A Red-Emitting Fluorescence Probe for Rapid Detecting Exogenous and Endogenous Peroxynitrite in Living Cells with High Sensitivity and Selectivity

: Peroxynitrite (ONOO − ) has been revealed to play crucial roles in many physiological and pathological processes, and many diseases were proven to be associated with its misregulated production. The development of ﬂuorescent probes meets the need for tracking ONOO − and gives a better understanding of its diverse mechanisms. In this work, a red-emitting ﬂuorescent probe BP-ONOO was synthesized via functionalization of the rhodol-like ﬂuorophore with a reactive site of hydrazide. The probe BP-ONOO exhibited high sensitivity, excellent selectivity, and short response time (less than 4 s) towards ONOO − under neutral or weak alkaline conditions. These attractive properties favor its application in real-time imaging of ONOO − in living cells, and the probe has been successfully applied for imaging the concentration levels of ONOO − in RAW 264.7 macrophage cells under drug stimulation.


Introduction
Peroxynitrite (ONOO − ), one of the highly reactive nitrogen species (hRNS) in living systems, is generated via a rapid radical-radical coupling interaction between nitric oxide radicals (•NO) and superoxide anion radicals (O 2 •− ) [1].Accumulating evidence suggests that ONOO − is highly prevalent in endothelial cells, macrophages, leukocytes, neutrophils, and nerve cells and plays a positive role in signal transduction and immunogenic response [2][3][4].On the other hand, ONOO − , possessing strong oxidability and nucleophilicity, is also considered to be cytotoxic because it can damage a magnitude of biomolecules and eventually cause cell necrosis or apoptosis [5,6].Many diseases, including neurodegenerative, diabetes, cardiovascular diseases, and cancers, have been reported to be associated with the misregulated production of ONOO − in vivo [7][8][9][10].Owing to its short half-life, low concentration, and high reactivity in living systems, it is still a challenge to fully understand the exact mechanisms and diverse roles of biological ONOO − [11].Thus, researchers are pursuing an effective technology that can monitor the concentration variation of ONOO − in physiological and pathological processes with high sensitivity and selectivity [12].
The fluorescent probe technique is distinguished by its inherent advantages, including simplicity, fast response, high sensitivity/selectivity, and noninvasiveness.It is widely recognized as a valuable tool for providing information on biological events with exceptional spatio-temporal resolution [13].Thus far, great effort has been made to develop probes for detecting ONOO − in the biosystems [12,13].Due to the oxidizing/nucleophilic nature of ONOO − , a variety of functional groups, generally comprising boronates [14], α-ketoamides [15], hydrazides [16], diphenyl phosphinates [17], C=C bonds [18], trifluoroketones [19], and other active moieties [20][21][22], were added as triggers to fluorophores.However, some of them suffered from a lack of selectivity for ONOO − against other reactive oxygen species (ROS)/reactive nitrogen species (RNS).When high concentrations (micromolar range) of these reactive species coexist with ONOO − (nanomolar range), they may seriously interfere with ONOO − detection by reacting with triggers with a similar oxidation mechanism [23,24].Meanwhile, fluorescent probes with emission spectra in the far-infrared to NIR regions have received great attention.Cheng et al. have screened a series of NIR fluorescent probes for imaging ONOO − to assess drug-induced hepatotoxicity [25], which features minimum phototoxicity to the biological samples, high imaging depth, and low auto-fluorescence interference from biomolecules.In view of the low concentration, the short half-life of ONOO − , and the existence of a variety of strong interferences in biological systems, it is still necessary to engineer fast-response, long-wavelength fluorescent probes with improved sensitivity and selectivity.
Previous studies have indicated that incorporating a hydrazide group into rhodaminetype fluorophores to form spirolactam has been proved to be an effective strategy [16,26,27].Probes designed using this approach can undergo a ring-opening process of Xanthene structures and exhibit an "off-on" signal change with the addition of ONOO − compared to other ROS/RNS.However, certain limitations exist in some of these probes regarding their selectivity, as they may also recognize ClO − and •OH [28].Therefore, we hypothesized that the selection of an appropriate fluorophore as well as recognition groups is critical.BP613 is a recently reported rhodol-like fluorophore [29] which possesses several desired properties, such as high fluorescence quantum yield, suitable pKa (5.1 in PBS) for biological applications, strong photostability, and acceptable biocompatibility.Motivated by this assumption that choosing the proper fluorophore may improve the selectivity toward ONOO − , we incorporated a hydrazide group as a reactive site into BP613 to synthesize BP-ONOO (Scheme 1).develop probes for detecting ONOO − in the biosystems [12,13].Due to the oxidizing/nucleophilic nature of ONOO − , a variety of functional groups, generally comprising boronates [14], α-ketoamides [15], hydrazides [16], diphenyl phosphinates [17], C=C bonds [18], trifluoroketones [19], and other active moieties [20][21][22], were added as triggers to fluorophores.However, some of them suffered from a lack of selectivity for ONOO − against other reactive oxygen species (ROS)/reactive nitrogen species (RNS).When high concentrations (micromolar range) of these reactive species coexist with ONOO − (nanomolar range), they may seriously interfere with ONOO − detection by reacting with triggers with a similar oxidation mechanism [23,24].Meanwhile, fluorescent probes with emission spectra in the far-infrared to NIR regions have received great attention.Cheng et al. have screened a series of NIR fluorescent probes for imaging ONOO − to assess drug-induced hepatotoxicity [25], which features minimum phototoxicity to the biological samples, high imaging depth, and low auto-fluorescence interference from biomolecules.In view of the low concentration, the short half-life of ONOO − , and the existence of a variety of strong interferences in biological systems, it is still necessary to engineer fast-response, long-wavelength fluorescent probes with improved sensitivity and selectivity.
Previous studies have indicated that incorporating a hydrazide group into rhodamine-type fluorophores to form spirolactam has been proved to be an effective strategy [16,26,27].Probes designed using this approach can undergo a ring-opening process of Xanthene structures and exhibit an "off-on" signal change with the addition of ONOO − compared to other ROS/RNS.However, certain limitations exist in some of these probes regarding their selectivity, as they may also recognize ClO − and •OH [28].Therefore, we hypothesized that the selection of an appropriate fluorophore as well as recognition groups is critical.BP613 is a recently reported rhodol-like fluorophore [29] which possesses several desired properties, such as high fluorescence quantum yield, suitable pKa (5.1 in PBS) for biological applications, strong photostability, and acceptable biocompatibility.Motivated by this assumption that choosing the proper fluorophore may improve the selectivity toward ONOO − , we incorporated a hydrazide group as a reactive site into BP613 to synthesize BP-ONOO (Scheme 1).4) high sensitivity and low detection limit; and (5) remarkable selectivity toward other ROS/RNS and bio-relevant analytes.Furthermore, the probe demonstrated minimal cytotoxicity and enabled imaging of exogenous and endogenous ONOO − in RAW 264.7 macrophage cells.

