A Benzil- and BODIPY-Based Turn-On Fluorescent Probe for Detection of Hydrogen Peroxide

Faced with rising threats of terrorism, environmental and health risks, achieving sensitive and selective detection of peroxide-based explosives (PEs) has become a global focus. In this study, a turn-on fluorescent probe (BOD) based on benzil (H2O2-recognition element) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivative (fluorophore) was developed to sensitively and specifically detect hydrogen peroxide (H2O2). The synthesized BOD had a very weak fluorescence due to intramolecular donor-excited photo-induced electron transfer (d-PET) effect; however, it could emit a strong fluorescence since H2O2 selectively oxidized the benzil moiety and released free BODIPY fluorophore (BOD-COOH). As a result, the proposed BOD detected H2O2 in linear detection ranged from 25 to 125 µM with a detection limit of 4.41 µM. Meanwhile, the proposed BOD showed good selectivity toward H2O2, which is not affected by other common reactive oxygen species (ROS) and ions from explosive residues. In addition, a blue shift from 508 to 498 nm was observed in the absorption spectra upon addition of H2O2. More importantly, the BOD was successfully applied for rapid detection of H2O2 vapor with good sensitivity (down to 7 ppb), which holds great potential for practical use in public safety, forensic analysis and environmental monitoring.


Introduction
In recent years, development of simple, sensitive and selective methods for the detection of explosives has become an increasingly critical public concern with rising threats in terrorism, homeland security and environmental pollution.In particular, among the conventional explosives, PEs have attracted widespread attention due to their easy synthesis and high explosive power [1].However, compared to nitroaromatic explosives, it is difficult to detect PEs because of their own characteristics of lack of nitro groups as well as minimal UV-vis absorption and non-fluorescence [2].It is well known that H 2 O 2 has a dual role as a starting synthetic material and a decomposition product of PEs, making it a signature compound for detecting PEs [3].Thus, achieving highly selective and sensitive detection of H 2 O 2 is of great significance in effective monitoring and early warning of PEs.
Currently, a variety of analytical methods have been developed for the sensitive detection of H 2 O 2 , including mass spectrometry [4], electrochemical detection [5], colorimetric and fluorimetric assay [6][7][8].Among them, fluorescence-based probes are the most preferred methods due to their low cost, rapid response, high sensitivity and selectivity [9,10].Usually, boronate ester chromophore-based fluorescent probes are used based on the cleavage of the boronate group by H 2 O 2 , leading to an increase in fluorescence intensity [11][12][13].However, these probes were reported to be susceptible to nitric oxide (NO), meaning the detection result can be disturbed by NO that decomposed by the nitro-aliphatic explosives [14].Therefore, in order to improve the selectivity for PE detection, it is crucial to ensure that the designed probes should be secured from disturbances like nitro-aliphatic and nitro-aromatic explosives as well as their decomposed products.Since Nagano et al. developed a novel fluorescence probe based on benzil chemistry and PET strategy to detect H 2 O 2 in 2011 [15], many benzil chemistry-based probes have been designed for detection of H 2 O 2 with a good reaction rate and high specificity [16,17].
As of recently, fluorescent probes based on BODIPY fluorophores hold great promise in explosive detection due to BODIPY's outstanding photophysical properties, such as high quantum yield and good photo stability [18].By conjugating the electron-donating group (EDG) or the electron-withdrawing group (EWG) to BODIPY, its photophysical properties can be fine-tuned through PET effect and charge-transfer intersystem crossing (ISC) process, resulting in an increase or suppression of fluorescence emission, which can be used for the construction of turn-on or turn-off fluorescent probes [15][16][17][18][19][20].Consequently, the combination of benzil chemistry and BODIPY fluorophores can be an appropriate candidate strategy for development of fluorescent probes to detect H 2 O 2 .For example, Li et al. developed a turn-on fluorescent probe (m-NBBD) for the visual detection of H 2 O 2 vapor with high sensitivity and specificity based on BODIPY combined with benzil moiety [21].Recently, in our previous study, a cancer cell-targeting fluorescence turn-on probe based on a benzil group and a BODIPY derivative was designed for detecting H 2 O 2 in biological systems, showing a high selectivity that can distinguish H 2 O 2 from common ROS [22].As a consequence, and inspired by the excellent selective response of benzil toward H 2 O 2 and great fluorescence features of BODIPY, in this study, we designed a turn-on fluorescent probe (BOD) for the detection of H 2 O 2 and H 2 O 2 vapor by combining the merits of benzil and BODIPY.
It was hypothesized that the proposed BOD could show almost no fluorescence owing to fluorescence quenching via the d-PeT process.Upon H 2 O 2 recognition, benzil could transform to benzoic acid, followed by the release of BODIPY dye, resulting in a fluorescence increment.Consequently, H 2 O 2 could be evaluated by measuring the BODIPY fluorescence, where benzil acted as the recognition group and BODIPY acted as the fluorophore.Meanwhile, the detection solution color changed from red to yellow-green, displaying potential for colorimetric assay.More importantly, BOD was further applied for the detection of H 2 O 2 vapor, which brought significant implications for the practice of PE detection.

