Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Simple Summary Spatially and temporally defined H2O2 signatures are essential parts of various signaling pathways. Therefore, monitoring H2O2 dynamics with high spatio–temporal resolution is significantly important to understand how this ubiquitous signaling molecule can control diverse cellular responses. In this study, we designed and characterized a Fluorescence Resonance Energy Transfer (FRET)-based genetically encoded H2O2 sensor that provides a powerful tool to monitor the spatio–temporal dynamics of H2O2 fluxes. We have used this sensor to monitor the flux of H2O2 in live cells under stress conditions. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively and would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling. Abstract Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.


Introduction
Reactive oxygen species (ROS) are short-lived and highly reactive molecules formed upon incomplete reduction in oxygen [1,2]. They are the key regulators of various biological processes like signaling and development in living organisms. The higher production of ROS was observed under biotic and abiotic stress conditions [3]. The ROS can lead to oxidation of lipids, proteins, and DNA, which causes oxidative stress and finally cell death [4,5]. Recently, H 2 O 2 has been observed as a specific component of numerous signaling pathways as well as a well-known toxic molecule among all the ROS present inside the cells [6]. Initially, the detection of ROS in cells, tissues, and organs were performed using colorimetric methods exploiting compounds such as nitroblue tetrazolium and diaminobenzidine, but these methods did not allow real-time measurements of ROS in the cells [7,8]. Later on, detection of ROS was achieved by using various fluorescent probes such as 20,70-dichlorofluorescein diacetate, dihydroethidium (DHE), dihydro-20,4,5,6,7,70-hexafluoro fluorescein but these probes are difficult to deliver into living cells and cause the toxicity [9]. Various H 2 O 2 -selective probes have been developed, which utilize a boronate-deprotection mechanism, and the detection approach depends on the selective H 2 O 2 -mediated conversion of aryl boronates to phenols [10,11]. Attachment of various fluorescent molecules with aryl boronates produces a fluorescent product when it reacts with H 2 O 2 . For investigating the changes in the endogenous level of H 2 O 2 , monoboronate-based probes have been developed Peroxyfluor-1 (PF-1), Peroxyfluor-2 (PF-2), Peroxyfluor-3 (PF-3), Peroxy Orange 1 (PO1), and Peroxy Yellow 1 (PY1). Although the H 2 O 2 localization studies have been improved through boronate-deprotection based probes, quantitative analysis of H 2 O 2 generation is still challenging by using these probes. Specifically, the concentration of the probe can affect the signal from the single-wavelength emitting probes. To deal with this issue, a monoboronate-based Ratio Peroxyfluor 1 (RPF)-1 probe was developed, which detects H 2 O 2 in a ratiometric manner and can potentially permit normalization to probe concentration. However, one disadvantage with this probe is that it cannot select one kind of ROS [12].
In recent years, single fluorescent protein probes have been developed, which allowed reversible in vivo detection of reactive oxygen species in cells [9,13]. These probes include roGFP [14] and HyPer [15], which are mainly based on oxidation-reduction processes between the H 2 O 2 and the reduced probe and show fluorescence upon oxidation. There are several disadvantages of single fluorescent protein (FP) probes, as these probes are more sensitive to some factors, i.e., pH and probe photobleaching. Therefore, to quantify the definite concentration of an analyte is not very reliable [16]. Therefore, there is a requisite to develop a simple, reliable, and non-invasive ratiometric method for the determination of H 2 O 2 in living cells. Genetically encoded fluorescent proteins (FPs) and sensitized emission Fluorescence Resonance Energy Transfer (FRET) (seFRET)-based sensors provide an alternate way to study the metabolite dynamics in living cells. seFRET measurements are simple, and having a high Spatio-temporal resolution, enable the tracking of fast molecular events (ms). Sensing of fast changes in metabolite dynamics and protein conformational changes is possible using the sensors based on FPs [17]. These kinds of sensors have been developed for non-invasive and specific detection of metal ions, amino acids, sugars, plant hormones [18][19][20][21].
In the present work, we developed a reliable tool, FLIP-H 2 O 2 that can measure the concentration of H 2 O 2 in a ratiometric manner and unlike the previously reported sensors, as their measurements are affected by auto-fluorescence [14], sensor protein concentrations, and excitation wavelength in lower visible region [16]. Some of them suffer from issues, such as cell toxicity [11,12], not being ideal for long term live cell imaging and pH sensitivity [16] FP and sensing domain-based sensors have several benefits in sensitivity and specificity over sensors made from traditional materials. They operate at a similar scale to natural biological process. All these factors make FLIP-H 2

