A Novel AIE Plus ESIPT Fluorescent Probe with a Large Stokes Shift

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General Research

A novel AIE plus ESIPT fluorescent probe with a large Stokes shift for cysteine and homocysteine: Application in cell imaging and portable kit Haohan Song, Yanmei Zhou, Haonan Qu, Chenggong Xu, Xiao Wang, Xiaoqiang Liu, Qingyou Zhang, and Xiaojun Peng Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b04643 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 21, 2018

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Industrial & Engineering Chemistry Research

A novel AIE plus ESIPT fluorescent probe with a large Stokes shift for cysteine and homocysteine: Application in cell imaging and portable kit Haohan Song,† Yanmei Zhou,*,†,‡ Haonan Qu,† Chenggong Xu,† Xiao Wang,† Xiaoqiang Liu,† Qingyou Zhang,† and Xiaojun Peng‡ †

Henan Joint International Research Laboratory of Environmental Pollution Control Materials,

College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, China ‡

State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024,

China * Corresponding author: Tel: +86-371-22868833-3422; Fax: +86-371-23881589; E-mail address: [email protected] (Y.M. Zhou)

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ABSTRACT: The application of fluorescent probes is limited due to the small Stokes shifts and aggregation-caused quenching (ACQ) effect when accumulated in cells. Herein, a novel colorimetric and turn-on fluorescent probe based on salicylaldehyde azine with both aggregationinduced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) properties for Cys/Hcy is proposed to solve these issues. This probe showed a large Stokes shift (148 nm), low cytotoxicity as well as outstanding photostability upon recognition and the response mechanism was confirmed by fluorescence spectroscopy, High Performance Liquid Chromatography (HPLC), Thin Layer Chromotography (TLC) and Transmission Electron Microscope (TEM). In addition to being used for cell imaging, a simple and user-friendly portable kit based on this probe was proposed as a new tool for the on-site inspection of more than ten micro-samples simultaneously, which could effectively prevent the occurrence of false positives and visual errors.


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1. INTRODUCTION Low molecular weight thiols, like cysteine (Cys) and homocysteine (Hcy), play significant roles in many biological processes.1-3 Nevertheless, abnormal levels of these biological thiols (bio-thiols) can cause various kinds of illness.4, 5 For instance, Cys is reported to link to many pathological symptoms such as slow growth in children, cardiovascular diseases, lethargy, hair depigmentation and hematopoiesis reduction.6-8 Also, Hcy is a risk factor for Alzheimer’s disease, inflammatory bowel disease, osteoporosis and neural tube defects.9-11 Thus, developing practical ways to detect and quantify Cys/Hcy is important in terms of early diagnosis and the study of pathology.12 Among various analytical techniques for Cys/Hcy detection, fluorescent detection method has drawn more and more attention owing to its hypersensitivity, easy operation, low cost and the capability of imaging in living biological systems.12-21 However, despite these advantages of traditional fluorescent probe, their applications are still restricted because of small Stokes shifts and aggregation-caused quenching (ACQ) effect when accumulated in cells.22, 23 As we all know, fluorescent probe possessing a large Stokes shift has the obviously separated excitation band and emission band, which can reduce the interferences of self-absorption or auto-fluorescence.24, 25 And, during the past few years, fluorophores with aggregation-induced emission (AIE) properties have attracted more and more attention, as it can show strong fluorescent after aggregation and can be applied to in situ imaging, drug delivery, and tumour diagnosis.23, 26-28 To date, many AIE fluorescent probes have been used for cell imaging due to the characteristic of wash-free, light-up imaging and in situ detection.29-31 And AIE fluorescent probe for bio-thiols detection has received more and more attention not only for its high emission in the aggregated or solid states but also for its high signal-to-noise ratio and excellent photostability.26,

31


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Therefore, it is of great significance to develop an AIE based fluorescent probe with a large Stokes shift for Cys/Hcy.31 Moreover, owing to their ACQ effect, it is hard to utilize these traditional fluorescent probes to sense Cys/Hcy in test papers and other portable kit.32-34 Fluorescent probes with AIE properties, by contrast, would be more excellent on account of their intense fluorescent emission in the aggregate state.32 Herein, a novel colorimetric and turn-on fluorescent probe (probe SATZ) based on salicylaldehyde azine (SA) with both AIE and excited-state intramolecular proton transfer (ESIPT) properties for Cys/Hcy is proposed to solve the above issues. Due to the good AIE properties of its mother molecule, probe SATZ was used to manufacture a simple and userfriendly portable kit to selectively and quantitatively detect Cys/Hcy on site. Furthermore, because of its ESIPT properties, probe SATZ showed a large Stokes shift (148 nm) upon recognition.35,

