A Novel Benzothiazole-Based Fluorescent AIE Probe for the Detection of Hydrogen Peroxide in Living Cells

Abstract

A benzothiazole-based derivative aggregation-induced emission (AIE) fluorescent ‘turn-on’ probe named 2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)benzo[d]thiazole (probe BT-BO) was developed and synthesized successfully for detecting hydrogen peroxide (H₂O₂) in living cells. The synthesis method of probe BT-BO is facile. Probe BT-BO demonstrates a well-resolved emission peak at 604 nm and the ability to prevent the interference of reactive oxygen species (ROS), various metal ions and anion ions, and good sensitivity. Additionally, the probe boasts impressive pH range versatility, a fast response time to H₂O₂ and low cytotoxicity. Finally, probe BT-BO was applied successfully to image A549 and Hep G2 cells to monitor both exogenous and endogenous H₂O₂.

1. Introduction

Reactive Oxygen Species (ROS) are highly reactive oxygen-containing compounds that play an important role in living cells [1,2]. Hydrogen peroxide (H₂O₂), one of the typical representatives of ROS, maintains a balance between oxidative and antioxidant systems in normal cells [3,4]. Nonetheless, once this balance is broken, for example, if the concentration of H₂O₂ is raised or the antioxidant capacity decreases, damage is caused to living cells, resulting in the destruction of nucleic acids, proteins and enzymes, and diseases [3,5,6,7,8]. During an inflammatory process, cells become activated and several mediators, including tissue factors, are released. In inflammatory responses, ROS can aggravate local tissue damage and lead to chronic inflammation [9]. In cancer therapy, H₂O₂ is a double-edged sword. On the one hand, the H₂O₂ level in tumor cells is higher than that in normal cells, which causes them to be in a state of oxidative stress, and provides a theoretical basis for some pro-oxidation drugs to treat cancer [10]. On the other hand, the excessive production and accumulation of H₂O₂ in cells can result in various diseases such as cancer, arthrophlogosis, asthma and cardiovascular diseases [11,12,13].

There are numerous methods for detecting H₂O₂, such as electron paramagnetic resonance (EPR)/electron spin resonance (ESR) techniques [14], chemiluminescence [15], th electrochemical method [16] and chromatography [17]. However, these methods exhibit limitations in applications, high costs and so on [18]. Currently, fluorescence spectroscopy has drawn the increasing attention of researchers and has been widely used in the field of detecting H₂O₂ in living cells, due to its high selectivity, high sensitivity, high efficiency, simple operation, good biocompatibility, wide range of application and real-time imaging [19,20,21,22,23]. Nevertheless, conventional organic fluorescent probe molecules have aggregation-caused quenching (ACQ) effects, i.e., fluorescence is partially or completely quenched [3,12,24,25,26]. The ACQ phenomenon results in low fluorescence signals, poor sensitivity, and easy photobleaching in biological detection [27]. In 2001, for the first time, Prof. Tang’s group found the aggregation-induced emission (AIE) effect of 1-methyl-1,2,3,4,5-pentaphenylsilole, which was ascribed to the limited rotations of rotatable meso moieties in closed spaces [28,29]. The principle of AIE is based on the restriction of intramolecular motion (RIM) or the restriction of intramolecular rotation (RIR). Molecules with AIE properties exhibit weak or even unobservable fluorescence features in dilute solutions, but they can fluoresce brightly when aggregated in a solution state [30], which effectively avoids the ACQ problem of traditional fluorescent molecules. Since 2001, a series of well-designed fluorescent probes that can specifically detect H₂O₂ have been developed. Tang et al. [18] explored a benzothiazole-based fluorescence probe for detecting hydrogen peroxide, which exhibited good sensitivity, selectivity and low cytotoxicity in HeLa cells. Liu et al. [26] developed an archetypal AIE luminogen (tetraphenylethene-based) H₂O₂ fluorescent probe, which shows high sensitivity, high selectivity, low cytotoxicity, and good cell membrane permeability. Zhong et al. [25] developed a new type of “turn-on” AIE probe based on excited-state intramolecular proton transfer (ESIPT), with phenylboronic acid pinacol ester-appended quinolinium used as the H₂O₂ recognition site due to the twisted intramolecular charge transfer (TICT) effect. Currently, boric acid or boric acid ester are widely used as H₂O₂-responsive elements [18,31,32]. ESIPT represents a distinctive four-stage photochemical process, characterized by fluorophores that predominantly reside in the electronic ground state, often manifesting as enols [18]. Upon light excitation, the charge distribution within this type of molecule is altered, leading to the enhanced acidity of the hydrogen bond donor in its enol form and a corresponding increase in the basicity of the hydrogen bond acceptor. Ultimately, phototautomerization leads to the excited enol form quickly converting to an excited keto structure. Upon returning to the ground state, reverse proton transfer (RPT) restores the original enol form [33]. 2-(2’-hydroxyphenyl)benzothiazole (HBT) is a common ESIPT fluorophore and its derivative has been widely used as a ESIPT fluorescent probe for detecting H₂O₂ [33,34].