Principle of the Benzil-and BODIPY-Based H 2 O 2 Assay
In this work, a benzil-and BODIPY-based turn-on fluorescent probe that employed benzil as H 2 O 2 -recognition group and BODIPY as fluorophore for the detection of H 2 O 2 as well as the recognition mechanism of the proposed fluorescent assays is developed and depicted in Scheme 1.Initially, the designed BOD was almost non-emissive, which was attributed to the occurrence of the d-PET effect from BODIPY fluorophore to nitrobenzene [15,22].In contrast, in the presence of H 2 O 2 , the benzil moiety could be specifically recognized and oxidized by H 2 O 2 , and a BODIPY derivative (BOD-COOH) could be formed and subsequently released, resulting in a turn-on fluorescence signal.Similar working principle had also been reported previously by many researchers [17,23].Additionally, the detection solution underwent a visual color change from red (BOD) to yellow-green (BOD-COOH), which had the potential for colorimetric analysis of H 2 O 2 .Therefore, highly sensitive and specific detection of H 2 O 2 can be successfully achieved based on the proposed benzil-and BODIPY-based probe through the fluorescence and colorimetric signal change.

Synthesis and Characterizations of the BOD
As shown in Scheme 2, BOD was synthesized according to our previous study [22].First, Compound (1) was synthesized through the reaction of 4-iodo-2-nitroaniline and chloroacetyl chloride in the presence of 4-dimethylaminopyridine (DMAP) and trimethylamine.Then, Compound (1) was reacted with tetramethylammonium azide to produce azide-modified Compound (2).Ethynyl-modified BODIPY of Compound (3) was synthesized based on the reported procedures by using 4-ethynylbenzaldehyde and 2,4dimethylpyrrole, where the reaction was conducted with stirring under nitrogen protection [24].Subsequently, Compound (4) was synthesized via Sonogashira coupling reaction of ( 2) and ( 3) by utilizing PdCl2(PPh3)2, CuI as catalysts and trimethylamine as a base.Finally, the BOD was obtained through the oxidation of (4) in the presence of PdCl2, and was characterized by 1 H NMR, 13 C NMR spectroscopies and high-resolution mass spectrometry (HR-MS) (Figures S1-S3).Scheme 2. Chemical structure and synthetic route of BOD.

Feasibility of the Benzil-and BODIPY-Based H2O2 Assay
The feasibility of the proposed strategy was demonstrated by detecting the absorption spectra and the fluorescence response signals of BOD before and after the addition of H2O2.As shown in Figure 1a, the BOD probe showed its characteristic absorption profile with an absorption maximum at about 508 nm; however, the addition of H2O2 to BOD induced a blue shift from 508 nm to 498 nm.More importantly, the absorption spectra of the product of reaction of BOD and H2O2 was similar to that of the BODIPY derivative (BOD-COOH), confirming that BOD indeed reacted with H2O2 and generated the product BOD-COOH.Likewise, from Figure 1b, the fluorescence intensity of BOD showed an about seven-fold increment after the reaction of BOD and H2O2, demonstrating the formation of the BODIPY dye BOD-COOH.In addition, the HR-MS analysis of the probe BOD was conducted before and after reacting with H2O2.As Scheme 1. Schematic representation of the benzil-and BODIPY-based turn-on fluorescent probe for the detection of H 2 O 2 (the X cross indicated that the process did not occur in the corresponding condition).

Synthesis and Characterizations of the BOD
As shown in Scheme 2, BOD was synthesized according to our previous study [22].First, Compound (1) was synthesized through the reaction of 4-iodo-2-nitroaniline and chloroacetyl chloride in the presence of 4-dimethylaminopyridine (DMAP) and trimethylamine.Then, Compound (1) was reacted with tetramethylammonium azide to produce azide-modified Compound (2).Ethynyl-modified BODIPY of Compound (3) was synthesized based on the reported procedures by using 4-ethynylbenzaldehyde and 2,4dimethylpyrrole, where the reaction was conducted with stirring under nitrogen protection [24].Subsequently, Compound (4) was synthesized via Sonogashira coupling reaction of ( 2) and (3) by utilizing PdCl 2 (PPh 3 ) 2 , CuI as catalysts and trimethylamine as a base.Finally, the BOD was obtained through the oxidation of (4) in the presence of PdCl 2 , and was characterized by 1 H NMR, 13 C NMR spectroscopies and high-resolution mass spectrometry (HR-MS) (Figures S1-S3).
Scheme 1. Schematic representation of the benzil-and BODIPY-based turn-on fluorescent probe for the detection of H2O2 (the X cross indicated that the process did not occur in the corresponding condition).

Synthesis and Characterizations of the BOD
As shown in Scheme 2, BOD was synthesized according to our previous study [22] First, Compound (1) was synthesized through the reaction of 4-iodo-2-nitroaniline and chloroacetyl chloride in the presence of 4-dimethylaminopyridine (DMAP) and trimethylamine.Then, Compound (1) was reacted with tetramethylammonium azide to produce azide-modified Compound (2).Ethynyl-modified BODIPY of Compound (3) was synthesized based on the reported procedures by using 4-ethynylbenzaldehyde and 2,4dimethylpyrrole, where the reaction was conducted with stirring under nitrogen protection [24].Subsequently, Compound (4) was synthesized via Sonogashira coupling reaction of ( 2) and (3) by utilizing PdCl2(PPh3)2, CuI as catalysts and trimethylamine as a base.Finally, the BOD was obtained through the oxidation of (4) in the presence of PdCl2 and was characterized by 1 H NMR, 13 C NMR spectroscopies and high-resolution mass spectrometry (HR-MS) (Figures S1-S3).
Scheme 2. Chemical structure and synthetic route of BOD.

