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Singlet oxygen is considered an important cell damaging agent due to its propensity to react with organic compounds. This drives the interest in developing methods for determination of 1O2. Simplicity of application and high sensitivity makes fluorescent probes a popular choice for in vivo 1O2 detection. Despite its proclaimed cell-impermeability, the commercially available Singlet Oxygen Sensor Green (SOSG) is widely applied to support assertions of 1O2 involvement in cell and tissue damage. Our investigation, however, demonstrate that different microbial species and cancer cells become fluorescent when exposed to SOSG under conditions which exclude generation of 1O2. Cells, permeabilized with chlorhexidine or by heat exposure under anaerobic conditions, exhibited SOSG fluorescence. Permeabilized cells could be stained with SOSG even 24 h post-permeabilization. Since SOSG is cell impermeable, the main factor that led to fluorescent staining was plasma membrane damage. Spectral analyses of different batches of SOSG revealed that SOSG endoperoxide (SOSG-EP) did not increase even after prolonged storage under the recommended conditions. The commercial preparations of SOSG, however, were not SOSG-EP free, which can produce erroneous results when SOSG staining is used as a proof of singlet oxygen production in vivo.

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Aquaculture is growing industry from the perspective of sustainable food fulfillment and county's economic development. Technology oriented aquafarming is the solution for effective water quality monitoring and high yield production. Internet of Things (IoT) integrated aquaculture can cater to such requirements. This research article introduces a comprehensive method aimed at seamlessly incorporate IoT sensors into aquafarming environments, utilizing Arduino boards and communication modules. The proposed method measures accurate water quality parameters, such as temperature, pH levels, and Dissolved Oxygen (DO), which are essential for maintaining optimal conditions for suitable aquaculture environment. This method enables the real-time collection of critical data points that are essential prevent fish diseases and mortality with low human intervention and maintenance cost. The key contributions of the methodology are mentioned below.•Design and development of a compact and efficient Printed Circuit Board (PCB) to achieve accurate sensor data readings and reliable communication in an aqua environment.•Prevent fish disease and mortality rate through data-driven decision incorporating correlation of DO, pH, and temperature sensor data.•Conducted instrument calibration checks and cross-validated automated system data with manual observations through repeatability tests to ensure precise measurements of sensor parameters.

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In recent years, significant attention has been directed toward advancing compact, point-of-care testing (POCT) devices to better deliver patient care and alleviate the burden on the medical care system. Common POCTs, such as blood oxygen sensors, leverage electrochemical sensing in their design. However, conventional electrochemical devices typically use Ag/AgCl reference electrodes, which are likely to release trace amounts of silver ions that contaminate the working electrode, causing rapid deterioration of the devices. This study proposes an effective reference electrode using graphene-coated porous silica spheres (G/PSS) with embedded Prussian blue (PB), denoted PB/G/PSS, designed specifically for small oxygen sensors. PB is a redox species that is an improvement over Ag/AgCl since it is significantly less water-soluble than AgCl. Since PB is an insulator, we dispersed PB in G/PSS, well-conductive mesoporous matrices, to ensure contact between PB clusters and the electrolytes. Moreover, the monodispersed, spherically shaped PB/G/PSS is an advantageous medium for fabricating POCT devices by screen printing. In this study, the open-circuit potential of the PB/G/PSS electrode remained stable within 30 mV for 31 days. The small oxygen sensor assembled through screen printing using PB/G/PSS demonstrated stable operation for several days or more. In contrast, a similar sensor with Ag/AgCl reference electrode rapidly deteriorated within a day. This PB/G/PSS reference electrode with improved stability is expected to be an excellent alternative to the Ag/AgCl system for small electrochemical-based POCT devices.