Based on the AIE-ESIPT principle, in this work, an aryl boric acid ester unit was introduced into a benzothiazole-based derivative fluorophore (HBT) to serve as the reactive group for H₂O₂. The new ‘turn on’ fluorescent probe, 2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)benzo[d]thiazole (denoted as probe BT-BO), was designed and synthesized successfully for the first time for the selective detection of H₂O₂. Probe BT-BO exhibited a well-resolved emission peak at 604 nm and the ability to prevent the interference of ROS, various metal ions and anion ions. Moreover, the probe possessed the excellent properties of a broad pH range, a fast response time to H₂O₂ and low cytotoxicity. Finally, we have successfully applied the probe BT-BO to image A549 and Hep G2 cells to monitor both exogenous and endogenous H₂O₂.

2. Results and Discussion

2.1. Synthesis and Structural Analysis of HBT and Probe BT-BO

The synthesis route of the fluorescent probe BT-BO used to monitor H₂O₂ is illustrated in Scheme 1. Probe BT-BO was synthesized via two simple routes: in step 1, salicylal (2-hydroxybenzaldehyde) was refluxed with 2-aminobenzenethiol in a anhydrous ethanol solution to obtain HBT (2-(2’-hydroxyphenyl)benzothiazole); in step 2, the arylboronate ester group was chosen as the recognition moiety to detect H₂O₂, which was refluxed with HBT to produce probe BT-BO (2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)benzo[d]thiazole) under a solution of anhydrous acetonitrile. The rational design of probe BT-BO is based on using the arylboronate ester group as a H₂O₂-responsive unit, which shows a “turn-on” fluorescent signal characteristic that recognizes and responds to H₂O₂ [29] (Scheme 2, Figure S1). Moreover, The ESIPT mechanism of HBT was verified by DFT calculations. Figure 1 shows a four-stage photochemical ESIPT process concerning the phototautomerization of the excited enol form to the excited keto form. The energy difference between the LUMO orbital and the HOMO orbital of the keto form (3.32 eV) is smaller than that of the enol form (4.58 eV), which confirmed the ESIPT process of HBT.

The structures of HBT and probe BT-BO were characterized by ¹H NMR, ¹³C NMR and MS (Figures S2–S7 in Supplementary Materials). The HR-MS spectra of HBT exhibit an intense ion peak at m/z = 228.0487 (M + H), which is ascribed to its corresponding ion [HBT + H]⁺, and match up well with the calculated m/z of 228.0483 of [M + H]⁺ (Figure S6). The same result was found for probe BT-BO, with an ion peak at an m/z value of 444.1801 for [BT-BO + H]⁺ and a calculated m/z value of 444.1805 (Figure S7). Moreover, the ¹H NMR and ¹³C NMR results also confirmed the structures of HBT and probe BT-BO; details can be found in Section 3.2 and Figures S2–S5 and Tables S2–S3 of the Supplementary Materials.

