Detection Of Superoxide Radicals Research Articles

One-step preparation of SnO2-AuNPs as nanocomposites on photoelectrodes to enhance photoelectrochemical detection of nitrite and superoxide

The integration of plasmonic metal nanoparticles and semiconductors improves the photoelectric conversion efficiency. We describe here a simple method for preparing photoelectrodes modified with nanocomposites of tin oxide (SnO2) and gold nanoparticles (AuNPs). This preparation process is more convenient, economical, and efficient than the previous methods. The nanocomposite of SnO2-AuNPs on indium tin oxide (ITO) conductive glass electrodes enhanced photoelectric conversion and increased conductivity. The electrodes prepared with SnO2-AuNPs nanocomposites were used as photoanodes in photoelectrochemical methods for the determination of nitrite (NO2−) and superoxide radical (O2−). The detection of superoxide radicals was achieved by stoichiometric conversion of superoxide radicals to nitrite, which overcomes a common problem of chemical instability of superoxide radicals. The photoelectrochemical method for the determination of NO2− had a detection limit of 0.1 nmol/L and a linear dynamic range from 10−9 to 10−5 mol/L. The method was successfully used to assess the photochemical generation of O2− by irradiating a photosensitizer, rose bengal (RB). The photoelectrochemical responses increased with the concentration of the photosensitizer and the time of photo irradiation (532 nm) as expected.

Detection of superoxide radical in all biological systems by Thin Layer Chromatography

The study presents a new method that detects O2•−, via quantification of 2-hydroxyethidium (2-ΟΗ-Ε+) as low as ∼30 fmoles by High-Performance Thin Layer Chromatography (HPTLC). The method isolates 2-ΟΗ-Ε+ after its extraction by the anionic detergent SDS (at 18-fold higher than its CMC) together with certain organic/inorganic reagents, and its HPTLC-separation from di-ethidium (di-Ε+) and ethidium (Ε+). Quantification of 2-OH-E+ is based on its ex/em maxima at 290/540 nm, and of di-E+ and E+ at 295/545 nm. The major innovations of the present method are the development of protocols for (i) efficient extraction (by SDS) and (ii) sensitive quantification (by HPTLC) for 2-OH-E+ (as well as di-E+ and E+) from most biological systems (animals, plants, cells, subcellular compartments, fluids). The method extracts 2-ΟΗ-Ε+ (by neutralizing the strong binding between its quaternary N+ and negatively charged sites on phospholipids, DNA etc) together with free HE, while protects both from biological oxidases, and also extracts/quantifies total proteins (hydrophilic and hydrophobic) for expressing O2•− levels per protein quantity. The method also uses SDS (at 80-fold lower than its CMC) to extract/remove/wash 2-ΟΗ-Ε+ from cell/organelle exterior membrane sites, for more accurate internal content quantification. The new method is applied on indicative biological systems: (1) artificially stressed (mouse organs and liver mitochondria and nuclei, ±exposed to paraquat, a known O2•− generator), and (2) physiologically stressed (cauliflower plant, exposed to light/dark).

Harnessing efficient in-situ H2O2 production via a KPF6/BiOBr photocatalyst for the degradation of polyethylene

• KPF 6 doped BiOBr were successfully synthesized using a simple one-pot hydrothermal method. • The photocatalytic H 2 O 2 production of 20 wt% KPF 6 /BiOBr is 1.96 times higher than pure BiOBr. • Plastic bags were decomposed by KPF 6 /BiOBr in conjunction with the in-situ generation of H 2 O 2 . • Hydroxyl radicals play a key role in the polyethylene degradation. • A potential mechanism for H 2 O 2 production and polyethylene degradation was proposed. The development of cost-effective dual-function photocatalytic materials to advance clean energy generation and storage combined with superior pollutant degradation performance has thus far remained challenging. For this study, potassium hexafluorophosphate (KPF 6 ) was introduced to modify BiOBr via a simple one-pot hydrothermal method, where uniformly dispersed KPF 6 on BiOBr nanosheets improved H 2 O 2 yields and the pollutant degradation performance. Such materials have proven to be critical for low bandgap and enhanced surface charge separation for efficient H 2 O 2 production. The optimized sample of 20 wt% KPF 6 /BiOBr showed a H 2 O 2 production rate of 53.36 mg·L –1 , which was much higher than that of pure BiOBr (27.19 mg·L –1 ). Nitroblue tetrazolium chloride (NBT) superoxide radical detection results suggested that two-step single-electron oxygen reduction was the primary pathway. More importantly, discarded polyethylene bags from supermarkets have been decomposed though the in-situ generation of H 2 O 2 using KPF 6 /BiOBr materials, and the mass loss of plastics was 2.33 times higher than that of the pure BiOBr. This research offers new perspectives toward the development of new functionalized materials for H 2 O 2 production, while improving capacities for the degradation of plastics.

