Rapid-Response and Highly Sensitive Boronate Derivative-Based Fluorescence Probe for Detecting H2O2 in Living Cells

Intracellular H2O2 monitoring is important and has driven researchers to pursue advancements for the rapid identification of H2O2, since H2O2 is short-lived in cell lines. An arylboronate derivative has been investigated as a chemospecific fluorescence recognition agent for H2O2. Triphenylimidazoleoxadiazolephenyl (TPIOP) boronate was contrived as a novel candidate for the rapid and sensitive recognition of H2O2. The probe was conjugated using the TPIOP functional group acting as an excellent fluorescent enhancer. The TPIOP group stimulated the polarization of C–B bond due to its extended π-conjugation, which included heteroatoms, and induced the production of rapid signal because of the highly polar C–B bond along with the corresponding boronate unit. While H2O2 reacts with TPIOP boronate, its nucleophilic addition to the boron generates a charged tetrahedral boronate complex, and then the C–B bond migrates toward one of the electrophilic peroxide oxygen atoms. The resulting boronate ester is then hydrolyzed by water into a phenol, which significantly enhances fluorescence through aggregation-induced emission. The TPIOP boronate probe responded to H2O2 rapidly, within 2 min, and exhibited high sensitivity with a limit of detection of 8 nM and a 1000-fold selectivity in the presence of other reactive oxygen species. Therefore, the developed TPIOP boronate chemodosimeter was successfully utilized to visualize and quantify intracellular H2O2 from human breast cancer (MCF-7) cells, as well as gaseous and aqueous H2O2 from environmental samples using Whatman paper strips coated with TPIOP boronate.


Introduction
Hydrogen peroxide (H 2 O 2 ) is a small yet important reactive oxygen species (ROS), which is present in biological systems, and exerts wide physiological and pathological effects [1]. Abnormal levels of H 2 O 2 in the human body can cause longterm damage to cells and organs and lead to significant neurodegeneration, oxidative stress, and cancer [2]. us, the far-ranging impingements of H 2 O 2 homeostasis encouraged scientists to construct sensitive, rapid response, and selective and accurate sensors for detecting H 2 O 2 . H 2 O 2 presents a unique inherent conflict of reactivity over the other ROS, because most of the other ROS are operated by one electron transfer pathway. Moreover, H 2 O 2 possesses amphiphilic reactivity; its labile O-O bond allows it to react as a two-electron electrophilic oxidant, whereas H 2 O 2 can also be a good nucleophile due to the α-effect of the adjacent nonbonding orbitals on its oxygen atoms [3,4]. Boronates present unique abilities for detecting amphiphilic substances and exhibit chemoselectivity for H 2 O 2 , while the aryl derivative bound to the boron atom converts to phenol when reacting with H 2 O 2 [3,4].
Although numerous aryl derivatives conjugated with boronate have been developed as H 2 O 2 chemosensors, their slow response times cause difficulties for tracking H 2 O 2 in situ . Since H 2 O 2 has a short half-life, the slow responses of H 2 O 2 chemosensors caused researchers to question the accuracy of the measured concentrations of H 2 O 2 from in situ cell lines and the environment. erefore, designing a novel molecule which would facilitate the fast detection of H 2 O 2 has been a challenge. Zhang et al. recently reported that the polarity of the C-B bond in boronate represented the key to achieving fast and sensitive recognition of H 2 O 2 [5][6][7]. ey determined that the extended conjugation of the π-electron system of tetraphenylethylene (TPE) combined with boronate compound could enhance the polarity of C-B bond, thus achieving rapid monitoring of H 2 O 2 through aggregation-induced emission (AIE) [5][6][7].
is finding opened a new window toward designing a fluorogenic probe for H 2 O 2 sensing through significantly increasing the C-B bond polarity. Incorporating heteroatoms into a fluorogenic probe using new structural design strategies for synthesizing intensive AIE luminogens caused large electron perturbations and increased the emission intensity of luminogens [31]. For example, imidazole rings present lone pairs of electron-rich nitrogen atoms, which can induce polarization by intramolecular charge transfer and have been widely exploited in the fields of biology and fluorescent sensors [32]. Many researches have recently been focusing on derivatives of phenylimidazole chemosensors based on their enhanced fluorescence properties. For example, Takagi et al. synthesized fused π-conjugated diphenylimidazole derivatives, which exhibited superior optical properties [33]. Based on these findings, we designed an imidazole containing boronate, which included a triphenyl group in the imidazole ring bridged with oxadiazolepheylboronate, and utilized it for H 2 O 2 detection. e additional oxadiazole ring was introduced to enhance the electron affinity associated with the electron transporting properties of the highly photoluminescent compound [34]. e newly synthesized triphenylimidazoleoxadiazolephenyl (TPIOP) boronate chemosensor was tested as a fast-response and highly sensitive chemosensor. e high degree of π-conjugation created an excellent electrostatic potential on the carbon atom bound to the boron atom. e boronate group acted as a recognition unit, and the extended triphenylimidazoleoxadiazole moiety significantly amplified the inherent fluorescence of the chemosensor compared to that of the earlier chemodosimeter containing a simple phenyl unit. As expected, the TPIOP boronate probe responded rapidly to H 2 O 2 , within 2 min of coming into contact with H 2 O 2 . When the probe was triggered using H 2 O 2 , the weakly fluorescent TPIOP boronate converted into triphenylimidazoleoxadiazolephenol (TPIOP-OH), which exhibited much stronger fluorescence, and the enhanced emission intensity was caused by the mechanistic pathway of AIE. e limit of detection (LOD) of H 2  − ), sodium sulfate (Na 2 SO 4 ), sodium nitrite (NaNO 2 ), and benzoyl peroxide (BPO) were obtained from AccuStandard (New Haven, CT, USA). e MCF-7 cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). All reagents were used as received without further purification.

