An AIEE Active Anthracene-Based Nanoprobe for Zn²⁺ and Tyrosine Detection Validated by Bioimaging Studies

Abstract

Novel anthracene-based Schiff base derivative (4-(anthracen-9-ylmethylene) amino)-5-phenyl-4H-1,2,4-triazole-3-thiol; AT2) is synthesized and utilized as an aggregation-induced emission-enhancement (AIEE) active probe to detect Zn²⁺ and Tyrosine. Ultraviolet-visible absorption/photoluminescence (UV-vis/PL) spectroscopy studies on the AIEE property of AT2 (in ethanol) with increasing water fractions (f_w: 0–97.5%) confirm the J-type aggregation. Excellent sensor selectivity of AT2 to Zn²⁺ and its reversibility with Tyrosine are demonstrated with PL interrogations. 2:1 and 1:1 stoichiometry and binding sites of AT2-Zn²⁺ and Tyrosine-Zn²⁺ complexes are elucidated from Job plots, HR-mass, and ¹H-NMR results. Nanomolar-level detection limits (LODs) of Zn²⁺ (179 nM) and Tyrosine (667 nM) and association constants (K_as) of 2.28 × 10⁻⁶ M⁻² (for AT2-Zn²⁺) and 1.39 × 10⁻⁷ M⁻¹ (for Tyrosine-Zn²⁺) are determined from standard deviation and linear fittings. Nanofiber formation in AIEE and aggregated/dispersed nanoparticles in the presence of the Zn²⁺/Tyrosine are supported by scanning-electron microscope (SEM), transmission-electron microscope (TEM), atomic-force microscope (AFM), and dynamic-light scattering (DLS) investigations. Density-functional theory (DFT) studies confirm an “On-Off” twisted intramolecular charge transfer/photo-induced electron transfer (TICT/PET) and “On-Off-On” PET mechanisms for AIEE and sensors, respectively. B16-F10 cellular and zebrafish imaging are conducted to support the applications of AIEE and sensors.

1. Introduction

One-pot synthesis of organic nanoprobes with aggregation-induced emission-enhancement (AIEE) features, along with their specific analyte-sensing capability, can be noted as trend-setting achievement in modern chemosensory research [1,2,3,4,5,6,7]. Schiff base derivatives are becoming attractive because of easy synthesis, analyte specificity, as well as AIEE properties [8,9,10,11,12]. Moreover, Schiff base derivatives, which exhibit high AIEE and specific analyte sensing via fluorescent response, can be effectively used in bioimaging and organic thin-film transistor (OTFT)-based sensors [13,14,15]. However, to achieve such highly fluorescent nanoprobes, it is essential to include the effective fluorophore units, such as naphthalene, anthracene, pyrene, other polyaromatic systems, etc. [16,17,18,19]. Similarly, if the probe is designed to detect specific metal ions, the receptor must have certain coordinative units like imines (-C=N), hydroxyl (-OH), thiol (-SH), etc. [20,21]. Recently, our group reported a pyrene-based Schiff base with “4-Amino-5-phenyl-4H-1,2,4-triazole-3-thiol” receptor, which has free thiol and imine groups (formed during the Schiff base reaction) to provide effective coordination with Zn²⁺ and to show reversibility with Tyrosine [15]. To further improve the above receptor, the Schiff base design can be modified by replacing the fluorophore that allows “turn-on” detection of Zn²⁺ and possesses AIEE property. To this track, the anthracene, which has the aforementioned optoelectronic properties, can be adopted to replace the pyrene unit in Schiff base synthesis [22,23,24]. Moreover, anthracene-based Schiff base probe may hold the nanostructural morphologies in AIEE and Zn²⁺ selectivity due to the π-π stacking and self-assembly nature, which has been demonstrated in in-vivo/in-vitro applications [25,26]. Thereby, anthracene-9-carboxaldehyde is refluxed with “4-Amino-5-phenyl-4H-1,2,4-triazole-3-thiol” to afford the Schiff base probe “(4-(anthracen-9-ylmethylene) amino)-5-phenyl-4H-1,2,4-triazole-3-thiol); AT2”, which has the AIEE property and also displays an “Off-On-Off” response to Zn²⁺ and Tyrosine.

Among transition metal ions, Zn²⁺ is essential for neural signal transmission and apoptosis and acts as a catalyst in enzymatic gene transcription and DNA synthesis [15,27,28,29]. Disorder of Zn²⁺ in human body may cause Alzheimer’s disease, metabolic disorder, infantile diarrhea, cerebral ischemia, epilepsy, etc. [30,31]. Therefore, many tactics have been proposed to detect Zn²⁺, such as inductively coupled plasma mass spectroscopy (ICP-MS), inductively coupled plasma-atomic emission spectrometry (ICP-AES), flame-atomic absorption spectrometry (FAAS), electrochemical methods, ion-selective membrane, and fluorescent assay tactics [32,33,34,35,36,37]. Among them, fluorescent detection of Zn²⁺ is well noted because of its applications in bioimaging studies [15,37,38], which allows us to utilize the Schiff base AT2 as the fluorescent probe for Zn²⁺ detection. In the same manner, Tyrosine plays a crucial role in neurochemicals (epinephrine, norepinephrine, and dopamine) production [39]. Disorders, such as viral infection, atherosclerosis, Parkinson’s disease, autoimmune disorders, tumors, and cancers, can also be attributed to the high concentration of Tyrosine [40]. Hence, reports on electrochemical, fluorescent, colorimetric, and light scattering tactics for Tyrosine detection are readily available [41,42,43,44] but further designs are still in demand. According to an earlier report [15], fluorescent detection of Zn²⁺ by AT2 Schiff base probe may lead to AT2-Zn²⁺-AT2* complex formation. If the complex can be reversible in the presence of Tyrosine, the probe can be regarded as an excellent one in terms of the multiple analyte sensing utility. To control the protein-Tyrosine phosphatase-β-activity, involvement of Zn²⁺ is crucial [45,46,47]. Thus, the Schiff base-Zn²⁺ complex may disintegrate in the presence of Tyrosine due to highly feasible Tyrosine-Zn²⁺ complex formation to restore the probe’s original signal, which may become useful in quantifying Tyrosine as well.