Photocatalytic H2O2 production using Ti3C2 MXene as a non-noble metal cocatalyst

Photocatalytic H2O2 production by O2 reduction is an environmental-friendly process for solar light conversion to chemical energy. In this work, Ti3C2 MXene was used as a non-noble metal cocatalyst to load on P25 as Ti3C2/TiO2 (TC/TO) photocatalysts for photocatalytic H2O2 synthesis. A H2O2 formation rate (179.7 μmol L−1 h−1) of the optimized 10 %-TC/TO composite was obtained to be over 21 folds as high as that of P25 under UV light. Radical quenching experiments and superoxide radical detection confirmed the superoxide radical as the primary intermediate, suggesting the O2 reduction in two-step single-electron indirect reaction. The higher activity of TC/TO can be attributed to the functions of Ti3C2 MXene in accelerating the separation and transfer of photogenerated electron-hole pairs, suppressing their recombination, and blocking the surface TiOOH formation. This work proves the promising roles of Ti3C2 MXene in the photocatalytic reaction and further expands their new applications in photocatalysis.

Superoxide radical scavenging by sodium 4,5-dihydroxybenzene-1,3-disulfonate dissolved in water: Experimental and quantum chemical studies

Tiron (sodium 4,5-dihydroxybenzene-1,3-disulfonate) is often used as a probe for detection of superoxide radicals. In this work, we have studied the interaction of Tiron with superoxide radicals dissolved in water solutions. The density functional theory (DFT) method was used for scrutinizing electron and energy characteristics of Tiron radicals in the systems modeling freshwater and salt water. In water solution at neutral pH, deprotonated form of superoxide radical, O2•-, dominates over the protonated form HO2•. Fully reduced (TH2) and fully oxidized (T) species of Tiron react with superoxide radicals, leading to the formation of the Tiron semiquinone radicals. DFT calculations show that the reactions of O2•- with TH2 and T in water solution will advance if these reactions are catalyzed by hydronium ions: TH2 + O2•- + H3O+ → TH• + H2O2 + H2O and T + O2•- + H3O+ → TH• + O2 + H2O. Electron paramagnetic resonance (EPR) and DFT data show that Tiron radicals dissolved in water solution with neutral pH exist predominantly in deprotonated state T•-. The constants of the hyperfine coupling between an unpaired electron and the hydrogen atoms of deprotonated radicals T•-, computed for freshwater and seawater solutions, are in a fairly good agreement with experimental constants derived from the EPR spectra of Tiron radicals.

New insights into the mechanisms of tartaric acid enhancing homogeneous and heterogeneous copper-catalyzed Fenton-like systems

The specific roles of tartaric acid (TA), as an eco-friendly ligand, in homogeneous and heterogeneous copper-catalyzed systems were systematically revealed and new mechanisms of TA enhancing the three Fenton-like processes were proposed to provide a theoretical significance in overcoming the deficiency of conventional Fenton processes. The results identified hydroxyl radical (•OH) as the main species responsible for the simultaneous decomposition of TA and metronidazole (MNZ) according to TOC removal. The ESR technique was used to detect superoxide radicals (•O2−), carbon-centered radical (•R) and hydrogen radical (•H) in the Cu2+/TA/H2O2 system, which contributed to the acceleration of the Cu2+/Cu+ redox cycle. The enhancing effect of TA on the homogeneous process was ascribed to the formation of a soluble complex with Cu2+, which favored the pH range extension, Cu+ oxidation, and radical generation. Moreover, the adsorption of TA on the catalysts surface promoted the consumption of H2O2, inducing •OH generation. The formed surface complex (≡Cu2+-TA) also accelerated the regeneration of ≡Cu+, which was confirmed by density functional theory (DFT) calculation and surface characterization analysis (SEM, XRD, and XPS). The possible degradation pathways of MNZ in TA-modified Fenton-like system were also clarified.