Feasibility of the Benzil-and BODIPY-Based H2O2 Assay
The feasibility of the proposed strategy was demonstrated by detecting the absorption spectra and the fluorescence response signals of BOD before and after the addition of H2O2.As shown in Figure 1a, the BOD probe showed its characteristic absorption profile with an absorption maximum at about 508 nm; however, the addition of H2O2 to BOD induced a blue shift from 508 nm to 498 nm.More importantly, the absorption spectra of the product of reaction of BOD and H2O2 was similar to that of the BODIPY derivative (BOD-COOH), confirming that BOD indeed reacted with H2O2 and generated the product BOD-COOH.Likewise, from Figure 1b, the fluorescence intensity of BOD showed an about seven-fold increment after the reaction of BOD and H2O2 demonstrating the formation of the BODIPY dye BOD-COOH.In addition, the HR-MS analysis of the probe BOD was conducted before and after reacting with H2O2.As Scheme 2. Chemical structure and synthetic route of BOD.

Feasibility of the Benzil-and BODIPY-Based H 2 O 2 Assay
The feasibility of the proposed strategy was demonstrated by detecting the absorption spectra and the fluorescence response signals of BOD before and after the addition of H 2 O 2 .As shown in Figure 1a, the BOD probe showed its characteristic absorption profile with an absorption maximum at about 508 nm; however, the addition of H 2 O 2 to BOD induced a blue shift from 508 nm to 498 nm.More importantly, the absorption spectra of the product of reaction of BOD and H 2 O 2 was similar to that of the BODIPY derivative (BOD-COOH), confirming that BOD indeed reacted with H 2 O 2 and generated the product BOD-COOH.Likewise, from Figure 1b, the fluorescence intensity of BOD showed an about seven-fold increment after the reaction of BOD and H 2 O 2 , demonstrating the formation of the BODIPY dye BOD-COOH.In addition, the HR-MS analysis of the probe BOD was conducted before and after reacting with H 2 O 2 .As described in Figure 2

Optimization of the Benzil-and BODIPY-Based H2O2 Assay Conditions
In order to achieve good performance of the proposed probe BOD for H2O2 detection, detection solution, incubation time and pH of solution were optimized.It is well known that BODIPYs are sensitive to the solvent [25]; therefore, absorption and fluorescence spectra of BOD were detected in various solvents to study if BOD could form aggregation that

Optimization of the Benzil-and BODIPY-Based H2O2 Assay Conditions
In order to achieve good performance of the proposed probe BOD for H2O2 detection, detection solution, incubation time and pH of solution were optimized.It is well known that BODIPYs are sensitive to the solvent [25]; therefore, absorption and fluorescence spectra of BOD were detected in various solvents to study if BOD could form aggregation that

