A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

Open AccessCCS ChemistryRESEARCH ARTICLE1 Aug 2021A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk Kai Xin†, Xinxing Li†, Yinghua Guo†, Youhuan Zhong†, Jungang Wang, Haotian Yang, Jie Zhao, Chunlei Guo, Yunxia Huang, Zuhai Lei, Yi-Lun Ying, Xiao Luo, Haolu Wang, Xuhong Qian, Wen Yang, Xiaowen Liang and Youjun Yang Kai Xin† State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 †K. Xin, X. Li, Y. Guo, and Y. Zhong contributed equally to this work.Google Scholar More articles by this author , Xinxing Li† Department of General Surgery, Changzheng Hospital, Shanghai 200003 †K. Xin, X. Li, Y. Guo, and Y. Zhong contributed equally to this work.Google Scholar More articles by this author , Yinghua Guo† State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 †K. Xin, X. Li, Y. Guo, and Y. Zhong contributed equally to this work.Google Scholar More articles by this author , Youhuan Zhong† Department of Molecular and Cellular Biochemistry, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 †K. Xin, X. Li, Y. Guo, and Y. Zhong contributed equally to this work.Google Scholar More articles by this author , Jungang Wang School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Haotian Yang The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, QLD 4102 Google Scholar More articles by this author , Jie Zhao State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Chunlei Guo State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Yunxia Huang State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Zuhai Lei State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Yi-Lun Ying School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Xiao Luo *Corresponding authors. E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Haolu Wang The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, QLD 4102 Google Scholar More articles by this author , Xuhong Qian State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Wen Yang *Corresponding authors. E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Department of Molecular and Cellular Biochemistry, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 Google Scholar More articles by this author , Xiaowen Liang *Corresponding authors. E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Department of General Surgery, Changzheng Hospital, Shanghai 200003 The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, QLD 4102 Google Scholar More articles by this author and Youjun Yang *Corresponding authors. E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000480 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging. Often, a small probe-product spectral separation leads to crosstalk, but the rational development of ratiometric probes with zero probe-product crosstalk remains challenging. Harnessing the recent progress on photophysical modulation of xanthenoid fluorochromes, we propose a powerful and versatile probe design principle, that is, “bridging-group modification,” and developed totalROX, a robust probe for monitoring the total cellular oxidative capacity. First, totalRox affirmatively detected the complete set of biorelevant highly oxidative species: per-acids (2 e−), radicals (1 e−), nitrosative (NO+) species, and singlet oxygen (1O2). Nonoxidative or mildly oxidative pro-oxidants, for example, O2•–, H2O2, NO, ONOO–, and ClO– were not detected. Second, the absorption/fluorescence maxima of the probe ( totalROX, λabs/λem = 425/525 nm) and the detection product ( Ox670, λabs/λem = 650/675 nm) were separated by ca. 225/150 nm, respectively, which eliminated probe-product crosstalk. Third, it renders the ratiometric signal with a single chromophore and is structurally succinct. TotalROX allowed quantitative analysis and was more sensitive than Amplex Red and CellROX Deep Red, two commercial probes for cell oxidative species. Bioimaging potentials of totalROX for monitoring cell redox status were exemplified in three different cell lines. Download figure Download PowerPoint Introduction Redox homeostasis is of vital importance to biological systems, and, depending on their nature and spatiotemporal flux, the roles of cellular oxidative species may