Reagents and Instrumentation
All chemical reagents were purchased from commercial suppliers and used without further purification. 1H and 13 C NMR spectra were obtained using a Bruker Advance 400 NMR spectrometer (internal standard: tetramethylsilane).Chemical shifts are expressed  4) high sensitivity and low detection limit; and (5) remarkable selectivity toward other ROS/RNS and bio-relevant analytes.Furthermore, the probe demonstrated minimal cytotoxicity and enabled imaging of exogenous and endogenous ONOO − in RAW 264.7 macrophage cells.

Reagents and Instrumentation
All chemical reagents were purchased from commercial suppliers and used without further purification. 1H and 13 C NMR spectra were obtained using a Bruker Advance 400 NMR spectrometer (internal standard: tetramethylsilane).Chemical shifts are expressed in ppm and coupling constants (J) are reported in hertz (Hz).High resolution mass spectra (HRMS) were collected on an AB SCIEX Triple TOF 5600+ mass spectrometer.UVvis absorption spectra were obtained on a SHIMADZU UV-1800 spectrophotometer with 10 mm light path cuvette.Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrofluorometer (Kyoto, Japan).The absorbances for Cell Counting Kit-8 (CCK-8) analysis were recorded on a SpectraMax M5 Reader (Molecular Devices).Fluorescence images were recorded on an FV3000-IX83 confocal microscope (Olympus).