Detection of Superoxide Radical in Adherent Living Cells by Electron Paramagnetic Resonance (EPR) Spectroscopy Using Cyclic Nitrones.

Spin trapping with cyclic nitrones coupled to electron paramagnetic resonance (EPR) enables the detection and characterization of oxygen-derived free radicals, such as superoxide and hydroxyl radicals, in living cells. Detection is usually performed on cell suspensions introduced in glass capillaries, gas-permeable tubing, or flat cells, even when cells normally require attachment for growth. However, radical production may be influenced by cell adhesion, while enzymatic or mechanical cell harvesting may damage the cells and alter their metabolic rates. Here, we describe the detection on adherent cells attached to microscope coverslip glasses. This method preserves cell integrity, ensures near physiological conditions for naturally adherent cells, and is relatively simple to set up. Up to 12 conditions can be screened in half a day using a single batch of culture cells.

Development of Gelatin-Alginate-TiO2-SOD Biosensor for the Detection of Superoxide Radicals

In this work, a biosensor that uses gelatin and alginate hydrogels in addition to titanium dioxide (TiO2) nanoparticles (NPs) as sensor matrix was developed in order to detect superoxide radicals (O2•-), which play role in carcinogenesis when present in excess levels. Parameters affecting the performance of the biosensor such as amount of gelatin-alginate ratio, amount of TiO2 NPs, concentration of SOD enzymes and glutaraldehyde cross-linker were investigated. Electrochemical Impedance Spectroscopy (EIS) and chronoamperometry were used as electrochemical technique for the development of biosensor as well as characterisation steps. The developed biosensor exhibited two linear ranges between 0.0009 mM – 0.125 mM and 0.25 mM – 2 mM which were utilized as calibration curves. Detection limit of the biosensor was found 0.9 μM, which was at appropriate level for the detection of O2•- in tumour samples. Finally, the constructed biosensor showed significant analytical performance such as high selection for O2•-, low detection limit, and long-term stability.

Triazine-based 2D covalent organic frameworks improve the electrochemical performance of enzymatic biosensors

Covalent organic frameworks (COFs) are crystalline nano/microporous materials assembled from organic molecules through covalent bonds. Having various advantages such as large surface area, fully conjugated structure, and being in atom-thick dimensions makes COFs a promising candidate for numerous applications such as energy storage, electrocatalysis, and electrochemical devices. Yet, their potential for facilitating biosensor design and bioelectrochemical processes has not extensively been investigated. Therefore, in this study, we harnessed the simplicity, enhanced conductive property, and organic nature of COFs in electrochemical enzymatic biosensor design that aimed to detect superoxide radicals as a model system. Two different triazine-based COFs, CTF-1 and TRITER-1, were successfully synthesized and characterized using Fourier-transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), X-ray diffraction (XRD), and transmission electron microscopy (TEM). Electrochemical studies demonstrated that CTF-1 improves the electrochemical performance of the enzymatic biosensors and is suitable for electrode design. Using the developed CTF-1-based biosensor that uses superoxide dismutase (SOD) as recognizing element, we measured the levels of superoxide anions, which are known to include in carcinogenesis process, with 0.5 nM detection limit.

