Search Results (29)

ROS (i.e., reactive oxygen species) scavenging is a key function of various Mn-based enzymes, including superoxide dismutases (SODs) and catalases, which are actively linked to oxidative stress-related diseases. In this study, we synthesized and characterized two novel Mn(III)-based synzymes (i.e., synthetic enzymes), designated C1 ([MnL1Cl(H₂O)]Cl·3H₂O) and C2 ([MnL2Cl₂]·2H₂O), which differ in the presence of a bridging aliphatic or aromatic group in the chelator. Using a range of analytical techniques, we found that the aromatic C2 bridge significantly influences the Mn(III) center’s cis-β configuration, unlike C1, which adopts a trans configuration. We then thoroughly evaluated the oxidation-reduction properties of C1 and C2, including their redox potentials (by cyclic voltammetry) and capacity to consume various ROS species (using DPPH, hydroxyl radical, hydrogen peroxide, and superoxide UV–visible spectrophotometric assays). The specific kinetics of the H₂O₂ dismutation process, as measured by a Clark-type electrode and time-resolved ESI-MS, revealed that both synzymes possess catalytic activity. Toxicological experiments using the Galleria mellonella larval model demonstrated the compounds’ innocuous nature towards higher eukaryotic organisms, while cytotoxicity assays confirmed their selective efficacy against lung cancer cells. Additional cytological assays, such as the thiobarbituric acid reactive substances assay and caspase-3 activity and p53 expression analysis, reported that C1 and C2 induce cytotoxicity against cancer cells via apoptosis rather than necrosis and behave very differently towards redox substances and ROS-regulating enzymes in vivo. These findings suggest that the structural differences between C1 and C2 lead to distinct redox properties and biological activities, highlighting the potential of these novel Mn(III)-based synzymes as therapeutic agents for the treatment of oxidative stress-related diseases, particularly lung cancer. Further studies are warranted to elucidate the underlying mechanisms of action and explore their clinical applications. Full article

The toxicity of emerging organic pollutants to photosystems of aquatic plants is still not well clarified. This study aimed to develop a novel ecotoxicological experimental protocol based on nanoscale electrochemical mapping of photosynthetic oxygen evolution of aquatic plants by scanning electrochemical microscopy (SECM). The protocol was also checked by confocal laser scanning microscopy (CLSM), the traditional Clark oxygen electrode method, and the chlorophyll fluorescence technique. The typical persistent organic pollutant 2,4-dichlorophenol (2,4-DCP) in a water environment and the common aquatic Microsorium pteropus (M. pteropus) were chosen as the model organic pollutant and tested plant, respectively. It was found that the SECM method could discriminate the responses of stoma micromorphology and spatial pattens of photosynthetic oxygen evolution on single stoma well. The shape of stoma blurred with increasing 2,4-DCP concentration, which was in good agreement with the CLSM images. The dose–response curves and IC₅₀ values obtained from the SECM data were verified by the data measured by the traditional Clark oxygen electrode method and chlorophyll fluorescence test. The IC₅₀ value of single-stoma oxygen emission of plant leaves exposed for 24 h, which was derived from the SECM current data (32,535 μg L⁻¹), was close to those calculated from the maximum photosynthetic efficiency (Fv/Fm) measured by the chlorophyll fluorescence test (33,963 μg L⁻¹) and the Clark oxygen electrode method photosynthetic oxygen evolution rate (32,375 μg L⁻¹). The 72 h and 96 h 2,4-DCP exposure data further confirmed the reliability of the nanoscale stoma oxygen emission mapping methodology for ecotoxicological assessment. In this protocol, the procedures for how to collect effective electrochemical data and how to extract useful information from the single-stoma oxygen emission pattern were well established. This study showed that SECM is a feasible and reliable ecotoxicological tool for evaluation of toxicity of organic pollutants to higher plants with a unique nanoscale visualization advantage over the conventional methods. Full article

The measurement of partial pressures of oxygen (O₂) and carbon dioxide (CO₂) is fundamental for evaluating a patient’s conditions in clinical practice. There are many ways to retrieve O₂/CO₂ partial pressures and concentrations. Arterial blood gas (ABG) analysis is the gold standard technique for such a purpose, but it is invasive, intermittent, and potentially painful. Among all the alternative methods for gas monitoring, non-invasive transcutaneous O₂ and CO₂ monitoring has been emerging since the 1970s, being able to overcome the main drawbacks of ABG analysis. Clark and Severinghaus electrodes enabled the breakthrough for transcutaneous O₂ and CO₂ monitoring, respectively, and in the last twenty years, many innovations have been introduced as alternatives to overcome their limitations. This review reports the most recent solutions for transcutaneous O₂ and CO₂ monitoring, with a particular consideration for wearable measurement systems. Luminescence-based electronic paramagnetic resonance and photoacoustic sensors are investigated. Optical sensors appear to be the most promising, giving fast and accurate measurements without the need for frequent calibrations and being suitable for integration into wearable measurement systems. Full article

