Aggregation induced emission enhancement (AIEE)

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 182 (2017) 58–66

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Aggregation induced emission enhancement (AIEE) characteristics of quinoline based compound — A versatile fluorescent probe for pH, Fe(III) ion, BSA binding and optical cell imaging Irulappan Manikandan a, Chien-Huei Chang b, Chia-Ling Chen b, Veerasamy Sathish a, Wen-Shan Li b,c,⁎, Mahalingam Malathi a,⁎⁎ a b c

Department of Chemistry, Bannari Amman Institute of Technology, Sathymangalam 638 401, India Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung 804, Taiwan

a r t i c l e

i n f o

Article history: Received 10 December 2016 Received in revised form 23 March 2017 Accepted 24 March 2017 Available online 25 March 2017 Keywords: Aggregation induced emission enhancement Fe(III) ion sensor pH sensor BSA binding Cell imaging

a b s t r a c t Novel benzimidazoquinoline derivative (AVT) was synthesized through a substitution reaction and characterized by various spectral techniques. Analyzing the optical properties of AVT under absorption and emission spectral studies in different environments exclusively with respect to solvents and pH, intriguing characteristics viz. aggregation induced emission enhancement (AIEE) in the THF solvent and ‘On-Off’ pH sensing were found at neutral pH. Sensing nature of AVT with diverse metal ions and bovine serum albumin (BSA) was also studied. Among the metal ions, Fe3+ ion alone tunes the fluorescence intensity of AVT probe in aqueous medium from “turn-on” to “turn-off” through ligand (probe) to metal charge transfer (LMCT) mechanism. The probe AVT in aqueous medium interacts strongly with BSA due to Fluorescence Resonance Energy Transfer (FRET) and the conformational change in BSA was further analyzed using synchronous fluorescence techniques. Docking study of AVT with BSA reveals that the active site of binding is tryptophan residue which is also supported by the experimental results. Interestingly, fluorescent AVT probe in cells was examined through cellular imaging studies using BT-549 and MDA-MB-231 cells. Thus, the single molecule probe based detection of multiple species and stimuli were described. © 2017 Elsevier B.V. All rights reserved.

1. Introduction There has been tremendous interest in the development of luminescent materials with great fundamental and technological implications are spurred among the researchers [1,2]. It is used for the development of optoelectronic materials with various device applications including organic light-emitting diodes and solid-state lasers [3,4]. In general, most of these luminogens are strongly emissive in dilute solutions, but they become weakly fluorescent after aggregation in the solid states [5]. The emission of many luminogens is totally or partly quenched upon aggregate formation due to the aggregation caused quenching (ACQ) effect [6–8]. To address this problem, a notable phenomenon, aggregation-induced emission (AIE) was coined by Tang and his coworkers in 2001 [9]. On the other hand, weakly emissive molecules

⁎ Correspondence to: W.-S. Li, Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan. ⁎⁎ Correspondence to: M. Malathi, Bannari Amman Institute of Technology, Sathymangalam 638 401, India. E-mail addresses: [email protected] (W.-S. Li), [email protected] (M. Malathi).

http://dx.doi.org/10.1016/j.saa.2017.03.055 1386-1425/© 2017 Elsevier B.V. All rights reserved.

are induced to emit efficiently through nanoaggregate formation, which are referred to as aggregation-induced emission enhancement (AIEE) [10]. Since the inception of these phenomena, a large number of AIE/AIEE active organic compounds have been developed [11–13] viz. siloles [14], tetraphenylethene (TPE) [15], malemide [16], benzothiadiazole [17], naphthalimide derivatives [18], 1-cyano-trans1,2-bis-(4′-methylbiphenyl)ethylene (CN-MBE) [19,20], 2,5-diphenyl1,4-distyrylbenzene (DPDSB) derivatives [21,22], triazine derivatives [23], boron-dipyrromethene (BODIPY) derivatives etc. [24,25]. Further, these AIE/AIEE based compounds are useful to bioimaging studies [26], sensor for proteins [27], detection of insulin and amyloid fibrils, which are responsible to neurodegenerative diseases [28,29]. Therefore, our aim to synthesize novel compounds having AIE/AIEE properties with interesting biological applications. (See Scheme 1.) In human physiological system pH plays a vital role in biological processes such as, cellular metabolism, enzymatic activity, ion transport, cell volume regulation, vesicle trafficking, cell membrane polarity, cellular signaling, cell activation, growth, and proliferation [30,31]. However, the abnormal intra cellular pH variation may affect the nervous system and to the formation of cancer as well as Alzheimer's disease [32,33]. Similarly iron is the one of the most important metal in our body,