To further verify the structure of probe BT-BO, we cultured a single crystal of BT-BO. A suitable crystal of BT-BO was grown by slowly evaporating the solution of THF at room temperature. Details of the crystallographic and structure refinement for BT-BO are given in Figure S8 and Table S1. The crystal structure and ¹H NMR, ¹³C NMR, and HR-MS all confirm that probe BT-BO forms a benzothiazole structure.

2.2. AIE Property of HBT

To verify the AIE property of HBT, different water fractions (f_w) in tetrahydrofuran (THF) solution were used to investigate the fluorescence behavior of HBT. As Figure 2a shows, photographs of HBT in water/THF mixtures were taken under visible light. However, when f_w was increased, a sapphire solution emerged; this then darkened slightly under ultraviolet light. In order to further quantify the phenomena, the emission spectra of HBT in different f_w in the THF solution were measured (Figure 2b). The fluorescence intensity at 604 nm gradually increased and then slightly lowered with an increase in the f_w value (Figure 2c). The above results indicate that HBT was a fluorescent material with AIE properties.

2.3. Spectral Properties of Probe BT-BO to H₂O₂

In order to investigate the fluorescence characteristics of probe BT-BO, probe BT-BO (5 μM) was treated with different concentrations of H₂O₂ in phosphate-buffered saline (PBS) solution (containing 2% THF, vol). We investigated the change in the fluorescence spectra signal of various concentrations of H₂O₂ (0~100 μM). As shown in Figure 3a, probe BT-BO exhibited weak fluorescence due to blocking from the aryl boric acid ester moiety, indicating the low fluorescence emission background of probe BT-BO itself. After adding various concentrations of H₂O₂, the fluorescence emission intensity at 604 nm was gradually increased, suggesting that H₂O₂ was indeed reacted with probe BT-BO. The fluorescence intensity at 604 nm for a H₂O₂ concentration of 100 μM was 20 times that for 0 μM of H₂O₂, which means that probe BT-BO has a high signal-to-noise ratio. The experimental results showed that the probe was a turn-on fluorescent probe, laying the foundation for its detection of H₂O₂ [33]. In addition, a relationship between the fluorescence intensity of probe BT-BO and the H₂O₂ concentration was fitted. The linear regression equation was y = 59.707 + 27.61x and the linear correlation coefficient R² was 0.9923, where y was the fluorescence intensity of probe BT-BO at 604 nm; x refers to the H₂O₂ concentration. The limit of detection (LOD) for H₂O₂ was estimated based on the following rule [8,18,35,36,37,38,39,40,41,42,43,44,45,46]:

LOD = 3σ/k

where σ is the standard deviation of blank fluorescence intensity measurements, which were detected 11 times, and k refers to the slope of the fitting curve (Figure 3b). The LOD value of probe BT-BO was determined to be 0.93 μM (3 × 8.54/27.61 = 0.93 μM), suggesting that probe BT-BO could be a good “turn-on” response sensor for determining H₂O₂.