Herein, one-pot synthesized anthracene-based Schiff base AT2 probe is firstly reported as Zn²⁺ sensors in ethanol via AT2-Zn²⁺-AT2* complex (Zn²⁺-induced excimer) formation with sensory reversibility in the presence of Tyrosine via “Off-On-Off” responses. The probe AT2 reveals a J-type head-to-tail aggregation via AT2-AT2* (excimer induced by H₂O) in AIEE with increasing water ratios (at f_w: 0–97.5%). Linear regression and nanomolar detection limits (LODs) of Zn²⁺ and Tyrosine sensors by AT2 are estimated from standard deviation and linear fittings of related PL studies. Decay profiles, binding mode, and stoichiometry are supplemented by time-resolved photoluminescence (TRPL), HR-Mass, and ¹H-NMR titrations. Nano-structural changes in the presence of Zn²⁺ and Tyrosine (AT2 nanoparticles aggregation and disaggregation) and AIEE (nanofibers formation at f_w: 0–97.5%) are exploited by SEM, TEM, AFM, and DLS inquiries. Existence of “On-Off” PET/TICT, RIR mechanism in AIEE of AT2, PET “On-Off-On” mechanism in the presence of Zn²⁺ and Tyrosine, HOMOs, LUMOs, and bandgaps of the sensory complexes are justified from DFT interrogations. Finally, AIEE and sensory responses of AT2 are authenticated via in-vitro/in-vivo (B16-F10 cellular and zebrafish) imaging studies. Figure 1 displays the table of content (TOC) schematic of AT2-based AIEE (at 0–97.5% f_w), Zn²⁺ and Tyrosine sensors, PET involvement, nanostructural changes, and their bioimaging applications described in this article.

2. Materials and Methods

General information on instruments, stock solutions, AIEE/sensory procedure, sample preparations, ¹H and ¹³C-NMR (nuclear magnetic resonance spectra) of AT2, high resolution (HR)-Mass of AT2, UV-Vis/PL-based sensory data, TRPL data, ¹H-NMR-titration, pH effect on sensors, DFT, SEM, TEM, AFM, MTT assay, and AIEE/time-dependent cellular images can be found in supporting information.

2.1. Synthesis of AT2 [3,15]

0.5 g (1 equivalent) of anthracene-9-carboxaldehyde and 466 mg (1 equivalent) of 4-Amino-5-phenyl-4H-1,2,4-triazole-3-thiol were mixed thoroughly in a well-cleaned round bottom flask with 100 mL of ethanol and refluxed for 24 h. The reaction was monitored through thin-layer chromatography (TLC) for final product (4-(anthracen-9-ylmethylene) amino)-5-phenyl-4H-1,2,4-triazole-3-thiol; noted as AT2) formation. The obtained crude AT2 was filtrated and kept in a hot vacuum oven to afford dried product. Recrystallization (twice) of the crude AT2 in absolute ethanol produced pure compound in the form of a greenish-yellow semi-fluffy solid. Yield: 0.857 g (93%). ¹H NMR (DMSO-d₆) δ/ppm: 14.40 (s, 1H (-SH)), 10.81(s, 1H (CH=N)), 8.89 (s, 1H-aromatic; Anthracene), 8.61 (dd, 2H-aromatic, J = 6.6 Hz), 8.19–8.22 (dd, 2H-aromatic; J = 6.4 Hz), 7.9–7.94 (dd, 2H- aromatic, J = 6.2 Hz), 7.53–7.64 (m, 7H-aromatic; Anthracene and phenyl); ¹³C NMR (DMSO-d₆) δ/ppm:167.62, 162.62, 149.52, 132.74, 131.28, 131.22, 130.77, 129.59, 129.31, 129.17, 128.45, 126.28, 126.11, 125.48, 123.39; m/z: calculated for C₂₅H₁₆N₄S: 380.11; found: 381.11 (M + H)⁺.

2.2. Computational Methods

Electronic structures of AT2 probe and AT2-Zn²⁺-AT2* complex were optimized (at ground states) via density functional theory (DFT) implemented in Gaussian16 program [48]. A well-established GGA hybrid functional B3LYP [49,50] in-line with effective core potential (ECP) including 6-31 + G(d,p) for carbon (C), hydrogen(H), nitrogen (N), oxygen (O), and sulphur (S) atoms and the LANL2DZ (Los Alamos National Laboratory 2 Double-Zeta) basis set for metal atom (Zn) were applied for all electronic structural calculations. By means of the solvent reaction field (SCRF) method with polarizable continuum model (PCM), optimization was performed in both gas and solvent phase (acetonitrile, DMSO, and ethanol). The presence of the PET mechanism in sensors and electronic cloud localization were recognized from molecular orbital analysis (HOMO-LUMO) and molecular electrostatic potential map (ESP), respectively. The optimized data for Tyrosine-Zn²⁺ complex were adopted from our earlier publication [15].

2.3. MTT Assay

The cytotoxicity and bio-compatibility of AT2 were authenticated by the methyl thiazolyl tetrazolium (MTT) test [51,52]. In a 96-well cell culture plates, B16F10 cell lines were grown and various concentrations (0, 20, 40, 60, 80, 100 μM) of AT2 were added to the wells. Those cells were incubated at 37 °C under 5% CO₂ for 24 h. Thereafter, each well was further incubated with 10 mL MTT (5 mg mL⁻¹) at 37 °C under 5% CO₂ for 4 h. After removal of the MTT solution, yellow precipitate (formazan) formation was revealed. The yellow precipitate was dissolved in 200 μL DMSO and 25 µL Sorensen’s glycine buffer (0.1 M glycine and 0.1 M NaCl). Each well’s absorbance at 570 nm was recorded by a Multiskan GO microplate reader. Finally, the viability of the cells was evaluated by the following equation:

Cell viability (%) = (Mean of absorbance value of treatment group)/(Mean of absorbance value of control group).