vary from signaling to damaging.1–3 The accumulation of cellular oxidative damages overtime impairs biological functions and is implicated in development of chronic inflammation, diabetes, degenerative disorders, cancers, and aging.4–13 Intensive efforts have been devoted to developing probes for a better understanding of the firmly established yet loosely delineated causation between oxidative damages and the associated pathological conditions.14–17 Mechanistically, cell oxidative injuries are not induced by a singular mechanism but by the cumulative results of radical (1 e−) chemistry, per-acid (2 e−) chemistry, singlet oxygen (1O2) chemistry, and nitrosative (NO+) chemistry (Figure 1).18 Therefore, a single probe capable of detecting all four groups of oxidative entities and assessing the cellular redox status is superior to the existing probes capable of detecting a subset (e.g., DCHF/DHR,19 APF,20 and CellROX21). As diverse as the fluorescent probes for reactive oxygen species are in the literature,22,23 such a one-for-all probe has not been reported to our knowledge. Figure 1 | A simplified oxidative network from oxygen to various biodamaging oxidative species. Download figure Download PowerPoint Ratiometric probes are highly cherished in bioimaging.24,25 With their self-calibration capability, artifacts due to uneven probe loading, uneven illumination, or probe photobleaching are mitigated, thus, these probes are advantageous for quantitative studies in complex biological milieu. Ideally, a ratiometric probe should meet the following four criteria. First, monochromophoric ratiometric probes are preferred. The size of such a probe is kept to a minimum and preferential photobleaching of the acceptor (A) over the donor (D) associated with the dual-chromophric fluorescence resonance energy transfer (FRET)-type ratiometric probes26 is not a concern. Second, the spectral separation between the probe and the detection product should be large. To avoid the probe-product crosstalk, the separation should be larger than the full width at half maximum (FWHM) of the probe emission band (Figure 2a). Ratiometric probes via protection/deprotection of the D or A of a D–π–A type fluorophore do not usually yield a large separation, exemplified by the classic example of Fura-2 for Ca2+.27 Third, the emission wavelength of the probe should be shorter than that of the detection product because accurate detection of the increase of a shorter wavelength emission in the presence of a long-wavelength emission is technically cumbersome. Many dye-hemicyanine hybrids are synthesized as ratiometric probes harnessing the reactivity toward nucleophilic addition or oxidative cleavage of the double bond.28,29 However, they typically furnish a shorter-wavelength emitting product. Fourth, both the probe and the product should be bright fluorophores. Based on excited-state intramolecular proton transfer (ESIPT), elegant ratiometric probes were reported.30,31 Yet, their disadvantage is the low-fluorescence quantum yield of ESIPT-type fluorophores. The limitations of the existing approaches for ratiometric probes prompted us to develop alternative design principles. Recently, enhancing of the electronegativity of the bridging atom or groups of the xanthenoid dyes dramatically lowers the highest occupied molecular orbital (HOMO)–lowest unoccupied molecular orbital (LUMO) energy gap, and hence redshifts the absorption/emission maxima.32–36 We envisage that this phenomenon can be judiciously harnessed to design ratiometric probes for cellular oxidative species, and developed totalROX. We note that the bridging-group modification is a novel probe design principle for the xanthenoid chromophores, complementing such existing principles as push–pull modification,37–39 conjugation disruption,40,41 covalent-assembly,42–45 and photo-induced electron transfer (PET)46–48 (Figure 2b). Figure 2 | (a) Probe-product separation and crosstalk. (b) Four probe design principles applicable to xanthenoid fluorochromes. (c) The structure of totalROX and its detection product (Ox670). Download figure Download PowerPoint Structurally, totalROX is a substituted anthracene derivative (Figure 2c). Anthracene has been