2.4. Selectivity, Effect of pH, Temperature and Response Time of Probe BT-BO

The selectivity and anti-interference capabilities of probe BT-BO were investigated using blank and various reactive oxygen species (ROS), namely glutathione (GSH), peroxynitrite (ONOO^–), tert-butyl hydroperoxide (TBHP), O₂⁻, HClO, ¹O₂, and H₂O₂; various metal ions, namely Ba²⁺, Fe²⁺, Fe³⁺, Ca²⁺, K⁺, Al³⁺, Mg²⁺, Na⁺, Cu²⁺; and various anion ions, namely N₃⁻, F⁻, HPO₄²⁻, SO₄²⁻, HSO₄⁻, Cl⁻, OH⁻, CN⁻, CO₃²⁻, HCO₃⁻, NO₃⁻, NO₂⁻, CH₃COO⁻ and blank. As illustrated in Figure 4a, the fluorescence intensity of probe BT-BO in the solution of H₂O₂ (100 μM) was significantly higher than that of potential interferences, even at a high concentration level (1000 μM). The response intensity of H₂O₂ was about 17~25 times that of other potential interferences. These results indicate that probe BT-BO has high selectivity for H₂O₂, which means that probe BT-BO could play an active role in endogenous H₂O₂ imaging in living cells.

For comparison, we have summarized some of the previously reported fluorescent probes used to compare the Limit of Detection (LOD), response time, selectivity, practical applications, and synthesis of BT-BO and previously reported fluorescent probes for detecting H₂O₂. Most of the LOD values obtained by previously reported fluorescent probes are around 0.13~5.3 μM, while our designed and synthesized probe possesses a LOD value of 0.93 μM; that is, BT-BO has comparability and high sensitivity for detecting H₂O₂ (Table S4). The comparison of the selectivity and anti-interference capabilities of the reported probes and BT-BO show that the lowest fluorescent response of H₂O₂ is around 2.26~13.75 better than interferences. However, this value for BT-BO is 17.02. The number of interference types investigated in the previously reported literature is 7~22, while the number in our work is 29. Moreover, we compare the synthesis steps and application cells between previous studies and this work. The number of synthesis steps and application cells used in the previously reported literature is 2~5 steps and 1~3 cells, respectively. Obviously, probe BT-BO also has comparability (two synthesis steps and two application cells). All in all, these preliminary experiments suggest that probe BT-BO could be a potential probe for detecting H₂O₂.

To investigate the effect of pH values on the experimental results, the fluorescence emission properties of probe BT-BO before and after adding H₂O₂ (100 μM) at different pH (5~9) values were studied (Figure 4b and Figure S9). The fluorescence intensity of probe BT-BO was weak and almost remained unchanged at a pH of 5~9, indicating that probe BT-BO was stabilized in the range of pH 5~9. While the fluorescence intensity of probe BT-BO + H₂O₂ demonstrated a volcanic curve, the maximal intensity was at a pH of 7. Consideration that the pH in the physiological environment was 7.4 and that probe BT-BO showed an optimal fluorescence intensity at a pH of 7, probe BT-BO could potentially detect endogenous H₂O₂ in living cells. What is more, the effect of the detection temperature of probe BT-BO was also evaluated. As Figure 4c shows, the fluorescence intensities of probe BT-BO when adding H₂O₂ were less influenced in the range of 26~34 °C, which shows another advantage of its utilization in living systems. The response time of probe BT-BO after adding H₂O₂ was investigated by measuring its fluorescence emission intensity (Figure 4d). After adding a 100 μM H₂O₂ solution, the fluorescence intensity increased up to 40 min, and then reached a plateau at 60 min. According to the first-order slope of the linear fitting curve (Figure 4d inner), the apparent reaction rate was 83.689 min⁻¹, suggesting that probe BT-BO possesses a fast response time to H₂O₂.

