Monitoring Of Reactive Oxygen Species Research Articles

Energy difference-driven ROS reduction for electrochemical tracking crop growth sensitized with electron-migration nanostructures

Aiming for sustainable crop productivity under changing climate conditions, it is essential to develop handy models for in-situ monitoring of reactive oxygen species (ROS). Herein, this work reports a simple electrochemical sensing toward hydrogen peroxide (H2O2) for tracking crop growth status sensitized with electron-migration nanostructure. To be specific, Cu-based metal-organic frameworks (MOFs) with high HOMO energy level are designed for H2O2 reduction on account of Cu(I)/Cu(II) redox switchability. Importantly, the sensing performance is improved by electrochemically reduced graphene oxide (GO) with ready to use feature. To overcome the shortcomings of traditional liquid electrolytes, conductive hydrogel as semi-solid electrolyte exhibits the adhesive property to the cut plant petiole surface. Benefitting from the preferred composite models and conductive hydrogel, the electrochemical sensing toward H2O2 with high sensitivity and good anti-interference against the coexistent molecules, well qualified for acquiring plant growth status.

(Invited) Electrochemical Microbiosensors for Real-Time Monitoring of Reactive Oxygen Species in Living Tissues

Understanding fundamental mechanisms involved in the progression and development of diseases is of great physiological and pathological importance. Such studies require the availability of effective methods to probe biochemical processes in real time and allow accurate detection and quantification of biomarkers and mediators of injury. Study of the role and mechanism of action of these mediators is also critical to develop therapies and assess pharmacological effects. However, the presence of these chemicals at very low concentrations and variable levels and the complexity of the biological environment pose a great challenge for their detection. Using custom designed microelectrodes, electrochemistry provides unique opportunities to detect reactive species in living tissues, providing direct and real time evidence of reactive oxygen (ROS) levels with high spatial resolution. This presentation will discuss our efforts to fabricate microbiosensing platforms for investigating fundamental physiological and biochemical mechanisms in vivo and in vitro, particularly those associated with ischemia and reperfusion injury. The influence of the microelectrode design parameters including surface coatings and electrode size, and the ability of the sensor to obtain quantitative spatial and temporal analytical data of the release and distribution of oxidative stress markers will be provided with examples of applications. Advantages and limitations of these systems for measurements in real biological environments will be discussed. Our results demonstrate the potential of electrochemical techniques to generate information about biomarkers and obtain fundamental biochemical-relevant information on physiology, metabolism and disease states in living systems.

Inhibition of Endothelial Inflammatory Response by HT-C6, a Hydroxytyrosol Alkyl Ether Derivative.

Hydroxytyrosol (HT) is a bioactive phenolic compound naturally present in olives and extra virgin olive oil (EVOO) which is described as an antioxidant, antitumoral and antiangiogenic molecule. Previous studies of semi-synthetic HT-derivatives presented the hydroxytyrosyl alkyl ether HT-C6 as one of the most potent derivatives studied in the context of antioxidant, anti-platelet and antiangiogenic assays, but its direct effect on inflammation was not reported. In this work, we use RT-qPCR measure of gene expression, protein analysis by Western-blot and immunofluorescence techniques, adhesion and migration functional assays and single-cell monitoring of reactive oxygen species (ROS) in order to explore in vitro the ability of HT-C6 to interfere in the inflammatory response of endothelial cells (ECs). Our results showed that HT-C6 strongly reduces the TNF-α-induced expression of vascular cell adhesion molecule 1 (VCAM1), intercellular cell adhesion molecule 1 (ICAM1), E-selectin (SELE), C-C motif chemokine ligand 2 and 5 (CCL2 and CCL5) in HUVECs, impairing the chemotactic and adhesion potential of these cells towards THP-1 monocytes in vitro. In this work, we define a mechanism of action underlying the anti-inflammatory effect of HT-C6, which involves the abrogation of nuclear factor kappa B (NF-κB) pathway activation in ECs. These results, together with the ability of HT-C6 to reduce ROS formation in ECs, point to this compound as a promising HT-derivative to be tested in the treatment of atherosclerosis.

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility, free thiol content and seasonality.

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.

A Three-Dimensional Electrochemical Biosensor Integrated with Hydrogel Enables Real-Time Monitoring of Cells under Their In Vivo-like Microenvironment.

