Fluorescent probes for <i>in vitro</i> and <i>in vivo</i> quantification of hydrogen peroxide
Hydrogen peroxide (H 2 O 2 ) plays essential roles in redox signaling and oxidative stress, and its dynamic concentration is critical to human health and diseases. Here we report the design, syntheses, and biological applications of HKPerox-Red and HKPerox-Ratio for quantitative measurement of H 2 O 2 . Both probes were successfully applied to detect endogenous H 2 O 2 fluxes in living cells or zebrafish, and biological effects of multiple stress inducers including rotenone, arsenic trioxide, and starvation were investigated. As H 2 O 2 is a common by-product for oxidase oxidation, a general assay was developed for ultrasensitive detection of various metabolites (glucose, uric acid, and sarcosine). Moreover, cellular H 2 O 2 measurements were achieved for the first time by combining flow cytometry with live cell calibration. This study provides a pair of unique molecular tools for advanced H 2 O 2 bio-imaging and assay development.
Introduction<!>Design and synthesis of HKPerox-Red and HKPerox-ratio<!>View Article Online<!>New assay development based on HKPerox-Red<!>Ratiometric detection of H 2 O 2 burst in As 2 O 3 treated leukaemia cells by using ow cytometry<!>Ratiometric uorescence imaging of starvation induced H 2 O 2 in living cells<!>Quantitative measurement of cellular H 2 O 2 by ow cytometry with live cell calibration<!>Conclusions
<p>Oxygen fuels energy production processes in aerobic organisms through respiration, while partial reduction of oxygen produces reactive oxygen species (ROS) as by-products of oxygen metabolism. 1 Among all ROS, hydrogen peroxide (H 2 O 2 ) stands out as the most signicant messenger molecule for redox signaling due to its relatively long half-life and diffusion range. 2 Cellular H 2 O 2 is constantly produced with a steady-state concentration ($0.1 mM) to signal cell growth and proliferation, whereas higher levels of H 2 O 2 (e.g. $100 mM) trigger cell growth arrest or apoptosis. 2b,3 Quantitative analysis of H 2 O 2 in biological content is key to understanding its multiple roles in redox signaling and oxidative stress. However, there is no method currently available for non-invasive, sensitive, and precise measurement of H 2 O 2 in intact cells, and previous H 2 O 2 quantications relied heavily on electrochemical analysis or peroxidase-based H 2 O 2 assay, where only extracellular H 2 O 2 secreted from cells could be measured accurately. 4 A powerful tool to investigate cellular H 2 O 2 uxes is uorimetry (e.g. confocal imaging and ow cytometry) due to its high sensitivity, non-invasiveness and spatio-temporal resolution. 5,6 While several genetically encoded H 2 O 2 sensors have been developed for H 2 O 2 detection with unparalleled dynamic subcellular precision, they may suffer from pH sensitivity, poor brightness, limited responses and selectivity toward H 2 O 2 . 7 Nearly all chemosensors for H 2 O 2 detection are based on smallmolecule dyes, including traditional dichloro-dihydro-uorescein diacetate (DCFH-DA, a commonly used unselective ROS probe) and selective probes, especially activity-based sensing, 5c including boronate oxidation, 5a,8 Baeyer-Villiger reaction, 5b,9 tandem Payne/Dakin reaction, 10 and very recently Mislow-Evans rearrangement. 11 Those intensity-based probes are very sensitive, but they are prone to signal uctuations arising from variations in probe uptake, distribution, or even optical inputs. Therefore, ratiometric sensing with an internal standard for calibration is more desirable for quantitative measurement of H 2 O 2 , 12 however, most of current ratiomeric probes (including both biosensors and chemosensors) still suffer from low selectivity, low sensitivity, as well as slow response, thereby limited biological applications. 