Optimization of the Benzil-and BODIPY-Based H 2 O 2 Assay Conditions
In order to achieve good performance of the proposed probe BOD for H 2 O 2 detection, detection solution, incubation time and pH of solution were optimized.It is well known that BODIPYs are sensitive to the solvent [25]; therefore, absorption and fluorescence spectra of BOD were detected in various solvents to study if BOD could form aggregation that could subsequently affect detection efficiency.As shown in Figure S4, the absorption and fluorescence spectra measurements of BOD were determined in DMSO, PBS (0.1 M, pH = 7.4, 5% DMSO) and PBS (0.1 M, pH = 7.4, 1% DMSO) buffer, respectively.The absorption spectrum of BOD in DMSO exhibited characteristic absorption maxima at about 498 nm, while the maximum absorption peaks of BOD in PBS (0.1 M, pH = 7.4, 5% DMSO) and (0.1 M, pH = 7.4, 1% DMSO) buffer were, respectively, at about 508 and 520 nm (Figure S4a).Moskalensky et al. found that BODIPY-based dyes aggregate in water (1% DMSO), which simultaneously showed a wide absorption peak (at about 500 nm) and two fluorescence peaks [26].As for the two fluorescence peaks, one at about 500 nm corresponded to the emission band of BODIPY in organic solvents, and one at about 650 nm corresponded to the formation of aggregation.However, the BOD only had one fluorescence peak at about 508 nm in both PBS (5% DMSO) and PBS (1% DMSO) buffer solutions (Figure S4b), indicating that the BOD was completely dissolved and no obvious aggregation phenomenon was observed in both PBS buffer solutions.In Figure S4a, the BOD shows wide absorption in the PBS buffer solution, which can be attributed to the influence of the contained benzil and azido group, and similar results have also been observed in the study of benzi by Mittal [27].In addition, Nagano et al. designed and synthesized a library of BODIPY-based environmental polarity sensors by utilizing photoinduced electron-transfer-controlled fluorescence on/off switching.They demonstrated that the fluorescence property can be well controlled by the PET mechanism, which depends on solvent polarity [28].Thus, as shown in Figure S4b, the fluorescence intensity of BOD in DMSO was stronger than that in the PBS (5% DMSO and 1% DMSO) buffer, which can be explained by the change in solvent polarity, resulting in a different PET process and fluorescence intensity.Consequently, the fluorescence emission of BOD was quenched mainly by the PET pathway in PBS (5% DMSO and 1% DMSO) buffer, which was not due to aggregation.
As for the optimization of incubation time, in Figure 3a, the BOD fluorescence increased on increasing the incubation time for the reaction of BOD and H 2 O 2 , with the fluorescence reaching a maximum at 60 min, and remained stable.Thus, 60 min was selected as the optimal incubation time.
Molecules 2024, 29, x FOR PEER REVIEW 5 of 14 could subsequently affect detection efficiency.As shown in Figure S4, the absorption and fluorescence spectra measurements of BOD were determined in DMSO, PBS (0.1 M, pH = 7.4, 5% DMSO) and PBS (0.1 M, pH = 7.4, 1% DMSO) buffer, respectively.The absorption spectrum of BOD in DMSO exhibited characteristic absorption maxima at about 498 nm, while the maximum absorption peaks of BOD in PBS (0.1 M, pH = 7.4, 5% DMSO) and (0.1 M, pH = 7.4, 1% DMSO) buffer were, respectively, at about 508 and 520 nm (Figure S4a) Moskalensky et al. found that BODIPY-based dyes aggregate in water (1% DMSO), which simultaneously showed a wide absorption peak (at about 500 nm) and two fluorescence peaks [26].As for the two fluorescence peaks, one at about 500 nm corresponded to the emission band of BODIPY in organic solvents, and one at about 650 nm corresponded to the formation of aggregation.However, the BOD only had one fluorescence peak at about 508 nm in both PBS (5% DMSO) and PBS (1% DMSO) buffer solutions (Figure S4b), indicating that the BOD was completely dissolved and no obvious aggregation phenomenon was observed in both PBS buffer solutions.In Figure S4a, the BOD shows wide absorption in the PBS buffer solution, which can be attributed to the influence of the contained benzil and azido group, and similar results have also been observed in the study of benzi by Mittal [27].In addition, Nagano et al. designed and synthesized a library of BODIPYbased environmental polarity sensors by utilizing photoinduced electron-transfer-controlled fluorescence on/off switching.They demonstrated that the fluorescence property can be well controlled by the PET mechanism, which depends on solvent polarity [28] Thus, as shown in Figure S4b, the fluorescence intensity of BOD in DMSO was stronger than that in the PBS (5% DMSO and 1% DMSO) buffer, which can be explained by the change in solvent polarity, resulting in a different PET process and fluorescence intensity.Consequently, the fluorescence emission of BOD was quenched mainly by the PET pathway in PBS (5% DMSO and 1% DMSO) buffer, which was not due to aggregation.
As for the optimization of incubation time, in Figure 3a, the BOD fluorescence increased on increasing the incubation time for the reaction of BOD and H2O2, with the fluorescence reaching a maximum at 60 min, and remained stable.Thus, 60 min was selected as the optimal incubation time.In addition, the effect of pH was also investigated by detecting the BOD fluorescence in the presence of H2O2 in PBS buffer from pH 5.0 to 8.0.After reaction with H2O2, the BOD fluorescence was pH-dependent and rose with increasing pH value as shown in Figure 3b, which can be attributed to the deprotonation of carboxylic acid (product BOD-COOH) to form carboxylate (−COO − ) in alkaline solution [21].In contrast, the fluorescence of free BOD remained stable between pH values of 5.0 and 8.0.Considering the generality of the proposed approach, therefore, physiological pH (pH = 7.4) in PBS buffer was chosen for the following experiments.In addition, the effect of pH was also investigated by detecting the BOD fluorescence in the presence of H 2 O 2 in PBS buffer from pH 5.0 to 8.0.After reaction with H 2 O 2 , the BOD fluorescence was pH-dependent and rose with increasing pH value as shown in Figure 3b, which can be attributed to the deprotonation of carboxylic acid (product BOD-COOH) to form carboxylate (−COO − ) in alkaline solution [21].In contrast, the fluorescence of free BOD remained stable between pH values of 5.0 and 8.0.Considering the generality of the proposed approach, therefore, physiological pH (pH = 7.4) in PBS buffer was chosen for the following experiments.

Photostability Study of the BOD Probe
Further, the photostability of the proposed BOD probe was studied since it is vital for optical-based sensing probes.In particular, PEs were usually detected by H 2 O 2 that generated by their photolysis.As a result, photostability is a critical requirement for the design of probes to detect H 2 O 2 .It was found that the fluorescence response of BOD remained stable in the presence of H 2 O 2 upon continuous UV irradiation at 365 nm for 180 min (Figure 4), indicating great photostability of the proposed BOD.
Molecules 2024, 29, x FOR PEER REVIEW

Photostability Study of the BOD Probe
Further, the photostability of the proposed BOD probe was studied since it for optical-based sensing probes.In particular, PEs were usually detected by H2 generated by their photolysis.As a result, photostability is a critical requirement design of probes to detect H2O2.It was found that the fluorescence response of B mained stable in the presence of H2O2 upon continuous UV irradiation at 365 nm min (Figure 4), indicating great photostability of the proposed BOD.