In a similar fashion, the half-maximal cell-inhibitory concentration (IC₅₀) value of AT2 was determined by plotting cell viability as a function of AT2 concentration (n = 3).

2.4. Procedure for Live Cell and Zebra Fish Imaging of AT2 [15]

2.4.1. Cellular Imaging of AIEE

The plated B16F10 cells (on 14 mm glass coverslips) were incubated with 10 % FBS at 37 °C and 5 % CO₂ for 24 h. The grown cells in DMEM were treated with DMSO-sterilized PBS (pH 7.4) dissolved by 20 μM of AT2 followed by incubation for 6 h at 37 °C. Next, the culture medium was removed and the cells were washed with PBS buffer (2 mL) before observation. Propidium iodide (PI; 5 μM; λ_ex/λ_em 493/636 nm) was added and incubated in pre-washed cells and kept at 37 °C for 15 min to achieve nuclear staining. Fluorescence images were recorded using a confocal microscope (Leica, Wetzlar, Germany, TCS-SP5-X AOBS). Emission of laser-excited cells was recorded between 430 and 480 nm at λ_ex = 425 nm.

2.4.2. Cellular Imaging of Zn²⁺ and Tyrosine

The well-grown B16F10 cells in DMEM media with 10% (v/v) FBS (fetal bovine serum) and penicillin/streptomycin (100 μg/mL) were incubated in 5% CO₂ incubator at 37 °C. The cells were then washed twice with cell culture medium followed by incubation with 20 μM of AT2 at 37 °C for 20 min. The exogenous pathway identification was carried out in a suspended culture medium via incubation of Zn²⁺ (50 μM) and Tyrosine (100 μM), respectively. Before examination, treated cells were washed with 0.1 M PBS (2 mL × 3). Propidium iodide (PI; 5 μM; λ_ex/λ_em 493/636 nm) was added and incubated in pre-washed cells and kept at 37 °C for 15 min to achieve nuclear staining. Confocal fluorescence images of cells were recorded using a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope (Wetzlar, Germany) with a 63 oil-immersion objective lens. Blue and green emissive cells were witnessed at 460 nm and 520 nm, correspondingly.

2.4.3. Zebrafish Imaging Studies

According to the guidelines of Animal Care and Use Committee of the National Yang Ming Chiao Tung University, the Zebrafish was treated first and kept under optimum breeding conditions at 28 °C. For mating, male and female zebrafish were housed in one tank at 28 °C for 14 h light/10 h dark period and laying of eggs was activated by light stimulation in the morning. Nearly all the eggs were fertilized. In 6-well plates, the zebrafish were grown in 5 mL of embryo medium added with 1-phenyl-2-thiourea (PTU) for 24 h at 30 °C. Anaesthetized (50 mg/L tricaine) 3-day old embryos of zebrafish were incubated with Zn²⁺ (50 μM) for 30 min at 28 °C in E3 media. Thereafter, the zebrafish were incubated with AT2 (20 μM) for 20 min at 28 °C and Tyrosine (100 μM) for 30 min at 28 °C, respectively. The excess Tyrosine and Zn²⁺ were removed by PBS. After washing with PBS to remove the remaining probes, the zebrafish was observed by a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope.

3. Results and Discussion

3.1. Synthesis and Characterization of AT2

As seen in Scheme 1, by refluxing Anthracene-9-carboxaldehyde and 4-Amino-5-phenyl-4H-1,2,4-triazole-3-thiol in ethanol for 24 h, the probe “[(4-(anthracen-9-ylmethylene) amino)-5-phenyl-4H-1,2,4-triazole-3-thiol); noted as AT2]” was obtained at a yield of 93%. The purity of the AT2 probe was attested using ¹H, ¹³C-NMR, and HR-Mass data, as displayed in Figures S1–S3 (Supporting Information; SI).

3.2. AIEE of AT2

Considering the solvent biocompatibility and effectiveness of Zn²⁺ sensory response as described in latter sections (Section 3.3 and Section 3.4), AT2 probe was firstly dissolved in ethanol solvent at 50 µM concentration followed by subjection to AIEE investigations via increasing the water fractions (f_w) from 0–97.5%. A weak fluorescence emission peak of AT2 (50 µM in ethanol; 0% f_w) was located at 456 nm (at λ_ex = 418 nm) with a quantum yield (Ф_f) value of 0.008. As shown in Figure 2A, enhanced emission was observed due to the AIEE effect with increased f_w from 0–60%. Moreover, the emission peak was also red-shifted from 456 nm to 470 nm. Due to solvent and precipitation effect, the AIEE of AT2 between 70–97.5% f_w was considerably affected by decreases in emission intensity. In related to the emission enhancement in AIEE, absorbance studies also showed red-shifted in absorbance from 396 nm to 407 nm, as seen in Figure S4A (SI). The red-shift in PL and absorbance peaks could be attributed to feasible J-type aggregation as illustrated in TOC (Figure 1) [3,15]. Similar to PL and absorption data, the Ф_f at 60% f_w reached a value of 0.142 (17-fold enhancement) as depicted in Figure 2B. Time-dependent Ф_f measurements on AIEE of AT2 confirmed that the aggregated state’s emission could be stable up to 6 h as shown in Figure S4B (SI). Figure 2C shows the emission enhancement of AT2 as a function of water fraction (0–97.5% f_w).