7,13 To address those challenges, we herein report a pair of new uorescent probes, HKPerox-Red and HKPerox-Ratio (Scheme 1). Both probes feature excellent selectivity and sensitivity for unambiguous H 2 O 2 detection and quantication in aqueous solution. Moreover, the red emissive HKPerox-Red allows imaging endogenous H 2 O 2 uxes in living cells and zebrash embryos, by virtue of its outstanding permeability. HKPerox-Red was further utilized to develop a general in vitro assay for ultrasensitive detection of various biomarkers and respective oxidases (glucose/glucose oxidase, uric acid/urate oxidase, sarcosine/sarcosine oxidase), since H 2 O 2 is quantatitively produced during those enzymatically catalyzed oxidation reactions. As a result of its nanomolar sensitivity, HKPerox-Red could be used for accurate measurement of glucose with 1000 times diluted serum (1 mL to 1 mL), which provides a new method for non-invasive glucose detection with high precision.</p><p>On the other hand, our novel ratiometric probe HKPerox-Ratio has been successfully applied to detect H 2 O 2 burst induced by arsenic trioxide (As 2 O 3 ) in multiple leukaemia cell lines. For the rst time, intracellular H 2 O 2 in response to starvation is visualized and quantied with HKPerox-Ratio by using both confocal microscopy and ow cytometry, establishing a useful protocol for H 2 O 2 ratiometric analysis with spatio-temporal precision. Our new data conrm the value of HKPerox series for advanced bio-imaging analysis (in vivo imaging, ratiometric imaging, and ow cytometry analysis) as well as the development of ultrasensitive chemical tools to interrogate H 2 O 2related physiology and pathology.</p><!><p>Our general strategy on developing red emissive and ratiometric H 2 O 2 probes is based on modulation of internal charge transfer (ICT) of uorophores that could be deprotected upon H 2 O 2 mediated Payne/Dakin reaction (activity-based sensing). As shown in Schemes 1 and 2a, an electron-withdrawing carbamate sensing moiety could efficiently interrupt the ICT process in the resorun derivative, making HKPerox-Red essentially non-uorescent. However, aer H 2 O 2 -mediated deprotection, the remaining electron-donating 7-methylamino group could restore the push-pull conjugating system in 3H-phenoxazin-3one scaffold, releasing a bright red emission. Similarly, HKPerox-Ratio was designed by modulating ICT on a biocompatible 4-amino-1,8-naphthalimide platform (see Scheme 2b): 12a,14 before the treatment of H 2 O 2 , the electron-withdrawing carbamate linker on the 4-position of 1,8-naphthalimide disrupts the ICT process to produce a blue emission; upon H 2 O 2 mediated deprotection, the carbamate linker is efficiently cleaved to release the electron-donating amine, and the ICT process is restored to provide a bright green emission.</p><p>Two general synthetic routes were envisioned to prepare HKPerox-Red and HKPerox-Ratio: (1) a general coupling reaction between halogen-or triate-substituted uorophores and the carbamate sensing moiety; (2) a general addition reaction between isocyanate-substituted uorophores and the benzyl alcohol sensing moiety. Specically, HKPerox-Red was synthesized from commercially available resorun in three steps: tri-ation, a cross-coupling between triate 1 and carbamate 2, and deprotection. HKPerox-Ratio was synthesized according to another approach (Scheme 2d) for amine-containing uorophore: 4-amino group of 1,8-naphthalimide uorophore was rst converted to an isocyanate by treatment with triphosgene, and the in situ generated isocyanate readily reacted with the benzyl alcohol sensing moiety 4 to form carbamate 5, which was deprotected to afford HKPerox-Ratio.