Sensitivity of the Benzil-and BODIPY-Based H2O2 Assay
In order to evaluate the H2O2 detection capability of the proposed BOD pro fluorescence spectra of the BOD were measured in the presence of H2O2 at differe centrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1, 2 mM).As the concentration of H2O2 in from 0 to 2 mM, the fluorescence emission peak of BOD was gradually enhanced 5a), which was consistent with the fact that H2O2 could selectively oxidize the benz ety of BOD and generate the BODIPY fluorophore BOD-COOH, subsequently resu a turn-on fluorescence signal with a bright green fluorescence under UV radiation 5d, upper panel).Furthermore, it was observed that the BOD fluorescence intensit nm had a good linear correlation to the concentration of H2O2 in the range of 0 to with a correlation coefficient of 0.9941 (Figure 5b), and a limit of detection (LOD) µM was determined according to 3σ/K (σ is the standard deviation and K is the s the curve of linear regression).

Sensitivity of the Benzil-and BODIPY-Based H 2 O 2 Assay
In order to evaluate the H 2 O 2 detection capability of the proposed BOD probe, the fluorescence spectra of the BOD were measured in the presence of H 2 O 2 at different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1, 2 mM).As the concentration of H 2 O 2 increased from 0 to 2 mM, the fluorescence emission peak of BOD was gradually enhanced (Figure 5a), which was consistent with the fact that H 2 O 2 could selectively oxidize the benzil moiety of BOD and generate the BODIPY fluorophore BOD-COOH, subsequently resulting in a turn-on fluorescence signal with a bright green fluorescence under UV radiation (Figure 5d, upper panel).Furthermore, it was observed that the BOD fluorescence intensity at 508 nm had a good linear correlation to the concentration of H 2 O 2 in the range of 0 to 125 µM with a correlation coefficient of 0.9941 (Figure 5b), and a limit of detection (LOD) of 4.41 µM was determined according to 3σ/K (σ is the standard deviation and K is the slope of the curve of linear regression).
In addition, the colorimetric sensing ability of the proposed probe BOD was evaluated by measuring UV-vis absorption spectra of the BOD with different concentrations of H 2 O 2 .The results showed that the characteristic absorption peak of BOD at 508 nm disappeared upon the addition of H 2 O 2 , while two new peaks appeared at ~540 nm and ~498 nm (Figure 5c).And the peak at ~540 nm disappeared gradually with the increase in H 2 O 2 concentration, but at the same time, the peak of BOD at ~498 nm displayed an enhancement.The generation of the new peak at ~540 nm might derive from the intermediate product of reaction between BOD and H 2 O 2 [15,21].Meanwhile, the color of the above BOD solution was clearly changed from red to yellow-green after H 2 O 2 treatment with a certain concentration (Figure 5d, bottom panel).As a result, H 2 O 2 can be visually detected by the naked eye when its concentration exceeds 0.5 mM.In addition, the colorimetric sensing ability of the proposed probe BOD was ev ated by measuring UV-vis absorption spectra of the BOD with different concentration H2O2.The results showed that the characteristic absorption peak of BOD at 508 nm dis peared upon the addition of H2O2, while two new peaks appeared at ~540 nm and ~ nm (Figure 5c).And the peak at ~540 nm disappeared gradually with the increase in H concentration, but at the same time, the peak of BOD at ~498 nm displayed an enhan ment.The generation of the new peak at ~540 nm might derive from the intermed product of reaction between BOD and H2O2 [15,21].Meanwhile, the color of the ab BOD solution was clearly changed from red to yellow-green after H2O2 treatment wi certain concentration (Figure 5d, bottom panel).As a result, H2O2 can be visually detec by the naked eye when its concentration exceeds 0.5 mM.

Selectivity of the Benzil-and BODIPY-Based H2O2 Assay
Selectivity is another important factor for fluorescent probes with reliable and h detection efficiency; therefore, the specificity of the BOD was then investigated by c paring H2O2 with other common ROS (superoxide radical (O2 •− ), 1 O2, •OH, •NO, ONO tert-butoxy radical (•O t Bu) and tert-butyl hydroperoxide (TBHP)) and common ions fr explosive residues (NO2 − , NO3 − and ClO − ).As displayed in Figure 6, only H2O2 was abl induce the highest fluorescence response when compared with other ROS and inter ences from the explosive residues.The results indicated that the proposed BOD is capa of detecting H2O2 with high specificity.[BOD] = 5 µM.