3.3. Optimum Conditions for AT2-Based Sensors

Before moving on to detailed sensory investigations, optimum conditions for investigations were determined as following: (1). To avoid the AIEE effect on sensors, metal ions and pH buffers were obtained from 10 mM stock solution and f_w was maintained below 10%; (2). Maximum concentration of metal ions and aminoacids was fixed as 25 µM as per earlier report [15]; (3). From the magnitude of PL enhancement and Ф_f interrogations on AT2 sensor response to Zn²⁺ in diverse solvents (see Figure S5A,B; SI; detailed in Section 3.4), the suitable solvent for all sensory studies was determined as ethanol.

3.4. Primary Sensory Results of AT2

Upon adding different metal ions (Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺, Pb²⁺, Al³⁺, Cs⁺, Na⁺, K⁺, Cd²⁺, Ca²⁺_, Zn²⁺, Hg²⁺, Cu²⁺, Ba²⁺, Sn²⁺, Fe²⁺, Cr³⁺, Cr⁶⁺, Y³⁺, Ga³⁺, Pd²⁺, and Fe³⁺) to AT2 (50 µM in ethanol), a red-shifted “turn-on’ PL emission at 520 nm (λ_ex = 418 nm) was visualized in the presence of Zn²⁺ as seen in Figure 3A. In fact, AT2 displayed a more moderate (10.2-fold) “turn-on” response enhancement than that of pyrene derivative (PT2; 196-folds) [15], which was attributed to the replacement of pyrene with anthracene moiety. However, in terms of fluorophore novelty and importance of Zn²⁺ detection in bio samples, this probe could still be an excellent alternative. During the initial investigations, metal ions, such as Cd²⁺, Hg²⁺, and Cu²⁺, showed negligible response (2–3 folds only; see Figure 3A), thereby attesting selectivity of Zn²⁺ by AT2. Towards reversibility investigations, 25 µM of amino acids [Ala: Alanine; Cys: Cysteine; H-Cys: Homo-Cysteine; Met: Methionine; Glu-acid: Glutamic acid; His: Histidine; Tyr: Tyrosine; Glu: Glucose; Thr: Threonine; AA: Ascorbic acid; Lys: Lysine; Phe-Ala: Phenyl Alanine; Orn: Ornithine; Gly: Glycine; Arg: Arginine; GSH: Glutathione; Ser: Serine; DA: Dopamine; Pro: Proline; Try: Tryptophan; Leu: Leucine; GA: Glutamine] from 10 mM stock solution were added to the above AT2 + Zn²⁺ sensory system in separate vials. Among the amino acids tested, only Tyrosine displayed reversibility as shown in Figure 3B, which was similar to the pyrene-based report [15].

During Zn²⁺ sensing, the initial Ф_f value of AT2 is increased from 0.008 to 0.103 (>12-fold), and the Ф_f value of AT2 + Zn²⁺ system was calculated as 0.011 in the presence of Tyrosine, thereby further supporting the reversibility. As shown in Figure S5A,B (SI), the magnitude of enhancement and Ф_f values of AT2 + Zn²⁺ in solvents, such as acetonitrile (ACN), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and DI-water are estimated as 8.1-, 9.2-, 4.7-, 1.2-, and 0.7-fold and 0.095, 0.098, 0.054, 0.011, and 0.01, respectively, which assure the use of ethanol as a solvent for sensory and AIEE investigations. Figure 4A,B show the selectivity photographs of AT2 and AT2 + Zn²⁺ sensory system under UV-lamp (λ_ex = 365 nm) in the presence of metal ions and amino acids.

It was noted that the original weak blue-emissive AT2 displayed a “turn-on” green emission in the presence of Zn²⁺ (see Figure 4A), which was then restored by adding Tyrosine (see Figure 4B). The above results were encouraging for detailed chemosensory investigations.

3.5. Interference Studies and Detection Limits

To study the selectivity of AT2 to Zn²⁺ and its reversibility with Tyrosine, single and dual interference experiments were conducted. For the single analyte studies, AT2 (50 µM in ethanol) + 25 µM of all metal ions were used and, for the dual interference interrogations, AT2 (50 µM in ethanol) + 25 µM of Zn²⁺ + 75 µM of other metal ions were consumed in PL measurements. As seen in Figure S6A, none of the metal ions showed emission enhancement at 520 nm except Zn²⁺, which indicated that the probe was highly selective in single analyte studies. In the results of dual interference studies, Cu²⁺, Hg²⁺, Cd²⁺, and Cr⁶⁺ showed mild but still evident (>5-fold enhancement) effects. Thus, the selective sensing of Zn²⁺ by AT2 was well demonstrated. For testing the single analyte reversibility, AT2 (50 µM in ethanol) + Zn²⁺ (25 µM from 10 mM stock in DI-water) system was mixed with 25 µM of aminoacids (from 10 mM stock in DI-water) and, for the dual studies on reversibility, interfering aminoacids were mixed with a ratio of 1:3 before PL investigations. Wherein, none of the aminoacids disrupted the reversibility of AT2 + Zn²⁺ in both single and dual interference studies as exploited in Figure S6B (SI).