Water oxidation is a multielectron complex reaction that produces molecular oxygen as the final product. The article addresses the lack of confirmation of oxygen product formation in electrochemical oxygen evolution reaction (OER) studies, despite the extensive research conducted on catalysts for water splitting. It critically evaluates the trend observed in many studies that solely rely on electrochemical methods for OER quantification without confirming the oxygen product via complementary analytical techniques. The omission of measuring evolved oxygen gas leaves a crucial gap in the quantification of the OER process and raises concerns about the validity and accuracy of reported results. Analytical techniques, such as gas chromatography, Rotating Ring-Disk Electrode (RRDE), fluorescence oxygen probes, Clark electrode, and volumetry are critically analyzed and described to ensure the reliability and credibility of voltammetry and bulk electrolysis to provide a more accurate assessment of the OER process. Full article

The use of hypoxic devices among athletes who train in normobaric hypoxia has become increasingly popular; however, the acute effects on heart and brain metabolism are not yet fully understood. This study aimed to investigate the mitochondrial bioenergetics in trained male and female Wistar rats after acute hypoxia training. The experimental plan included exercising for 30 min on a treadmill in a Plexiglas cage connected to a hypoxic generator set at 12.5% O₂ or in normoxia. After the exercise, the rats were sacrificed, and their mitochondria were isolated from their brains and hearts. The bioenergetics for each complex of the electron transport chain was tested using a Clark-type electrode. The results showed that following hypoxia training, females experienced impaired oxidative phosphorylation through complex II in heart subsarcolemmal mitochondria, while males had an altered ADP/O in heart interfibrillar mitochondria, without any change in oxidative capacity. No differences from controls were evident in the brain, but an increased electron transport system efficiency was observed with complex I and IV substrates in males. Therefore, the study’s findings suggest that hypoxia training affects the heart mitochondria of females more than males. This raises a cautionary flag for female athletes who use hypoxic devices. Full article

The extracellular purinergic agonist uridine diphosphate glucose (UDP-G) activates chemotaxis of human neutrophils (PMN) and the recruitment of PMN at the lung level, via P2Y14 purinergic receptor signaling. This effect is similar to the activation of PMN with N-formyl-methionyl-leucyl-phenylalanine (fMLP), a mechanism that also triggers the production of superoxide anion and hydrogen peroxide via the NADPH oxidase system. However, the effects of UDP-G on this system have not been studied. Defects in the intracellular phagocyte respiratory burst (RB) cause recurrent infections, immunodeficiency, and chronic and severe diseases in affected patients, often with sepsis and hypoxia. The extracellular activation of PMN by UDP-G could affect the RB and oxidative stress (OS) in situations of inflammation, infection and/or sepsis. The association of PMNs activation by UDP-G with OS and RB was studied. OS was evaluated by measuring spontaneous chemiluminescence (CL) of PMNs with a scintillation photon counter, and RB by measuring oxygen consumption with an oxygen Clark electrode at 37 °C, in non-stimulated cells and after activation (15 min) with lipopolysaccharides (LPS, 2 µg/mL), phorbol myristate acetate (PMA, 20 ng/mL), or UDP-G (100 μM). The stimulation index (SI) was calculated in order to establish the activation effect of the three agonists. After stimulation with LPS or PMA, the activated PMNs (0.1 × 10⁶ cells/mL) showed an increase in CL (35%, p < 0.05 and 56%, p < 0.01, SI of 1.56 and 2.20, respectively). Contrariwise, the stimulation with UDP-G led to a decreased CL in a dose-dependent manner (60%, 25 μM, p < 0.05; 90%, 50–150 μM, p < 0.001). Nonetheless, despite the lack of oxidative damage, UDP-G triggered RB (SI 1.8) in a dose-dependent manner (38–50%, 100–200 μM, p < 0.0001). UDP-G is able to trigger NADPH oxidase activation in PMNs. Therefore, the prevention of OS and oxidative damage observed upon PMN stimulation with UDP-G indicates an antioxidant property of this molecule which is likely due to the activation of antioxidant defenses. Altogether, LPS and UDP-G have a synergistic effect, suggesting a key role in infection and/or sepsis. Full article