Selectivity of the Benzil-and BODIPY-Based H 2 O 2 Assay
Selectivity is another important factor for fluorescent probes with reliable and high detection efficiency; therefore, the specificity of the BOD was then investigated by comparing H 2 O 2 with other common ROS (superoxide radical (O 2 •− ), 1 O 2 , •OH, •NO, ONOO − , tert-butoxy radical (•O t Bu) and tert-butyl hydroperoxide (TBHP)) and common ions from explosive residues (NO 2 − , NO 3 − and ClO − ).As displayed in Figure 6, only H 2 O 2 was able to induce the highest fluorescence response when compared with other ROS and interferences from the explosive residues.The results indicated that the proposed BOD is capable of detecting H 2 O 2 with high specificity.
Additionally, reduction in azide-functionalized fluorophores to amines can be utilized for the detection of hydrogen sulfide (H 2 S) [29][30][31][32]; therefore, the BOD was treated with H 2 S to further study whether there is any possible interference of H 2 S with the detection performance of the proposed strategy.As shown in Figure S5a, the BOD showed a relatively weak fluorescence emission in the presence of H 2 S (Figure S5a), while the introduction of H 2 O 2 induced a large fluorescent enhancement (about 12-fold) (Figure S5b).The reason for this result may be that the PET effect from BODIPY to benzil moiety still remains after the reduction in the azide group as the BOD in the study was constructed by modifying BODIPY with an azide-substituted benzil group, which cannot recover the BODIPY fluorescence, further confirming the selectivity of the proposed BOD for H 2 O 2 detection.Additionally, reduction in azide-functionalized fluorophores to amines can lized for the detection of hydrogen sulfide (H2S) [29][30][31][32]; therefore, the BOD was with H2S to further study whether there is any possible interference of H2S with tection performance of the proposed strategy.As shown in Figure S5a, the BOD a relatively weak fluorescence emission in the presence of H2S (Figure S5a), while troduction of H2O2 induced a large fluorescent enhancement (about 12-fold) (Figu The reason for this result may be that the PET effect from BODIPY to benzil moi remains after the reduction in the azide group as the BOD in the study was cons by modifying BODIPY with an azide-substituted benzil group, which cannot reco BODIPY fluorescence, further confirming the selectivity of the proposed BOD fo detection.•OH, •NO, ONOO − , •O t Bu, and TBHP) and common ions from explosive residues (NO 2 − , NO 3 − and ClO − ).F was the fluorescence intensity of the BOD at 508 nm with the addition of different testing species, F 0 was the fluorescence intensity of the BOD at 508 nm without the addition of the testing species; [BOD] = 5 µM.

Detection of H 2 O 2 Vapor Based on the Proposed BOD Probe
It is well known that trace detection of vapor emanated from explosives is a significant practical application for explosive monitoring.Therefore, the practical applicability of the proposed BOD probe was examined by detecting H 2 O 2 vapor.We found that the BODcoated thin-layer chromatography (TLC) plate with the image of SMU (an abbreviation of Shanxi Medical University) emitted an obvious green fluorescence under H 2 O 2 vapor (Figure 7a), suggesting the success of detection of H 2 O 2 vapor based on the BOD.Then, the response time of BOD toward H 2 O 2 vapor was tested, as shown in Figure 7b; a bright green emission could be observed 20 min after exposure to H 2 O 2 vapor and the green fluorescence increased gradually with exposure time, which can be clearly distinguished from background emission (without H 2 O 2 treatment), indicating a great potential in H 2 O 2 vapor rapid detection.
What is more, we studied the sensitivity of the BOD probe for the detection of H 2 O 2 vapor by being exposed to different concentrations of H 2 O 2 vapor from 0.8 to 31 ppb (Figure 7c).It was observed that the fluorescence emission of BOD became stronger progressively with increasing the concentration of H 2 O 2 , and the fluorometric change can be easily observed by the naked eye when H 2 O 2 vapor concentration was above 7 ppb, suggesting the detection range of 0.8 to 31 ppb with the limit of detection of 7 ppb.All these results demonstrated the high sensitivity of the proposed probe for the detection of H 2 O 2 vapor, implying the proposed assay has great potential for practical applications, especially in rapid on-site detection of PEs.
Although the proposed method showed enough sensitivity, there is still great potential to improve detection time.Nagano et al. developed a series of fluorescence probes based on benzil chemistry and photo-induced electron transfer (PET) strategy to detect H 2 O 2 .They found that the reactivity between benzil and H 2 O 2 could be adjusted by modification of the benzene ring of benzil [15].They synthesized five derivatives with various electron-donating or -withdrawing substituents on the benzil moiety, and the results showed that compounds with strongly electron-withdrawing groups of the cyano group and the nitro group exhibited a rapid fluorescence increase even at low concentrations of H 2 O 2 .In addition, they designed the probe by using the water-soluble fluorophore carboxyfluorescein.Thus, the cyano group, fluorescein and rhodamines can be utilized to construct benzil-based fluorescent probes to further optimize the measurement time for the detection of H 2 O 2 .
vapor (Figure 7a), suggesting the success of detection of H2O2 vapor based on the BO Then, the response time of BOD toward H2O2 vapor was tested, as shown in Figure 7b bright green emission could be observed 20 min after exposure to H2O2 vapor and t green fluorescence increased gradually with exposure time, which can be clearly disti guished from background emission (without H2O2 treatment), indicating a great potent in H2O2 vapor rapid detection.What is more, we studied the sensitivity of the BOD probe for the detection of H2 vapor by being exposed to different concentrations of H2O2 vapor from 0.8 to 31 ppb (Fi ure 7c).It was observed that the fluorescence emission of BOD became stronger progre sively with increasing the concentration of H2O2, and the fluorometric change can be easi observed by the naked eye when H2O2 vapor concentration was above 7 ppb, suggestin the detection range of 0.8 to 31 ppb with the limit of detection of 7 ppb.All these resu demonstrated the high sensitivity of the proposed probe for the detection of H2O2 vapo implying the proposed assay has great potential for practical applications, especially rapid on-site detection of PEs.
Although the proposed method showed enough sensitivity, there is still great pote tial to improve detection time.Nagano et al. developed a series of fluorescence prob based on benzil chemistry and photo-induced electron transfer (PET) strategy to dete H2O2.They found that the reactivity between benzil and H2O2 could be adjusted by mo ification of the benzene ring of benzil [15].They synthesized five derivatives with vario electron-donating or -withdrawing substituents on the benzil moiety, and the resu showed that compounds with strongly electron-withdrawing groups of the cyano grou and the nitro group exhibited a rapid fluorescence increase even at low concentrations
As for the selectivity of the fluorescence assay, O 2 − , NO 3 − and ClO − were used.Based on the published procedures for the preparation of ROS [22,[33][34][35], the ROS stock solutions were first prepared as follows.O 2