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It was precisely these two characteristics that made it suitable for in situ

imaging of Cys in cells. Furthermore, the response mechanism was confirmed via fluorescence spectroscopy, High Performance Liquid Chromatography (HPLC), Thin Layer Chromotography (TLC) and Transmission Electron Microscope (TEM). 2. EXPERIMENTAL SECTION 2.1. Materials and instrumentations. All chemicals and commercial solvents were purchased from suppliers and used without further purification. All reagents in the optical spectroscopic studies were HPLC grade. TLC analyses were carried out using silica gel plate GF 254 and column chromatographic purifications were carried out on silica gel (200-300 mesh). NMR spectra were recorded on a Bruker DMX-300 spectrometer and MS spectra were measured on Bruker AmaZon SL. All pH measurements were performed with a Jingke PHS-3D digital pHmeter. UV-Vis absorption spectra were recorded with a U-4100 spectrophotometer and


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fluorescence spectra were obtained on an Edinburgh FS5 spectrofluorometer. HPLC experiments were measured using a HPLC system (Agilent Technologies 1260 Infinity) with a Zorbax SBC18 column (5 µm, 4.6×150mm). TEM photos were taken with a JEM-2100 electron microscope. Cell imaging was performed with a confocal laser scanning microscope (Zeiss LSM710). 2.2. Synthesis 2.2.1. Synthesis of SA.The synthesis of SA was performed according to the reported literature procedures (Scheme 1).37 1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 2H), 9.01 (s, 2H), 7.70 (dd, J = 7.7, 1.7 Hz, 2H), 7.41 (ddd, J = 8.6, 7.4, 1.7 Hz, 2H), 6.98 (t, J = 7.9 Hz, 4H).

Scheme 1. The Synthetic route of probe SATZ and SA. 2.2.2.

Synthesis

of

Compound

1.

Salicylaldehyde

(209

µL,

2

mmol),

2,4-

dinitrobenzenesulfonyl chloride (533 mg, 2 mmol) and triethylamine (283 µL) were dissolved in 10 mL dichloromethane, and the mixture was stirred overnight at room temperature. Then, removing the solvent under reduced pressure and the solid obtained was purified by


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chromatography on a silica gel column to give Compound 1. (311 mg, yield 44%) 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.14 (d, J = 2.3 Hz, 1H), 8.65 (dd, J = 8.7, 2.3 Hz, 1H), 8.33 (d, J = 8.7 Hz, 1H), 7.97 (dd, J = 7.6, 1.8 Hz, 1H), 7.76 (td, J = 7.8, 1.9 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.29-7.23 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 188.46, 152.15, 149.32, 148.59, 136.76, 134.21, 131.06, 130.83, 129.47, 129.36, 128.13, 123.71, 121.68. APCI-MS: m/z, calcd: 352.28, found: 351.55 ([M－H]-). 2.2.3. Synthesis of probe SATZ. Compound 1 (141 mg, 0.4 mmol) was dissolved in 10 mL absolute ethanol. Hydrazine hydrate (10 mg, 0.2 mmol) was added into above solution, and the mixture was stirred overnight at room temperature. Precipitates were filtered and washed with absolute ethanol to obtain probe SATZ. (68 mg, yield 49%) 1H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J = 2.2 Hz, 2H), 8.65-8.59 (m, 2H), 8.53 (s, 2H), 8.26 (d, J = 8.7 Hz, 2H), 8.08 (dd, J = 7.7, 1.8 Hz, 2H), 7.65 (td, J = 7.8, 1.9 Hz, 2H), 7.58-7.53 (m, 2H), 7.30 (dd, J = 8.2, 1.2 Hz, 2H). 13

C NMR (101 MHz, DMSO-d6) δ 156.71, 152.05, 148.61, 148.35, 134.10, 131.15, 129.34,

129.03, 128.05, 127.03, 123.85, 121.61. APCI-MS: m/z, calcd: 700.02, found: 701.15 ([M+H]+). 2.3. General spectra measurements. Stock solution of probe SATZ (2 mM) was prepared in DMSO and solutions of various analytes (1 mM) were prepared in distilled water. All the detection experiments were measured in 10 mM PBS buffer-DMSO (pH = 7.4, v/v, 9:1) at room temperature. The fluorescence spectra were measured by fluorescence spectrometer. Unless otherwise mentioned, the excitation wavelength was set to 401 nm, with excitation and emission slit width 5 nm/5 nm. 2.4. Preparation of portable kit and detection method. Silica gel plates were cut into 4x2 cm dimension. Probe SATZ (100 µM) was dissolved in dichloromethane, and silica gel plates