Instrumentation.
All UV-Vis absorption spectra were measured in the 300-800 nm range, using polystyrene cells of 1 mm path lengths on an S-3100 spectrophotometer (Sinco, Seoul, Republic of Korea). Fluorescence spectra were recorded using a LS-45 luminescence spectrometer (PerkinElmer, Waltham, MA, US). Both 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance 600 MHz spectrometer (Billerica, MA, US). e mass spectra were obtained in positive mode using a Synapt G2 high-resolution mass spectrometer (HR-MS) (Waters, Milford, MA, US). e pH of the solution was adjusted using a HI 2210 pH meter (Hanna Instruments, Woonsocket, RI, USA). Fluorescence images were acquired using a LSM 700 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with a 63× oil immersion objective lens and a diode laser as light source. Cytotoxicity tests were performed using a Spectramax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Spectroscopy Measurements.
e TPIOP boronate solution was prepared using 10 mM HEPES buffer at pH 7.4 containing 2 vol% DMSO. e final concentrations of the TPIOP boronate solution used to carry out UV-Vis and fluorescence measurements were 20 and 2 μM in 10 mM HEPES buffer at pH 7.4 containing 2 vol% of DMSO, respectively. In addition, 15 mM solutions of ROS such as KO 2 , NO 3 − , TBHP, mCPBA, HOCl, ClO 4 − , SO 4 2− , NO 2 − , NO 3 − , and BPO (benzoyl peroxide) were prepared using double distilled water.

Computational Methods.
Quantum calculations using the density functional theory (DFT) were carried out for the abovementioned probe molecule. e generalized gradient approximation method involved the Becke three-parameter plus Lee-Yang-Parr (B3LYP) functional, while the basis set was the Pople 6-31 + G(d,p) one [33][34][35]. e intrinsic solvent model was considered using the c-pcm model, the dielectric constant being 78.39. All calculations were carried out using the Q-Chem 4.3 program. Graphical representation for the calculated results was obtained using the IQmol software [36,37].

Paper Strip Test.
For the paper strip-based detection of H 2 O 2 , Whatman paper (8 mm diameter) samples were first immersed into 1 μM TPIOP boronate solution for 5 min and subsequently dried in air. e dried papers were exposed to different concentrations of H 2 O 2 for 2 min. en, the papers were illuminated under a UV lamp (365 nm), and fluorescence images were obtained.