Three-dimensional (3D) cell culture can better reproduce the in vivo cell environment and has been extensively used in fields such as tissue engineering, drug screening, and pathological research. Despite the tremendous advancement of 3D cultures, an analysis technique that could collect real-time information of the biological processes therein is sorely lacking. Electrochemical sensing with fast response and high sensitivity has played a vital role in real-time monitoring of living cells, but most current sensors are based on planar electrodes and fail to perfectly match the 3D cell culture matrix. Herein, we developed a robust 3D electrochemical sensor based on functionalized graphene foam (GF), which could be integrated with hydrogels for the 3D culture and in situ monitoring of cells for the first time. Specifically, platinum nanoparticles (Pt NPs) electrodeposited on GF (GF/Pt NPs) conferred the prominent electrochemical sensing performance, and the anti-fouling coating of poly(3,4-ethylenedioxythiophene) (PEDOT) endowed the GF/Pt NPs electrode with greatly improved stability. As a proof of concept, collagen hydrogel with microglia seeded in was filled into the interspace of the 3D GF/Pt NPs/PEDOT sensor to establish an integrated platform, which allowed the successful real-time monitoring of reactive oxygen species released from microglia in the collagen matrix. Given the versatility, our proposed biosensor in conjunction with various 3D culture models will serve as an excellent tool to provide biochemical information of cells under their in vivo-like microenvironment.

Noble metal alloy nanoparticles coated flexible MoS2 paper for the determination of reactive oxygen species

The monitoring of reactive oxygen species (ROS) in biological system has been occupying research hotspots recently, since the biochemical alterations caused by the overproduction of ROS are the main incentives of diseases and accelerated aging process. In this work, we proposed an effective and simple strategy for the construction of high-performance ROS electrochemical sensor. Noble metal alloy nanoparticles (AuPt nanoparticles) with high catalytic activity were spontaneously coated on the freestanding metallic molybdenum disulfide (MoS2) paper independent of any auxiliary conditions. Results have found that the abundant defects and electrons distributed on the metallic MoS2 paper could provide active sites for the nucleation and growth of noble metal nanoparticles. Besides, the excellent mechanical properties of the MoS2 paper promote the formation of flexible sensors. The fabricated MoS2 paper-based sensor was demonstrated to detect ROS with the advantages of wide linear range, prominent selectivity and flexibility, satisfactory detection stability, as well as simple and convenient preparation process. Furthermore, the desirable results obtained in the real sample experiments operated in plant extract pave the way for further real-time monitoring of plant physiological status to provide valuable information for guidance during plant growth.

AOP1, a New Live Cell Assay for the Direct and Quantitative Measure of Intracellular Antioxidant Effects.

Taking advantage of Light Up Cell System (LUCS) technology, which allows for fine monitoring of reactive oxygen species (ROS) production inside live cells, a new assay called Anti Oxidant Power 1 (AOP1) was developed to specifically measure ROS and/or free-radical scavenging effects inside living cells. This method is quantitative and EC50s obtained from AOP1 dose-response experiments were determined in order to classify the intracellular antioxidant efficacy of 15 well known antioxidant compounds with different hydrophilic properties. Six of them (epigallocatechin gallate, quercetin, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethoxyquin, resveratrol) gave EC50s in the range of 7–64 μM, four (Trolox, catechin, epicatechin, EUK134) in the range of 0.14 to 1 mM, and 5 (sulforaphane, astaxanthin, α- and γ-tocopherols, vitamin E acetate) showed only partial or no effect. Interestingly, effects with measurable EC50s were observed for compounds with hydrophilic properties (LogP ≤ 5.3), while all antioxidants known to act at the plasma membrane level (LogP ≥ 10.3) had partial or no effect. Sulforaphane, a hydrophilic but strict Keap1/Nrf2 pathway enhancer, did not show any effect either. Importantly, AOP1 assay captures both antioxidant and prooxidant effects. Taken together, these results led us to the conclusion that AOP1 assay measures antioxidant effect of compounds that selectively enter the cell, and act as free radical scavengers in the cytosol and/or nucleus level.

In Vitro and In Vivo Electrochemical Measurement of Reactive Oxygen Species After Treatment with Anticancer Drugs.

In vivo monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in real-time. ROS levels were measured in vivo inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.