A flexible pH sensor based on using iridium oxide (IrO_x) as the sensing film was developed by the roll-to-roll (R2R) process. The inert and biocompatible properties of IrO_x make it a desired metal oxide for pH-sensing applications. The flexible substrates being continuously processed by the R2R technique provides the advantages of scalability, reconfigurability, resiliency, on-demand manufacturing, and high throughput, without the need for vacuum systems. Potential sweeps by cyclic voltammetry across the IrO_x film against commercial and planar Ag/AgCl electrodes validated the reversible electrochemical mechanisms. Multiple IrO_x electrodes showed similar output potentials when continuously tested in the pH range of 2–13, indicating good fabrication uniformity. For practical applications, planar IrO_x/Ag-AgCl pairs developed on polyimide substrates were tested, with a good linear fit within pH 2–13, achieving Nernstian responses of around −60.6 mV/pH. The pH sensors showed good repeatability when analyzed with hysteresis, drift, fluctuation, and deviation as the stability factors. The selectivity of the interference ions and the effect of temperature were studied and compared with the reported values. The electrodes were further laminated in a process compatible with the R2R technique for packaging. The flexible sensors were tested under flat and curved surface conditions. Tests in artificial sweat and viscous solutions were analyzed in the Clarke error grid, showing reliable pH-sensing performance. The materials used during the manufacturing processes were sustainable, as the active materials were in small amounts and there was no waste during processing. No toxic chemicals were needed in the fabrication processes. The cost-effective and efficient materials and the fabrication process allow for rapid production that is necessary for disposable and point-of-care devices. Flexible electronics provide a platform for device and sensor integration and packaging, which enables Internet-of-things (IoT) network applications. Full article

Express assessment of the biochemical activity of microorganisms is important in both applied and fundamental research. A laboratory model of a microbial electrochemical sensor formed on the basis of the culture of interest is a device that provides rapidly information about the culture and is cost effective, simple to fabricate and easy to use. This paper describes the application of laboratory models of microbial sensors in which the Clark-type oxygen electrode was used as a transducer. The formation of the models of the reactor microbial sensor (RMS) and the membrane microbial sensor (MMS) and the formation of the response of biosensors are compared. RMS and MMS are based on intact or immobilized microbial cells, respectively. For MMS, the response of biosensor is caused both by the process of transport of substrate into microbial cells and by the process of the initial metabolism of substrate; and only initial substrate metabolism triggers the RMS response. The details of the application of biosensors for the study of allosteric enzymes and inhibition by substrate are discussed. For inducible enzymes, special attention is paid to the induction of microbial cells. This article addresses current problems related to implementation of the biosensor approach and discusses the ways how to overcome these problems. Full article

Catalase is a key antioxidative enzyme, and a deficiency or malfunction of catalase is hypothesized to be related to various diseases. To investigate catalase activity, it is important to use reliable methods and experimental protocols enabling consistent fallouts. One major problem, however, is that the activity values obtained with different techniques and procedures can vary to a large extent. The aim of this work was to identify experimental conditions that provide similar catalase activity values with two different methods based on either spectrophotometry or chronoamperometry. The investigated parameters include the concentration of catalase and its substrate (H₂O₂), as well as the effect of deoxygenation of the catalase medium by nitrogen (N₂). Within the frame of investigated conditions, we show that spectrophotometry is strongly affected by the catalase concentration, whereas chronoamperometry is shown to be more dependent on the substrate concentration. Deoxygenation leads to elevated catalase activity values in the case of chronoamperometry, whereas it shows no influence on the results obtained with spectrophotometry. In particular, in the case of low substrate concentrations (i.e., low catalase reaction rates), higher and more accurate results are obtained with deoxygenation in the case of chronoamperometry measurements due to minimized oxygen escape. The effect of deoxygenation, giving rise to elevated catalase activity values, however, is not statistically significant at high substrate concentrations, implying that the protocol can be simplified by excluding this step as long as the other parameters are optimized. Finally, by comparing the two methods at different experimental conditions, we identified protocols resulting in similar results, i.e., 10 mM H₂O₂ and catalase activity of 4–5 U/mL. Based on this work, improved consistency of catalase activity data obtained with different methodologies and in different labs is expected. Full article