I. Manikandan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 182 (2017) 58–66

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Scheme 1. Synthesis of AVT.

especially Fe(III) plays an crucial role in the human metabolism like oxygen transport and acts as a cofactor in many enzymatic electron transfer as well as oxidation reactions [34]. The deficiency of iron in hemoglobin leads to anemia, whereas the high content of iron in the human body have been associated with liver diseases, heart attack, diabetes mellitus, osteoarthritis, osteoporosis, oxidative stress and neurodegenerative diseases [35]. Above all, serum albumins are important constituents of plasma in the blood. Especially, bovine serum albumin (BSA), has been used as a model protein in numerous biochemical studies because of its structural homology with human serum albumin (HSA) and facilitates the metabolism, transport and distribution of exogenous and endogenous substances [36]. It is a heart shaped protein of single polypeptide chain that consists of 583 amino acid residues with 17 disulfide bonds and one free SH group [37]. Recently, the advances in recognizing biology at the molecular level are assisted by the use of chemical probes, including small-molecule fluorophores [38]. Noninvasive visualization and investigation of interactions among proteins, biomolecules, and enzymes in living cells is an important goal for biologists. Additionally fluorescence probes are powerful tools for this purpose [39]. Therefore, the design and development of fluorescent materials would be very useful imaging tools and might have diagnostic value in disease treatment [40]. Single molecular probes based the detection of multiple species or stimuli are the fascinating area of research for chemical as well as biological sensing [41]. Due to its high sensitivity and selectivity, a burst of research activity is more concentrated towards fluorescence based sensing techniques [42]. With these ideas in our mind, we develop a single probe with diverse fluorescent sensing of the biologically significant pH, metal ions and molecules. Additionally, the AVT fluorescence was studied under cell imaging studies as a dye. To the best of our knowledge, this is the first report on the probe has been five-in-one fluorescence sensor features in a single compound. 2. Experimental Section 2.1. Materials and Methods

with 1:1 M ratio were added and the resulting mixture was refluxed with constant stirring for about 3 h. The solvent was removed under reduced pressure. The crude product was transferred to a beaker containing crushed ice and neutralized using dil. HCl solution. The yellow precipitate was filtered, dried and adsorbed for the silica (60– 120 mesh) column chromatography. The pure product was obtained in the 24% petroleum ether and ethyl acetate medium. Mp: 187 oC. IR (cm− 1): 3070 cm−1 aromatic(C\\H), 1425 cm− 1 aliphatic (NCH2), 1027 cm−1 (−CH2OH). 1H NMR (CDCl3, 400 MHz, TMS): 2.91 (S, 1H), 3.24–3.32 (m, 2H), 4.22–4.28 (m, 2H), 7.26–7.32 (m, 2H), 7.43–7.86 (m, 6H), 7.91–8.10 (m, 2H). ESI-MS m/z: 322.100 (M + H)+. 2.3. AIEE Studies The stock solution of AVT in THF was prepared at 1 × 10−4 M. The absorption and emission spectral properties of AVT (20 μL) were studied with varying concentrations of water (0 to 90%) at pH 7. The SEM study of AVT was analyzed at 10:90 (v/v) proportion of THF:water content. 2.4. pH Sensor Study AVT probe stock solution at 1 × 10−4 M was prepared in 10% aqueous ethanol medium for absorption and emission spectral titration with respect to pH. The various pH of the AVT probe solution was adjusted by dilute HCl and NaOH solution and the resulting pH was monitored under digital pH meter. 2.5. Metal Binding Studies The metal binding studies of AVT with metal ions in 1:1 M ratio were performed in 10% aqueous ethanol as solvent medium. The stock solution was prepared at the concentration of 1 × 10−4 M for absorption and the resulting spectrum was measured in range from 200 to 600 nm at 303 K. For the emission spectral studies AVT probe at 1 × 10−5 M with were carried out at the excited wavelength of 267 nm and the spectrum were recorded from the range of 300 nm to 650 nm. The fluorescence quenching constant was calculated using Stern– Volmer equation,