Chemicals, Vectros and Strains
All chemicals were purchased from Sigma-Aldrich (USA) unless otherwise specified. pDONR, pYES-DEST52 and pcDNA3.1(-) vectors were purchased from Thermo Fischer Scientific (USA). pGWF1 was purchased from Addgene (Watertown, MA, USA). E. coli strain DH5 alpha and BL21(DE3) from New England Biolabs (Beverly, MA, USA) were used for cloning and expression purpose. Saccharomyces cerevisiae (strain BY4742), kindly gifted by Yeast Resource Center (YRC), Washington, USA and HeLa cells purchased from ATCC (USA) were used for live cell imaging. Restriction enzymes were procured from New England Biolabs and Life technologies (USA). BP and LR clonase and other ligase enzymes were purchased from Thermo Fischer Scientific (USA). Nickel NTA (nitrilotriacetic acid) affinity chromatography columns, Imidazole, Ni 2+ -His bind resin were obtained from Novagen (USA).

Plasmid Construction
The regulatory domain (RD) of OxyR, a transcription factor of Escherichia coli, was found to sense H 2 O 2 . Therefore, it was used as the recognition element for developing the H 2 O 2 nanosensor. The crystal structure of the protein was retrieved from RCSB-PDB (Research Collaboratory for Structural Bioinformatics-Protein Data Bank) (PDB ID-1I69, Supplementary Figure  S1). The DNA sequence of the RD was extracted from the Kyoto Encyclopedia of Genes and Genomes (KEGG). ECFP and mVenus sequences were taken from addgene. ECFP and mVenus share a significant spectral overlap and Forster radius, which make this pair a good FRET pair. The nucleotide sequences of RD were amplified using E. coli K12 genomic DNA and gateway primers. 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CGA GAT GGC AAG CCA GCA GGG-3 that introduces attB1 site at the 5 end of the RD gene was used as forward primer. 5 -GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC AAC CGC CTG TTT TAA AAC TTT-3 that introduces the attB2 site was used as the reverse primer. The amplified RD nucleotide sequence was cloned in the pDONR222 vector by using gateway cloning approach (BP clonase) and the entry clone was generated (Supplementary Figure S2). We selected pGWF1, a gateway destination plasmid, as a bacterial expression vector (addgene). The property of this vector is that it already contains ECFP and mVenus fluorescent tags and gateway attachment sites. The entry clone generated during BP reaction was further used for sub-cloning in the pGWF1 vector, and results in the development of the pGWF1_ECFP_RD_mVenus construct (Supplementary Figure S3) along with in-frame 6xHis tag). The construct was checked by restriction digestion and expected bands observed on agarose gel (Supplementary Figure S4). Nucleotide sequence fidelity was confirmed by Sanger sequencing (Supplementary Figure S5). The developed FRET nanosensor for H 2 O 2 detection was named as FLIP-H 2 O 2. The whole construct (ECFP-RD-mVenus) was further sub-cloned in pYES-DEST52 (Supplementary Figure S6) and pcDNA3.1 (Supplementary Figure S7) for expression in Saccharomyces cerevesie and in mammalian cells, respectively.

