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A color-tailored fluorogenic sensor for hydrogen peroxide

Nature Chemical Biology (2025)Cite this article

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Abstract

H2O2 is an important signaling molecule and redox regulator of normal cellular metabolism and a major element of oxidative stress. Here we report HyPerFLEX (HyPer with flexible fluorogen excitation), a sensor from the HyPer family designed for high-precision H2O2 monitoring in living cells. HyPerFLEX combines the redox-sensitive OxyR domain from Neisseria meningitidis and circularly permuted fluorogenic protein Y-FAST, yielding oxygen-independent fluorescence upon oxidation of OxyR by H2O2. HyPerFLEX enables imaging H2O2 dynamics in living cells, with tunable spectra from green to far red for multicompartment imaging, even under prolonged hypoxia. It surpasses HyPer7 in detecting ultralow H2O2 concentrations, such as during early glucose-stimulated insulin production, and can measure H2O2 levels in the highly oxidizing endoplasmic reticulum lumen. These advanced features and broad compatibility make HyPerFLEX a powerful tool for studying oxidative stress and cellular signaling.

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Fig. 1: Design of HyPerFLEX.
Fig. 2: HyPerFLEX reacts specifically with H2O2 in the nanomolar range through a two-phase association model.
Fig. 3: Kinetics of HyPerFLEX H2O2 response and the roles of C121 and F130.
Fig. 4: HyPerFLEX performance in mammalian cells.
Fig. 5: Multisensor and multiparameter imaging of H2O2 in mammalian cells.
Fig. 6: Ratiometric two-photon imaging of chemogenetically induced H2O2 dynamics in acute brain slices using the HyPerFLEX-UnaG sensor.

Data availability

This paper does not report original code. Data are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This research was funded by the Russian Science Foundation (RSF), grant 23-75-30023 (V.V.B., D.I.B., E.S.P. and D.I.M.). Optimization and production of viral gene delivery systems were supported by the Center for Precision Genome Editing and Genetic Technologies for Biomedicine (V.V.B. grant 075-15-2019-1789 to Pirogov Russian National Research Medical University). Development of the multiphoton imaging approach was supported by RSF grant 22-72-10044 (A.S.C. and A.A.L.), the Development Program of Moscow State University and the National Project ‘Science and Universities’ (A.A.L. and A.B.F.). J.M. is supported by a VIB grant.

Author information

Author notes

  1. Valery V. Pak

    Present address: Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

  2. These authors contributed equally: Ekaterina S. Potekhina, Dina I. Bass, Daria Ezeriņa, Daria D. Fleckenstein.

Authors and Affiliations

  1. Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency, Moscow, Russia

    Ekaterina S. Potekhina, Dmitry I. Maltsev, Aleksandr A. Moshchenko & Vsevolod V. Belousov

  2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

    Ekaterina S. Potekhina, Dina I. Bass, Daria D. Fleckenstein, Vera A. Sysoeva, Dmitry I. Maltsev, Valery V. Pak, Anatolii I. Sokolov, Ivan N. Myasnyanko, Mikhail S. Baranov & Vsevolod V. Belousov

  3. Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia

    Ekaterina S. Potekhina, Dina I. Bass, Dmitry I. Maltsev & Vsevolod V. Belousov

  4. VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, Brussels, Belgium

    Daria Ezeriņa & Joris Messens

  5. Brussels Center for Redox Biology, Vrije Universiteit Brussel, Brussels, Belgium

    Daria Ezeriņa & Joris Messens

  6. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Daria Ezeriņa & Joris Messens

  7. Physics Department, Lomonosov Moscow State University, Moscow, Russia

    Artem S. Chebotarev, Andrei B. Fedotov & Aleksandr A. Lanin

  8. Laboratory of Medicinal Substances Chemistry, Institute of Translational Medicine, Pirogov Russian National Research Medical University, Moscow, Russia

