repeatability of acid-base discoloration under three cycles. The well acid-base discoloration repeatability of TPEBZMZ-based nanofibrous film suggests its good potential on fluorescent film sensors application. These results demonstrated that electrospinning acts as a simple and efficient method to prepare TPEBZMZ-based nanofibrous fluorescent film with good reversible acid-induced discoloration property, which provides an effective strategy for developing fluorescent film sensors.

CONCLUSION
In conclusion, we have developed an AIE molecule (TPEBZMZ) containing tetraphenylethylene (TPE) and benzimidazole fragments, which is synthesized from 2-cyanomethylbenzimidazole and 4-(1,2,2-tristyryl) benzaldehyde. TPEBZMZ powder shows obvious AIE and fluorescent sensing properties and exhibits force-and acidinduced discoloration phenomena. More importantly, the TPEBZMZ-based fluorescent nanofibrous film is fabricated by electrospinning the solution of TPEBZMZ blended with polylactic acid (PLA), exhibiting a good nanofiber film structure and well reversible acid-induced discoloration properties. This work demonstrates a simple strategy to achieve stimulus-responsive fluorescent film, which will benefit the development of fluorescent film sensors.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author/s.