Activatable selenium-containing fluorescent apoptotic agent for biosensing and tracing cancer cell apoptosis

Activation of cancer cell apoptosis and understanding its working mechanism play a critical role in formulating effective cancer treatment strategies. Herein, we report the design, synthesis, and biological evaluations of a selenium-containing fluorescent apoptotic agent MPSE. MPSE enabled the tracking and imaging of cancer cell apoptosis process. Its emission at 550 nm turned off in the presence of thioredoxin reductase (TrxR) in mitochondria, by forming a non-emissive compound MP. This emission “turn-off” phenomenon allows the monitoring of reactive oxygen species (ROS) induced mitochondria damages. As dysfunctional mitochondria were removed by lysosomes, MP entered lysosomes and its protonation led to bright emissions peaked at 475 nm. The new emission enables the tracking of lysosome activation during the apoptosis process. With the help of MPSE, along with other biological techniques, we have revealed three signal transduction pathways that activate cancer cell apoptosis and inhibit tumor cell proliferation. Our results showed that MPSE was highly useful in elucidating the working mechanism of theranostic-based optical apoptotic agents. We hope that understanding this mechanism will greatly facilitate the development of new cancer treatments.

In vivo therapeutic response monitoring by a self-reporting upconverting covalent organic framework nanoplatform.

The real-time and in situ monitoring of reactive oxygen species (ROS) generation is critical for minimizing the nonspecific damage derived from the high doses of ROS required during the photodynamic therapy (PDT) process. However, phototherapeutic agents that can generate ROS-related imaging signals during PDT are rare, hampering the facile prediction of the future therapeutic outcome. Herein, we develop an upconverting covalent organic framework (COF) nanoplatform via a core-mediated strategy and further functionalized it with a singlet oxygen reporter for the efficient near-infrared activated and in situ self-reporting of PDT. In this work, the COF photodynamic efficacy is greatly improved (12.5 times that of irregular COFs) via tailoring the size. Furthermore, this nanoplatform is able to not only produce singlet oxygen for PDT, but it can also emit singlet oxygen-correlated luminescence, allowing the real-time and in situ monitoring of the therapeutic process for cancer cells or solid tumors in vivo via near-infrared luminescence imaging. Thus, our core-mediated synthetic and size-tailored strategy endows the upconverting COF nanoplatform with promising abilities for high-efficacy, deep-tissue, precise photodynamic treatment.

Reactive Oxygen Correlated Chemiluminescent Imaging of a Semiconducting Polymer Nanoplatform for Monitoring Chemodynamic Therapy.

In chemodynamic therapy (CDT), real-time monitoring of reactive oxygen species (ROS) production is critical to reducing the nonspecific damage during CDT and feasibly evaluating the therapeutic response. However, CDT agents that can emit ROS-related signals are rare. Herein, we synthesize a semiconducting polymer nanoplatform (SPN) that can not only produce highly toxic ROS to kill cancer cells but also emit ROS-correlated chemiluminescent signals. Notably, the efficacy of both chemiluminescence and CDT can be significantly enhanced by hemin doping (∼10-fold enhancement for luminescent intensity). Such ROS-dependent chemiluminescence of SPN allows ROS generation within a tumor to be optically monitored during the CDT process. Importantly, SPN establishes an excellent correlation of chemiluminescence intensities with cancer inhibition rates in vitro and in vivo. Thus, our nanoplatform represents the first intelligent strategy that enables chemiluminescence-imaging-monitored CDT, which holds potential in assessing therapeutic responsivity and predicting treatment outcomes in early stages.

Synthesis and Chemiluminescent Properties of Amino-Acylated luminol Derivatives Bearing Phosphonium Cations.

The monitoring of reactive oxygen species in living cells provides valuable information on cell function and performance. Lately, the development of chemiluminescence-based reactive oxygen species monitoring has gained increased attention due to the advantages posed by chemiluminescence, including its rapid measurement and high sensitivity. In this respect, specific organelle-targeting trackers with strong chemiluminescence performance are of high importance. We herein report the synthesis and chemiluminescence properties of eight novel phosphonium-functionalized amino-acylated luminol and isoluminol derivatives, designed as mitochondriotropic chemiluminescence reactive oxygen species trackers. Three different phosphonium cationic moieties were employed (phenyl, p-tolyl, and cyclohexyl), as well as two alkanoyl chains (hexanoyl and undecanoyl) as bridges/linkers. Synthesis is accomplished via the acylation of the corresponding phthalimides, as phthalhydrazide precursors, followed by hydrazinolysis. This method was chosen because the direct acylation of (iso)luminol was discouraging. The new derivatives’ chemiluminescence was evaluated and compared with that of the parent molecules. A relatively poor chemiluminescence performance was observed for all derivatives, with the isoluminol-based ones being the poorest. This result is mainly attributed to the low yield of the fluorescence species formation during the chemiluminescence oxidation reaction.

Conductive Polymer Coated Scaffold to Integrate 3D Cell Culture with Electrochemical Sensing.