routinely used to trap 1O2, but is generally unreactive toward other oxidative species. Therefore, two electron-donating diethylamino groups were installed at C-2 and C-7 to promote its reactivity toward oxidation. The carboxyethyl (–CH2CH2COOH) moiety improved aqueous solubility of the probe. The o-tolyl group at C-10 was introduced to promote the fluorescence brightness of the proposed detection product ( Ox670), which is the first xanthenoid dye with a –CR(OH)– as the bridging group. Experimental Methods All chemical reagents were purchased from Chinese suppliers, for example, Aladdin (Shanghai, China); Energy (Shanghai, China); Titan (Shanghai, China); TCI Shanghai; Macklin (Shanghai, China); Sinopharm hemical Reagent (Shanghai, China); Meryer (Shanghai, China); Adamas-beta (Shanghai, China); ZhongHe Chemistry(Shanghai, China); and Xushuo Biology (Shanghai, China). Coumarin 153 (98%) was purchased from ZhongHe Chemistry. Cy5 (95%) was purchased from Xushuo Biology. The analytical grade organic solvents, EtOAc, MeOH, CH2Cl2, and petroleum ether were purchased and used without further purification. Anhydrous tetrahydrofuran (THF) for reactions was obtained by distillation over benzophenone-sodium. Anhydrous CH2Cl2 was obtained by drying over CaH2. The 1H NMR and 13C NMR spectra were collected on a Bruker AV-400 spectrometer. The chemicals shifts of the NMR spectra were referenced to the residual solvent peaks in the unit of ppm. The high-resolution mass spectrometry (HRMS) spectra were acquired on a Micromass GCT spectrometer. The UV–vis absorption spectra were collected on a SHIMADZU UV-2600 UV–vis spectrophotometer. Fluorescence spectra were obtained from a PTI-QM4 steady-state fluorimeter with a 75 W Xenon arc-lamp and a model 810 PMT (voltage = 950 V). The excitation and emission slits were set to 2 nm. All absorption and fluorescence spectra data were corrected in 50 mM phosphate buffer containing 1% dimethylformamide (DMF) at pH 7.4. The molar absorptivity was calculated by fitting the absorption spectra of a serial dilution of dye solutions with the Beer–Lambert equation. The fluorescence quantum yield of totalROX and Ox670 was measured per the reported literature with coumarin 15349 and Cy550 as a reference. Fluorescence titration studies were conducted by adding various reactive species stock solution with different concentrations to totalROX solution. Results and Discussion Spectral studies The UV–vis absorption and emission spectra of totalROX were acquired in phosphate buffer (50 mM, pH = 7.4) containing 1% DMF. It had a broad absorption band (350–540 nm) peaking at 425 nm (ɛ = 5100 cm−1 M−1). A green emission with a maximum at 537 nm (φ = 0.34) was measured upon excitation at 425 nm. Into a solution of totalROX (5 μM, neutral phosphate buffer) was added an aliquot of hypochlorite (ClO−) and the solution turned light aqua-blue (λabs = 650 nm, ɛ = 42,600 cm−1 M−1). Excitation of the solution at 650 nm led to a deep-red emission band (λem = 670 nm, φ = 0.40). The detection product was characterized to be Ox670 ( Supporting Information Figures S1–S11). The NMR spectra showed that the MeOH analogue of Ox670 was isolated ( Supporting Information Figures S37–S39) due to the use of MeOH as a cosolvent for chromatographic isolation. Since both totalROX and Ox670 are fluorescent, a ratiometric signal is attainable. In addition, their absorption/emission maxima are widely separated by ca. 225/150 nm, respectively, highlighting the strength of the “bridging-group modification” approach for ratiometric probes with minimal probe-product crosstalk. The oxidative potential of totalROX was 0.24 V in CH3CN ( Supporting Information Figure S12). The UV–vis absorption and fluorescence titrations were carried out to study the reactivity of totalROX toward various oxidative substrates implicated in oxidative damages, including O2•−, H2O2, nitric oxide (NO) in the absence/presence of O2, the conjugate acid/base pairs of peroxynitrous acid/peroxynitrite (HOONO/ONOO−), hypochlorous acid/hypochlorite (HOCl/ClO−), HO•, and 1O2 (Figure 3). A stock solution of KO2 in anhydrous DMF was added into totalROX (5 μM) in phosphate buffer (50 mM and pH = 7.4) with 1% DMF. The spectral changes