Synergistic effect of reduced graphene oxide/azo-polymer layers on electrochemical performance and application as nonenzymatic chemiresistor sensors for detecting superoxide anion radicals

The present paper describes a nonenzymatic chemiresistor sensor based on an azo-polymer and reduced graphene oxide (rGO) as a resistive platform for superoxide radical detection. The sensorial platform was prepared on layer-by-layer-assembled films of poly(azo-Bismarck Brown Y) and reduced graphene oxide by cyclic voltammetry. The nanocomposite film exhibited interesting synergetic properties based on the redox properties of the azo-polymer combined with the good electronic conductivity and stability of graphene. The electrical conductivity mechanism of the graphene-polymer was analyzed by electrochemical impedance and compared to that of an electrode coated with only polymer. The conductance properties of the interlayer formed by π-π stacking between the conjugated structure of the polymer chains and the structure of the graphene sheet are dependent on the applied potential of the system. The performance of the poly(azo-BBY)/rGO film as a chemiresistor material for the sensing of superoxide anions was evaluated by impedance measurements at the applied potential of +0.30 in PBS (7.4). The charge transfer resistance values change substantially in the presence of superoxide in solution. The effect of superoxide on the resistivity of the device is attributed to changes in the oxidation state of the polymer. The impedance measurements in real time (chrono-impedance) with the poly(azo-BBY)/rGO sensor with different superoxide concentrations revealed good linearity behavior between the real impedance and the superoxide anion concentration (0.12–2.6 ​mmol ​L−1) with a detection limit of 81.0 μmol L−1.

Ti3C2 Mxene/porous g-C3N4 interfacial Schottky junction for boosting spatial charge separation in photocatalytic H2O2 production

The development of efficient photocatalysts for the production of hydrogen peroxide (H2O2) is a promising strategy to realize solar-to-chemical energy conversion. Graphitic carbon nitride (g-C3N4) presents giant potential for photocatalytic H2O2 production, but the sluggish charge separation depresses its photocatalytic performance. Herein, an interfacial Schottky junction composed of Ti3C2 nanosheets and porous g-C3N4 nanosheets (TC/pCN) is constructed by a facile electrostatic self-assembly route to significantly boost the spatial charge separation to promote the activation of molecular oxygen for H2O2 production. As the optimal sample, TC/pCN-2 possesses the highest H2O2 production rate (2.20 μmol L−1 min−1) under visible light irradiation (λ > 420 nm), which is about 2.1 times than that of the porous g-C3N4. The results of superoxide radical detection and rotating disk electrode measurement suggest that the two-step single-electron reduction of oxygen is the predominant reaction step during this photocatalytic H2O2 production process. The enhanced photocatalytic performance is ascribed to the formation of Schottky junction and subsequent built-in electric field at their interface, which accelerate the spatial charge separation and restrain the charge recombination. This work provides an in-depth understanding of the mechanism of photocatalytic H2O2 production, and gives ideas for the design of highly active materials for photocatalytic H2O2 production.

Synthesis and properties of a series of β-cyclodextrin/nitrone spin traps for improved superoxide detection.

Three new DEPMPO-based spin traps have been designed and synthesized for improved superoxide detection, each carrying a cyclodextrin (CD) moiety but with a different alkyl chain on the phosphorus atom or with a long spacer arm. EPR spectroscopy allowed us to estimate the half-life of the superoxide spin adducts which is close to the value previously reported for CD-DEPMPO (t1/2 ≈ 50-55 min under the conditions investigated). The spectra are typical of superoxide adducts (almost no features of the HO˙ adduct that usually forms with time for other nitrone spin traps such as DMPO) and we show that at 250 μM, the new spin trap enables the reliable detection of superoxide by 1 scan at the position opposite to the corresponding spin trap without the CD moiety. The resistance of the spin adducts to a reduction process has been evaluated, and the superoxide spin adducts are sensitive to ascorbate and glutathione (GSH), but not to glutathione peroxidase/GSH, reflecting the exposed nature of the nitroxide moiety to the bulk solvent. To understand these results, 2D-ROESY NMR studies and molecular dynamics pointed to a shallow or surface self-inclusion of the nitrone spin traps and of nitroxide spin adducts presumably due to the high flexibility of the permethyl-β-CD rim.