All the chemicals were procured from Sigma Aldrich and used without further purification. Infrared spectrum was recorded on a PerkinElmer FT-IR spectrometer PARAGON 1000 and 1H NMR spectrum on Bruker AMX-400 FTNMR spectrometer. FAB-MS data were obtained using a JEOL, JMS-700 double focusing mass spectrometer. Melting point of the samples has been observed by using sigma melting point apparatus. UV–visible spectra were studied using Perkin-Elmer lambda 35 spectrophotometer. Fluorometric analyses have been carried out using Shimadzu spectrofluorometer RF-5301 (1 cm path length quartz cuvette). The morphology of the nanoaggregates were studied using scanning electron microscopy (SEM) (Carl Zeiss) equipped with energy dispersive X-ray analysis (EDX).

where, Io and I are the fluorescence intensity of AVT before and after addition of metal ion, Ksv is the quenching constant and [Q] is the quencher (metal ion) concentration [43–45]. Emission quantum yield of AVT probe was measured using quinine sulphate in 0.1 N H2SO4 as a standard solution [46]. To evaluate the detection efficiency of the probe towards metal species, the limit of detection was calculate based on 3δ/k, where δ is the standard deviation of the blank solution and k is the slope of the calibration plot [47].

2.2. Synthesis of 2-3-(1H-benzo[d]imidazol-2-ylthio)ethanol (AVT)

2.6. Protein Binding Studies

To the refluxing solution of NaH (80%) (0.1 g, 3.45 mmol) in THF (50 mL), 2-mercapto ethanol (0.1 mL, 1.15 mmol) and 3-(Hbenzo[d]imidazol-2-yl)-2-chloroquinoline (BIQ) (0.35 g, 1.15 mmol)

The excitation wavelength of BSA at 275 nm and the respective emission at 340 nm were monitored during the protein binding studies and 10% aqueous ethanol medium was the solvent environment. The

Io =I ¼ 1 þ K sv ½Q ;

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DMEM medium (GIBCO) containing 10% FBS. All media contained 100 U per mL penicillin and 100 U per mL streptomycin.

2.8.2. MTT Cell Viability Assay BT549 and MDA-MB-231 were plated in 96 well plates and incubated at 37 °C overnight. The cultured cells were treated with gradient concentrations of compound AVT (final concentrations: 0, 10, 20, 30, 40, 50 μM) for 24 h and viable cells were measured by a MTT (3-(4,5dimethylthiahiazol-2-y1)-2,5-diphenyltetrazolium bromide) (sigma) assay. Cells were incubated with 100 μL MTT solution (1 mg/mL) for 4 h at 37 °C, and then the medium containing with non-metabolized MTT was aspirated. Formazan crystals were dissolved in 100 μL DMSO and absorbance was measured at 540 nm.

Fig. 1. UV–vis spectra of AVT (20 μM) in THF and THF/Water (10:90, v/v).