were not obvious even though the dose of O2•− was as high as 100 eq (Figures 3a1–3a5). This result was anticipated since O2•− is reductive rather than oxidative.51 The decrease of the absorption band of the probe at 425 nm and the minor increase of the product absorption band at 650 nm was not caused by O2•−, but by HOO•, which is a potent 1 e− oxidizing agent. H2O2 (up to 100 eq) was added to totalROX and reacted quite sluggishly (Figures 3b1–3b5). In 500 s, no noticeable spectral changes were observed. NO did not react with totalROX in the absence of O2 ( Supporting Information Figure S14). This is desirable since NO is not oxidative and does not contribute to total cellular oxidative capacity. In contrast, the addition of NO under aerobic conditions quickly induced a dramatic signal change in less than 60 s (Figures 3c1–3c5). The absorption/emission of the detection product rose abruptly, while the absorption/emission of totalROX decreased. It took approximately 4 eq of NO to fully activate the signal. Likely, N2O3 is the NO+ species accounting for the oxidation of the probe in this scenario. We then tested the reactivity of OONO− and ClO− toward totalROX at different pH. First, they did not oxidize the probe in aqueous buffer at a high pH value of 12 ( Supporting Information Figure S22). At pH = 12, both OONO− (pKa = 6.8) and ClO− (pKa = 7.5) predominantly exist as anions and only a negligible amount is in their oxidative conjugative acid form, that is, HOONO and HOCl, respectively. However, at neutral pH of 7.4, addition of OONO− (Figures 3d1–3d5) and OCl− (Figures 3e1–3e5) instantaneously oxidized totalROX. Obviously, the conjugative acids oxidized the probe. Into a solution of totalROX and H2O2 (100 μM) was added an aliquot of Fe(ClO4)2 in H2O. The •OH generated in situ via the well-known Fenton mechanism quickly oxidized totalROX (Figures 3f1–3f5). Irradiation of totalROX with an infrared photosensitizer ECXf52 readily oxidized totalROX into Ox670. This confirmed the reactivity of totalROX toward 1O2 (Figures 3g1–3g5). The lower detection limits of totalROX toward HOONO, HOCl, and NO/O2 were estimated to be low nM range. Figure 3 | Spectral titration data of totalROX (5 μM) by various oxidative species in phosphate buffer (50 mM at pH = 7.4) with 1% DMF as a cosolvent. Column 1: The UV–vis absorption titration with different dose of oxidative species. Column 2: The emission titration of the probe channel (totalROX, λex = 425 nm). Column 3: The emission titration of the product (Ox670, λex = 650 nm). Column 4: The change of the fluorescence intensity at 525 nm (blue) and 675 nm (red), respectively, with respect to the dose of oxidizing species. Column 5: The kinetic trace of formation of Ox670 upon addition of various oxidative species. Rows a–h are titration data of (a) O2•− at 300 eq, (b) H2O2 at 100 eq, (c) NO with air at 2.0 eq, (d) HOONO at 2.0 eq, (e) HClO at 2.0 eq, (f) HO•, and (g) 1O2, respectively. Download figure Download PowerPoint Comparisons with commercial probes Detection sensitivity, either in the form of the dissociation constant (KD) for equilibrium-based sensors or the limit of detection (LOD) for activity-based probes, is an important parameter to evaluate a probe’s overall performance and must be reliably acquired for substrates, whose steady-state concentration does not decay via any other mechanism but the reaction with the probe. However, this property cannot be obtained with the highly oxidative species, whose presence is usually transient. We adopted an alternative method to qualitatively assess the probe detection sensitivity: a side-by-side comparison with two known probes, Amplex Red with horseradish peroxidase (HRP) and CellROX Deep Red. Xanthine oxidase with xanthine (XO/X) was the standardized source of oxidative species. It is known that O2•− is generated by XO/X and acts as a reductant instead of an oxidant. However, we emphasize that O2•− in the presence of XO is oxidative because O2•− coordinates to the metal centers of XO to afford a metal-based peroxyl radical, which is highly oxidative and elicits 1 e− oxidative chemistry. TotalROX in phosphate buffer was mixed with XO (0.4 mU) and xanthine (0.05 mM), and the ratio