    Anatolii I. Sokolov, Ivan N. Myasnyanko & Mikhail S. Baranov

  9. Russian Quantum Center, Moscow, Russia

    Andrei B. Fedotov

  10. Life Improvement by Future Technologies (LIFT) Center, Moscow, Russia

    Aleksei M. Zheltikov, Aleksandr A. Lanin & Vsevolod V. Belousov

Authors
  1. Ekaterina S. Potekhina
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  2. Dina I. Bass
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  3. Daria Ezeriņa
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  8. Valery V. Pak
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  17. Vsevolod V. Belousov
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Contributions

D.D.F., V.A.S. and V.V.P. designed and mutated the cpFAST and HyPerFLEX proteins and performed the preliminary HyPerFLEX in vitro characterization. E.S.P. and D.I.B. designed the cellular research, сreated and tested the cell0localized versions of HyPerFLEX, carried out the hypoxia experiment and multiparametric cellular imaging and analyzed the data from imaging. J.M. and D.E. designed and performed the in vitro experiments with purified HyPerFLEX (pH dependence, sensitivity and specificity), characterized the kinetic parameters of the reaction, investigated the H2O2-mediated oxidation mechanism and performed the structural predictions using AlphaFold2. M.S.B., I.N.M. and A.I.S. synthesized the fluorogens. A.S.C., A.B.F., A.M.Z. and A.A.L. performed the two-photon imaging and analyzed the data. A.A.M. carried out the lentivirus production. E.S.P. and D.I.M. performed the experiments with mice. V.V.B. directed the research. E.S.P., D.I.B., J.M., D.E., D.D.F., A.A.L. and V.V.B. wrote the paper.

Corresponding authors

Correspondence to Joris Messens or Vsevolod V. Belousov.

Ethics declarations

Competing interests

E.S.P., D.I.B. and V.V.B. have filed a patent application as inventors under the jurisdiction of the Russian Federation for the use of the nucleotide and amino acid sequences of the HyPerFLEX sensor (patent application 2024111459). The other authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks Marc Fransen and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 HyPerFLEX design and optimization.

Color coding: NmOxyR (blue), cpFAST (gray), linkers (pink), permutation linker (GTGG) and amino acids 98-100 (TLT) (orange).

Extended Data Fig. 2 HyPerFLEX reacts with Η2Ο2 in the lower nanomolar range.

a, Titration of HyPerFLEX with H2O2. The signal intensity, corresponding to the excitation maximum (Fig. 2a), was normalized to the intensity with no H2O2 and plotted against the log2 of the H2O2 concentration. Data were fitted to a sigmoidal dose-response curve, with the dashed line representing the 95% confidence interval. The R2 value, indicating the goodness of fit, is displayed. b, The linear part of the curves from Fig. 2c, used to estimate of the limit of detection (LOD) of HyPerFLEX in combination with each fluorogen. Data represent the mean of at least n = 3 experiments, with error bars indicating the standard error. The 95% confidence interval is indicated with dashed lines.

Source data

Extended Data Fig. 3 The kinetics of the H2O2 response of HyPerFLEX depend on the protonation state of Cys121, with Phe130 being essential for its functioning.

a, HyPerFLEX is a dimer, as determined by size exclusion chromatography. The elution profile and calibration curve are shown, with HyPerFLEX represented by the red point. b, The KD of fluorogen binding to HyPerFLEX is in the low micromolar range for all fluorogens, regardless of oxidation state. For each fluorogen, intensity at the excitation peak was normalized to the maximum and minimum intensity. KD and Hill coefficient (h) values were obtained by fitting the data to a “specific binding with Hill slope’ model. The mean +/- SEM of at least n = 3 independent experiments is shown. The 95% confidence interval is indicated with dashed lines. c, Anti-HyPer7 western blot for HyPerFLEX and its mutants, along with the corresponding post-transfer Ponceau stain. d, The F130A mutation does not affect the secondary structure of HyPerFLEX or structural changes upon oxidation. CD spectra of reduced and oxidized HyPerFLEX WT and F130A are shown. The data are the mean of n = 3 independent experiments. e, The F130A mutant displays a similar KD for WT HyPerFLEX for HBR-3,5-DOM. The intensity at the excitation peak was normalized to the maximum and minimum intensity. KD and Hill coefficient (h) values were obtained by fitting the data to a “specific binding with Hill slope’ model. The mean +/- SEM of n = 2 independent experiments is shown. The 95% confidence interval is indicated with dashed lines. f, Diagnostic residual plots from fitting the reaction curves of HyPerFLEX with Η2Ο2 to one-phase or two-phase exponential models (Fig. 3e, f). g, kobs values vs Η2Ο2 concentration obtained from fitting response curves of the HyPerFLEX C261S mutant to at least a 10x higher Η2Ο2 concentration. The mean of n = 3 independent experiments +/- SEM, is shown. h, Diagnostic residual plots from fitting the reaction curves of HyPerFLEX C261S mutant exposed to a 10x lower molar excess of Η2Ο2 to a one-phase exponential model (Fig. 2f).