To identify detection limits (LODs) of Zn²⁺ and Tyrosine, individual PL titrations were performed as follows. Upon the addition of 0–35 µM Zn²⁺ (from 10 mM stock in DI-water) to AT2 (50 µM in ethanol), a red-shifted peak was observed at 520 nm as displayed in Figure 5A. The intensity of “turn-on” emission peak at 520 nm began to saturate after 25 µM of Zn²⁺ (see inset of Figure 5A), which suggested feasible 2:1 stoichiometry of AT2 to Zn²⁺. After adding 0–35 µM of Tyrosine in AT2 + Zn²⁺ system, quenching of fluorescence and restoration of AT2′s origin PL emission are observed, as shown in Figure 5B. At 30 µM concentration (see inset of Figure 5B), PL emission of AT2 has completely recovered, thereby suggesting feasible 1:1 stoichiometry of Tyrosine-Zn²⁺ coordination. From the above titrations (Figure 5A,B), linear regression and standard deviation plots of [Zn²⁺] as a function of I/I₀ and [Tyr] as a function of I₀/I at 520 nm are displayed in Figure 5C,D. From both plots, the LODs (3σ/slope) of Zn²⁺ and Tyrosine are estimated as 179 nM (Y = 0.34433x + 0.99997; R² = 0.99771; n = 3) and 667 nM (Y = 0.05553x + 0.98001; R² = 0.98085; n = 3), respectively. These nanomolar LODs attest the effectiveness of the AT2 probe in selective discrimination of Zn²⁺ and Tyrosine. Subsequently, UV-Vis investigations on AT2 to Zn²⁺ and its reversibility with Tyrosine revealed red-/blue-shifted quenched/restored peaks around 401 nm and 396 nm, correspondingly, as seen in Figure S7A (SI). Separate titrations of AT2 and AT2 + Zn²⁺ with Zn²⁺ and Tyrosine (0–35 µM for both) (see Figure S7B,C; SI), demonstrated similar results to the PL-based sensory responses.

3.6. TRPL, pH Effect and Stoichiometry

To further support PL-mediated sensing of Zn²⁺ and Tyrosine, TRPL investigations were conducted as follows. In contrast to the pyrene-based report [15,53], decay profiles and average decay constants of AT2, AIEE, and its sensory responses consisted of four decay constants, namely τ₁ (fastest), τ₂ (second fastest), τ₃ (slowest), and τ₄ (second slowest) with weighted factors A_1, A₂, A₃, and A₄, respectively. For AT2 (in ethanol at 0% f_w), the major contribution was from τ₁ (97.9%) and the average decay constant (τ_av) is 0.1001 ns. On the other hand, τ_av with a value of 0.2021 ns was attained for AT2@60% f_w, which was contributed from all four components (τ₁ (93.3%), τ₂ (5.8%), τ₃ (0.6%), and τ₄ (0.3%)). It indicated the distinct AIEE nature rather than that of the pyrene-based report. TRPL-fitting plots of AT2@0% f_w and AT2@60% f_w are depicted in Figure S8A (SI). In the presence of Zn²⁺, a τ_av value of AT2 (in ethanol) was estimated as 0.2329 ns, which was contributed largely from τ₁ (80%), τ₂ (17.9%), and τ₃ (2%) due to AT2 + Zn²⁺ complex formation. The above complex formation was restored in the presence of Tyrosine and displayed a τ_av value of 0.1003 ns, which had similar contributions as in the case of AT2@0% f_w. TRPL fitting plots of AT2 + Zn²⁺ and AT2 + Zn²⁺ + Tyrosine were shown in Figure S8B (SI) with all the fitting parameters of AIEE and sensors delivered in Table S1 (SI). To validate the effectiveness of Zn²⁺ and Tyrosine response, pH effects were attested by using freshly prepared “1–14” pH buffers [54,55]. As shown in Figure S9 (SI), both sensors displayed high selectivity between pHs 5–10, which suggested the feasible sensing utility of AT2 in mild acidic, neutral, and mild basic conditions.

Stoichiometry of sensory complexes must be evaluated to justify the exact binding mode as well as the mechanism. For AT2 + Zn²⁺ and AT2 + Zn²⁺ + Tyrosine sensory systems, mole fractions (X= {[AT2]/[AT2] + [Zn²⁺]} and X= {[Zn²⁺ in AT2 + Zn²⁺ complex]/[Tyrosine] + [Zn²⁺ in AT2 + Zn²⁺ complex]}) were plotted as a function of X*(I − I₀) and X*(I₀ − I), correspondingly. As shown in Job plots (Figure S10A,B; SI), AT2 to Zn²⁺ (in AT2 + Zn²⁺) and Tyrosine to Zn²⁺ (in AT2 + Zn²⁺ + Tyrosine) passed through Ca. 0.67 and Ca. 0.51, which suggest 2:1 and 1:1 stoichiometry, respectively. The 2:1 stoichiometry of AT2 to Zn²⁺ could be attributed to Zn²⁺-induced excimer (AT2-Zn²⁺-AT2*) formation and the 1:1 stoichiometry corresponded to Tyrosine-Zn²⁺ complex (here Zn²⁺ were derived from mentioned excimer to restore AT2 as well). To support the estimated stoichiometry, HR-Mass interrogations were performed. As seen in Figure S11 (SI), mass peak at m/z = 838.8378 corresponded to [AT2-Zn²⁺-AT2*]⁺ + H₂O − 2, thereby confirming the 2:1 stoichiometry of AT2 to Zn²⁺. Upon adding Tyrosine to AT2 + Zn²⁺, two mass peaks at m/z = 381.1161 and m/z = 226.9513 were assigned to AT2 (M + H)⁺ and [Tyr + Zn²⁺]-H₂O (M + 2H)⁺ configurations (see Figure S12; SI), thereby attesting the 1:1 stoichiometry of Tyrosine to Zn²⁺ and reversibility of AT2 probe.

3.7. Association Constants and Nano-Morphological Interrogations

After confirming the 2:1 and 1:1 stoichiometry of AT2 to Zn²⁺ and Tyrosine-Zn²⁺ complexes, association constants (K_as) were derived as follows [3,15]. By using the following equation (Equation (1)), K_a of AT2 + Zn²⁺ complex can be estimated.

α²/(1 − α) = 1/2K_aC_F [Zn²⁺]

(1)

where C_F = total concentration of AT2 exist in the complex, α = free AT2: total concentration of AT2 (ratio) which is determined from the following Equation (Equation (2)).