Protein Expression and Purification
For the expression of recombinant construct, pGWF1_ECFP_RD_Venus construct was transformed to E.coli. The transformed cells were grown in Luria Bertini (LB) medium (HiMedia, India) containing antibiotic (ampicillin 100 µg/mL) at 37 • C up to the OD 600 0.6. The expression of sensor protein in bacterial cells was induced by using 1mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) (Himedia, India) and grown at 21 • C for 48 h under dark conditions due to the sensitivity of fluorophores for visible light. To harvest the expressed bacterial cells, the culture was centrifuged for 10 min at 4500 rpm. Then, supernatant was discarded. Resuspension of the pellet was done in 20 mM Tris-Cl buffer (pH 8.0) with 2-mercaptoethanol (5 mM, to keep the thiol group of the RD domain in reduced form), followed by bacterial cell lysis through ultrasonication and then centrifuged at 7000 rpm for 20 min to remove the cell debris. For the purification of nanosensor protein, supernatant was syringe filtered for the removal of remaining impurities and then loaded in a Ni-NTA resin packed column and incubated at 4 • C for 4 h for the efficient binding of the recombinant His-tagged protein with resin. Washing of the column was done with buffer containing Tris-Cl (20 mM) and imidazole (20 mM). Elution buffer (Tris-Cl-20 mM; imidazole-200 mM) was used to elute the sensor protein. The purified nanosensor protein was incubated overnight at 4 • C for proper folding to its native conformation. The purity and molecular weight of nanosensor protein was checked by SDS-PAGE analysis (Supplementary Figure  S8).

In Vitro Characterization and Ligand Binding Affinity of Nanosensor Protein
The emission spectrum of the nanosensor was monitored by using an excitation filter 430 nm/20 nm for ECFP and taking emission intensities of the nanosensor in the range of 400 to 600 nm using the spectrofluorometer (LS50B Perkin Elmer, USA). For other measurements of ECFP and mVenus emission intensities, 480 nm/20 nm and 530 nm/25 nm filters of microplate reader (Synergy H1, Biotek, USA) were used, respectively. Certain in vitro tests were also performed to check the stability and specificity of nanosensor protein in different buffer systems PBS (Phosphate buffer saline), MOPS (3-(N-morpholino) propanesulfonic acid), Tris-Cl, and TBS (Tris-buffered saline). Furthermore, the stability of nanosensor protein was also checked in pH from 5.0-8.0 in the presence of H 2 O 2 (1 µM) and the absence of H 2 O 2 . The PBS buffer at pH 7.0 was found to be suitable and selected for further analysis. The FRET ratio was recorded after adding the 20 µL of ligand with 180 µL of the diluted protein sample. The ligand titration curve was obtained by titrating purified protein with different concentration of H 2 O 2, ranging from nanomolar to millimolar. The affinity of nanosensor protein (K d ) was determined by using the change in mVenus/ECFP ratio (FRET ratio) after binding with H 2 O 2 by applying the ligand titration curves to the equation: , where S is saturation; r is ratio; r apo is ratio in the absence of H 2 O 2 ; r sat is ratio at saturation with H 2 O 2 .

In Vivo Characterization in Bacterial Cells
The pGWF1_ECFP_RD_Venus transformed bacterial cells (E.coli BL21) were grown in LB medium at 37 • C for in vivo analysis of the nanosensor. Expression of the nanosensor protein was induced by adding 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) to the cultured bacterial cells, and then further culture for 48 h at 21 • C. The bacterial cells were harvested through centrifugation, and the pellet was dissolved in PBS buffer (pH 6.5). Various level of salt treatment (0, 100 mM and 200 mM NaCl) and arsenic (sodium arsenate) treatment (10 µM) were given to produce H 2 O 2 inside the bacterial cells. The 180 µL bacterial cells expressing nanosensor protein was transferred to 96-well plate and the FRET ratio was recorded for 420 s at regular interval of 60 s under normal conditions after the addition of 20 µL of different concentrations of H 2 O 2 (0 mM, 0.5 mM and 1 mM). FRET ratio was also recorded under stress conditions (with arsenic and NaCl). Initially, fluorescence emission of acceptor and donor was measured for baseline correction and then measurement of the FRET ratio was done after 2 min of the addition of 10 µM arsenic (sodium arsenate) and 100 mM and 200 mM NaCl. The measurements were taken for further 18 min at every 2 min internals.