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were immersed in above solution. In a short time, silica gel plates were taken out from the solution and dried in air. The portable kit was successfully prepared, and the detection method is as follows. Trace amounts of sample solution was added on portable kit by capillary (0.3×100 mm). After the solvent has completely evaporated, the fluorescence of portable kit was observed by a 365 nm UV light. 2.5. Procedure of HPLC. The retention time of all samples was measured at a wavelength of 301 nm, and using a 70:30 (v/v) methanol: water solution as the mobile phase at 30 °C with a flow rate of 1.5 mL/min. 2.6. Cell culture and fluorescence imaging. Cell culture and fluorescence microscope imaging were performed according to the literature procedure.24, 38, 39 The images were collected upon excitation at 405 nm, and the signals were collected using an emission filter at 520-600 nm.22, 40-43 3. RESULTS AND DISCUSSION The AIE properties of SA. We firstly investigated whether SA exhibits AIE properties by monitoring the fluorescence intensity of SA with the increase of water volume fraction in the PBS buffer-DMSO system where PBS buffer and DMSO do duty for the poor solvent and the good solvent, respectively.22,

32, 33, 40, 44

As shown in Figure 1a, the fluorescence was almost

completely quenched in the good solvent. As the ratio of PBS buffer in solution gradually increases, the fluorescence intensity increased sharply when the water volume fraction is more than 80%. When the water volume fraction was 99%, the fluorescence intensity reaches the maximum. This result is in accord with the photos captured under irradiation by a 365 nm UV light. Then, the phenomenon of SA aggregates in the poor solvent was tested by illuminating a


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red laser through the solution and observing the Tyndall effect.37, 45 As shown in Figure 1b, SA dissolved in the good solvent didn't show the Tyndall effect, which suggested that no aggregates were formed. Nevertheless, it is clear to observe the Tyndall effect in the poor solvent, showing the formation of SA nanostructures. According to the above results, we could infer that the formation of nanostructures may be the reason for its fluorescence, and SA exhibits good AIE properties.45

Figure 1. (a) The fluorescence intensity change of SA (10 µM) in DMSO with increasing water volume content. (b) Tyndall effect and fluorescence of SA solutions. Response to Cys and Hcy. The time-dependent experiment displayed that the fluorescence intensity of probe SATZ towards Cys got to the maximum value at about 12 min, and then become mild (Figure S1). At the same time, the fluorescence intensity of probe SATZ solution with Hcy increased slowly in 12 min, and its intensity is lower than that with Cys. For GSH, the fluorescence intensity hardly increases for 18 min. Thus, all the following spectra measurements were performed at 12 min. Then, the selectivity of probe SATZ over all kinds of potential distractions was investigated by absorption spectra and fluorescence spectra, respectively. As shown in Figure 2a, upon addition of Cys, an obvious absorption peak appeared around 353 nm. Meanwhile, upon addition of Hcy and GSH, the same absorption peak was appeared but to a


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lesser degree. Delightedly, upon addition of other potential interferences, the absorption peak around 353 nm didn't show any noticeable changes. Similar results were shown in Figure 2b. Almost no fluorescent emission (ΦF = 0.026) could be detected for the probe alone, and upon addition of Cys, the fluorescence intensity of probe SATZ solution showed a remarkable increase (ΦF = 0.115) at 547 nm. Meanwhile, upon addition of Hcy, the fluorescence intensity of probe SATZ solution showed a slightly weaker fluorescence. In addition, no remarkable fluorescence intensity change was observed upon addition of other potential interferences. These results implied that probe SATZ has high selectivity for Cys and Hcy over other potential interferences. Significantly, the turn-on emission of probe SATZ for Cys with a large Stokes shift (λex = 399 nm, λem = 547 nm, ∆λ = 148 nm) is highly favorable for biomedical imaging because it can avoid self-quenching background levels from the excitation and scattered light (Figure S2).