In the last decade, there has been continuous competition between two methods for detecting the concentration of dissolved oxygen: amerometric (Clark electrode) and optical (quenching of the phosphorescence of the porphyrin metal complex). Each of them has obvious advantages and disadvantages. This competition is especially acute in the development of biosensors, however, an unbiased comparison is extremely difficult to achieve, since only a single detection method is used in each particular study. In this work, a microfluidic system with synchronous detection of the oxygen concentration by two methods was created for the purpose of direct comparison. The receptor element is represented by Saccharomyces cerevisiae yeast cells adsorbed on a composite material, previously developed by our scientific group. To our knowledge, this is the first work of this kind in which the comparison of the oxygen detection methods is carried out directly. Full article

Modern life quality is strongly supported by the advances made in biosensors, which has been attributed to their crucial and viable contribution in point-of-care (POC) technology developments. POC devices are exploited for the fast tracing of disease progression, rapid analysis of water, and food quality assessment. Blood glucose meters, home pregnancy strips, and COVID-19 rapid tests all represent common examples of successful biosensors. Biosensors can provide great specificity due to the incorporation of selective bio-recognition elements and portability at significantly reduced costs. Electrochemical biosensor platforms are one of the most advantageous of these platforms because they offer many merits, such as being cheap, selective, specific, rapid, and portable. Furthermore, they can be incorporated into smartphones and various analytical approaches in order to increase their sensitivity and many other properties. As a very broad and interdisciplinary area of research and development, biosensors include all disciplines and backgrounds from materials science, chemistry, physics, medicine, microbiology/biology, and engineering. Accordingly, in this state-of-the-art article, historical background alongside the long journey of biosensing construction and development, starting from the Clark oxygen electrode until reaching highly advanced wearable stretchable biosensing devices, are discussed. Consequently, selected examples among the miscellaneous applications of nanobiosensors (such as microbial detection, cancer diagnosis, toxicity analysis, food quality-control assurance, point of care, and health prognosis) are described. Eventually, future perspectives for intelligent biosensor commercialization and exploitation in real-life that is going to be supported by machine learning and artificial intelligence (AI) are stated. Full article

The generation of reactive oxygen species presents a destructive challenge for the skin organ and there is a clear need to advance skin care formulations aiming at alleviating oxidative stress. The aim of this work was to characterize the activity of the antioxidative enzyme catalase in keratinocytes and in the skin barrier (i.e., the stratum corneum). Further, the goal was to compare the activity levels with the corresponding catalase activity found in defatted algae biomass, which may serve as a source of antioxidative enzymes, as well as other beneficial algae-derived molecules, to be employed in skin care products. For this, an oxygen electrode-based method was employed to determine the catalase activity and the apparent kinetic parameters for purified catalase, as well as catalase naturally present in HaCaT keratinocytes, excised stratum corneum samples collected from pig ears with various amounts of melanin, and defatted algae biomass from the diatom Phaeodactylum tricornutum. Taken together, this work illustrates the versatility of the oxygen electrode-based method for characterizing catalase function in samples with a high degree of complexity and enables the assessment of sample treatment protocols and comparisons between different biological systems related to the skin organ or algae-derived materials as a potential source of skin care ingredients for combating oxidative stress. Full article

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 10⁵ cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 10⁷ cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions. Full article

A new catalase amperometric biosensor for hydroperoxides detection has been built as part of research aimed at the development of biosensors based on layered double hydroxides (LDH) used as support for enzyme immobilization. The fabricated device differs from those developed so far, usually based on an LDH enzyme nanocomposite adsorbed on a glassy carbon (GC) electrode and cross-linked by glutaraldehyde, since it is based on an amperometric gas diffusion electrode (Clark type) instead of a GC electrode. The new biosensor, which still uses LDH synthesized by us and catalase enzyme, is robust and compact, shows a lower LOD (limit of detection) value and a linearity range shifted at lower concentrations than direct amperometric GC biosensor, but above all, it is not affected by turbidity or emulsions, or by the presence of possible soluble species, which are reduced to the cathode at the same redox potential. This made it possible to carry out accurate and efficient determination of H₂O₂ even in complex or cloudy real matrices, also containing very low concentrations of hydrogen peroxide, such as milk and cosmetic products, i.e., matrices that would have been impossible to analyze otherwise, using conventional biosensors based on a GC–LDH enzyme. An inaccuracy ≤ 7.7% for cosmetic samples and ≤8.0% for milk samples and a precision between 0.7 and 1.5 (as RSD%), according to cosmetic or milk samples analyzed, were achieved. Full article