Cytotoxicity Assay of Probe
HeLa cells were cultured in RPMI-1640 medium containing 10% (v/v) fetal bovine serum (FBS) and 100 µg/mL penicillin-streptomycin in a humidified 37 • C, 5% CO 2 incubator.The cells were dispersed in a 96-well plate at a density of 5000 cells per well and maintained at the above condition for 24 h.Then, HeLa cells were incubated with various concentrations of BP-ONOO (0, 2, 5, 10, 15, and 20.0 µM) for 24 h; the media were then replaced with fresh media containing 10% CCK-8 and incubated for another 30 min.Then, the absorbance at 450 nm was measured, in which each concentration of BP-ONOO was performed three times.

Fluorescence Imaging Study for Endogenous and Exogenous ONOO −
RAW 264.7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 100 µg/mL penicillin-streptomycin in a humidified 37 • C, 5% CO 2 incubator.Cells were seeded in glass-bottom dishes and incubated for one night before the imaging test.
For exogenous ONOO − detection, the adherent cells were rinsed three times with DMEM, pretreated with 10 µM BP-ONOO in culture media at 37 • C for 30 min, washed three times with DMEM, and incubated with various concentration of SIN-1 (0 µM, 20 µM, 50 µM, 100 µM, and 200 µM) for 30 min in serum-free DMEM at 37 • C. The cells were washed three times with PBS and the confocal imaging test was performed.
For endogenous ONOO − detection, the cells were stimulated with 1 µg/mL lipopolysa ccharide (LPS) and 1 µg/mL phorbol myristate acetate (PMA) for 6 h and 18 h respectively, washed three times using culture media, and incubated with 10 µM BP-ONOO in DMEM at 37 • C for 30 min.The cells were washed three times with PBS, then subjected to confocal imaging experiment.For the inhibition test, RAW 264.7 cells were treated with 1 µg/mL LPS and 1 µg/mL PMA in the presence of 200 µM minocycline, washed, and incubated with 10 µM BP-ONOO in DMEM in a sequential manner before their fluorescence images were finally acquired.For BP-ONOO, emission was collected at 600-700 nm (excited at 570 nm).

Synthesis of Probe
BP613 was prepared by reported method [29].Subsequently, BP-ONOO was prepared via the condensation reaction between BP613 and hydrazine monohydrate using NHS and EDCI as condensation reagent (Scheme 2).Both BP613 and BP-ONOO was purified by silica gel column chromatography and confirmed by NMR and HRMS (Figures S1-S6).

Spectral Responses of BP-ONOO for ONOO −
Figure 1 shows the UV-vis absorption spectra and FL spectra of BP-ONOO before and after adding ONOO − in PBS (20 mM, pH = 7.4) at room temperature.The absorption spectrum of the free probe (10 µM) exhibits an ambiguous band in the visible region, rendering the solution colorless.Correspondingly, the fluorescence emission spectrum demonstrates an exceptionally low background with no discernible fluorescence signal.These observations can be attributed to the presence of a spirolactam structure in BP-ONOO, which disrupts the conjugated structure of the fluorophore [31].Upon adding 40 µM ONOO − , there is a noticeable change in solution color from colorless to purple, along with the appearance of an absorption band centered at 570 nm (Figure 1a).Simultaneously, a remarkable red-emitting fluorescence is observed with a significant enhancement peak at 613 nm (Figure 1b).These alterations indicate that BP-ONOO can serve as a red-emitting turn-on probe for ONOO − .Scheme 2. Synthetic route for BP-ONOO.