between the fluorescence intensities of the oxidized product ( Ox670, λex/λem = 640/680 nm) and the probe ( totalROX, λex/λem = 425/537 nm) was collected by a plate reader (Figure 4a). The fluorescence turn-on ratio from totalROX was higher when a smaller dose of totalROX was used (1 or 5 μM), compared to the fluorescence turn-on ratio obtained with the use of a higher dose of totalROX at 10 or 50 μM. This seemingly counterintuitive result is rationalized by the two-step oxidation mechanism of totalROX by radical species ( Supporting Information Figure S1). TotalROX is oxidized into a radical intermediate by the first radical. Presumably, this intermediate exhibits a higher oxidative potential than totalROX, and therefore, its further oxidation to the final Ox670 by a second radical is not favored when there is a large excess of totalROX. In comparison, fluorescence enhancement with the Amplex Red/HRP system showed a good correlation with Amplex Red in the range of 1–10 μM (Figure 4b). This experiment qualitatively delineated the high sensitivity of totalROX toward highly reactive radical species compared with an existing commercial probe (Amplex Red) for oxidative species. Then, a mixture of totalROX (10 μM) and xanthine (0.05 mM) was titrated with XO, and the fluorescence turn-on (Figure 4c) was found to be in excellent agreement with the results from Amplex Red/HPR (Figure 4d), confirming the potential of totalROX for quantitative studies. We also found that the presence of superoxide dismutase (SOD) eliminated the fluorescence turn-on by disproportionating superoxide with a high kinetic rate (1 × 109 M−1 s−1) proving that totalROX is oxidized by metal peroxyl radical (Figure 4e). In comparison, SOD did not inhibit but slightly promoted the fluorescence turn-on from Amplex Red/HRP (Figure 4f). CellROX Deep Red was another reference probe chosen for comparison. CellROX Deep Red and totalROX have almost identical absorption/emission maxima upon oxidation facilitating a side-by-side comparison under the same microscopic setup. We noted that totalROX exhibited higher fluorescence intensity (Figure 4g) and higher resistance toward photobleaching (Figure 4h). Figure 4 | (a) Fluorescence turn-on ratio of totalROX (1, 5, 10, or 50 μM) incubated with XO/X (0.4 mU/0.05 mM) for 15 min. (b) Fluorescence intensity of Amplex Red (1, 5, 10, or 50 mM) and HRP (100 mU) incubated with XO/X (0.4 mU/0.05 mM). (c) Fluorescence turn-on ratio of totalROX (10 μM) incubated with XO/X for 15 min. (d) Fluorescence intensity of Amplex Red (10 μM) and HRP (100 mU) incubated with XO/X. (e) Fluorescence turn-on ratio of totalROX (10 μM), XO (0.4 mU), and xanthine (0.05 mM) in the absence and presence of SOD (50 mU) for 15 min, respectively. (f) Fluorescence intensity of Amplex Red/HRP in the absence and presence of SOD (50 mU) for 15 min, respectively. (g) Average fluorescence intensity of cells treated with totalROX and CellROX Deep Red. (h) Photobleaching kinetics of cellular totalROX and CellROX Deep Red. (i) Subcellular localization of totalROX. The epi-fluorescence images of Ox670 (λex/λem = 620 ± 30/700 ± 38 nm) with MitoTracker Green (λex/λem = 480 ± 20/527 ± 15 nm), LysoTracker Green (λex/λem = 480 ± 20/527 ± 15 nm), and PEX14:EGFP (λex/λem = 480 ± 20/527 ± 15 nm). Scale bar = 5 μm. (j) Ratiometric confocal cell imaging of HCT116 cells treated by menadione (50 μM) with totalROX. Scale bar: 10 μm. **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint In vitro ratiometric fluorescence imaging The cytotoxicity of totalROX was assessed by incubating human colon cancer cells (HCT116) with up to 40 μM of probe for 24 h. TotalROX was readily membrane-permeable. The MTT assay yielded a viability of over 95% ( Supporting Information Figure S23) suggesting totalROX was not cytotoxic up to 40 μM. Subcellular localization of the totalROX was investigated. TotalROX (5 μM) was first incubated with HeLa cells for 2 h, during which time it was partially oxidized. A colocalization study with MitoTracker Green, LysoTracker Green, and a peroxisomal membrane protein (PEX14) fused with enhanced green fluorescent protein (EGFP) revealed that totalROX predominantly localized in mitochondria (Figure 