Figure 2. (a) Absorption spectra of probe SATZ (10 µM) with various analytes (100 µM). (b) Fluorescence spectra of probe SATZ (20 µM) with various analytes (200 µM). We then evaluated the sensing capability of probe SATZ in response to Cys. As shown in Figure 3a, upon the addition of different concentrations of Cys, the absorption peak at 353 nm was considerably enhanced, which is the reason of color changes from colorless to yellow (Figure 3d). Similarly, as shown in Figure 3b, the fluorescence intensity of probe SATZ solution


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was also gradually enhanced, and its fluorescence intensity at 547 nm was plotted versus the Cys concentrations (Figure 3c). This plot gave a good linear relationship in the range of 0-200 µM of Cys, with a detection limit of 2.843 µM (LOD = 3 σ/slope: σ is the standard deviation of blank measurement).6, 37, 46, 47

Figure 3. (a) Absorption spectra of probe SATZ (10 µM) in the presence different concentrations of Cys (from 0 to 100 µM). (b) Fluorescence spectra of probe SATZ (20 µM) in the presence different concentrations of Cys (from 0 to 400 µM). (c) The fluorescence intensity changes of probe SATZ (20 µM) as a function of Cys concentration. Inset: Linear relationship between fluorescence intensity of probe SATZ and the Cys concentration. λem = 547 nm. (d) The photographs of probe SATZ (20 µM) in the absence and presence of Cys (200 µM) under daylight or a 365 nm UV light.


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To investigate whether probe SATZ possesses the ability to sense Cys in intricate living biological systems, we also researched the anti-interference capacity of probe SATZ. As shown in Figure 4a, probe SATZ exhibited a well response to Cys even in the presence of other potential interferences. Moreover, probe SATZ showed a wonderful response to Cys at physiological pH. (Figure 4b) These results suggested that probe SATZ has the potential to image in intricate living biological systems.

Figure 4. (a) The fluorescence intensity of probe SATZ (20 µM) toward Cys (200 µM) in the presence of other potential interferences (200 µM). (b) Profile of pH dependence of the fluorescence intensity of SATZ (20 µM) at 547 nm in the absence (black line) and presence (red line) of Cys (200 µM). Next, we evaluated the performance of probe SATZ for detecting Cys in living cells. As the key parameters for imaging applications, the cytotoxicity and photostability was investigated. As the results shown in Figure S10, the cell viabilities are all over 90% even at the concentration of 50 uM, indicating the feasibility of probe SATZ for imaging in live cells. In order to research the photostability of probe SATZ upon recognition, the photostability of reaction product (SA) was compared with 4-Methylumbelliferone (Figure S11).48 Upon exposure to a 15 W 365 nm UV light for 2 h, the fluorescence intensity of SA keeps about the same intensity, while the


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fluorescence of 4-Methylumbelliferone drop by more than 20%. These results suggested that probe SATZ is suitable for detecting Cys in living cells. As shown in Figure 5a, strong fluorescence could be visualized in PC12 cells upon addition of probe SATZ.9, 24 But, in control experiment, the PC12 cells pre-incubated with N-ethylmaleimide (NEM, a scavenger of biothiols) exhibited almost no fluorescence signal (Figure 5b).9, 11, 49 After the addition of various concentrations of Cys (0 µM, 10 µM, 40 µM), different degrees of fluorescent signal could be visualized in PC12 cells pre-incubated with NEM (Figure 5c-d). The result indicated that probe SATZ possesses the potential to detect Cys in intricate living cells.


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Figure 5. Confocal fluorescence images of PC12 cells. (a) PC12 cells were incubated with probe SATZ (10 µM) for 30 min. (b-d) PC12 cells were pre-incubated with NEM (0.5 mM) for 30 min and then further treated with different concentrations of Cys (0 µM, 10 µM, 40 µM) and probe SATZ (10 µM) for 30 min, respectively. To further expand the function of probe SATZ, taking the good performance of AIE properties, we designed a simple and user-friendly portable kit to detect Cys and Hcy on site.35, 50


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Carefully adding the aqueous solution of various analytes to the portable kit, and we could observe the emission of the spot under a 365 nm UV light. Figure 6a showed that the addition of Cys and Hcy triggered yellow-emitting spot on the portable kit, and the other didn't. This result indicated that the portable kit could specifically recognize Cys and Hcy and it inspired us to further investigate the possibility of quantitative detection. As shown in Figure 6b, the fluorescence intensity of the spot was gradually enhanced upon the addition of different concentrations of Cys (0-10 mM). These phenomena demonstrated the broad adaptability of this portable kit on site.