H 2 O 2 Dynamics in Yeast under Normal and Stress Conditions
Live cell imaging of FLIP-H 2 O 2 expressing yeast cells, and dynamics of H 2 O 2 in the yeast cells under stress conditions using FLIP-H 2 O 2 were carried out in the presence of arsenic and salt treatment (200 mM) for 10 min as per the protocol of Mohsin and Ahmad [19]. Saccharomyces cerevesie strain BY4742 was transformed with gateway cloned pYES-DEST-ECFP_RD_mVenus. The dual emission intensity ratio (mVenus(Y)/ECFP(C) ratio) was monitored by using confocal microscope (SP5, Leica Microsystems, Wetzlar, Germany) which is controlled by LAS-AF (Leica Application Suite-Advanced Fluoresence) software (Leica, Germany).

Monitoring of H 2 O 2 Changes in Mammalian Cells
Live cell imaging and measurement of the change in the level of the H 2 O 2 in the mammalian cells (HeLa cells) expressing FLIP-H 2 O 2 was carried out as per Ahmad et al. [22].

Designing, Construction and Spectral Analysis of FLIP-H 2 O 2
In this study, a genetically encoded nanosensor was developed for non-invasive real-time measurement of H 2 O 2 in living cells by using bright fluorophores and advanced fluorescence imaging. The regulatory domain (RD) of OxyR, a member of the LysR family of bacterial transcription factor, was used as a ligand sensory domain (Supplementary Figure S1). Previous studies reported that the regulatory domain binds to H 2 O 2 and an intramolecular disulphide bond is formed between Cys-199 and Cys-208 due to which structural changes occur in the regulatory domain that fetches both termini close to each other [23]. The RD of OxyR in reduced and oxidized form was shown in supplemental Figure S9. Significant conformational changes occurred in RD due to the disulphide bond formation between specific cysteines residues (Supplementary Figure S10). The two variants of green fluorescent protein were ligated with the H 2 O 2 sensory domain, RD, at the N and C-terminus, respectively. For the development of these genetically encoded FRET sensors, the prerequisite is that the ligand sensory domain must be undergoing the appropriate conformational changes, which is translated the ligand binding into the FRET [24]. The sensing element, RD, fulfils this criterion of FRET as there was significant variation in the emission intensity of acceptor and donor fluorophore with the addition of the H 2 O 2 . The schematic mechanism of FLIP-H 2 O 2 sensor is represented in Figure 1A

In Vitro Characterization of FLIP-H2O2 Nanosensor
Fluorescent proteins are pH-sensitive, and their stability depends on the pH and composition of the buffer. To test the pH sensitivity and stability of nanosensor, fluorescence measurements were performed in different buffers (PBS, MOPS, Tris-Cl, and TBS) and at various pH ranges (5.0-8.0). The nanosensor protein exhibit the optimum stability in the PBS buffer (pH 7.0), as with this buffer the least changes in the FRET ratio were observed (Figure 2A). In the absence of H2O2, the purified protein showed no change in FRET ratio. However, after the addition of H2O2 to the purified sensor protein, which is suspended in PBS buffer, the variation in FRET ratio was found (Figure 2A). Therefore, PBS buffer (pH 7.0) was selected to carry out further investigation of nanosensor. Specificity of the nanosensor was measured in vitro with various oxidants. The addition of oxidants such as super-