Spectral Responses of BP-ONOO for ONOO −
Figure 1 shows the UV-vis absorption spectra and FL spectra of BP-ONOO before and after adding ONOO − in PBS (20 mM, pH = 7.4) at room temperature.The absorption spectrum of the free probe (10 µM) exhibits an ambiguous band in the visible region, rendering the solution colorless.Correspondingly, the fluorescence emission spectrum demonstrates an exceptionally low background with no discernible fluorescence signal.These observations can be attributed to the presence of a spirolactam structure in BP-ONOO, which disrupts the conjugated structure of the fluorophore [31].Upon adding 40 µM ONOO − , there is a noticeable change in solution color from colorless to purple, along with the appearance of an absorption band centered at 570 nm (Figure 1a).Simultaneously, a remarkable red-emitting fluorescence is observed with a significant enhancement peak at 613 nm (Figure 1b).These alterations indicate that BP-ONOO can serve as a red-emitting turn-on probe for ONOO − .The response of BP-ONOO to ONOO − exhibited remarkable rapidity, as demonstrated by the swift fluorescence enhancement reaching a plateau within 4 s (shown in Figure 2a).The characteristic confers significant advantages for real-time detection of transient ONOO − in living systems.We further investigated the influence of pH on the detection process.As shown in Figure 2b, the free probe solution (10 µM) exhibited negligible fluorescent signal variation when the pH ranged from 6 to 10, indicating its insensitivity to changes in pH.However, the addition of ONOO − resulted in a significant enhancement in fluorescence signal within the pH range of 7 to 9, indicating that the probe exhibits optimal performance under neutral and weak alkaline conditions.The response of BP-ONOO to ONOO − exhibited remarkable rapidity, as demonstrated by the swift fluorescence enhancement reaching a plateau within 4 s (shown in Figure 2a).The characteristic confers significant advantages for real-time detection of transient ONOO − in living systems.We further investigated the influence of pH on the detection process.As shown in Figure 2b, the free probe solution (10 µM) exhibited negligible fluorescent signal variation when the pH ranged from 6 to 10, indicating its insensitivity to changes in pH.However, the addition of ONOO − resulted in a significant enhancement in fluorescence signal within the pH range of 7 to 9, indicating that the probe exhibits optimal performance under neutral and weak alkaline conditions.
The sensing mechanism of ONOO − was investigated by analyzing the high-resolution mass spectrometry of the reaction mixture containing BP-ONOO and equivalent ONOO − .Figure S7 shows the emergence of a new mass peak at m/z = 446.1154,which corresponds to the calculated mass peak of BP613 (446.1154 for M + ), indicating the formation of BP613 during the reaction.Therefore, it can be reasonably speculated that the response mechanism is attributed to the activation of a carbonyl group in a spirolactam moiety, consistent with the design strategy employed for the well-known rhodamine-type fluorophores.The response mechanism is illustrated in Scheme 1, where non-fluorescent BP-ONOO reacts with ONOO − to generate BP613 and produce a fluorescence signal output.The sensing mechanism of ONOO − was investigated by analyzing the high-resolution mass spectrometry of the reaction mixture containing BP-ONOO and equivalent ONOO − .Figure S7 shows the emergence of a new mass peak at m/z = 446.1154,which corresponds to the calculated mass peak of BP613 (446.1154 for M + ), indicating the formation of BP613 during the reaction.Therefore, it can be reasonably speculated that the response mechanism is attributed to the activation of a carbonyl group in a spirolactam moiety, consistent with the design strategy employed for the well-known rhodamine-type fluorophores.The response mechanism is illustrated in Scheme 1, where non-fluorescent BP-ONOO reacts with ONOO − to generate BP613 and produce a fluorescence signal output.