α = I − I₀/I₁ − I₀

(2)

At any given Zn²⁺ concentration, PL intensity at 520 nm is assigned as I. In the absence and presence of Zn²⁺, the PL maxima intensity at 520 nm is noted as I₁ and I₀, correspondingly. As shown in Figure S13A (SI), K_a of AT2 to Zn²⁺ is estimated as 2.28 × 10⁻⁶ M⁻² by linearly fitting the plot of α²/(1 − α) as a function of 1/[Zn²⁺] (slope of the linear plot). On the other hand, linear fitting of 1/I − I₀ as a function of 1/[Tyrosine] (see Figure S13B; SI) via Benesi-Hildebrand plot (suitable for 1:1 complexes) reveals a K_a value of Tyrosine to Zn²⁺ as 1.39 × 10⁻⁷ M⁻¹ (slope of the linear plot) [56]. The higher association constant value of Tyrosine to Zn²⁺ than that of AT2 to Zn²⁺ well supports reversibility studies.

In the following, nano-morphological changes were studied to justify AT2 as a nanoprobe. As visualized in SEM images (Figure 6A,B), AT2@0% f_w and AT2@60% f_w form dispersed nanoparticles and nanofibers, which suggests that aggregation/assembly of AT2 nanoparticles leads to nanofibers formation in the presence of H₂O.

SEM images in Figure S14A–L (SI) show the aggregation of AT2 nanoparticles at 0–100% f_w. Up to 80% AIEE-mediated growth of nanofibers can be seen in Figure S14A–I of SI. Thereafter, precipitation of AT2 in a less fibric environment began to take place (see Figure S14J–L; SI). However, for AT2 + Zn²⁺ complexation, Zn²⁺ induced nanoparticles aggregation, but was restored to its original configuration in the presence of Tyrosine as displayed in Figure 6C,D. To support the above-observed nano-morphological changes, TEM experiments were conducted. Results from TEM investigations also demonstrated similar nanostructural features for AT2@0% f_w (dispersed nanoparticles), AT2@60% f_w (nanofibers), AT2 +Zn²⁺ (Zn²⁺ induced nanoparticles’ aggregation), and AT2 +Zn²⁺ + Tyrosine (nanoparticles dispersion due to AT2 reversibility provided by Tyrosine) as explored in Figure 7A–D.

Moreover, nanofiber growth via AT2 nanoparticles aggregation at 0–97.5% f_w (see Figure S15A–H; SI) also showed a similar trend as in SEM studies. Therefore, AFM studies on AT2 at 0, 30, 60, and 90% f_w also displayed growth of nanofibers from dispersed AT2 nanoparticles at 0% f_w and solidification at 90% f_w as shown in Figure S16A–D (SI). Similarly, for AT2 + Zn²⁺ and AT2 + Zn²⁺ + Tyrosine, AFM scanning results reveal Zn²⁺-induced particles’ aggregation and Tyrosine-induced nanoparticles’ dispersion (reversibility) as seen in Figure S16E,F (SI).

To further clarify the nanostructural changes, DLS studies were carried out. For AT2@0% f_w, AT2@60% f_w, AT2 +Zn²⁺, and AT2 +Zn²⁺ + Tyrosine, their particle sizes were estimated as 66.8 ± 42.7 nm (nanoparticles), 17,662.8 ± 12,080.6 nm (nanofibers in AIEE), 253.7 ± 63.2 nm (Zn²⁺ induced AT2 aggregation), and 90.1 ± 70.2 nm (Tyrosine-induced reversibility of AT2), respectively, as depicted in Figures S17–S20 (SI). Based on SEM, TEM, AFM, and DLS interrogations, the nano-level changes during AIEE and sensory studies of AT2 were well-justified, thereby confirming AT2 as a nanoprobe. However, compared to the earlier pyrene-based report [15], there was no gelation formation (up to 30 min) during AIEE even though nanofibers were formed. This might be due to the presence of diverse fluorophores, which justified the use of anthracene towards distinct nanoprobe development parallel to existing nanomaterials [57,58].

3.8. ¹H-NMR Titrations and DFT Optimizations on Sensors

To verify the exact binding mode of AT2 to Zn²⁺, ¹H-NMR titrations were conducted. The AT2 (20 mM; 1 equiv., in d₆-DMSO) was titrated with increasing Zn²⁺ concentrations (0–12 mM; 0–0.6 equiv. in deuterated ethanol), which revealed changes in the original ¹H-NMR peaks, described as follows. With increasing Zn²⁺ concentrations, the free thiol (-SH) peak of AT2 at 14.38 ppm slowly vanished accompanied with upfield-shifting of the -CH peak (-CH=N) from 10.79 to 10.69 ppm, as explored in Figure S21A–D (SI). The upfield-shifted -CH=N proton also validated participation of the N atom in -CH=N rather than N atom in triazole unit. In relation to the above changes, a majority of the aromatic proton peaks between 8.89 to 7.53 ppm were also mildly upfield-shifted and broadened due to excimer formation and involvement of anthracene and phenyl rings in the complex structure. Moreover, the complete disappearance of the -SH proton at 14.38 ppm and the broadening of spectra at 0.6 equiv. confirmed the 2:1 stoichiometry between AT2 to Zn²⁺. These observations on the mode were supported by the DFT optimizations described below.

DFT optimizations (by B3LYP/6 – 31 + G(d,p) in gas phase) were carried out to resolve the exact binding mode and mechanism. Two possible structures for AT2-Zn²⁺-AT2* were proposed as depicted in Figure 8A and Figure S22A (SI).