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Well plates are widely used in biological experiments, particularly in pharmaceutical sciences and cell biology. Its popularity stems from its versatility to support a variety of fluorescent markers for high throughput monitoring of cellular activities. However, using fluorescent markers in traditional well plates has its own challenges, namely, they can be potentially toxic to cells, and thus, may perturb their biological functions; and it is difficult to monitor multiple analytes concurrently and in real-time inside each well. This paper presents a fully instrumented microphysiological system with integrated sensors (IMSIS) with a similar well format. Each well in the microphysiological system has a set of sensors for monitoring multiple metabolic analytes in real-time. The IMSIS platform is supported by integrated bioelectronic circuits and a graphical user interface for easy user configuration and monitoring. The system has integrated microfluidics to maintain its microphysiological environment within each well. The IMSIS platform currently incorporates O2, H2O2, and pH sensors inside each well, allowing up to six wells to perform concurrent measurements in real-time. Furthermore, the architecture is scalable to achieve an even higher level of throughput. The miniaturized design ensures portability, suitable for small offices and field applications. The IMSIS platform was successfully used to monitor in real-time the mitochondrial functions of live bovine embryos in O2 consumption, H2O2 release as an indication of ROS production, and extracellular acidity changes before and after the introduction of external substrates.

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The galvanic dissolved oxygen sensor finds widespread applications in multiple critical fields due to its high precision and excellent stability. As its core sensing components, the oxygen-permeable membrane, electrode, and electrolyte significantly impact the sensor's performance. To systematically investigate the comprehensive effects of these core sensing components on the performance of galvanic dissolved oxygen sensors, this study selected six types of oxygen-permeable membranes made from two materials (Perfluoroalkoxy Polymer (PFA) and Fluorinated Ethylene Propylene Copolymer (FEP)) with three thicknesses (0.015 mm, 0.03 mm, and 0.05 mm). Additionally, five concentrations of KCl electrolyte were configured, and four different proportions of lead-tin alloy electrodes were chosen. Single-factor and crossover experiments were conducted using the OxyGuard dissolved oxygen sensor as the experimental platform. The experimental results indicate that under the same membrane thickness conditions, PFA membranes provide a higher output voltage compared to FEP membranes. Moreover, the oxygen permeability of FEP membranes is more significantly affected by temperature. Furthermore, the oxygen permeability of the membrane is inversely proportional to its thickness; the thinner the membrane, the better the oxygen permeability, resulting in a corresponding increase in sensor output voltage. When the membrane thickness is reduced from 0.05 mm to 0.015 mm, the sensor output voltage for PFA and FEP membranes increases by 86% and 74.91%, respectively. However, this study also observed that excessively thin membranes might compromise measurement accuracy. In a saturated, dissolved oxygen environment, the sensor output voltage corresponding to the six oxygen-permeable membranes used in the experiment exhibits a highly linear inverse relationship with temperature (correlation coefficient ≥ 98%). Meanwhile, the lead-tin ratio of the electrode and electrolyte concentration have a relatively minor impact on the sensor output voltage, demonstrating good stability at different temperatures (coefficient of variation ≤ 0.78%). In terms of response time, it is directly proportional to the thickness of the oxygen-permeable membrane, especially for PFA membranes. When the thickness increases from 0.015 mm to 0.05 mm, the response time extends by up to 2033.33%. In contrast, the electrode material and electrolyte concentration have a less significant effect on response time. To further validate the practical value of the experimental results, the best-performing combination of core sensing components from the experiments was selected to construct a new dissolved oxygen sensor. A performance comparison test was conducted between this new sensor and the OxyGuard dissolved oxygen sensor. The results showed that both sensors had the same response time (49 s). However, in an anaerobic environment, the OxyGuard sensor demonstrated slightly higher accuracy by 2.44%. This study not only provides a deep analysis of the combined effects of oxygen-permeable membranes, electrodes, and electrolytes on the performance of galvanic dissolved oxygen sensors but also offers scientific evidence and practical guidance for optimizing sensor design.

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Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical in vivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor ß (IκBß). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBß oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBß oxomer formation did not occur via an IκBß asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBß ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBß from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBß contributes to the hypoxia-dependent regulation of inflammation.