Cell Culture, Cytotoxicity Tests, and Confocal Microscopy
Imaging.
e MCF-7 cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin. e cells were maintained in an incubator at 37°C in 5% CO 2 environments. For the cytotoxicity tests, the cells were seeded in a 96-well plate containing culture media. After overnight culture, the cells were incubated using different concentrations of TPIOP boronate. To measure the viability of the cells, 0.5 mg/mL MTT medium was added to each of the cells for 4 h and the produced formazan was dissolved in 0.1 mL DMSO and analyzed using a Spectramax microwell plate reader. e cytotoxic effects of TPIOP boronate were calculated using the following equation: where OD(sample) and OD(control) are the optical densities of the sample and control, respectively. For live cells imaging, cells were seeded in 35 mm glass-bottomed dishes containing culture media. After overnight culture, the MCF-7 cells were incubated with 10 μM TPIOP boronate for 30 min, washed with DBS, and incubated with 10 μM H 2 O 2 for 30 min. Fluorescence images were acquired using an LSM 700 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with a 63× oil immersion objective lens and a diode laser as light source [38].

Spectral Studies of TPIOP Boronate.
e detailed synthesis of TPIOP boronate is represented in Scheme 1.
e TPIOP boronate probe was characterized using 1 H NMR, 13 C NMR, and HR-MS techniques (Figures S1A, S2A, and S3A, respectively). We initially evaluated the photophysical properties of the TPIOP boronate probe, and its ability to track H 2 O 2 was investigated using HEPES buffer solution (10 mM, pH 7.4 containing 2 vol% DMSO). e optical properties of TPIOP boronate were analyzed, and the results are represented own in Figure 1. e 2 μM solution of TPIOP boronate in 10 mM HEPES buffer solution (10 mM, pH 7.4 containing 2 vol% of DMSO) exhibited an excitation maximum centered at 346 nm. e TPIOP boronate probe presented weak fluorescence at 467 nm with a Stokes shift of 121 nm (Figure 1). After the probe was triggered using H 2 O 2 , a significant turn-on fluorescent enhancement was observed at 467 nm, and the fluorescence was also visually observed using an UV lamp, as shown in Figure 2(a). e TPIOP boronate probe contained TPIOP and a boronic ester moiety in its structure. It is well known that the boronate group undergoes oxidation in the presence of H 2 O 2 , and the oxidation of boronate is caused by the enhanced nucleophilicity of H 2 O 2 due to the α-effect, imparted by the adjacent nonbonding orbitals on its oxygen atoms and its weak O-O bonds. e nucleophilic addition of H 2 O 2 to the boron atom results in a charged tetrahedral boronate complex, which subsequently undergoes a 1,2-insertion where the C-B bond migrates to one of the electrophilic peroxide oxygen atoms. e resulting borate ester is then hydrolyzed by water into phenol (Scheme 2) [6,10,11]. e conversion of TPIOP boronate into TPIOP-OH was supported by 1 H NMR, 13 C NMR, and HR-MS data ( Figures S1B, S2B, and S3B, respectively). e 1 H NMR of TPIOP boronate presented a methyl singlet peak at 1.07 ppm, which disappeared after TPIOP boronate reacted with H 2 O 2 . en, a new peak appeared at 6.25 ppm, which was attributed to phenolic -OH proton ( Figure S1B). e 13 C NMR spectrum contained a methyl peak which appeared at 25.5 ppm and disappeared after the conversion of boronate into phenol ( Figure S2B). After injecting H 2 O 2 into the TPIOP boronate sample, a new m/z value was observed at 457.49 [M] + , which corresponded to the molecular weight of TPIOP-OH ( Figure S3B).
is demonstrated that while reacting with H 2 O 2 , TPIOP boronate was converted into TPIOP-OH (Scheme 2), and the yield was found to be 78%. e conformational change from hydrophobic to hydrophilic form during the conversion of TPIOP boronate into TPIOP-OH led to aggregation. Intramolecular rotation associated with this conversion was restricted due to physical constraints, which blocked the nonradiative relaxation and commenced the radiative decay. erefore, the intensity of the emission was enhanced based on an AIE mechanism.   A computational study was utilized to elucidate the characteristics of the C-B and B-O chemical bonds in TPIOP boronate. e polarities of the C-B and B-O bonds were relatively high given the electronegativities of the individual atoms ( Figures S4 and S5), and the C-B bond was the most labile site for the H 2 O 2 (Scheme 2). is was caused by extensively delocalized π-electrons throughout the TPIOP group. Moreover, the presence of heteroatoms increased the electron perturbation throughout TPIOP in half. erefore, the electron density of the carbon atom bound to boron was high, which would lead to the high polarity of the C-B bond. is leads to the activation of the reaction centers during oxidation reaction of boronate in the TPIOP boronate probe, which allowed for the rapid identification of H 2 O 2 . To achieve rapid H 2 O 2 detection using TPIOP boronate, we examined the time-dependent fluorescent kinetics, as shown in Figure 2(b). Upon adding H 2 O 2 to TPIOP boronate, the fluorescence intensity at 467 nm increased rapidly with time and achieved its maximum within 2 mins, which intimated that TPIOP boronate was an effective fluorescent probe able to detect H 2 O 2 very rapidly. is was due to the presence of active reaction centers (C and B) in TPIOP boronate, which were provided by the TPIOP moiety.