Figure 6. (a) The photograph of the Portable Kit after exposure to various analytes (10 mM) under a 365 nm UV light. (b) The photograph of the Portable Kit after exposure to different concentrations of Cys (0-10 mM) under a 365 nm UV light. Proposed mechanism. According to the previously reported references, we guessed that the 2,4-dinitrobenzenesulfonate group of probe SATZ was cleaved by sulfydryl to give the SA.32, 5154

And isomerization of SA molecules would be happened via excited-state intramolecular proton

transfer, resulting in a large Stokes shift (148 nm) upon recognition.22, 32-36, 40 The schematic


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diagram of proposed response mechanism was shown in Scheme 2, and further experiments were carried out to demonstrate our conjecture. Firstly, the responsive mechanism was extrapolated on the basis of fluorescence spectroscopy. As shown in Figure 7a, the fluorescence spectra of the reaction mixture was consistent with that of SA, indicating the formation of SA in the certain degree.1 Subsequently, we detected the HPLC peaks of probe SATZ + Cys (0 equiv., 5 equiv., 20 equiv., respectively) and SA. As shown in Figure 7b, the retention time of probe SATZ alone was 10.480 min. After reaction with different concentrations of Cys, a new peak could be observed at 7.930 min, which was consistent with the retention time of SA (7.930 min); meanwhile, the peak of probe SATZ at 10.480 min decreased, gradually. These results confirmed that SA was the product generated from probe SATZ reacted with Cys, which was in accord with our proposed response mechanism.14, 55-57 A similar result could also be monitored using TLC (petroleum ether: dichloromethane = 2:1 as developing solvent). As shown in Figure 7c, a yellow-emitting compound was appeared, which implied that SA was formed. The above results strongly supported the supposition that Cys could induce the formation of SA.

Scheme 2. Proposed response mechanism of probe SATZ towards Cys and Hcy.


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In addition, the morphology of probe SATZ before and after reaction with Cys was investigated. As shown in Figure 7d, the probe SATZ solution itself exhibited a obvious aggregate state.58,

59

But, due to the quenching effect of 2,4-dinitrobenzenesulfonyl, the AIE

properties of probe SATZ were inhibited and the probe SATZ solution have no fluorescence signal.51-53 Meanwhile, addition of Cys to probe SATZ solution would induce the release of SA and the formation of a larger size aggregate state (Figure 7b-d). Thus we speculated that the formation of SA aggregate state was the cause of fluorescence enhancement, and AIE properties would be activated upon recognition.59 These facts were in accord with the AIE properties of SA (Figure 1a).

Figure 7. (a) Fluorescence spectra comparison of probe SATZ (blue line, 20 µM), SA (cyan line, 10 µM), and the reaction mixture (red line) of probe SATZ (20 µM) with Cys (200 µM) in 10 mM PBS buffer-DMSO (pH = 7.4, v/v, 9:1). (b) HPLC chromatogram analysis. (A) Probe SATZ;


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(B) Probe SATZ + Cys (5 equiv.); (C) Probe SATZ + Cys (20 equiv.); (D) SA. Conditions: mobile phase, methanol/H2O (v/v, 70:30); flow rate, 1.5 mL/min; detection wavelength, 301 nm. (c) TLC image of probe SATZ, the reaction of probe SATZ with Cys, and SA. (Developing solvent: petroleum ether/dichloromethane, v/v, 2/1). (d) TEM images of SATZ (20 µM) before (left) and after (right) reaction with Cys (200 µM) in 10 mM PBS buffer-DMSO (pH = 7.4, v/v, 9:1). 4. CONCLUSION In summary, we designed and synthesized a novel colorimetric and turn-on fluorescent probe with both AIE and ESIPT properties for the detection of Cys/Hcy. Probe SATZ possessed good sensing performance, low cytotoxicity as well as outstanding photostability upon recognition, and the response mechanism was confirmed by multiple characterization methods. In addition to being used for cell imaging, a simple and user-friendly portable kit based on probe SATZ was proposed to detect Cys and Hcy on site. A 4x2 cm portable kit could be used for the inspection of more than ten samples simultaneously, which can effectively prevent the occurrence of false positives and visual errors when compared with traditional test papers. In future, AIE-based probes like probe SATZ can be used to manufacture such portable kits, which will provide new tools for selective and quantitative detection of micro-samples. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. (Cytotoxicity Assay, fluorescence quantum yields, supplemental spectra, 1H NMR,

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C NMR,

and MS spectra.)


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AUTHOR INFORMATION Corresponding Author E-mail address: [email protected] (Y.M. Zhou); Tel: +86-371-22868833-3422; Fax: +86-371-23881589; ORCID Yanmei Zhou: 0000-0001-8203-245X Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors thank the National Natural Science Foundation of China (21776061, 21576071 and U1504215), the State Key Laboratory of Fine Chemicals (KF1514), the program for Science & Technology Innovation Team in Universities of Henan Province (19IRTSTHN029), the program for Science & Technology Innovation Talents in Universities of Henan Province (19HASTIT037) and the Foundation of International Science and Technology Cooperation of Henan Province (No. 162102410012).


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A Novel AIE Plus ESIPT Fluorescent Probe with a Large Stokes Shift

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