Remarkable progresses have been made in electrochemical monitoring of living cells based on one-dimensional (1D) or two-dimensional (2D) sensors, but the cells cultured on 2D substrate under these circumstances are departed from their three-dimensional (3D) microenvironments in vivo. Significant advances have been made in developing 3D culture scaffolds to simulate the 3D microenvironment yet most of them are insulated, which greatly restricts their application in electrochemical sensing. Herein, we propose a versatile strategy to endow 3D insulated culture scaffolds with electrochemical performance while granting their biocompatibility through conductive polymer coating. More specifically, 3D polydimethylsiloxane scaffold is uniformly coated by poly(3,4-ethylenedioxythiophene) and further modified by platinum nanoparticles. The integrated 3D device demonstrates desirable biocompatibility for long-term 3D cell culture and excellent electrocatalytic ability for electrochemical sensing. This allows real-time monitoring of reactive oxygen species release from cancer cells induced by a novel potential anticancer drug and reveals its promising application in cancer treatment. This work provides a new idea to construct 3D multifunctional electrochemical sensors, which will be of great significance for physiological and pathological research.

Interference-free surface-enhanced Raman scattering nanosensor for imaging and dynamic monitoring of reactive oxygen species in mitochondria during photothermal therapy

Highly reliable detection, imaging, and monitoring of reactive oxygen species (ROS) at subcellular organelles are critical for understanding the biological roles of ROS and learning the pathogenesis of some diseases. In this study, we presented an interference-free surface-enhanced Raman scattering (SERS)-active nanoprobe for the intracellular ROS detection. This nanoprobe was designed as an Au core-Ag shell nanoparticle (Au@Ag NPs) with a SERS reporter (4-mercaptobenzonitrile) resided in the inner of the core-shell. Intracellular ROS is able to etch the Ag shell and dramatically decreases the SERS intensity of the SERS reporters. It is worth mentioning that the Raman band of the reporter we used in this study locate in the cellular Raman-silent region (1800–2800 cm−1), which eliminates interference possibility from cellular molecules. The shell can also further protect the reporters from the interference of mediums. We detected ROS at subcellular organelle level, e.g. mitochondria, by modifying the surface of the nanoprobes with a mitochondria-targeting peptide. And for the first time, the SERS-based monitoring of ROS at mitochondria during the photothermal therapy process was validated and the profiles of the ROS distribution were disclosed by SERS mapping.

Tunable Plasmonic Cavity for Label-free Detection of Small Molecules.

Owing to its high sensitivity and high selectivity along with rapid response time, plasmonic detection has gained considerable interest in a wide variety of sensing applications. To improve the fieldwork applicability and reliability of plasmonic detection, the integration of plasmonic nanoparticles into optical devices is desirable. Herein, we propose an integrated label-free detection platform comprising a plasmonic cavity that allows sensitive molecular detection via either surface-enhanced Raman scattering (SERS) or plasmon resonance energy transfer (PRET). A small droplet of metal ion solution spontaneously produces a plasmonic cavity on the surface of uncured poly(dimethylsiloxane) (PDMS), and as PDMS is cured, the metal ions are reduced to form a plasmonic antennae array on the cavity surface. Unique spherical feature and the integrated metallic nanoparticles of the cavity provide excellent optical functions to focus the incident light in the cavity and to rescatter the light absorbed by the nanoparticles. The optical properties of the plasmonic cavity for SERS or PRET are optimized by controlling the composition, size, and density of the metal nanoparticles. By using the cavity, we accomplish both 1000-fold sensitive detection and real-time monitoring of reactive oxygen species secreted by live cells via PRET. In addition, we achieve sensitive detection of trace amounts of toxic environmental molecules such as 5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-isothiazol-3-one (CMIT/MIT) and bisphenol A, as well as several small biomolecules such as glucose, adenine, and tryptophan, via SERS.

Reactive oxygen and iron species monitoring to investigate the electro-Fenton performances. Impact of the electrochemical process on the biodegradability of metronidazole and its by-products

In this study, the monitoring of reactive oxygen species and the regeneration of the ferrous ions catalyst were performed during electro-Fenton (EF) process to highlight the influence of operating parameters. The removal of metronidazole (MTZ) was implemented in an electrochemical mono-compartment batch reactor under various ranges of current densities, initial MTZ and ferrous ions concentrations, and pH values. It was found that under 0.07 mA cm−2, 0.1 mM of ferrous ions and pH = 3, the efficiency of 100 mg L−1 MTZ degradation and mineralization were 100% within 20 min and 40% within 135 min of electrolysis, respectively. The highest hydrogen peroxide and hydroxyl radical concentrations, 1.4 mM and 2.28 mM respectively, were obtained at 60 min electrolysis at 0.07 mA cm−2. Improvement of the biodegradability was reached from 60 min of electrolysis with a BOD5/COD ratio above 0.4, which was reinforced by a respirometric study, that supports the feasibility of coupling electro-Fenton and biological treatment for the metronidazole removal.