Sensitive Detection of H 2 O 2 Using TPIOP Boronate.
e initial detection ability of TPIOP boronate for H 2 O 2 was analyzed using UV-Vis absorbance studies. We added so-  Figure 3). e LOD was estimated to be 8 nM (signal to noise ratio (S/ N) � 3), which was significantly lower than the reported range (Table 1).
is revealed that the TPIOP boronate probe was able to detect nanomolar level concentration of Considering the best performance of TPIOP boronate, we also extended the study to create solid state H 2 O 2 sensors using paper strips. We determined that depending on its concentration; H 2 O 2 renders Whatman paper emissive (inset, Figure 3). is demonstrated that the paper strips coated with TPIOP boronate could potentially act as environmental H 2 O 2 detector.

DFT Calculations on Molecular Orbitals of TPIOP Boronate.
To analyze the sensitivity of the abovementioned probe, the frontier molecular orbitals in TPIOP boronate were calculated and are represented in Figure 4. e splitting of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels occurred at the opposite ends of the molecule: HOMO was located at the opposite end of the boronate group, whereas LUMO was located near the C-B bond. As can be seen from the LUMO of TPIOP boronate, π orbitals extended from the biphenyl moiety to the boron atom. It is well known that the mechanism for sensing H 2 O 2 of TPIOP boronate involves the oxygen atom of H 2 O 2 interacting with the C-B bond through a Lewis acidic reaction. e LUMO level of the probe in this reaction was lowered by HOO − , which facilitated the breaking of the C-B bond. When HOMO located near LUMO, the possibility of electron transitions would be high and the interaction with HOOwould be obstructed by the electron-rich area near the C-B bond. e electron transitions might be hindered by the splitting of the frontier orbitals, which is responsible for the high sensitivity of TPIOP boronate for H 2 O 2 . e HOMO/LUMO  Journal of Analytical Methods in Chemistry  transition energy was quantitatively calculated employing time-dependent DFT using optimized conformations and DFT calculations (Figure 4). It can be seen that the energy gap increased from 3.022 to 3.188 eV after TPIOP boronate reacted with H 2 O 2 . ese findings coincided with the experimental data obtained using UV spectroscopy ( Figure S6A).  (Figure 5(a)), and the detection of H 2 O 2 was not hindered even in the presence of ROS solutions 1000× more concentrated than the H 2 O 2 solution ( Figure 5(b)). ese findings were also confirmed by the fluorescence images in the insets of Figures 5(a) and 5(b) and indicated that TPIOP boronate can also be used to visually identify and discriminate the presence of H 2 O 2 from wide ranges of ROS pools under UV radiation. e bar chart representation of the fluorescence response of TPIOP boronate in the presence of various ROS is shown in Figure 5(c). From the above studies, we concluded that TPIOP boronate was 1000 times more sensitive to H 2 O 2 than to other ROS when other ROS were present. Also, it was clearly observed that the TPIOP boronate probe presented rapid and nanomolar level sensitivity toward H 2 O 2 .