excitation and emission slit widths were kept at 5 and scan rates were maintained constant for all of the experiments. A stock solution of BSA was prepared in 1 × 10 4 M HEPES (pH ̶ 7.4) and stored in the dark at 4 °C for further use. For synchronous fluorescence spectra the same concentrations of BSA and the compound AVT were used and the resulting spectra were measured at different Δλ values (difference between the excitation and emission wavelengths of BSA 15 and 60 nm respectively. 2.7. Docking Analysis The crystal structure of Bovine serum albumin (4F5S.pdb) was downloaded from the protein data bank (PDB database). The polar hydrogen atoms were added and water molecules were removed from the protein pdb file. The structure of AVT was optimized using Gaussian 03 W software. Ligand docking was carried out using Auto dock 1.5.6 using Lamarckian Genetic Algorithm (GA). 2.8. Cellular Imaging Studies 2.8.1. Cell Culture BT-549 breast cancer cells (ATCC) were grown adherently and maintained in RPMI 1640 medium (GIBCO) containing 10% FBS and 0.023 IU/mL insulin. MDA-MB-231 cells (ATCC) were maintained in

2.8.3. Fluorescent Detection of Probes in Live Cells Breast cancer cells (3 × 104 cells/well, BT549 and MDA-MB-231 cells) were seeded on chambered cover glass (Thermo Scientific™) and incubated at 37 °C in humidified air with 5% CO2 for 24 h. After adherent cells were treated with 10 μM and 20 μM fluorescent compound, in DMSO kept for 24 h and dropped NucRed Live 647 Ready Probes reagent (Thermo Fisher Scientific) directly about 30 min to stain living cell nucleus. Zeiss LSM 510 META NLO DuoScan confocal microscopy equipped a IR pulse laser source was used with excitation at 780 nm for chemical compounds and a He-Ne laser device with excitation at 638 nm for NucRed Live 647 Ready Probes reagent. The live cells were observed with an ×40 oil-objective.

2.8.4. Photo Fading Behavior of Probes in Live Cells MDA-MB-231 cells were plated in chambered cover glass and incubated at 37 °C for 24 h. After adherent cells were treated with 20 μM fluorescence compound in DMSO for 24, 48 and 72 h. We used Zeiss LSM 510 META NLO DuoScan confocal microscopy with an IR pulse laser source with excitation at 780 nm for chemical compounds. The live cells were observed with an ×40 oil-objective.

3. Results and Discussion A nucleophilic substitution reaction of 3-(H-benzo[d]imidazol-2-yl)2-chloroquinoline (BIQ) with 2-mercapto ethanol (1:1 M ratio respectively) was performed in the THF solvent medium using sodium hydride as base (Scheme 1). The pure product AVT probe was isolated through silica column chromatography and the structure was characterized by 1 H NMR, IR, mass spectral analysis (Figs. S1–S3).

Fig. 2. (a) Emission spectra of AVT (20 μM) with various fraction of THF and water, insert: photograph of AVT with various fraction of THF and water mixture (colorless to blue color) under illumination of 365 nm UV lamp. (b) Dependence of I/I0 ratios of AVT with various fraction of THF and water mixture (λex = 267 nm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Fig. 3. SEM image of AVT in THF/Water solution with 1:9 proportion of THF:water content.

3.1. Aggregation Induced Emission Enhancement Characteristics of AVT The compound AVT is soluble in organic solvents such as THF, DMSO, ethanol etc. and insoluble in water. The AIEE property of AVT was analyzed using UV–visible spectral technique in THF and THF:water mixture (Fig. 1). In the THF medium, AVT exhibited two absorption peaks at 267 and 317 nm. The peak appeared in high energy region ascribed to n-π* electronic transition. In THF:water mixture (1:9) volume, the absorbance increases without any considerable shift in wavelength and the peak is extended with a tail, which confirms the formation of nanoparticle suspension due to Mie effect [48]. The emission spectral study of AVT was performed in THF:water mixture with varying water volumes (λex = 267 nm) In general, the compound AVT displayed a weak luminescence spectrum at 420 nm in THF solvent medium. However an enormous enhancement of emission intensity (~ 50 fold) was observed after incremental addition of water volume in THF (Fig. 2a). Hence it is presumed that the compound AVT should undergo aggregation in the presence of high volume of water (1:9 volume ratio) in THF. Further, the plot of emission intensity against the solvent composition clearly distinguishes the molecular solutions from the nanoaggregates (Fig. 2b). The time dependent emission spectra of AVT in THF:water mixture (1:9 volume ratio) revealed that the constant emission intensity up to 1 h which confirms the stability of nanoaggregation (Fig. S4). Additionally, a visible color change from colorless to blue was noted after irradiation under UV light (Fig. 2a inset). Even though the THF solution of AVT is faint luminescence in nature, the solid state emission spectrum of the compound AVT was planned to