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Extended Data Fig. 4 HyPerFLEX performance in mammalian cells.

a-c, Dynamics of HyPerFLEX response in HeLa cells to varying external H2O2 concentrations using fluorogens HBR-3,5-DOM (a), N871b (b), and 3a (c). d-f, Response dynamics in HeLa cells for HyPerFLEX variants in highly oxidative compartments. The HyPerFLEX response was measured in the intermembrane space of mitochondria (d), the cytosolic side of ER membrane (e), and luminal side of the ER membrane (f) after adding different H2O2 concentrations. N871b was used as the fluorogen. In all panels, mean values for n cells were normalized to baseline fluorescence, error bars indicate standard deviation, and n values are specified in the plot.

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Extended Data Fig. 5 Multisensor and multiparameter imaging of H2O2 in mammalian cells.

a, Single-color controls for HyperRed-DAO, HyPerFLEX(3a), and HyPer7 co-expression experiments. HeLa Kyoto cells expressing a single sensor were imaged using the same optical settings, exposure times, and fluorescence detection parameters as those used in the co-expression experiment shown in Figs. 5d and g. Each graph shows the mean and SD for 10 cells. b, Confocal fluorescent images of HeLa Kyoto co-expressing HyPerRed-DAO-ERlum and HyPerFLEX(3a)-ERlum. Representative images are shown. Scale bars: 50 μm. Right panel shows H2O2 detection by HyPerFLEX(3a)-ERlum in cells expressing HyPerRed-DAO-ERlum. Peroxide was formed by DAO after D-Ala addition. c, Fluorescent images of Hela Kyoto co-expressing HyPerRed-DAO-ERlum, HyPerFLEX(3a)-mito and HyPer7-NES. Representative images shown. Scale bars: 50 μm. H2O2 dynamics in various compartments of HeLa Kyoto cells expressing HyPerRed-DAO-ERlum, HyPer7-NES (green) and HyPerFLEX(3a)-mito (violet). Light green and light purple lines show the dynamics of individual cells. Dark lines show mean values. H2O2 production in the ER lumen was induced by D-Ala. Mean value of 15-18 cells shown. d, Confocal fluorescent images of MIN6 co-expressing jRCaMP1a and mitochondrial HyPerFLEX(3a)-mito or cytoplasmic HyPerFLEX(3a)-NES. Representative images shown. Scale bars: 50 μm. e, Single-color controls for HyPerFLEX(3a) and jRCaMP1a co-expression experiments. MIN6 cells expressing a single sensor were imaged using the same optical settings, exposure times, and fluorescence detection parameters as in the co-expression experiment shown in Figs. 5h, i. Each graph shows the mean and SD for 11 (red fluorescence) and 14 (far red fluorescence) cells. f, g H2O2 detection in the mitochondria and cytosol of pancreatic β-cells during glucose stimulation. Glucose stimulation induces an increase of H2O2 levels in the mitochondrial matrix (detected with HyPerFLEX(3a)-mito (f violet)) and cytosol (detected with HyPerFLEX(3a)-cyto (g violet)) of MIN6 mouse insulinoma β-cells. HyPer7 (green), co-transfected with an identical to HyPerFLEX localization signal, showed no change in peroxide levels. Cytosolic calcium increase, preceding insulin release, was detected using jRCaMP1a (red). Mean values and standard deviation (SD) for 15 cells. All imaging experiments represent one of three biological replicates. At the end of each experiment, 100 μM H2O2 was added to confirm HyPer probes functionality under experimental conditions.