</p><p>Reactivity and selectivity of HKPerox-Red and HKPerox-Ratio toward H 2 O 2 With both probes in hand, we rst investigated the spectroscopic properties of HKPerox-Red and HKPerox-Ratio in potassium phosphate buffer (0.1 M, pH 7.4, 100 mM CCl 3 CN). Due to its excellent water-solubility, both probes could form a homogenous solution in the buffer without additional cosolvent. As shown in Fig. 1a, an obvious absorption peak of HKPerox-Red was observed at 580 nm aer H 2 O 2 treatment. At the same time, a bright red emission at 602 nm appeared with increasing amounts of H 2 O 2 in a dose-dependent manner (0-100 mM). The resulting uorescent product was detected as a resorun derivative by UPLC-MS analysis (Scheme 2a and ESI †), which conrms that tandem Payne/Dakin reaction was successfully extended to this scaffold. As shown in Fig. 1c, the uorescence intensity at 602 nm was linearly correlated with H 2 O 2 concentration ranging from 0 to 30 mM. The detection limit was estimated to be as low as 4.8 nM (3s/k) with this standard calibration curve, which could be used as an ultrasensitive assay for H 2 O 2 quantication. The major challenges for H 2 O 2 sensors are actually sensitivity and selectivity: the reactivity of H 2 O 2 is milder than those highly reactive species, thus competing responses from other ROS seem to be inevitable with previously reported strategies. However, HKPerox-Red is highly sensitive toward H 2 O 2 , and a >150-fold enhancement in uorescence intensity was observed upon treatment with H 2 O 2 , while other potential competing ROS/RNS including 1 O 2 , ROOc, TBHP, NO, O 2 c À , $OH, ONOO À , and HOCl, only triggered negligible changes (Fig. 1d).</p><p>In addition, reaction kinetics of HKPerox-Red were investigated. Time courses of uorescence intensities of HKPerox-Red (10 mM) at 602 nm upon treatment with 100 mM H 2 O 2 were recorded. The reaction was almost completed within 10 min (Fig. S1, ESI †), meaning that a short period of incubation is enough to trigger a signicant uorescence response. At pH 8.0, this reaction could be further accelerated, which is consistent with the mechanism of tandem Payne/Dakin reaction. The reaction rate constant was calculated to be 1.3 Â 10 À2 s À1 , based on a pseudo rst order model by mixing 1 mM HKPerox-Red with 1 mM H 2 O 2 (Fig. 1e and f).</p><p>As shown in Fig. 2a, the absorption peak at 375 nm of HKPerox-Ratio was shied to 425 nm upon H 2 O 2 oxidation, which is consistent with restoration of ICT process in this ratiometric probe. 12a,14 To our delight, subsequent uoresence measurments revealed an isosbestic point on the uorescence spectra of HKPerox-Ratio (Fig. 2b), indicating its clean conversion. The green uorescent compound was also conrmed by UPLC-MS analysis to be the cleavage product 4-amino-1,8naphthalimide (Scheme 2b and ESI †). The ratios of uorescence emission intensities at 540 nm and 475 nm (F 540 /F 475 ) were proportional to H 2 O 2 concentrations ranging from 0 to 100 mM. The selectivity of HKPerox-Ratio was also tested, which exhibited a 12.7-fold increase in F 540 /F 475 toward H 2 O 2, while other ROS/RNS gave negligible ratio changes (Fig. 2d). This reaction also proceeded rapidly to complete in 30 min (Fig. S2, ESI †). Collectively, these results demonstrate excellent watersolubility, sensitivity, selectivity of HKPerox-Ratio toward H 2 O 2 .</p><p>Encouraged by their excellent performances toward H 2 O 2 in chemical system, we further performed biological evaluations of HKPerox-Red and HKPerox-Ratio in living cells. The cytotoxicities of HKPerox-Red and HKPerox-Ratio were assessed in RAW264.7 macrophages, and no obvious toxicity was observed up to 20 mM aer 24 h incubation (Fig. S3 and S4, ESI †).</p><!><p>Molecular imaging of endogenous H 2 O 2 production in live cells and in vivo using HKPerox-Red</p><p>We went on to apply HKPerox-Red in live cell imaging to detect endogenous H 2 O 2 production with confocal microscopy. RAW264.7 macrophages were co-incubated with HKPerox-Red (4 mM) in Hank's Balanced Salt Solution (HBSS) for 30 min before imaging. As expected, the uorescence signal in PMA treated cells was signicantly stronger than that of untreated cells (Fig. 3a). This PMA induced H 2 O 2 burst could be effectively inhibited by the addition of NADPH oxidase (NOX) inhibitor DPI, and quantications based on imaging showed a clear 7fold enhancement of uorescence intensities (Fig. 3b). Endogenous H 2 O 2 bursts can be robustly visualized in stimulated macrophages with HKPerox-Red in a selective manner.