Between them, the preferred complex model (Figure 8A) possessed a larger overlapping percentile of anthracene (>60%) than that of excimer and the unconventional complex model (Figure S22A; SI) (displays < 30% overlapping of anthracene). DFT-optimized structures of both preferred and unconventional models are shown in Figure 7B and Figure S22B (SI), correspondingly. As seen in Figure 8C,D, electrostatic potential (ESP) of the AT2-probe showed the electron cloud located over anthracene moiety, which was delocalized over the complex center in the preferred complex model of AT2-Zn²⁺-AT2*. The HOMO, LUMO, and band gap (E_g) of the AT2-probe were calculated as −6.02 eV, −2.91 eV, and 3.11 eV, respectively, as depicted in Figure 8E. Likewise, HOMO, LUMO, and E_g for the formed preferred excimer complex were established as −5.84 eV, −3.01 eV, and 2.83 eV, individually, as shown in Figure 8F. All the HOMOs (at 0, −1, and −2 levels) and LUMOs (at 0, +1, and +2 levels) of AT2-probe and the preferred complex model of AT2-Zn²⁺-AT2* were displayed in Figures S23 and S24 (SI). Compared to the preferred model, the unconventional complex model had HOMO, LUMO, and E_g of −5.69 eV, −2.64 eV, and 3.05 eV, correspondingly, as seen in Figure S25 and Table S2 (SI). Since E_g of above model did not show any significant change, therefore, it was named as the unconventional one. HOMO, HOMO-1, HOMO-2, LUMO, LUMO+1, and LUMO+2 of the unconventional complex model are shown in Figure S25 (SI). As shown in Figure S26 (SI), Zn²⁺---S and Zn²⁺---N bond lengths were found to be 2.36 Å and 2.20 Å for the preferred complex and 2.50 Å and 2.09 Å for the unconventional model, respectively. The above observations on bond-lengths are consistent with the earlier pyrene-based report [15], which also clarified the preferred complex model. For Tyrosine-based reversibility, HOMOs and LUMOs of Tyrosine-Zn²⁺ optimized model were adopted from the earlier report [15]. DFT-based optimizations of AT2 and AT2-Zn²⁺-AT2* (preferred and conventional models) are also conducted in ACN, DMSO, and ethanol phases, which revealed similar results and support gas phase investigations as summarized in Table S2 (SI). From the DFT studies, it was noted that, during the initial phase, AT2-probe triggered the photo-induced electron transfer (PET) between anthracene to phenyl ring attached in the triazole unit, however, this process was inhibited during the Zn²⁺ coordination. After adding Tyrosine, PET was reactivated due to the effective coordination of Tyrosine to Zn²⁺. Thereby, PET “On-Off-On” mechanism was proposed for the AT2-based sensors. In other words, electron transfer was initially allowed between HOMO and HOMO-1 of AT2 but was prohibited between HOMO-1 and LUMO due to larger E_g of AT2 (3.11 eV). On the contrary, electron transfer was allowed between HOMO-1 and LUMO and prohibited between HOMO and HOMO-1 levels in the AT2 + Zn²⁺ complex. Due to lower E_g of AT2-Zn²⁺-AT2* (2.83 eV), these transferred/excited electrons quickly decayed to HOMO level, thereby enhancing photoluminescence intensity. AT2-probe was restored by adding Tyrosine to extract Zn²⁺ from AT2-Zn²⁺-AT2* complex via the PET “On-Off-On” mechanism. In the AIEE studies, increasing f_w induced AT2-AT2* formation and prohibited the PET/TICT of the AT2-probe supported by RIR. Thus, the PET “On-Off” mechanism is responsible for the AIEE of AT2. Based on the results in earlier reports [3,15] and UV-Vis and PL measurements in this study, the formation of J-type aggregation in AIEE is well-attested.

Thereby, based on UV-Vis/PL and DFT interrogations, the PET “On-Off-On” and the PET “On-Off” mechanisms are proposed for the AT2-based sensors and AIEE, respectively, as displayed in Figure 9A,B. The optical properties, HOMOs, LUMOs, E_g, Ф_f values, and TRPL constants for the AT2-based sensors and AIEE are delivered in

Table 1. HOMOs, LUMOs, band gaps, and optical properties of probe AT2 and its AIEE and sensory complexes.

Composition	HOMO (eV)	LUMO (eV)	Band Gap (eV)	λabs (nm)	λem (nm)	Φf	τ (ns)
Probe AT2	−6.02	−2.91	3.11	396	456	0.008 a	0.1001
AT2@60% fW	ND	ND	ND	407	470	0.142 a	0.2021
AT2 + Zn2+	−5.84 b	−3.01 b	2.83 b	401	520	0.103 a	0.2329
	−5.69 c	−2.64 c	3.05c
AT2 + Zn2+ + Tyr	ND	ND	ND	396	520	0.011 a	0.1003
Tyr + Zn2+	−5.47 d	−2.70 d	2.77d	ND	ND	ND	ND

ND: Not detected: ^a Fluorescein in ethanol (Φ_f = 0.79) was used as a standard reference; ^b DFT-optimized data for preferred complex model; ^c DFT-optimized data for unconventional complex model; ^d DFT-optimized HOMO, LUMO, and bandgap data were obtained from Ref. [15].

.

3.9. MTT Assay and Bioimaging

Bioimaging capability of AT2 towards AIEE and sensory studies (Zn²⁺ and Tyrosine detection) were established from in-vitro/in-vivo (live cell/zebra fish) imaging interrogations. Methyl thiazolyl tetrazolium (MTT) assay was carried out to examine cytotoxicity and 50% cell survival (IC₅₀) of AT2. Upon incubation at diverse concentrations of AT2 (0, 20, 40, 60, 80 and 100 µM, respectively), exceptional viability (>75%) was demonstrated even at 80 µM concentration, as seen in Figure S27A (SI). Likewise, IC₅₀ value was determined as 149 µM (Figure S27B; SI) based on cytotoxic measurements, which confirmed the biocompatibility of AT2. After six hours’ incubation of AT2 in the B16-F10 cell line, blue emissive cell lines with a PI-dye overlay were observed as seen in Figure S28 (SI). In fact, the existing water content inside cell lines induced AIEE of AT2, therefore, blue emissive images were visualized. Due to a weaker AIEE effect than that of the pyrene-based probe (PT2) [15], cellular incubation after one hour did not display any emission, which was also supported by sensory-cellular imaging studies. However, these observations well-demonstrated the AIEE-based in-vitro imaging studies of AT2.