Selectivity and Interference
ese results obtained using the TPIOP boronate encouraged us to further investigate the possibility of using it for fluorescence imaging in living cells. Before considering the biological applications of TPIOP boronate, its sensitivity in terms of its fluorescent behavior was tested as a function of pH. e fluorescence emission intensity at 467 nm was monitored before and after adding 15 μM H 2 O 2 to a 2 μM TPIOP boronate probe solution and was plotted against the pH ( Figure S7A). e fluorescence images observed for TPIOP boronate at different pH levels and the fluorescence responses after adding H 2 O 2 to it are illustrated in Figure S7B. It can be noticed that the emission intensity is usually high in basic media (pH 7-12), but it reached its maximum at pH 7-8. Hence, the probe solution was maintained at the physiological pH of 7.4 throughout all the experiments.

Cytotoxicity of TPIOP Boronate and Its H 2 O 2 Detection in
Live MCF-7 Cells. Since the TPIOP boronate probe exhibited excellent sensitivity (nM level) for the detection of H 2 O 2 , we hypothesized that it would be possible to detect H 2 O 2 in cell lines using TPIOP boronate. Before using the TPIOP boronate probe for live cell imaging, its biocompatibility was tested using an MTT assay. e bioimaging applications of TPIOP boronate for detecting H 2 O 2 were demonstrated using living MCF-7 cells. e cytotoxicity of the probe was low, as shown in Figure S8, which reveals that the MCF-7 cells were able to survive concentration of TPIOP boronate up to 100 μM. We chose a 10 μM solution of TPIOP boronate for staining the probe. Living MCF-7 cells stained with TPIOP boronate were used to detect H 2 O 2 , and live images were recorded using a Zeiss LSM 700 confocal microscope. Images of H 2 O 2 in the MCF-7 cells were recorded after the TPIOP boronate probe    (Figure 6(b)). e increase in the emission intensity of the probe after interacting with the live MCF-7 cells was due to H 2 O 2 and is illustrated in Figure S9. ese results demonstrated that the TPIOP boronate probe in our study would be amenable for livecell H 2 O 2 imaging.

Conclusions
e findings of our current study indicated that a novel TPIOP boronate chemodosimeter containing highly polar C-B bond was designed, synthesized, characterized, and utilized for H 2 O 2 detection. Increasing the polarity of the C-B bond can increase the reactivity of TPIOP boronate with H 2 O 2 . is was achieved by introducing a TPIOP moiety using CPB. Adding H 2 O 2 to TPIOP boronate triggered the formation of TPIOP-OH, and then the fluorescence intensity was increased via the AIE mechanism. erefore, the abovementioned TPIOP boronate probe could become a fluorescent tool for the rapid, selective, and sensitive monitoring of H 2 O 2 . e lowest LOD of 8 nM was achieved using TPIOP boronate at physiological pH level, which was below the range reported in the literature. In addition, paper strips coated with TPIOP boronate were used for on-site naked-eye H 2 O 2 detecting experiments using UV radiation. Furthermore, TPIOP boronate exhibited low cytotoxicity and was utilized as fluorescent marker for detecting H 2 O 2 in living cells [35].

Data Availability
e data used to support the findings of this study are included within the article and the supplementary information file(s).

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper. Figure S1: 1H NMR spectra of (A) TPIOP boronate and (B) TPIOP-OH. Figure S2: 13C NMR spectra of (A) TPIOP boronate and (B) TPIOP-OH. Figure S3: HR-MS of (A) TPIOP boronate and (B) TPIOP-OH. Figure S4: molecular model of TPIOP boronate and its atomic charges. Figure  S5: electrostatic potential diagram of TPIOP boronate. Figure S6: UV-Vis spectra for different concentration of H 2 O 2 in (A) 20 μM TPIOP boronate and (B) its corresponding linearity plot. Figure S7: (A) fluorescence intensity at 467 nm for 2 μM TPIOP boronate before and after reacting with H 2 O 2 and (B) fluorescence images obtained for 2 μM TPIOP boronate. Figure S8: cytotoxicity test results of MTT assay. Figure S9