2.5. Cytotoxicity and Cellular Imaging

In order to investigate the probe’s performance in different cellular environments, the cytotoxicity and biocompatibility of probe BT-BO were evaluated by MTT assay using human pulmonary carcinoma A549 cells and human hepatocellular carcinoma Hep G2 cells. A series of different concentrations of BT-BO (0, 5, 10, 15, 20, 25, 30, 35 and 40 μM) were added to a cell culture medium. It can be clearly seen that there was no significant inhibition of cell growth with an increase in the BT-BO concentration for both A549 and Hep G2 cells (Figure 5a,b, respectively). The cell viability of A549 and Hep G2 cells measured by the MTT assay was over 94% and 91% at a BT-BO concentration of 30 μM, respectively, indicating that the designed probe possessed low-cytotoxicity cultured cells. Therefore, BT-BO, at a low concentration level (30 μM), was applied for the fluorescence imaging of A549 and Hep G2 cells. Furthermore, it was essential to explore the long-term stability of probe BT-BO in biological systems. Biological systems were simulated under a solution of 0.3% DMSO in PBS with probe BT-BO at 37 °C for 24 h. As shown in Figure S10, the purity of BT-BO (C₂₆H₂₇NO₃SB, m/z: calcd. 444.1805 [M + H]⁺, found 444.160) was 99.7%. When probe BT-BO was dissolved in the simulated biological system (0.3% DMSO in PBS) at 37 °C for 24 h, the purity of BT-BO was slightly reduced, but still maintained at 95.2%. The above results indicate that the synthesized probe, BT-BO, possesses high purity and long-term stability in biological systems.

Furthermore, to continue delving into the biological applications of probe BT-BO, two typical human cancer cells (A549 cells and Hep G2 cells) were used to study the applicability of BT-BO in living systems. As shown in Figure 6a,g, untreated A549 and Hep G2 cells both exhibit no fluorescence themselves. When the cells were cultured with BT-BO only, there was negligible fluorescence on their own (Figure 6c,i). However, after treating A549 and Hep G2 cells with PMA (a well-known kind of inducer for endogenous H₂O₂ generation) for 30 min and subsequently incubating them with probe BT-BO, a green fluorescence was dimly discernible and observed (Figure 6b,h, respectively); this suggested that endogenous H₂O₂ could be detected by probe BT-BO. To further investigate the detectability of probe BT-BO on A549 cells with exogenous H₂O₂, a range of H₂O₂ (5, 50, 100 μM) concentrations were introduced into A549 and Hep G2 cells (Figure 6d,e,f,j–l, respectively). Clearly, the fluorescence of both A549 and Hep G2 cells became stronger along with the increase in the H₂O₂ concentration. The above cellular imaging results indicate that probe BT-BO could detect both endogenous and exogenous H₂O₂ and could be a potential probe for the detection of H₂O₂ in living cells.

3. Materials and Methods

3.1. Materials and Measurements

2-hydroxybenzaldehyde, 2-aminobenzenethiol and 4-bromomethylphenylboronic acid pinacol ester were purchased from Accela ChemBio Co., Ltd. (Shanghai, China). 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and formic acid were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Anhydrous ethanol, anhydrous acetonitrile, potassium carbonate, hydrochloric acid and sodium hydroxide were purchased from TCI (Shanghai, China) Development Co., Ltd. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and penicillin were purchased from Invitrogen Thermo Fisher Scientific—CN (Shanghai, China).

The ¹H NMR and ¹³C NMR spectra were measured on a Bruker Avance III 400 MHz narrow-cavity liquid NMR spectrometer (Bruker, Bremen, Germany). Thin-layer chromatography (TLC) was carried out on a silica gel (GF254, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). The fluorescence spectra were obtained using a fluorescence spectrophotometer (F-2710, Hitachi, Hiroshima-shi, Japan) under 700 V, EX 2.5 nm and EM 2.5 nm.