In Vitro Characterization of FLIP-H 2 O 2 Nanosensor
Fluorescent proteins are pH-sensitive, and their stability depends on the pH and composition of the buffer. To test the pH sensitivity and stability of nanosensor, fluorescence measurements were performed in different buffers (PBS, MOPS, Tris-Cl, and TBS) and at various pH ranges (5.0-8.0). The nanosensor protein exhibit the optimum stability in the PBS buffer (pH 7.0), as with this buffer the least changes in the FRET ratio were observed (Figure 2A). In the absence of H 2 O 2 , the purified protein showed no change in FRET ratio. However, after the addition of H 2 O 2 to the purified sensor protein, which is suspended in PBS buffer, the variation in FRET ratio was found ( Figure 2A). Therefore, PBS buffer (pH 7.0) was selected to carry out further investigation of nanosensor. Specificity of the nanosensor was measured in vitro with various oxidants. The addition of oxidants such as super-oxide anion, nitric oxide and peroxinitrite did not cause any significant change in the FRET ratio of sensor protein ( Figure 2B). To find out the saturation limit and K d of nanosensor protein, a titration experiment was conducted with nanosensor protein by adding the H 2 O 2 from nanomolar to millimolar series ( Figure 3). The calculated affinity (K d ) of sensor with the H 2 O 2 was found to be 247 µM. FRET ratio measurement approach based on donor and acceptor fluorophore emission intensities has previously been used for the monitoring of antioxidants and amino acids [25,26]. In case of Hyper, authors showed sub-micro molar affinity for H 2 O 2 in bacterial cells and higher affinity in HeLa cells and reported that in living cells at least 5 µM H 2 O 2 is required to induce the fluorescence changes [15]. Exact K d value was not mentioned in this case, however we expect this value to be around 8 µM. K d reported in the literature for these systems is comparable with K d value reported in our case and it is in this range. Limit of detection reported in case of the other H 2 O 2 sensor was in the range of 1 to 200 µM [12,27,28]. FLIP-H 2 O 2 showed a detection range from sub-micromolar to micromolar range (~0.1 to 563 µM). Sensor based on FRET offers higher dynamic range in terms of detection. For superior performance at nanomolar level, its limit of detection can further be improved in future. A comparative analysis of major H 2 O 2 detection method has been presented in Supplementary Table S1. protein exhibit the optimum stability in the PBS buffer (pH 7.0), as with this buffer the least changes in the FRET ratio were observed (Figure 2A). In the absence of H2O2, the purified protein showed no change in FRET ratio. However, after the addition of H2O2 to the purified sensor protein, which is suspended in PBS buffer, the variation in FRET ratio was found (Figure 2A). Therefore, PBS buffer (pH 7.0) was selected to carry out further investigation of nanosensor. Specificity of the nanosensor was measured in vitro with various oxidants. The addition of oxidants such as super-oxide anion, nitric oxide and peroxinitrite did not cause any significant change in the FRET ratio of sensor protein ( Figure 2B). To find out the saturation limit and Kd of nanosensor protein, a titration experiment was conducted with nanosensor protein by adding the H2O2 from nanomolar to millimolar series ( Figure 3). The calculated affinity (Kd) of sensor with the H2O2 was found to be 247 µM. FRET ratio measurement approach based on donor and acceptor fluorophore emission intensities has previously been used for the monitoring of antioxidants and amino acids [25,26]. In case of Hyper, authors showed sub-micro molar affinity for H2O2 in bacterial cells and higher affinity in HeLa cells and reported that in living cells at least performance at nanomolar level, its limit of detection can further be improved in future. A comparative analysis of major H2O2 detection method has been presented in Supplementary Table  S1.