Fig. 5. Emission spectra of AVT (20 μM) with the addition of various concentration of FeCl3 (0–120 μM).

correlate with liquid state. The spin coated plate of AVT was studied under the solid state analysis which exhibited a very strong greenish blue luminescence at 420 nm (Fig. S5). In UV light, it illuminated as bright green color. The combined optical studies revealed the aggregation induced emission enhancement property of AVT sustained by its water insoluble nature. The scanning electron microscopy (SEM) analysis confirms the formation of nanoaggregates (Fig. 3). It indicates the nanoparticles are distorted spherical shape with the size of approximately 100 nm. The aggregation induced emission originates from the restriction of intramolecular rotation (RIR) of benzimidazole moiety present in the AVT [49]. The compound AVT exhibits active intramolecular rotation in THF medium effectively initiates nonradiative relaxation of excited AVT molecule. Hence a faint luminescence is observed. However, the molecular aggregate formation of AVT in THF:water (10:90, v/v) mixture restricts the intramolecular rotation (RIR) of benzimidazole moiety and block nonradiative relaxation channel and inducts radiation decay with bright luminescence. The quantum yield value of AVT in THF is 0.035 and it increases up to 55% for the THF:water mixture in 1:9 volume ratio. To check the external conversion for the RIR process mainly for viscosity, the THF solution of AVT was examined using varying concentration of ethylene glycol (0–90%) (Fig. S6). For each addition of ethylene glycol, the emission intensity is enhanced drastically akin to AIEE property of AVT. Increasing viscosity impedes the intramolecular rotation of peripheral benzimidazole moiety and activate the radiative transitions and thereby enhanced the fluorescence intensity [50].

Fig. 4. (a) Fluorescence spectra of AVT (20 μM) at different pH values in aqueous ethanol. (b) pH dependence of AVT (I/Imax) at 303 K at different pH values.

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Fig. 6. (a) Fluorescence spectra of BSA (20 μM) with the addition of AVT (20–100 μM). (b) The overlap of UV absorption spectrum of AVT with the emission spectrum of BSA.

3.2. pH Sensing Absorption spectra of AVT solution (20 μM) in different pH were recorded at room temperature (Fig. S7). In lower pH values AVT showed a strong absorption peak at 267 nm which decreased gradually with slight blue shift to the tune of 3 nm from 267 nm to 264 nm during the subsequent increase of pH level. During the course of pH titration, a new peak was observed at 354 nm and its absorbance was increased with the successive addition of pH. This peak is due to the photo induced electron transfer between the quinoline and benzimidazole ring [51]. The protonation and deprotonation of some heteroatom-containing groups will response to emission at various pH levels [52]. The emission pH titration of AVT was presented in Fig. 4a and b. AVT showed an intense luminescent in the acidic solution revealed that the protonation of heteroatoms in AVT compound inhibits the photo induced electron transfer (PET) reaction which leads a radiative relaxation process of excited molecules. Hence, the compound AVT shows fluorescence in acidic pH range [53,54]. Under high pH conditions, fluorescence quenching was observed with blue shift of 13 nm from 425 to 412 nm. It is believed that the base induced deprotonation of heteroatoms in AVT provides availability of lone pair of electrons for PET process and leads to nonradiative molecular relaxation which was responsible for the fluorescence quenching of AVT (Fig. S8).