3.2. Synthesis of the Fluorescence Probe BT-BO

3.2.1. Synthesis of 2-(2’-Hydroxyphenyl)Benzothiazole (HBT)

The 2-hydroxybenzaldehyde (244.2 mg, 2 mmol), anhydrous ethanol (10 mL) and 2-aminobenzenethiol (275.4 mg, 2.2 mmol) were added to a round-bottom flask (50 mL) in sequence. Then, two drops of formic acid, to be used as a catalyst, were added to the solution above. The temperature of the mixed solution was raised to 90 °C, and the solution was heated at 90 °C to reflux for 2.5 h. The progress of the reaction was monitored using thin-layer chromatography (TLC). After the reaction was finished, the obtained precipitate was filtrated and washed with cold anhydrous ethanol 5 times and dried under an infrared lamp to give HBT (2-(2’-hydroxyphenyl)benzothiazole), a faint yellow product (350 mg, yield of 76.5%). ¹ H NMR (400 MHz, DMSO-d₆) δ 11.62 (1H, s, OH), 8.19 (1H, d, J = 8.0 Hz, ArH), 8.16 (1H, d, J = 8.0 Hz, ArH), 8.08 (1H, d, J = 8.0 Hz, ArH), 7.56 (1H, t, J = 8.0 Hz, ArH), 7.43–7.48 (2H, m, ArH), 7.11 (1H, d, J = 8.0 Hz, ArH), 7.04 (1H, t, J = 8.0 Hz, ArH). ¹³C NMR (100 Hz, DMSO-d₆) δ 165.9, 156.8, 151.9, 134.6, 132.9, 129.0, 126.9, 125.6, 122.6, 122.4, 120.2, 118.7, 117.5. HR-MS: m/z (ESI⁺): C₁₃H₁₀NOS, calcd. 228.0483 [M + H]⁺, found 228.0487.

3.2.2. Synthesis of 2-(2-((4-(4,4,5,5-Tetramethyl-1,3,2-Dioxaborolan-2-Yl)Benzyl)Oxy)Phenyl)Benzo[d]Thiazole (Probe BT-BO)

The HBT (229 mg, 1.0 mmol) was added into a flask of anhydrous acetonitrile (5 mL). After homogeneously mixing the solution, 4-(Bromomethyl) benzene boronic acid pinacol ester (356.4 mg, 1.2 mmol) and K₂CO₃ (165.6 mg, 1.2 mmol) were added into the solution above. The mixture was heated at 90 °C to reflux for 4 h and then cooled to room temperature. A yellow solid (probe BT-BO, (2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)benzo[d]thiazole, 339.9 mg, yield of 76.4%) was obtained by filtrating and purifying the solution above. ¹ H NMR (400 MHz, CDCl₃, TMS) δ 8.58 (1H, d, J = 8.0 Hz, ArH), 8.11 (1H, d, J = 8.0 Hz, ArH), 7.92 (1H, d, J = 8.0 Hz, ArH), 7.89 (2H, d, J = 8.0 Hz, ArH), 7.58 (2H, d, J = 8.0 Hz, ArH), 7.50 (1H, t, J = 8.0 Hz, ArH), 7.43 (1H, t, J = 8.0 Hz, ArH), 7.38 (1H, t, J = 8.0 Hz, ArH), 7.16 (1H, t, J = 8.0 Hz, ArH), 7.11 (1H, t, J = 8.0 Hz, ArH), 5.38 (2H, s, CH₂), 1.35 (12H, s, CH₃).¹³C NMR (100 Hz, DMSO-d₆) δ 162.6, 156.3, 152.0, 140.0, 135.9, 135.1, 132.7, 129.5, 127.8, 126.7, 125.4, 122.9, 122.3, 122.0, 121.8, 114.3, 84.1, 70.6, 25.1. HR-MS: m/z (ESI⁺): C₂₆H₂₇NO₃SB, calcd. 444.1805 [M + H]⁺, found 444.1801.