</p><p>We next explored the applications of HKPerox-Red to in vivo imaging of zebrash. To perform molecular imaging of H 2 O 2 produced in living zebrash, the desirable probe should be both lipophilic and water-soluble to penetrate through sh skin by simple co-incubation. HKPerox-Red, our resorun-derived H 2 O 2 probe, featuring small-molecular weight, good lipophilicity, excellent water-solubility, and most importantly, ultrasensitivity, is very promising for such study.</p><p>In our initial study, zebrash embryos 6 hours post fertilization (hpf) were acquired to co-incubate with HKPerox-Red (including 100 mM CCl 3 CN in 1 mL E3 buffer) or HKPerox-2, our previous rhodol-base probe, 10a at room temperature for 30 min to monitor zebrash development process. Endogenous H 2 O 2 produced during cell proliferation was clearly visualized by HKPerox-Red since the red uorescence was detected inside zebrash embryo (Fig. 4), while there was no uorescence signal in the yolk part of the fertilized egg or outside of the embryo. In contrast, HKPerox-2 could not penetrate through the embryo membrane via co-incubation, and the resulting uorescence signals were only found outside the embryo. Thus, HKPerox-Red could efficiently stain zebrash embryo by simple coincubation, and H 2 O 2 production during cell proliferation in early stage of embryo development was visualized.</p><p>Rotenone is a widely used pesticide that interferes with the electron transport chain during mitochondrial respiration. Previous study reveals that long term exposure to rotenone is associated with higher risk of Parkinson's disease in farm workers, 15 while their models for rotenone challenging mainly focused on isolated mitochondria or cell lines, 16 and the imaging reagents used to quantify ROS burst were traditional unselective probes. Thus, we applied HKPerox-Red to evaluate the effect of rotenone on zebrash embryos. 72 hpf zebrash embryos were incubated with HKPerox-Red (20 mM) for 20 min at room temperature, then challenged with or without rotenone (50 mM) in E3 buffer supplemented with 100 mM CCl 3 CN for 15 min. Basal H 2 O 2 production and distribution could be visualized as a weak uorescence signal (Fig. 4c). However, this signal was signicantly stronger in rotenone treated zebrash, which indicates acute H 2 O 2 burst in living embryos. Distributions of H 2 O 2 burst could be found in the brain and gut regions of zebrash embryos, which may further contribute to oxidative damage of proteins and the initiation of neurodegenerative diseases.</p><!><p>As H 2 O 2 is a biologically important oxidizing reagent, its production and scavenging proles are of great interest in health products, nutrition, and food industry. 17 Moreover, H 2 O 2 is one of the most common products of enzyme catalyzed oxidations, thus a series of metabolites including glucose, cholesterol, uric acid, and sarcosine could be converted to H 2 O 2 quantitively with corresponding oxidases or enzyme mimetics. 18 For example, in Amplex Red Glucose/Glucose Oxidase assay, glucose could be oxidized to glucuronolactone in the presence  of glucose oxidase and oxygen, and one equivalent of H 2 O 2 is produced and quantied by Amplex Red in this process. However, this resulting uorescence could be bleached by extra amounts of H 2 O 2 in presence of horseradish peroxidase (HRP), and Amplex Red is also vulnerable to photo oxidation. 