Internalization and Phytotoxic Effects of CuO Nanoparticles in Arabidopsis thaliana as Revealed by Fatty Acid Profiles.

Internalization and phytotoxic effects of CuO nanoparticles (nCuO) in plants were studied at the cellular level. Arabidopsis thaliana was hydroponically challenged by nCuO (100 mg/L), as compared to Cu2+ ions (1.2 mg/L), to account for nCuO dissolution for 96 h and 28 days to monitor Cu accumulation in the plant as well as the fatty acid (FA) profiles of the plant cell membrane. Under the same growing conditions, the nCuO exposure resulted in more Cu accumulation than did the Cu2+ exposure. Multiple microscopic techniques confirmed the internalization and sequestration of nCuO in root cell vacuoles, where transformation of Cu(II) to Cu(I)Cl occurred. Short and long exposures (96 h versus 28 days) to both nCuO and Cu2+ elevated FA saturation degrees in plant cells through oxidative stress, as verified by in situ detection of superoxide radicals, with conversions mostly from C18:3, C16:3, and C18:2 to C16:0. Only the long exposure to nCuO significantly brought about an additional elevation of FA saturation degree in root cells. These results demonstrated that the acute effects of plant exposure to nCuO were mainly produced from the stress of Cu2+ ions released from nCuO dissolution, while the chronic effects in roots were significantly developed by the nCuO particle stress. The findings in this work are novel and may offer significant implications in better understanding nanoparticle-induced phytotoxicity and potential risks in ecosystems.

Advanced Electrochemical in Vitro Detection of Superoxide Radicals with Fully Integrated Microsensor System

An electrochemical sensing system for in vitro measurement of superoxide radical is presented. The approach of the system is based on a fully integrated sensor setup in conventional tissue culture containment, utilizing novel direct oxidation of ROS on polymer covered gold microelectrode. The concept was shown to provide stable and sensitive measurement procedure without the need for enzymatic recognition elements. Sensor performance was investigated from artificial enzymatic superoxide production, with a sensitivity of 2247 AM-1m2 and outstanding high selectivity for main interfering substances. Online monitoring of superoxide release from human breast carcinoma cell line T-47D was successfully demonstrated.

Copper–zinc alloy nanoparticle based enzyme-free superoxide radical sensing on a screen-printed electrode

In this paper, amperometric enzyme-free sensors using superoxide dismutase (SOD) enzyme as a catalyst for the dismutation reaction of superoxides into oxygen and hydrogen peroxide, enabling superoxide radical detection have been described. For this purpose, the surfaces of screen-printed platinum electrodes have been modified with gelatin composites of CuO, ZnO and CuZn nanoparticles with the expectation of an increase in catalytic effect toward the dismutation reaction. SOD containing electrodes were also prepared for comparative studies in which glutaraldehyde was used as a cross-linker for the immobilization of SOD to the nanocomposite materials. Electrochemical measurements were carried out using a screen-printed electrochemical system that included potassiumferrocyanide (K4[Fe(CN)6]) and potassiumferricyanide (K3[Fe(CN)6]) as the redox probes. The results revealed that the enzyme-free detection method using CuZn nanoparticles can determine superoxide radicals with high performance compared to other detection methods prepared with different nanoparticles by mimicking the active region of superoxide dismutase enzyme. The anodic (ksa) and cathodic (ksc) electron transfer rate constants and the anodic (αa) and cathodic (αc) transfer coefficients were evaluated and found to be ksa=6.31s−1 and αa=0.81, ksc=1.48s−1 and αc=0.19 for the gelatin–CuZn–SOD electrode; ksa=6.15s−1 and αa=0.79, ksc=1,63s−1 and αc=0.21 for the enzyme-free gelatin–CuZn electrode. The enzyme-free electrode showed nearly 80% amperometric performance with respect to the enzyme containing electrode indicating the superior functionality of enzyme-free electrode for the detection of superoxide radicals.