3.3. Liquid Chromatography–Mass Spectrometry

Experiments to determine the probe’s long-term stability in simulated biological systems were performed using a Waters high-performance liquid chromatography–tandem mass spectrometer (Milford, MA, USA) and quadrupole detector (model: liquid phase 2695–2996, mass spectrometry ZQ2000, Waters, Milford, MA, USA). The mobile phase comprised formic acid:water (1:100, vol:vol) and formic acid:acetonitrile (1:100, vol:vol). The liquid chromatography conditions used were as follows: Waters Sunfire C18 column (4.6 × 100 mm, 3.5 μm); column temperature, 30 °C; detection wavelengths, 214 nm and 254 nm; flow rate, 0.8 mL/min; and injection volume, 10 μL. The mass spectral conditions were as follows: electric spray ionization (ESI) source, negative ion ionization mode; capillary voltage, 3.0 kV; cone hole voltage, 10 V; source temperature, 140 °C; ionization temperature, 350 °C; ionization gas flow rate, 800 L/h; and cone hole gas flow rate, 50 L/h [47]. The detection molecular weight range was 360~560 m/z, and substances were quantitatively analyzed based on MS ES+ signals. Biological systems were simulated under a solution of 0.3% DMSO in PBS with probe BT-BO (10 μM) at 37 °C for 24 h.

3.4. Cytotoxic Assay

The cytotoxicity of probe BT-BO towards A549 and Hep G2 cells was investigated by the standard 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) method. Typically, about 5 × 10⁴ A549 cells per well were seeded in 96-well plates for 24 h, and incubated with certain concentrations of probe BT-BO (0, 5, 10, 15, 20, 25, 30, 35 and 40 μM) at 37 °C for another 12 h. Then, 100 μL of the MTT solution was added into each well, followed by incubation under 5% CO₂ at 37 °C in the dark for another 4 h. After this, the culture medium was removed and 100 μL of DMSO was added to dissolve the formed formazan. Each well was analyzed by the micro plate reader (Biotek Epoch, Winooski, VT, USA) at an absorbance of 490 nm. The cell viability was measured as a percentage of the control cells (blank) by taking the mean value of three replicate wells.

3.5. Cell Image Experiments

A549 and Hep G2 cells were chosen to study the living cell imaging. For a typical round, A549 and Hep G2 cells were cultured in DMEM on 35 mm dishes and incubated with penicillin (1%, vol/vol) and FBS (10%, vol/vol) at 37 °C in a 5% CO₂ atmosphere for 24 h. All cell culture medium was removed and washed by PBS three times before the cell image experiments. For the endogenous H₂O₂ imaging experiment, A549 and Hep G2 cells were pretreated by using the activator PMA (phorbol-12-myristate-13-acetate, 1 µM) for 30 min and then treated with probe BT-BO (30 μM) for another 30 min. Subsequently, probe BT-BO was washed by PBS three times. For the detection of exogenous H₂O₂, A549 and Hep G2 cells were incubated with probe BT-BO (30 μM) for 30 min at 37 °C; the cells were then washed with PBS three times to remove excess probe BT-BO. Subsequently, H₂O₂ (0, 5, 50 and 100 μM) was introduced into cells for 30 min. Cell images were collected by an inverted fluorescence microscope (Nikon Ts2-FL, Tokyo, Japan) using blue bandpass filters with excitation wavelengths of 470 nm at 30 W, and a time frame of 10 ms~2 s. Images were captured at 40 × magnification (Table S4).

3.6. DFT Calculations

The structure of the compounds was optimized by using Chem3D Pro 14.0 to obtain the optimal structures. Then, theoretical calculations were performed using the B3LYP/6–31 + G method.

4. Conclusions

In summary, a novel “turn-on” fluorescent probe, namely 2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)benzo[d]thiazole (probe BT-BO), with high sensitivity to H₂O₂ in living cells was designed and synthesized successfully for the first time via a two-step simple chemical reaction with a high yield of 76.4% and purity of 99.7%. Probe BT-BO exhibited a well-resolved emission peak at 604 nm and the ability to prevent the interference of ROS, various metal ions, and anion ions. Moreover, the probe possessed the excellent properties of a broad pH range, a fast response time to H₂O₂ and low cytotoxicity. Finally, we successfully applied probe BT-BO to image A549 and Hep G2 cells to monitor both exogenous and endogenous H₂O₂. Overall, it is believed that the designed 2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)benzo[d]thiazole could be a potential probe for the diagnosis of H₂O₂-related diseases in living systems.