4i). Menadione is known to enhance the cell oxidative capacity.53 We investigated the fluorescence change of totalROX in menadione-induced cellular oxidative stress. HCT116 cells were incubated with totalROX (10 μM) for 30 min, and then rinsed with buffer. The fluorescence cell images of totalROX via the green channel (λex = 488 nm, λem = 530 ± 20 nm) and Ox670 via the red channel (λex = 640 nm, λem = 670 ± 15 nm) were acquired. The green fluorescence of the probe channel was strong (Figure 4j, top), and the near-infrared (NIR) emission of the product channel was negligible (Figure 4j, middle). The ratio of the two channels was calculated, and the values were typically low except for some hotspots. Then, menadione (50 μM) was added to the cell culture to trigger the generation of oxidative species, and cell images of both channels were collected every 60 s. Over the course of 14 min, the fluorescence intensity of the probe channel steadily decreased and that of the product channel steadily increased (Figure 4j, bottom). The ratio images of the two channels also exhibited an obvious increase of total cellular oxidative capacity upon treatment with menadione. We employed CellROX Deep Red Reagent to verify the menadione-induced rise of oxidative stress in HCT116 cells by flow cytometry ( Supporting Information Figure S24). To further exhibit the potentials of totalROX for imaging cellular oxidative status, we also used totalROX to monitor the rise of oxidative stress in hepatocytes (PH5CH8 cells) treated with acetaminophen (APAP) ( Supporting Information Figure S25) and HCT116 cells treated with cisplatin ( Supporting Information Figure S14). The rise of oxidative stress levels in both these two cell models were also verified by CellROX Deep Red, in good agreement with their literature pharmacology of acetaminophen and cisplatin. Conclusion We have devised a novel design principle for ratiometric probes, via modulating the electron-donating capability of the bridging group of xanthenoid dyes. TotalROX is the first probe that is readily oxidized by all four groups of biorelevant oxidizing species, including 2 e−, 1 e−, NO+ species, and 1O2, but not by superoxide radical anion, hydrogen peroxide, NO, hypohalides, and peroxynitrite. Hence, totalROX is a superior probe to monitor the total cellular oxidative capacity. Importantly, totalROX is ratiometric, and the absorption/emission maxima of the detection product ( Ox670) red-shifted by 225/150 nm, respectively, to completely avoid the probe-product crosstalk. The detection mechanism was thoroughly investigated via mass spectrometry. TotalROX exhibits higher detection sensitivity and photostability than two commercial probes. TotalROX was showcased to readily respond to the rise of oxidative stress in human colorectal tumor cells (HCT116) and hepatocytes cells (PH5CH8) treated with menadione or cisplatin. We expect totalROX to find practical applications in cell-based biological studies. Future endeavors of this project will focus on development of organelle- or protein-targeting analogs. Supporting Information Supporting Information is available. Conflict of Interest There is no conflict of interest to report. Funding Information This work is supported by the National Natural Science Foundation of China (nos. 21822805, 21908065, 31871430, 81802979, 818800585, and 8180032537), Young Medical Talents Training Program of Shanghai (2018), the Commission of Science and Technology of Shanghai Municipality (no. 18430711000), the Australian National Health and Medical Research Council (nos. APP1126091 and APP1125794), the Science and Technology Commission of Shanghai Municipality for the Shanghai International Cooperation Program (18430711000), and the China Postdoctoral Science Foundation (nos. 2019M651427 and 2020T130197), W. Yang acknowledges the financial support of the innovative research team of high-level local universities in Shanghai. References 1. Calabrese V.; Cornelius C.; Mancuso C.; Lentile R.; Stella A. M.; Butterfield D. A.Redox Homeostasis and Cellular Stress Response in Aging and Neurodegeneration.Methods Mol. Biol.2010, 610, 285–308. Google Scholar 2. Ray P. 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