6f,19 Similar to Amplex Red in both excitation and emission wavelengths, HKPerox-Red can be a novel molecular tool compatible with current assays using Amplex Red, yet it is more stable, sensitive, and HRP independent. In HKPerox-Red glucose assay, various amounts of glucose (0-40 mM, nal concentrations) were added into potassium phosphate buffer, and then 1 U mL À1 glucose oxidase and HKPerox-Red (10 mM; with 100 mM CCl 3 CN) were added subsequently into the solution. Resulting solutions were incubated for 30 min at 37 C before uorescence measurement. As shown in Fig. 5a, H 2 O 2 produced by glucose oxidation could be sensitively detected with HKPerox-Red, and a relationship between glucose concentrations and uorescence emissions at 602 nm was observed. The detection limit of glucose was calculated to be as low as 34 nM (3s/k) according to the calibration curve.</p><p>To prove the practical applications of this novel assay, nondiabetic urine and serum samples were analyzed by HKPerox-Red glucose assay, and only 1 mL serum is needed due to the ultra-sensitivity. In this experiment, 1 mL deproteinized serum was diluted with 1 mL potassium phosphate buffer (pH 7.4, 0.5% DMF) containing 1 U mL À1 glucose oxidase, 10 mM HKPerox-Red and 100 mM CCl 3 CN. Quantications were based on serum samples spiked with 3 mM glucose as internal standard, and the glucose concentration in serum sample was quantied as 4.38 AE 0.32 mM. Therefore, with tiny amount of sample, HKPerox-Red holds great potential to measure serum glucose in a fast, accurate, and high-throughput assay. The ultrasensitivity also makes it possible to conduct non-invasive glucose evaluation with samples like sweat, saliva or tears. More importantly, HKPerox-Red enables a general assay for biologically important metabolites detection with suitable oxidases. As a proof of concept, uric acid/urate oxidase assay and sarcosine/sarcosine oxidase assay were developed by using HKPerox-Red (Fig. 5c and d), which provide practical methods for disease diagnosis based on metabolite screening. A clinical study with HKPerox-Red is on-going to use urinary sarcosine as potential biomarker for prostate cancer diagnosis.</p><!><p>Next, we sought to explore ratiometric H 2 O 2 sensing with our HKPerox-Ratio. Although some H 2 O 2 ratiometric uorescent probes have been developed for this purpose, very few of them are sensitive enough to be used in ow cytometry, and none of them is suitable for quantifying endogenous H 2 O 2 in living cells. 13 We rstly applied HKPerox-Ratio in ow cytometry to detect H 2 O 2 uxes in leukemia cells challenged with As 2 O 3 , a very effective approach to treat acute promyelocytic leukemia. 20 As leukemia cells are suspension cells, they are not suitable for confocal imaging; however, ow cytometry enables quantitative analysis of a large population of suspension cells rapidly. Moreover, with a suitable ratiometric probe, multiple uoresence emissions could be detected simultaneously by ow cytometry, 21 and the green to blue emission ratio (F green /F blue ) reported by HKPerox-Ratio is expected to be a more reliable ratiometric indicator of cellular H 2 O 2 .</p><p>For this purpose, U-937, NB4 and THP-1 leukemia cells were selected for the treatment with As 2 O 3 for 24 h, and then untreated cells and As 2 O 3 challenged cells were co-incubated with HKPerox-Ratio (10 mM) for 30 min before ow cytometry analysis. Over 10 000 cells were analysed, and F green /F blue in each individual cell was calculated by a FlowJo soware. As shown in Fig. 6a-c, As 2 O 3 -treated leukemia cells could be clearly distinguished from those untreated cells by F green /F blue ratio (xaxis), which correlates with H 2 O 2 levels. Further quantications in Fig. 6d showed a signicant enhancement in F green /F blue ratio in all three cell lines (1.9-fold for U-937, 1.7-fold for NB4, and 2.8-fold for THP-1), indicating a robust cellular H 2 O 2 ux. This As 2 O 3 -challenged H 2 O 2 burst may be responsible for differentiation or apoptosis of leukemia cells. 