ion alone, AVT signaled a notable increase of absorption intensity (267 nm) and appearance of a new peak at 351 nm indicates the Fe3+ ion was strongly interacting with AVT. Additionally, a naked eye detection of color change from colorless to yellowish brown solution was observed (Fig. S10). Because of the Fe3+ ion is coordinated with oxygen, the excited state is more stable than the ground state, which leads to drastic change in the absorption and emission spectra [55,56]. Interestingly, the emission spectra of AVT showed a significant fluorescence quenching after the addition of Fe3+ ion, while the other metal ions displayed only a negligible change (Fig. S11a and b). This effect reveals that AVT possesses fluorescence turn-off sensing behavior with high selectivity towards Fe3 + ion. To have a better understanding of the fluorescence spectral response of AVT with Fe3 + ions, a titration with various concentration of Fe3 + were further conducted (Fig. 5). The emission titration study showed that the emission intensity was decreased with the increasing concentrations of Fe3+ ions from 10 μM to 120 μM. This fluorescence quenching of AVT is due to the energy transfer from the ligand (probe) to the metal d-orbital and/or ligand to metal charge transfer (LMCT) [57–59]. The quenching efficiency of AVT with Fe3+ ion was found to be 89% with the Stern–Volmer quenching constant of 8.5 × 104 M−1 and the detection limit was 0.4 μM. The sensitivity, selectivity and the detection limit is quite high compared to other reports [47]. 3.4. BSA Binding Study

3.3. Detection for Fe3+ Ions The absorption spectra of AVT (20 μM) with several metal ions Cd2+, Co2 +, Fe2 +, Fe3 +, Zn2 +, Hg2 +, Pb2 +, Ni2 + and Mg2 + (1 × 10− 4 M) were recorded in aqueous ethanol medium (Fig. S9). An intensive absorbance peak of AVT was found at 267 nm. In the presence of Fe3 +

The interaction nature of AVT with biomolecule was elucidated by performing an absorption spectral titration by keeping BSA as in stable concentration and changing the concentration of AVT (Fig. S12). After the successive addition of AVT, the absorbance was increases without any shift and it was confirmed that the AVT strongly binds on BSA

Fig. 7. Synchronous spectra of BSA (10 μM) in the absence and presence of AVT (20–100 μM) in the wavelength difference of Δλ = 15 nm (a) and Δλ = 60 nm (b).


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Fig. 8. (a) Docking view of AVT with nearest residues of BSA, the residues involved in hydrogen bonding are represented by dotter line. (b) The BSA molecule was represented using solid ribbon and AVT structure was represented as stick model.

molecule through ground state charge transfer complex formation [60]. Similarly emission titration was carried out with excitation at 280 nm (Fig. 6a). The concentration of BSA was fixed at 20 μM and the concentration of AVT varied from 20 to 100 μM. During the emission titration, a new luminescence band was generated at 438 nm in addition with the quenching of BSA characteristic emission peak at 340 nm. The fluorescence intensity of the new band increases with an increase in the concentration of the compound AVT. Since the inner filter effect is negligible, the fluorescence quenching of BSA and generation of a new emission peak revealed that a fluorescence resonance energy transfer (FRET) takes place from the donor of BSA to the acceptor compound AVT [61]. In general, the FRET is a non-radiative relaxation which is used to monitor the proximity and the relative orientation of the guest [62]. However, a radiative FRET is also observed due to the overlap of emission spectrum of donor with absorption spectrum of acceptor. In current system, a strong overlap of the fluorescence spectrum of the tryptophan (Trp) of BSA with the absorption spectrum of AVT is occurred (Fig. 6b). 3.5. Synchronous Fluorescence Spectroscopy Studies Synchronous fluorescence spectra were used to characterize the interaction between the fluorescent probe and proteins [63]. The fluorescence of BSA is mainly due to the presence of tyrosine and tryptophan residues. Hence, the spectroscopic methods are usually applied to study the conformation of protein. In synchronous fluoroscopy according to Miller, the difference between excitation wavelength and emission wavelength (Δλ = λemi − λexc) reflects the different nature of chromophores. When a Δλ of 15 nm is used, the obtained synchronous fluorescence spectrum indicates the spectral property of tyrosine residues, whereas a Δλ of 60 nm indicates tryptophan residues [64]. The maximum emission wavelengths of tryptophan and tyrosine residue in the protein molecule are related to the polarity of their surroundings and the changes of emission maximum wavelengths can reflect changes of protein conformation. The effect of AVT on the synchronous fluorescence spectrum with Δλ = 60 nm and Δλ = 15 nm is shown in Fig. 7. A blue shift was obtained when Δλ is equal to 60 nm reveals that there must be a slight change in the conformation of BSA. It occurs mainly due to the change in polarity around the tryptophan residues i.e., decrease in hydrophobicity [65]. At the same time in the emission of tyrosine has no change on it. The role of tryptophan in binding was further confirmed from docking studies.