In the presence of Zn²⁺ with AT2, the incubated B16-F10 cell lines displayed a strong green channel image, however, the green color disappeared via incubating with Tyrosine as depicted in Figure 10. The complete disappearance of the green channel image in the presence of Tyrosine suggested the reversibility of the AT2 probe. Due to the weak AIEE effect of AT2, no blue emission could be observed. According to the earlier reports [59,60], it was noted that B16-F10 cell lines could release the Tyrosine via a time-dependent incubation. During the time-dependent incubation of AT2 + Zn²⁺, the green channel images were observed to vanish gradually by means of intercellular Tyrosine release (see Figure S29; SI). These observations also suggested feasible Tyrosine-Zn²⁺ complex formation via trapping Zn²⁺ from AT2 + Zn²⁺ complex and recovering back to the AT2 probe subsequently. From time-dependent cellular imaging studies, it can be concluded that the B16-F10 cell lines released Tyrosine slowly so that AT2 was able to display green emission in the beginning. However, cells with excess Tyrosine may hinder the AT2 to Zn²⁺ complex via strong affinity of Tyrosine to Zn²⁺. Thus, cellular imaging studies of AT2 to Zn²⁺ were limited by the Tyrosine concentrations in cell lines. In the following, the in-vivo imaging capability of AT2 to Zn²⁺ and Tyrosine were demonstrated via zebrafish imaging studies. 3-day old embryos of zebrafish were firstly incubated with Zn²⁺ (50 µM) for 30 min, which did not show any fluorescence in blue or green channels. A strong green fluorescence was observed, as shown in Figure 11, after adding AT2 followed by incubating for 20 min. After incubating these zebrafish samples with Tyrosine for 20 min, the green channel fluorescence began to fade and finally turned into a mild blue channel fluorescence due to the AIEE of AT2. In zebrafish samples with higher f_w, the blue emissive channel of AIEE could be observed within 1.5 h. From both cellular and zebrafish imaging studies, it was concluded that AT2 could be effectively used in AIEE, Zn²⁺, and the Tyrosine sensor tuned in-vitro/in-vivo imaging studies. Similar to cellular imaging studies, zebrafish imaging was also limited by Tyrosine concentrations. In-vivo samples/tissues with excess initial Tyrosine concentrations could affect Zn²⁺ imaging by AT2, therefore, careful monitoring on primary Tyrosine concentrations was mandatory. Finally, the use and performance of AT2-probe towards Zn²⁺ and Tyrosine detection were reliable and comparable to earlier reports [38,42,43,44,59,60,61,62,63,64,65,66,67,68,69,70,71,72] in terms of linear range, LODs and applications as displayed in Tables S3 and S4 (SI). Note that AT2-based sequential detection of Zn²⁺ and Tyrosine was considered as exceptional in terms of one-pot synthesize, linear range nanomolar LODs, and bioimaging applications.

4. Conclusions

An anthracene-based Schiff base probe (4-(anthracen-9-ylmethylene) amino)-5-phenyl-4H-1,2,4-triazole-3-thiol; AT2) was synthesized via one-pot reaction and its AIEE and PL “Off-On-Off” sensory utilities to Zn²⁺ and Tyrosine were systematically demonstrated. In the AIEE, the red-shifted UV-vis/PL peaks of AT2 (in ethanol) with increasing f_w (0–97.5%) confirmed the J-type aggregation via AT2-AT2* excimer formation. Furthermore, AT2 (in ethanol) revealed highly selective sensitivity to Zn²⁺ (0.5 equiv.) via “Turn-On” green emission, which was restored with Tyrosine (0.5 equiv.) via displaying the “Turn-Off” emission of the probe. From the Job plot, HR-mass, ¹H-NMR, and DFT studies, the binding modes, 2:1 and 1:1 stoichiometry of AT2-Zn²⁺-AT2* excimer and Tyrosine + Zn²⁺ complexes were clearly justified. Uniqueness of sensory responses was further supported by TRPL and pH-effect investigations. By means of standard deviation and linear fittings, association constants of AT2 to Zn²⁺ and Tyrosine to Zn²⁺ were determined as 2.28 × 10⁻⁶ M⁻² and 1.39 × 10⁻⁷ M⁻¹, respectively. Individual titrations of AT2 with Zn²⁺ and Tyrosine exhibited a linear regression between 0–35 µM and LODs of 179 nM and 667 nM (by 3σ/slope), correspondingly. From SEM, TEM, AFM, and DLS interrogations, the nano-morphological changes authenticated AT2 as a nanoprobe. The involvement of PET “On-Off-On” in Zn²⁺ and Tyrosine detection were verified by DFT studies. Similarly, feasible “On-Off” PET/TICT, and an RIR mechanism in AIEE were proposed based on UV/PL results. Moreover, adopting AT2-based AIEE and its sensors (towards Zn²⁺ and Tyrosine) in cellular and zebrafish imaging studies authenticated the potential in-vitro and in-vivo applications in many biological samples. However, the initial excess Tyrosine concentrations in cells or tissues may have affected Zn²⁺ selectivity by AT2 via direct Tyrosine-Zn²⁺ coordination, therefore, careful optimization was mandatory. Finally, optimizations of AT2-based OTFT devices for detection of Zn²⁺ and Tyrosine in solid state are currently under research.