Quantitative Analysis of ONOO − Using BP-ONOO
The response of BP-ONOO towards ONOO − was subsequently evaluated through a fluorescence titration experiment.As shown in Figure 3a, the fluorescence intensity exhibited a gradual increase with the addition of increasing amounts of ONOO − , reaching a plateau after approximately two equivalents of ONOO − were added.At saturation, the fluorescence intensity was enhanced up to 140-fold.The concentration-response curve is presented in Figure 3b, demonstrating that the fluorescence intensity enhancement at 613 nm displayed linearity with respect to the concentration of ONOO − within the range from 0 to 22.5 µM (linear equation: y = 28.4983× [ONOO − ] (µM) + 4.9982, R 2 = 0.9981).The detection limit of BP-ONOO for ONOO − was estimated to be as low as 18 nM according to the 3σ criteria (defined as 3σ/slope, where σ is the standard deviation of blank solution), suggesting the high sensitivity for ONOO − detection.Hence, considering the relatively low concentration (nM-µM) of peroxynitrite in a complicated biological environment [13], we anticipate that BP-ONOO, exhibiting exceptional sensitivity, can serve as a potent tool for precise quantitative analysis of peroxynitrite within biological systems (Table S1).

Quantitative Analysis of ONOO − Using BP-ONOO
The response of BP-ONOO towards ONOO − was subsequently evaluated through a fluorescence titration experiment.As shown in Figure 3a, the fluorescence intensity exhibited a gradual increase with the addition of increasing amounts of ONOO − , reaching a plateau after approximately two equivalents of ONOO − were added.At saturation, the fluorescence intensity was enhanced up to 140-fold.The concentration-response curve is presented in Figure 3b, demonstrating that the fluorescence intensity enhancement at 613 nm displayed linearity with respect to the concentration of ONOO − within the range from 0 to 22.5 µM (linear equation: y = 28.4983× [ONOO − ] (µM) + 4.9982, R 2 = 0.9981).The detection limit of BP-ONOO for ONOO − was estimated to be as low as 18 nM according to the 3σ criteria (defined as 3σ/slope, where σ is the standard deviation of blank solution), suggesting the high sensitivity for ONOO − detection.Hence, considering the relatively low concentration (nM-µM) of peroxynitrite in a complicated biological environment [13], we anticipate that BP-ONOO, exhibiting exceptional sensitivity, can serve as a potent tool for precise quantitative analysis of peroxynitrite within biological systems (Table S1).

Selectivity of BP-ONOO
In order to assess the feasibility of the designed fluorescent probe in real sample analysis, various interferences were selected to investigate the selectivity of probe BP-ONOO, including reactive oxygen/nitrogen species (H 2 O 2 , ClO − , ROO • , TBHP, • NO, • OH, O 2 •− , and ONOO − ), common anions (S 2− , S 2 O 3 2− , SO 3 2− , HSO 3 − , SCN − , NO 2 − , F − , Br − , and I − ), metal ions (Ag + , Ba 2+ , Fe 2+ , Fe 3+ , Pb 2+ , Zn 2+ , Hg 2+ , Mg 2+ , Ni 2+ , Ca 2+ , Cu 2+ , and Al 3+ ) as well as other biologically important substances (Vc, ATP, Lys, His, Glu, Gly, and GSH).As shown in Figure 4, the addition of 10 equivalents of interferences did not result in any significant fluorescence enhancement, which is highly promising.In contrast, the introduction of ONOO − led to a distinct increase in fluorescence intensity.These findings collectively demonstrate the excellent selectivity of BP-ONOO towards ONOO − , thereby highlighting its highly desirable attributes as an effective probe.Fluorescence intensity (at 613 nm) and increased concentration of ONOO − from 0 to 22.5 µM was linearly fitted with the inserted equation.The spectrum was recorded after standing for 30 min at room temperature.Each measurement was performed in triplicate and the data are expressed as the mean ± SD. λex = 570 nm, slit width: dex = dem = 5 nm.

Cytotoxicity and Cell Imaging of BP-ONOO
Prior to the application of the fluorescence probe for exogenous and endogenous ONOO − imaging, we initially investigated the cytotoxicity of BP-ONOO.