Real-Time In Vivo Monitoring of Reactive Oxygen Species in Guard Cells.

The intra-/intercellular homeostasis of reactive oxygen species (ROS), and especially of superoxides (O2.-) and hydrogen peroxide (O2.-) participate in signalling cascades which dictate developmental processes and reactions to biotic/abiotic stresses. Polyamine oxidases terminally oxidize/back convert polyamines generating H2O2. Recently, an NADPH-oxidase/Polyamine oxidase feedback loop was identified to control oxidative burst under salinity. Thus, the real-time localization/monitoring of ROS in specific cells, such as the guard cells, can be of great interest. Here we present a detailed description of the real-time in vivo monitoring of ROS in the guard cells using H2O2- and O2.- specific fluorescing probes, which can be used for studying ROS accumulation generated from any source, including the amine oxidases-dependent pathway, during development and stress.

Au nanoparticle@silica@europium coordination polymer nanocomposites for enhanced fluorescence and more sensitive monitoring reactive oxygen species

Au nanoparticle (Au NP)@SiO2@TDA-Eu nanocomposites were prepared by a two-step process: Au NP@SiO2 nanocomposites were prepared by a modified onepot process. Then the europium coordination polymer was deposited on the surface of the Au NP@SiO2 by mixing 2,2’-thiodiacetic acid [S(CH2COO)22-, TDA] and Eu(NO3)3·6H2O in ethanol via a hydrothermal method. The maximum fluorescent enhancement factor of the nanocomposites was 6.81 at 30 nm thickness of silica between the core of the Au NP and the shell of TDA-Eu. The prepared nanocomposites exhibit more sensitive monitoring of reactive oxygen species.

Myoglobin and Polydopamine-Engineered Raman Nanoprobes for Detecting, Imaging, and Monitoring Reactive Oxygen Species in Biological Samples and Living Cells.

Highly reliable detection, imaging, and monitoring of reactive oxygen species (ROS) are critical for understanding and studying the biological roles and pathogenesis of ROS. This study describes the design and synthesis of myoglobin and polydopamine-engineered surface-enhanced Raman scattering (MP-SERS) nanoprobes with strong, tunable SERS signals that allow for specifically detecting and imaging ROS sensitively and quantitatively. The study shows that a polydopamine nanolayer can facilitate the modification of Raman-active myoglobins and satellite Au nanoparticles (s-AuNPs) to a plasmonic core AuNP (c-AuNP) in a controllable manner and the generation of plasmonically coupled hot spots between a c-AuNP and s-AuNPs that can induce strong SERS signals. The six-coordinated Fe(III)-OH2 of myoglobins in plasmonic hotspots is reacted with ROS (H2 O2 , •OH, and O2- ) to form Fe(IV)O. The characteristic Raman peaks of Fe(IV)O from the Fe-porphyrin is used to analyze and quantify ROS. This chemistry allows for these probes to detect ROS in solution and image ROS in cells in a highly designable, specific, and sensitive manner. This work shows that these MP-SERS probes allow for detecting and imaging ROS to differentiate cancerous cells from noncancerous cells. Importantly, for the first time, SERS-based monitoring of the autophagy process in living cells under starvation conditions is validated.

Ratiometric Fluorescent Silicon Quantum Dots-Ce6 Complex Probe for the Live Cell Imaging of Highly Reactive Oxygen Species.

The monitoring of reactive oxygen species (ROS) in living cells remains challenging because of the complexity, short half-life, and autofluorescence of biological samples. In this work, we designed a ratiometric fluorescent probe for the detection and imaging of ROS, which was constructed from silicon quantum dots (Si QDs) with chlorin e6 (Ce6) through electrostatic attraction and showed well-resolved dual fluorescence emission signals (490 and 660 nm). Sensitive and selective biosensing of hydroxyl radical (•OH) was demonstrated on the basis of fluorescence quenching of the Si QDs and Ce6 as an internal reference to avoid environmental interference, with a detection limit of ∼0.97 μM. The endogenous release of •OH was also monitored and imaged in living cells.