•−
was prepared from the enzymatic reaction of 23.6 mU/L xanthine oxidase and 1.0 mM hypoxanthine in the presence of catalase (25 µg/mL, which is used as a scavenger of H 2 O 2 ). 1 O 2 was generated through the reaction of 1.0 mM OCl − (5-fold excess) and 200 µM H 2 O 2 .
•OH was produced by the Fenton reaction of 1.0 mM Fe(ClO 4 ) 2 with 200 µM H 2 O 2 in which Fe(ClO 4 ) 2 was in excess.•O t Bu was produced by reaction of 1.0 mM Fe(ClO 4 ) 2 with 200 µM TBHP.•NO was obtained from sodium nitroferricyanide.ONOO − was synthesized by the reaction of NaNO 2 with H 2 O 2 in an acidic solution, and its concentration was determined by the absorbance peak at 302 nm.To a solution of BOD (5 µM) in a PBS (0.1 M, pH = 7.4, 5% DMSO) buffer, 500 µM H 2 O 2 , 500 µM of O 2 •− , 1 O 2 , •OH, •NO, ONOO − , •O t Bu and TBHP, 100 mM of NO 2 − , NO 3 − and ClO − were added at 37 • C, respectively.After incubation of 60 min, fluorescence intensities of the BOD were recorded at 508 nm with excitation at 475 nm (all fluorescence spectra were recorded using the Tianmei FL970 fluorescence spectrophotometer with the same detection parameters as described above).

Detection of H 2 O 2 Vapor
In order to detect the H 2 O 2 vapor, the BOD solution was drop-coated onto a silica gel TLC plate (2.8 (length) × 2.3 (width) cm) with about 1.0 cm radius circle and subsequently dried for 10 min.The experiment was performed by hanging the prepared BOD-coated TLC plate in the saturated vapor of H 2 O 2 generated in a 100 mL bottle, where approximately 20 mL of H 2 O 2 solution (diluted down to various concentrations) was put in and sealed for 12 h to reach the equilibrium vapor pressure.The equilibrium vapor pressure corresponding to a specific diluted concentration of H 2 O 2 solution was deduced from the reported literatures [21,36,37].Thus, various diluted concentrations (3.5 wt%, 1.4 wt%, 0.7 wt%, 0.35 wt%) of H 2 O 2 solution were obtained by diluting the commercial 30 wt% H 2 O 2 solution with pure water, which produce saturated (equilibrium) vapor pressures of H 2 O 2 of 10.5 ppm, 4.0 ppm, 1.9 ppm and 1.0 ppm, respectively.Then, the above H 2 O 2 solutions were diluted with water to obtain the final test vapor with lower concentrations (0.8 ppb, 7 ppb, 15 ppb and 31 ppb).After exposure to the vapor for different time intervals, the fluorescence images of the TLC plates were obtained on a gel imaging system under the excitation at 365 nm.The H 2 O 2 vapor concentration was calibrated by using the following Equation (1) since a binary system of H 2 O/H 2 O 2 was employed as a solution: where y, Y, X, Ps, and P were defined as the molar fraction in the vapor phase, the activity coefficient, the molar fraction in the liquid phase, the vapor pressure of H 2 O 2 (1.801 mmHg), and atmospheric pressure (1 atm), respectively [38].Moreover, the activity coefficient of H 2 O 2 was calculated from the following Equation (2) (Margules' equation): After incubation, the fluorescence images of the TLC plates were obtained on gel imaging system (Bio-Rad, Hercules, CA, USA) under the excitation at 365 nm.