Cytotoxicity and Cell Imaging of BP-ONOO
Prior to the application of the fluorescence probe for exogenous and endogenous ONOO − imaging, we initially investigated the cytotoxicity of BP-ONOO. Figure 5   The capability of BP-ONOO to monitor exogenous and endogenous ONOO − was evaluated in live RAW 264.7 macrophage cells, which are recognized as model cells for generating ROS/RNS during inflammatory and immunological processes [32,33].
For imaging exogenous ONOO − (Figure 6), SIN-1 (3-morpholinosydnonimine hydrochloride, an ONOO − generator) was added at concentrations of 20 µM, 50 µM, 100 µM, and 200 µM.As shown in Figure 6(A2), no significant fluorescence was observed with only the probe BP-ONOO present.However, upon addition of SIN-1, a notable dosedependent increase in fluorescence intensity was observed (B2-E2 in Figure 6).These results demonstrate that BP-ONOO can be utilized for imaging the external source of ONOO − .The capability of BP-ONOO to monitor exogenous and endogenous ONOO − was evaluated in live RAW 264.7 macrophage cells, which are recognized as model cells for generating ROS/RNS during inflammatory and immunological processes [32,33].
For imaging exogenous ONOO − (Figure 6), SIN-1 (3-morpholinosydnonimine hydrochloride, an ONOO − generator) was added at concentrations of 20 µM, 50 µM, 100 µM, and 200 µM.As shown in Figure 6(A2), no significant fluorescence was observed with only the probe BP-ONOO present.However, upon addition of SIN-1, a notable dosedependent increase in fluorescence intensity was observed (B2-E2 in Figure 6).These results demonstrate that BP-ONOO can be utilized for imaging the external source of ONOO − .For endogenous ONOO − imaging, the cells were stimulated with lipopolysaccharide (LPS, 1 µg/mL) and phorbol myristate acetate (PMA, 1 µg/mL) for 6 h and 18 h respectively.Subsequently, the cells were incubated with BP-ONOO.As shown in Figure 7, a significant increase in intracellular fluorescence intensity (B2 and C2) was observed For endogenous ONOO − imaging, the cells were stimulated with lipopolysaccharide (LPS, 1 µg/mL) and phorbol myristate acetate (PMA, 1 µg/mL) for 6 h and 18 h respectively.Subsequently, the cells were incubated with BP-ONOO.As shown in Figure 7, a significant increase in intracellular fluorescence intensity (B2 and C2) was observed after treatment with LPS and PMA compared to the probe-only as control (A2).For the inhibition test, the RAW 264.7 cells were pre-treated with 1 µg/mL LPS and 1 µg/mL PMA in the presence of 200 µM minocycline (an ONOO − scavenger), followed by incubation with BP-ONOO for 30 min.Remarkably, there was a significant decrease in cellular fluorescence intensity observed (D2), providing compelling evidence that the probe effectively monitors endogenous ONOO − levels within living cells.
Finally, we investigated the sub-cellular distribution of BP-ONOO in HeLa cells.Commercially available LysoTracker Green, MitoTracker Green, and Hoechst 33342 were utilized to co-stain with BP-ONOO.As shown in Figure S8, after incubation with SIN-1 for 30 min, the fluorescence location of BP-ONOO was distributed throughout the cell.These results suggest that BP-ONOO can image ONOO − produced by various organelles and is mainly distributed in the cytoplasm.

Scheme 1 .
Scheme 1. Response mechanism of probe BP-ONOO to ONOO − .BP-ONOO exhibited numerous advantages for sensing ONOO − in biosystems, including: (1) a fluorescent turn-on signal change with a maximum excitation/emission at 570/613 nm; (2) excellent performance in aqueous solutions and physiological pH; (3) rapid response time (less than 4 s); (4) high sensitivity and low detection limit; and (5) remarkable selectivity toward other ROS/RNS and bio-relevant analytes.Furthermore, the probe demonstrated minimal cytotoxicity and enabled imaging of exogenous and endogenous ONOO − in RAW 264.7 macrophage cells.