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Epilepsy is a prevalent neurological disorder with a complex pathogenesis and unpredictable nature, presenting limited treatment options in >30 % of affected individuals. Neurometabolic abnormalities have been observed in epilepsy patients, suggesting a disruption in the coupling between neural activity and energy metabolism in the brain. In this study, we employed amperometric biosensors based on a modified carbon fiber microelectrode platform to directly and continuously measure lactate and oxygen dynamics in the brain extracellular space. These biosensors demonstrated high sensitivity, selectivity, and rapid response time, enabling in vivo measurements with high temporal and spatial resolution. In vivo recordings in the cortex of anaesthetized rats revealed rapid and multiphasic fluctuations in extracellular lactate and oxygen levels following neuronal stimulation with high potassium. Furthermore, real-time measurement of lactate and oxygen concentration dynamics concurrently with network electrical activity during status epilepticus induced by 4-aminopyridine (4-AP) demonstrated phasic changes in lactate levels that correlated with bursts of electrical activity, while tonic levels of lactate remained stable during seizures. This study highlights the complex interplay between lactate dynamics, electrical activity, and oxygen utilization in epileptic seizures.

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The intracellular concentrations of oxygen and reactive oxygen species (ROS) in living cells represent critical information for investigating physiological and pathological conditions. Real-time measurement often relies on genetically encoded proteins that are responsive to fluctuations in either oxygen or ROS concentrations. The direct binding or chemical reactions that occur in their presence either directly alter the fluorescence properties of the binding protein or alter the fluorescence properties of fusion partners, mostly consisting of variants of the green fluorescent protein. Oxygen sensing takes advantage of several mechanisms, including (i) the oxygen-dependent hydroxylation of a domain of the hypoxia-inducible factor-1, which, in turn, promotes its cellular degradation along with fluorescent fusion partners; (ii) the naturally oxygen-dependent maturation of the fluorophore of green fluorescent protein variants; and (iii) direct oxygen binding by proteins, including heme proteins, expressed in fusion with fluorescent partners, resulting in changes in fluorescence due to conformational alterations or fluorescence resonance energy transfer. ROS encompass a group of highly reactive chemicals that can interconvert through various chemical reactions within biological systems, posing challenges for their selective detection through genetically encoded sensors. However, their general reactivity, and particularly that of the relatively stable oxygen peroxide, can be exploited for ROS sensing through different mechanisms, including (i) the ROS-induced formation of disulfide bonds in engineered fluorescent proteins or fusion partners of fluorescent proteins, ultimately leading to fluorescence changes; and (ii) conformational changes of naturally occurring ROS-sensing domains, affecting the fluorescence properties of fusion partners. In this review, we will offer an overview of these genetically encoded biosensors.

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BACKGROUND AND AIMS: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). METHODS: hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. RESULTS: The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1ß, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. CONCLUSIONS: Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.

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The chemoresistive properties of multilayer titanium-containing Ti2CTx and Ti3C2Tx MXenes, synthesized by etching the corresponding MAX phases with NaF solution in hydrochloric acid, and the composites based on them, obtained by partial oxidation directly in a sensor cell in an air flow at 150 °C, were studied. Significant differences were observed for the initial MXenes, both in microstructure and in the composition of surface functional groups, as well as in gas sensitivity. For single Ti2CTx and Ti3C2Tx MXenes, significant responses to oxygen and ammonia were observed. For their partial oxidation at a moderate temperature of 150 °C, a high humidity sensitivity (T, RH = 55%) is observed for Ti2CTx and a high and selective response to oxygen for Ti3C2Tx at 125 °C (RH = 0%). Overall, these titanium-containing MXenes and composites based on them are considered promising as receptor materials for low temperature oxygen sensors.

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tris-[(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)] dichloride (Ru(DPP)3Cl2), a fluorescent sensor which is sensitive to the amount of oxygen in the sample, was applied using the fluorescent optical respirometry (FOR) technique. The oxygen in the samples quenches the fluorescence. The fluorescence intensity depends on the metabolic rate of the viable microorganisms. The effect of DMSO and plant extracts on bacteria was determined by FOR. It was shown that the MIC values obtained by FOR were consistent with the results of the MIC determinations using the method of serial dilutions; at the same time, the effects of concentrations lower than the growth-inhibitory concentrations on microbial cells were demonstrated. The FOR method enables the detection of multiplying bacteria in sterile and non-sterile pharmaceutical preparations in real time, which significantly shortens the time required to obtain results and allows the introduction of repair processes in the production. This method also allows for quick, unambiguous detection and the counting of the viable cells of aerobic microorganisms in non-sterile pharmaceuticals.