22 To conclude, HKPerox-Ratio is a promising probe to detect H 2 O 2 uxes via ow cytometry in a ratiometric manner, which provides an ideal platform to screen novel apoptosis inducer for leukemia therapy.</p><!><p>As H 2 O 2 is believed to be an important mediator during autophagy in response to nutrient shortage, 23 we would like to identify and quantify endogenous H 2 O 2 production during this process with ratiometric probe HKPerox-Ratio. RAW264.7 macrophages were co-incubated with HKPerox-Ratio (5 mM) in were collected simultaneously with an excitation at 405 nm (Fig. 7a and b). As expected, the blue emission was signicantly stronger than the green emission in untreated cells, and the merged Fig. 7c showed the predominant blue emission. In Fig. 7d, a heatmap of green-to-blue emission ratio (F green /F blue ) was generated by a Ratio Plus plugin in ImageJ soware. Basal H 2 O 2 concentrations and distributions were reported as a small value of F green /F blue ($0.2), which is consistent with low H 2 O 2 levels in resting cells. To mimic starvation induced stress, RAW264.7 cells were incubated HBSS (protein free) for 24 h, and then confocal images were taken under the same optical settings. Upon starvation challenge, blue emission in cells decreased signicantly, while green emission increased due to H 2 O 2 burst (Fig. 7e and f). The merged image in Fig. 7g showed a predominant green emission, and the ratiometric heatmap (Fig. 7h) conrmed a signicant increase of F green /F blue ($1.0) inside the cells. Quantication based on confocal images was performed to show a clear 5-fold overall increase of F green /F blue in starvationchallenged cells compared to resting cells (Fig. 7i), which demonstrated the excellent sensitivity of HKPerox-Ratio in cell imaging. The green signal and blue signal overlapped well with an overlap coefficient of 0.85 (Fig. S5, ESI †), which shows a good co-localization of HKPerox-Ratio and its cleavage product in cells. Collectively, those data demonstrate that HKPerox-Ratio can be robustly applied in ratiometric imaging of endogenous H 2 O 2 uxes with spatio-temporal precision.</p><!><p>Calibration curves of previous ratiometric probes were largely established in a cell-free solvent/buffer mixture, which may not be applicable for precise quantication of cellular H 2 O 2 . 13 Therefore, we further applied HKPerox-Ratio in ow cytometry for quantitative measurement of H 2 O 2 uxes during starvation. As shown in Fig. 8a and b, a calibration curve with excellent linearity (R 2 ¼ 0.993) was obtained by adding known amounts of H 2 O 2 (0, 10, 20, 40, and 80 mM H 2 O 2 ) into the probe solution and then co-incubating with cells for 30 min before ow cytometry measurement. The green to blue emission ratio (F green /F blue ) in each group of cells was analysed, and those cell populations could be distinguished in Fig. 8a. The detection limit of HKPerox-Ratio by ow cytometry measurement was estimated to be as low as 1.8 mM (3s/k) in this cellular assay. A standard curve (y ¼ 0.372 + 0.010x) with live cell calibration was thus established for RAW264.7 cells, enabling the direct measurement of intracellular H 2 O 2 concentrations.</p><p>As shown in Fig. 8c and d, starvation induced H 2 O 2 was detected and quantied with HKPerox-Ratio. Our data showed that 3 h starvation already induced a discernible increase in F green /F blue ratio, corresponding to 7.9 mM H 2 O 2 according to the calibration curve. This rapidly elevated cellular H 2 O 2 levels during starvation suggest that treatment of cells in protein-free buffers (such as HBSS and phosphate-buffered saline) may stimulate unexpected ROS burst, thus longtime incubation in those buffers should be avoided.</p><p>Moreover, H 2 O 2 uxes in living RAW264.7 cells reached its maximum concentration (34 mM) aer 14 h starvation, and longer period of starvation (e.g. 24 h) did not induce higher levels of H 2 O 2 . This endogenous H 2 O 2 burst is sufficient to signal subsequent pathological processes, such as growth arrest or apoptosis. 