Intracellular H2O2 Flux Monitoring in Bacterial Cells
For in vivo H2O2 flux analysis, the E. coli BL21 cells were transformed with pGWF1-ECFP_RD_mVenus and allowed to express in the cytosol. The harvested bacterial cells were resuspended in PBS buffer and then resuspended bacterial cells were transferred to microplate wells and supplemented with H2O2. After a fixed interval, the emission intensities of both fluorophores were recorded using fluorescence plate reader. An increase in the FRET ratio from 1.53 to 1.70 and 1.53 to 1.75 after the addition of 0.5 mM and 1 mM of H2O2, respectively (Figure 4). Previously, the

Intracellular H 2 O 2 Flux Monitoring in Bacterial Cells
For in vivo H 2 O 2 flux analysis, the E. coli BL21 cells were transformed with pGWF1-ECFP_RD_mVenus and allowed to express in the cytosol. The harvested bacterial cells were resuspended in PBS buffer and then resuspended bacterial cells were transferred to microplate wells and supplemented with H 2 O 2 . After a fixed interval, the emission intensities of both fluorophores were recorded using fluorescence plate reader. An increase in the FRET ratio from 1.53 to 1.70 and 1.53 to 1.75 after the addition of 0.5 mM and 1 mM of H 2 O 2, respectively ( Figure 4). Previously, the measurement of metabolite and ion accumulation was also performed inside the bacterial cells using FRET-based nanosensors [26,29].

Monitoring of H 2 O 2 in Yeast and Mammalian Cells
Real-time confocal imaging data obtained from sensor transformed yeast cells indicated that the FLIP-H 2 O 2 was expressed in the cytosol ( Figure 5A) and allowed monitoring of H 2 O 2 inside the cells. After one minute of addition of H 2 O 2 , FLIP-H 2 O 2 expressing yeast cells showed an increase in the FRET ratio from 1.42 to 1.55 ( Figure 5B). Live cell imaging was also performed earlier for the monitoring of 2-oxoglutarate and ajmalacine in yeast cells using FRET-based 2-oxoglutarate and ajmalacine nanosensors [30,31].
In the case of yeast, nanosensor showed a kind of fluorescence quenching which was reflected in the form of initial low fluorescence intensity. Nanosensor had reacted to the H 2 O 2 fast, so it reached to the saturation quickly after addition in these cells. Confocal imaging of transiently transfected HeLa cells with FLIP-H 2 O 2 showed that FLIP-H 2 O 2 distributed predominantly in the cytoplasm of cultured cells ( Figure 6A). Non-invasive analysis of transfected HeLa cells showed an increase in the FRET ratio from 1.51 to 1.66 and 1.50 to 1.62 by the addition of H 2 O 2 and NaCl respectively ( Figure 6B). NaCl induced H 2 O 2 production was also reported in endothelial cells where it was observed that the extracellular volume expands due to increased salt intake and induces the activation of transforming growth factor-β (TGF-β) [32]. However, TGF-β increases endothelial NADPH (nicotinamide adenine dinulceotide phosphate) oxidase-4 [33], an enzyme that induces the production of H 2 O 2 [34].  In the case of yeast, nanosensor showed a kind of fluorescence quenching which was reflected in the form of initial low fluorescence intensity. Nanosensor had reacted to the H2O2 fast, so it reached to the saturation quickly after addition in these cells. Confocal imaging of transiently transfected HeLa cells with FLIP-H2O2 showed that FLIP-H2O2 distributed predominantly in the cytoplasm of cultured cells ( Figure  6A). Non-invasive analysis of transfected HeLa cells showed an increase in the FRET ratio from 1.51 to 1.66 and 1.50 to 1.62 by the addition of H2O2 and NaCl respectively ( Figure 6B). NaCl induced H2O2 production was also reported in endothelial cells where it was observed that the extracellular volume expands due to increased salt intake and induces the activation of transforming growth factor-β (TGF-β) Biology 2020, 9, x 9 of 12 [32]. However, TGF-β increases endothelial NADPH (nicotinamide adenine dinulceotide phosphate) oxidase-4 [33], an enzyme that induces the production of H2O2 [34].