Conclusions
In summary, we developed a turn-on fluorescent probe BOD to sensitively and specifically detect H 2 O 2 .The proposed BOD was designed by integrating benzil moiety with BODIPY derivative, in which benzil was employed as an H 2 O 2 recognition group and BODIPY was used as fluorophore.The fluorescence and absorbance emission of the BOD could be tuned by adjusting H 2 O 2 concentration, so H 2 O 2 can be detected through the fluorescence and colorimetric signal change with high sensitivity and selectivity.More importantly, the probe BOD exhibited fast response (down to 20 min) and high sensitivity (down to 7 ppb) for the detection of H 2 O 2 vapor, providing great potential for real-time in-field detection and monitoring of PEs.

3 of 13 14 Scheme 1 .
Scheme 1. Schematic representation of the benzil-and BODIPY-based turn-on fluorescent probe for the detection of H2O2 (the X cross indicated that the process did not occur in the corresponding condition).
, after incubation of the BOD and H 2 O 2 for 2 h at 37 • C, the HR-MS analysis showed peaks at m/z = 367.1443and 598.1849, which can be assigned to BOD-COOH ([M − H] − ) and BOD ([M − H] − ), respectively, further confirming the reaction of the BOD and H 2 O 2 , and subsequently the generation and release of the BOD-COOH product.Therefore, these results suggested that the proposed BOD probe for the detection of H 2 O 2 is feasible as it can simultaneously generate fluorescence and colorimetric signal change.Molecules 2024, 29, x FOR PEER REVIEW 4 of 14 described in Figure 2, after incubation of the BOD and H2O2 for 2 h at 37 °C, the HR-MS analysis showed peaks at m/z = 367.1443and 598.1849, which can be assigned to BOD-COOH ([M − H] − ) and BOD ([M − H] − ), respectively, further confirming the reaction of the BOD and H2O2, and subsequently the generation and release of the BOD-COOH product.Therefore, these results suggested that the proposed BOD probe for the detection of H2O2 is feasible as it can simultaneously generate fluorescence and colorimetric signal change.

Figure 2 .
Figure 2. HR-MS analysis showing peaks of the BOD and BOD-COOH after the addition of H2O2 in a negative ion mode.

Figure 2 .
Figure 2. HR-MS analysis showing peaks of the BOD and BOD-COOH after the addition of H2O2 in a negative ion mode.

Figure 2 .
Figure 2. HR-MS analysis showing peaks of the BOD and BOD-COOH after the addition of H 2 O 2 in a negative ion mode.

Figure 5 .
Figure 5. (a) Fluorescence response spectra of the proposed BOD probe with different concentrat of H2O2 in the range of 0-2 mM; (b) The relationship between the BOD fluorescence intensity at nm and different concentrations of H2O2 in the range of 0 to 125 µM (0, 25, 50, 75, 100, 125 µM UV−vis absorption spectra of the BOD with different concentrations of H2O2; (d) Upper panel photographs of the BOD before and after addition of H2O2 under a UV lamp; Bottom panel photographs of the BOD before and after addition of H2O2 under visible-light irradiation.[BO 5 µM.

Figure 5 .
Figure 5. (a) Fluorescence response spectra of the proposed BOD probe with different concentrations of H 2 O 2 in the range of 0-2 mM; (b) The relationship between the BOD fluorescence intensity at 508 nm and different concentrations of H 2 O 2 in the range of 0 to 125 µM (0, 25, 50, 75, 100, 125 µM); (c) UV−vis absorption spectra of the BOD with different concentrations of H 2 O 2 ; (d) Upper panel: the photographs of the BOD before and after addition of H 2 O 2 under a UV lamp; Bottom panel: the photographs of the BOD before and after addition of H 2 O 2 under visible-light irradiation.[BOD] = 5 µM.

Molecules 2024 ,Figure 6 .
Figure 6.Selectivity assay of fluorescence responses of the BOD to H2O2 and other ROS (O •OH, •NO, ONOO − , •O t Bu, and TBHP) and common ions from explosive residues (NO2 − , N ClO − ).F was the fluorescence intensity of the BOD at 508 nm with the addition of differen species, F0 was the fluorescence intensity of the BOD at 508 nm without the addition of th species; [BOD] = 5 µM.

Figure 6 .
Figure 6.Selectivity assay of fluorescence responses of the BOD to H 2 O 2 and other ROS (O 2 •− , 1 O 2 ,

Figure 7 .
Figure 7. (a) The emissive response of the BOD-coated TLC plate with the image of SMU (an abb viation of Shanxi Medical University) before and after being exposed to H2O2 vapor under UV ir diation (365 nm); (b) The emissive response of the BOD-coated TLC plate toward H2O2 vapor w different exposure time under UV irradiation (365 nm); (c) The emissive response of the BOD-coat TLC plate toward H2O2 with different concentrations of H2O2 vapor under UV irradiation (365 nm

Figure 7 .
Figure 7. (a) The emissive response of the BOD-coated TLC plate with the image of SMU (an abbreviation of Shanxi Medical University) before and after being exposed to H 2 O 2 vapor under UV irradiation (365 nm); (b) The emissive response of the BOD-coated TLC plate toward H 2 O 2 vapor with different exposure time under UV irradiation (365 nm); (c) The emissive response of the BOD-coated TLC plate toward H 2 O 2 with different concentrations of H 2 O 2 vapor under UV irradiation (365 nm).