A novel functional imidazole fluorescent ionic liquid: simple and efficient fluorescent probes for superoxide anion radicals

Novel imidazole fluorescent ionic liquids with anthracene groups (ImS-FILA) were synthesized for the first time to act as fluorescent probes. They were developed for the determination of superoxide anion radicals (O2 (•-)) in an aqueous system. O2 (•-) was produced by pyrogallol autoxidation. The fluorescence of ImS-FILA was quenched by superoxide anion radicals. The π-bond structure of the fluorescent molecules was oxidized and damaged. This method is very simple and sensitive. The linear range of sensitivity was 1-70μM ImS-FILA, and the detection limit for reactive oxygen species was 0.1μM. This method was used to detect superoxide radicals in papaya and garlic, with satisfactory results. Further work is needed to demonstrate the utility of this method in detecting reactive oxygen species in a biological aqueous system.

Detection of superoxide radicals in tomato plants exposed to salinity, drought, cold and heavy metal stress using CMC-G-SOD biosensor

A novel highly sensitive electrochemical carboxymethylcellulose-gelatin-superoxide dismutase biosensor was used for the determination of superoxide radicals enhancement in tomato plants exposed to salinity, drought, cold and heavy metal stress. The variations in superoxide radicals depending on abiotic stress was determined using biosensor. The superoxide radical production with regard to control rapidly was increased in tomato plants exposed to salinity, drought, cold and heavy metal stress. The superoxide radical enhancement in tomato plants exposed to salinity, drought, cold and heavy metal stress was successfully determined using carboxymethylcellulose-gelatin-superoxide dismutase biosensor.

A novel nano-interfaced superoxide biosensor

Abstract A nano-interfaced biosensor for superoxide (O2 −) was developed based on superoxide dismutase (SOD) immobilized on iron oxide nanoparticles (nano Fe3O4) coated on a gold electrode surface. Nano Fe3O4 were synthesized via hydrothermal method and superoxide dismutase enzyme (SOD) was attached to it by covalent linking. FT-IR, FE-SEM and XRD have been used for morphological and structural characterization of nano Fe3O4. The modified electrode was used to detect superoxide radicals generated using sodium hydroxide and dimethyl sulfoxide. The cyclic voltammetry studies of the SOD/nano Fe3O4/Au bioelectrode exhibited an electrochemical reduction behavior for increasing concentrations of SOD at a scan rate of 0.1 V/s in a cell containing 5 mL phosphate buffer solution (pH 7.4). Amperometric studies revealed that the nano-interfaced bioelectrode displayed a sharp response time of 4 s with a limit of detection (LOD) as 3.46 nM and limit of quantification as (LOQ) as 11.5 nM. The response is found to be linear in the range of 0.2–1.4 μM superoxide. The sharp response time is attributed to the fact that a rapid and direct electron transfer between the SOD tagged nano Fe3O4 and the gold electrode without any redox mediator. The biosensor also exhibited good reproducibility, stability and shelf life.

Photosynthetic electron flow to oxygen and diffusion of hydrogen peroxide through the chloroplast envelope via aquaporins

Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Enhanced superoxide and hydrogen peroxide detection in biological assays

Superoxide reductase (SOR) is an enzyme that converts superoxide into hydrogen peroxide at a twofold higher yield than canonical superoxide dismutases (SOD). Superoxide radical detection was investigated using the Amplex red (AR)/peroxidase system to measure the difference in hydrogen peroxide production yield in the presence of SOR or SOD. We found that reduced SOR reacts with the AR oxidation intermediate, a one-electron reduced AR • radical, by reducing this intermediate back to the initial AR leuco compound. Ascorbate also quenched this radical in a concentration-dependent manner and could be used to compete efficiently with SOR; at concentrations of ascorbate higher than 5 µM, SOR no longer interfered with the detection of H 2O 2. By using xanthine/xanthine oxidase as a superoxide-generating system, it was possible to successfully quantify superoxide and hydrogen peroxide in vitro using the AR/peroxidase/SOR system, either by visible absorption or by fluorescence emission, with a considerable low detection limit of 10 nM/min. The use of enzymes with diffusion-limited reactivity toward superoxide substantially increases specificity and detection threshold for superoxide and turns this approach into a powerful system to detect ROS in biological systems.