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Extended Data Fig. 6 Design and characterization of a pseudoratiometric HyPerFLEX version.

a, Brightness of fluorescent proteins at 3-photon excitation (1250 nm). Fluorescence intensity of various fluorescent proteins expressed in HeLa cells under 3-photon excitation is shown. b, Fluorescence dynamics of HyPerFLEX-N871b and UnaG in HeLa cells after adding 7.5 μM H2O2 were measured for the HyPerFLEX-UnaG fusion construct and the original HyPerFLEX. Data represent n = 40–50 cells. c, Fluorescence ratio dynamics of HyPerFLEX-UnaG in HeLa cells after adding varying H2O2 concentrations, using N871b as the external fluorophore. Data represent n = 40–60 cells. d, Maximal response of HyPerFLEX-UnaG to increasing H2O2 concentrations in HeLa cells. Mean values normalized to baseline fluorescence. Data represent n = 40–60 cells. e, Multiphoton excitation fluorescence dynamics in HeLa cells for HyPerFLEX-UnaG and original HyPerFLEX (with 3a fluorogen) after adding 100 μM H2O2 under multiphoton excitation. Data represent n = 20–40 cells. f, HoloUnaG spectra: absorbance, fluorescence excitation, and emission spectra. Absorbance spectrum includes an additional peak at ~400 nm, most probably due to residual free bilirubin in the sample. In b–e, mean values for n cells are normalized to baseline fluorescence, error bars represent standard deviation (SD), and n values are specified in the plot.

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Extended Data Fig. 7 Ratiometric two-photon imaging of chemogenetically induced H2O2 dynamics in acute brain slices using the HyPerFLEX-UnaG sensor.

a, Two-photon excitation fluorescence ratiometric imaging of neurons ex vivo. Images of UnaG (green) and HyPerFLEX-HBR3,5-DOM (red) at depths of 50 µm, 100 µm, 150 µm, 200 µm, and 250 µm below the brain slice surface after fluorogen and D-norvaline addition. b, Depth dependence of two-photon-excited HyPerFLEX signal in brain tissue using HBR-3,5-DOM (orange triangles) and N871b (red circles) fluorogens plotted against tissue depth. The gray line represents typical exponential signal decay due to scattering. c, Intensity dynamics of the sensor’s pseudoratiometric signal. Changes in HBR-3,5-DOM (orange triangles) and UnaG (cyan boxes) fluorescence after fluorogen (fgn arrow) and D-Norvaline (D-Norval arrow) addition in a flow system. d, HyPerFLEX/UnaG fluorescence ratio in control brain slices. Mean (black solid lines) and individual (colored lines) responses of the HyPerFLEX(HBR-3,5-DOM)/UnaG fluorescence ratio at various depths. e, HyPerFLEX/UnaG fluorescence ratio in neurons. Mean (black solid lines) and individual (colored lines) responses from neurons in three brain slices. f, Two-photon excitation fluorescence images. Representative images from the UnaG and HyPerFLEX channels before and after HBR-3,5-DOM fluorogen addition. All images and data represent typical experimental results.

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Extended Data Table 1 Optical properties of reduced and oxidized HyPerFLEX complexes with various fluorogens
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Extended Data Table 2 Localization signals used with HyPerFLEX
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Extended Data Table 3 Parameters for single-photon fluorescence imaging microscopy
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Supplementary information

Supplementary Information

Supplementary Table 1 (primers used for HyPerFLEX design, cloning and mutagenesis) and Methods (Britton–Robinson buffers, pH titration and fluorogen synthesis).

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Potekhina, E.S., Bass, D.I., Ezeriņa, D. et al. A color-tailored fluorogenic sensor for hydrogen peroxide. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02036-6

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