24  Due to variations of cellular environment, independent calibration curves should be obtained in different cell types before ratiometric H 2 O 2 quantication. We further applied this protocol to HeLa cells, and a new calibration curve with excellent linearity (y ¼ 0.606 + 0.013x, R 2 ¼ 0.988) was obtained with HKPerox-Ratio by using ow cytometry (Fig. S6, ESI †). Note that the calibration curve obtained by using a uorospectrometer in chemical system (Fig. 2c, y ¼ 0.670 + 0.053x) is very different from those obtained by using ow cytometry with live cell calibration. This result indicates that calibration curves established in a cell-free solvent/buffer mixture are not applicable to cellular H 2 O 2 measurement; however, with a suitable ratiometric probe, ow cytometry provides an ideal high-throughput platform for quantitative analysis. One potential risk from live cell calibration may arise from different cell permeabilities of HKPerox-Ratio and its cleavage product. A control experiment is recommended to co-stain the cells with both compounds to conrm their similar permeability (Fig. S7, ESI †). To the best of our knowledge, HKPerox-Ratio is the rst ratiometric probe that can be used to quantify cellular H 2 O 2 level by ow cytometry, which makes it an invaluable tool for not only qualitative detection, but also quantication in redox biology.</p><!><p>During past decades, great efforts have been made to develop novel H 2 O 2 sensors, and diverse roles of H 2 O 2 in mediating various biological processes have been uncovered. However, unambiguous quantitative analysis of H 2 O 2 in vitro and in vivo still represents a substantial challenge even to date. In this work, we have developed a pair of highly selective and sensitive uorescent probes for precise measurement of H 2 O 2 . Our unique strategy relies on tandem Payne/Dakin reaction, which provides a general and biocompatible strategy to distinguish H 2 O 2 from other potential competing ROS. HKPerox-Red and HKPerox-Ratio have been successfully applied to evaluate the effects of PMA, As 2 O 3 , rotenone, and starvation in various models.</p><p>In particular, HKPerox-Red with outstanding permeability could be used in zebrash imaging by simple co-incubation, and rotenone induced oxidative stress was also successfully visualized in vivo. Moreover, the outstanding performance of HKPerox-Red has been exploited to develop new assays for H 2 O 2 scavenging capacity or H 2 O 2 -based quantication for metabolites including glucose, uric acid and sarcosine, which are regarded as diagnostic biomarkers for diabetes, gout, and prostate cancer, respectively. The intrinsic sensitivity of our novel strategy ensures its robust application for in vitro quan-tication of H 2 O 2 and related metabolites.</p><p>Even more challenging in the redox biology eld is to measure intracellular H 2 O 2 accurately. In this study, we have developed HKPerox-Ratio, a small-molecule ratiometric probe, that allows quantitative detection of H 2 O 2 by measuring the ratio of the blue and green emission. HKPerox-Ratio could be used in various cell lines with simple co-incubation, and the signicant signal changes triggered by intracellular H 2 O 2 were robustly captured by both ow cytometry and confocal imaging. Therefore, As 2 O 3 -induced H 2 O 2 uxes in multiple leukemia cells were readily detected. HKPerox-Ratio further enables the rst quantitative detection of endogenously generated H 2 O 2 in living cells by using ow cytometry and live cell calibration curve.</p><p>We anticipate that both probes provide important molecular tools to study H 2 O 2 physiology and pathology, and more efforts are on-going to apply HKPerox series probes and tandem Payne/ Dakin reaction in advanced bio-imaging, drug screening, and disease diagnostics. View Article Online</p>