Scheme 1 .
Scheme 1. Response mechanism of probe BP-ONOO to ONOO − .BP-ONOO exhibited numerous advantages for sensing ONOO − in biosystems, including: (1) a fluorescent turn-on signal change with a maximum excitation/emission at 570/613 nm; (2) excellent performance in aqueous solutions and physiological pH; (3) rapid response time (less than 4 s); (4) high sensitivity and low detection limit; and (5) remarkable selectivity toward other ROS/RNS and bio-relevant analytes.Furthermore, the probe demonstrated minimal cytotoxicity and enabled imaging of exogenous and endogenous ONOO − in RAW 264.7 macrophage cells.

Chemosensors 2023 , 12 Scheme 2 .Figure 1
Scheme 2. Synthetic route for BP-ONOO.3.2.Spectral Responses of BP-ONOO for ONOO −Figure1shows the UV-vis absorption spectra and FL spectra of BP-ONOO before and after adding ONOO − in PBS (20 mM, pH = 7.4) at room temperature.The absorption spectrum of the free probe (10 µM) exhibits an ambiguous band in the visible region, rendering the solution colorless.Correspondingly, the fluorescence emission spectrum

Figure 3 .
Figure 3. (a) Fluorescence spectra response of BP-ONOO (10 µM) toward different concentration of ONOO − (0-45 µM) in PBS (20 mM, pH = 7.4).(b) Fluorescence intensity (at 613 nm) changes of BP-ONOO under the varied concentration of ONOO − (0-45 µM).Fluorescence intensity (at 613 nm) and increased concentration of ONOO − from 0 to 22.5 µM was linearly fitted with the inserted equation.The spectrum was recorded after standing for 30 min at room temperature.Each measurement was performed in triplicate and the data are expressed as the mean ± SD. λex = 570 nm, slit width: dex = dem = 5 nm.

Figure 3 .
Figure 3. (a) Fluorescence spectra response of BP-ONOO (10 µM) toward different concentration of ONOO − (0-45 µM) in PBS (20 mM, pH = 7.4).(b) Fluorescence intensity (at 613 nm) changes of BP-ONOO under the varied concentration of ONOO − (0-45 µM).Fluorescence intensity (at 613 nm) and increased concentration of ONOO − from 0 to 22.5 µM was linearly fitted with the inserted equation.The spectrum was recorded after standing for 30 min at room temperature.Each measurement was performed in triplicate and the data are expressed as the mean ± SD. λ ex = 570 nm, slit width: d ex = d em = 5 nm.

Figure 3 .
Figure 3. (a) Fluorescence spectra response of BP-ONOO (10 µM) toward different concentration of ONOO − (0-45 µM) in PBS (20 mM, pH = 7.4).(b) Fluorescence intensity (at 613 nm) changes of BP-ONOO under the varied concentration of ONOO − (0-45 µM).Fluorescence intensity (at 613 nm) and increased concentration of ONOO − from 0 to 22.5 µM was linearly fitted with the inserted equation.The spectrum was recorded after standing for 30 min at room temperature.Each measurement was performed in triplicate and the data are expressed as the mean ± SD. λex = 570 nm, slit width: dex = dem = 5 nm.
demonstrates that approximately 90% of the cells survived at a probe concentration of 20 µM, indicating the low cytotoxicity of BP-ONOO.Chemosensors 2023, 11, x FOR PEER REVIEW 8 of 12demonstrates that approximately 90% of the cells survived at a probe concentration of 20 µM, indicating the low cytotoxicity of BP-ONOO.

Figure 5 .
Figure 5. HeLa cell viability after treatment with different concentrations of BP-ONOO after 24 h; results of cytotoxicity assays were obtained via CCK-8 assay.

Figure 5 .
Figure 5. HeLa cell viability after treatment with different concentrations of BP-ONOO after 24 h; results of cytotoxicity assays were obtained via CCK-8 assay.

Figure 5 .
Figure 5. HeLa cell viability after treatment with different concentrations of BP-ONOO after 24 h; results of cytotoxicity assays were obtained via CCK-8 assay.