lies within the hydrogen bonding distance with tryptophan 134 (2.5 Å) and Lys 131 (2.21 Å). Docking results illustrate that the ethylene hydroxyl functional group in the compound AVT interacts with imidazole azide-N functional group of tryptophan residue. Docking analysis ensure that the H-bonding bonding formation between the AVT and BSA certainly change the polarity around the fluorescent chromophores of BSA. The docking studies of AVT with BSA protein corroborate that the experimental results observed in the fluorescence and synchronous fluorescence spectral studies. 3.7. Cell Toxicity of Probe In order to use the compound AVT as fluorescence probe for applications in living cells, the minimum cytotoxic requirement is crucial to empirical development to establish suitable protocols for different cells. Therefore, the cytotoxicity of the probe AVT on human breast cancer cells (BT-549 and MDA-MB-231) was evaluated using MTT assays in different concentrations from 10 to 50 μM for over a period of 24 h (Fig. 9). Interestingly, the AVT compound displays no toxicity (up to 50 μM) to MDA-MB-231 cells under the experimental conditions. Notably, AVT compound was observed to have less cytotoxicity towards human breast cancer cells BT-549, after only 24 h treatment with increasing concentrations (0–50 μM).Thus, the compound AVT exhibits low cytotoxicity with cell viabilities N 70% at concentrations of up to 20 μM, suggesting that it is a biocompatible probe and may has potential applications in live cell imaging studies. 3.8. Fluorescence Imaging of Live Cells To validate the potential applications of the compound AVT probe in live cell imaging, we first conducted preliminary cell microscopy experiments in both BT549 and MDA-MB-231 cell lines whether the probe

3.6. Docking Studies AVT was docked to BSA to determine the theoretical binding free energy of AVT and it was found to be −5.9 kJ/mol at 298 K. The docking view of AVT with protein BSA was shown in Fig. 8. The bounded AVT

Fig. 9. The cytotoxicity of fluorescent probe AVT in BT549 and MDA-MB-231for 24 h.

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Fig. 10. Live MDA-MB-231 cells were treated with (a) DMSO control and 10 μM and 20 μM fluorescence compound (b) AVT; for 24 h. Nuclei were stained with nucred live ready probe reagent (red) for 30 min. The cells were observed under a confocal microscope. Scale bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

AVT has fluorescent signal in living cell by fluorescent microscope. As shown in Fig. S13, AVT probe (10 μM and 20 μM) were easily excited by UV wavelength light (λexc = 365 nm) in living cells and displayed blue emission collected from the blue channel (λem = 450 nm). These experiments suggest that the rapid internalization of AVT probe by the cells allows imaging study with live cells. With this in mind, we evaluate the applicability of AVT probe for its ability to penetrate cell membranes and their molecular mode of action involved in live cell imaging using

confocal laser scanning microscopy. In control experiments of live BT549 cells, the red fluorescence indicated that the cells were stained by nucred live ready probes reagent (NucRed®, red staining), the nucleus marker (Fig. S14a). Representative fluorescence images of live cells that were treated 10 μM or 20 μM AVT respectively, on 8-well plates for 24 h and subsequently stained by NucRed® (red, acell-permeant nuclear stain) after 30 min of incubation (Fig. S14b). As shown in Fig. S14, the BT549 cells incubated with the probe AVT displays intracellular

Fig. 11. LiveMDA-MB-231 cells were treated with 20 μM compound AVT for 24, 48, and 72 h. The cells were observed under Zeiss confocal microscope and experimental conditions were the same as described above. Scale bar = 20 μm.