In Vivo H2O2 Flux Monitoring under Stress Conditions
To analyze and monitor the effect of abiotic stress conditions on the generation of H2O2, NaCl and arsenic were provided externally and fluorescence intensity changes were monitored in a ratiometric manner in FLIP-H2O2 expressing yeast and bacterial cells using microplate reader. For the generation of abiotic stress, we treated bacterial and yeast cells with different concentrations of NaCl and arsenic, separately. FRET ratio was monitored at the interval of 2 min for 18 min. In the first 2 min, no significant

In Vivo H 2 O 2 Flux Monitoring under Stress Conditions
To analyze and monitor the effect of abiotic stress conditions on the generation of H 2 O 2 , NaCl and arsenic were provided externally and fluorescence intensity changes were monitored in a ratiometric manner in FLIP-H 2 O 2 expressing yeast and bacterial cells using microplate reader. For the generation of abiotic stress, we treated bacterial and yeast cells with different concentrations of NaCl and arsenic, separately. FRET ratio was monitored at the interval of 2 min for 18 min. In the first 2 min, no significant change in the FRET ratio was observed because the expression of stress producing enzymes and synthesis of H 2 O 2 in bacterial and yeast cells takes time. In arsenic and salt treated cells, an increase in the FRET ratio was observed after 2 min, which was increased up to 10 min and then become saturated (Figures 7 and 8A,B). At the saturation point, ascorbate was added to check and measure the effect of this antioxidant on cellular H 2 O 2 . Nanosensor reports the decreased level of H 2 O 2 inside the bacterial cells in the presence of ascorbate (Figure 7), which indicate its antioxidant property. Experimental results have indicated the generation of H 2 O 2 after exposure to arsenic in Chinese hamster ovary cells [35]. Biology 2020, 9, x 10 of 12

Conclusions
Understanding how living cells cope with environmental change requires a Spatio-temporal perspective. The development and adaptation of a FRET nanosensor provides the capacity to visualize changes in the transport and accumulation of metabolites and small molecules at the cellular level, which is required to identify the regulatory switches in the metabolic network. Flux monitoring of H2O2 in the cytosol of living bacterial, yeast and mammalian cells demonstrate that the FLIP-H2O2 is an excellent tool for non-invasive real-time monitoring of H2O2 in any cell type. Moreover, attachment of sub-cellular

Conclusions
Understanding how living cells cope with environmental change requires a Spatio-temporal perspective. The development and adaptation of a FRET nanosensor provides the capacity to visualize changes in the transport and accumulation of metabolites and small molecules at the cellular level, which is required to identify the regulatory switches in the metabolic network. Flux monitoring of H2O2 in the cytosol of living bacterial, yeast and mammalian cells demonstrate that the FLIP-H2O2 is an excellent tool for non-invasive real-time monitoring of H2O2 in any cell type. Moreover, attachment of sub-cellular targeting signal to the FLIP-H2O2 will facilitate in vivo monitoring of H2O2 in key cellular organelles, such

Conclusions
Understanding how living cells cope with environmental change requires a Spatio-temporal perspective. The development and adaptation of a FRET nanosensor provides the capacity to visualize changes in the transport and accumulation of metabolites and small molecules at the cellular level, which is required to identify the regulatory switches in the metabolic network. Flux monitoring of H 2 O 2 in the cytosol of living bacterial, yeast and mammalian cells demonstrate that the FLIP-H 2 O 2 is an excellent tool for non-invasive real-time monitoring of H 2 O 2 in any cell type. Moreover, attachment of sub-cellular targeting signal to the FLIP-H 2 O 2 will facilitate in vivo monitoring of H 2 O 2 in key cellular organelles, such as chloroplasts, mitochondria. Therefore, live imaging approaches coupled with automated image-analysis algorithms are revealing new levels of dynamism and plasticity of the cellular response. Together, these tools will offer a more comprehensive understanding of environmental responses in cellular organisms.