fluorescence emission (blue) in a dose-dependent manner, indicating direct visualization of AVT internalized by cells. The above showed cell images, that the probe AVT could homogeneously localize in the cytoplasm, which was differentiated from the presence of a NucRed® nuclear localization signal. Similarly, MDA-MB-231cells stained by10 μM or 20 μM AVT emit blue fluorescence emission in a dose-dependent manner (Fig. 10b) as compared to a control NucRed® probe (Fig. 10a). As far as the autofluorescence of dead, damage or other stress cell concerned, we did not observe any alternative and specific signals from the full wavelength and the whole cells had the same signal without difference under the similar cell morphology. In Fig. 9, we see AVT displayed no cytotoxic property till 30 μM in MDA-MB231 cells and also exhibited very low cytotoxic at concentration of 20 μM towards BT549 cells. Taken together, the results suggest that the AVT probe was internalized by human breast cancer cell lines and verified its potential as fluorescent dye through cell imaging with live-cell fluorescence microscopy.

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We are also grateful for financial support of this work provided by the Academia Sinica (MOST 105-0210-01-13-01 and MOST 106-0210-0115-02) and the National Science Council (NSC 102-2325-B-001-025 and MOST 103-2113-M-001-024-MY3). Instrumentation support was provided by the Chemical Biology Facility in Institute of Chemistry at Academia Sinica, Taiwan. Experiments and data analysis were performed in part through the use of the confocal microscope at Division of Instrument Service of Academia Sinica and with the assistance of Shu-Chen Shen. Appendix A. Supplementary Data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.saa.2017.03.055.

References 3.9. Photostability of Probe Development of cell-staining fluorophore has gained unexpected attention recently, because these materials have been widely used in live cell imaging, biological quantification and medical diagnosis [66,67]. However, they frequently suffer from low photo stability during laser scanning and retard their further applications as biocompatible fluorophore in living cells. To gain insights into the photo stability of AVT probe involved in live cell imaging, the imaging performance of the probe was investigated for different time periods, 24, 48, and 72 h using confocal laser scanning microscopy (Fig. 11). The intensity of fluorescence in MDA-MB-231 cells increased as treatment time extended up to 48 h and gradually decayed at 72 h. Notably, the analysis of living cells implied that the uptake of AVT probe occurs mostly at 48 h of incubation and this action provides urgently needed for the long-term observation of live cells up to 72 h. The findings indicate that AVT probe display significant photo stability, which suggests that this probe with low cytotoxicity may be potential candidate in live cells as a fluorescent tool for exposing targets and dynamic mechanisms of action. 4. Conclusion The present work is the first example to demonstrate the concept of five-in-one probe AVT having AIEE character, pH and Fe3+ ion sensor, BSA binding and application in the cellular imaging studies. We have successfully synthesized and characterized the compound AVT. The fluorescence efficiency of the compound AVT have readily tuned by adding water to its THF solution indicates AIEE-active nature. It shows high sensitive and selective on-off emission with respect to variation in the pH. The metal sensing property of the compound AVT were analyzed with large number of transition metal ions by absorption and emission spectral techniques. The fluorescence quenching of AVT upon titration with Fe3+ ion illustrates its strong binding ability and selectivity towards Fe3+ ion. Besides, binding interaction between AVT and BSA were analyzed by spectroscopic and molecular modeling techniques. The combined experimental and theoretical results exemplified that the compound AVT strongly interacts with BSA. The confocal microscope cell images indicate that AVT can be utilized as fluorescence probe for cellular imaging of BT-549 and MDA-MB-231 cells. Moreover, MTT assay showed that the compound AVT has no cytotoxicity within 50 μM concentration range. Therefore, it is worthwhile to mention that, AVT opens up new vistas for the development of research towards multi applications in a single probe based fluorescent sensors in the forthcoming years. Acknowledgments This work was supported under DST-Fast-Track young scientist program by Science and Engineering Research Board (SR/FT/CS-51/2011).

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