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An optical sensor system based on wavelength modulation spectroscopy (WMS) was developed for atmospheric oxygen (O2) detection. A distributed feedback (DFB) laser with butterfly packaging was used to target the O2 absorption line at 760.89 nm. A compact multi-pass gas cell was employed to increase the effective absorption length to 3.3 m. To ensure the stability and anti-interference capability of the sensor in field measurements, the optical module was fabricated with isolation of ambient light and vibration design. A 1f normalized 2f WMS (WMS-2f/1f) technique was adopted to reduce the effect of laser power drift. In addition, a LabVIEW-based dual-channel lock-in amplifier was developed for harmonic detection, which significantly reduced the sensor volume and cost. The detailed detection principle was described, and a theoretical model was established to verify the effectiveness of the technique. Experiments were carried out to obtain the device's sensing performances. An Allan deviation analysis yielded a minimum detection limit of 0.054% for 1 s integration time that can be further improved to 0.009% at ~60 s. Finally, the reliability and anti-interference capability of the sensor system were verified by the atmospheric O2 monitoring.

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The current status of microbiological testing methods for the determination of viable bacteria in complex sample matrices, such as food samples, is the focus of this review. Established methods for the enumeration of microorganisms, particularly, the 'gold standard' agar plating method for the determination of total aerobic viable counts (TVC), bioluminescent detection of total ATP, selective molecular methods (immunoassays, DNA/RNA amplification, sequencing) and instrumental methods (flow cytometry, Raman spectroscopy, mass spectrometry, calorimetry), are analyzed and compared with emerging oxygen sensor-based respirometry techniques. The basic principles of optical O2 sensing and respirometry and the primary materials, detection modes and assay formats employed are described. The existing platforms for bacterial cell respirometry are then described, and examples of particular assays are provided, including the use of rapid TVC tests of food samples and swabs, the toxicological screening and profiling of cells and antimicrobial sterility testing. Overall, O2 sensor-based respirometry and TVC assays have high application potential in the food industry and related areas. They detect viable bacteria via their growth and respiration; the assay is fast (time to result is 2-8 h and dependent on TVC load), operates with complex samples (crude homogenates of food samples) in a simple mix-and-measure format, has low set-up and instrumentation costs and is inexpensive and portable.

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Indirect calorimetry (IC) is considered the gold standard for measuring resting energy expenditure (REE). This review presents an overview of the different techniques to assess REE with special regard to the use of IC in critically ill patients on extracorporeal membrane oxygenation (ECMO), as well as to the sensors used in commercially available indirect calorimeters. The theoretical and technical aspects of IC in spontaneously breathing subjects and critically ill patients on mechanical ventilation and/or ECMO are covered and a critical review and comparison of the different techniques and sensors is provided. This review also aims to accurately present the physical quantities and mathematical concepts regarding IC to reduce errors and promote consistency in further research. By studying IC on ECMO from an engineering point of view rather than a medical point of view, new problem definitions come into play to further advance these techniques.

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Enzymatic O2 sensors transduce the availability of O2 within the cell into a physiological, typically adaptive response. One such O2-sensing enzymatic family is the N-terminal cysteine dioxygenases in plants (plant cysteine oxidases [PCOs]). In vitro kinetic studies have determined the O2-sensing capacity of PCOs. Here we describe the rationale and experimental protocol for an assay with which the O2 sensitivity of Arabidopsis thaliana PCOs (AtPCOs) can be measured. We explain each step from the recombinant protein synthesis of AtPCOs to the steady-state kinetic assays of AtPCOs for primary substrate and O2 from which kinetic parameters can be derived. The same techniques can be applied to other N-terminal cysteine thiol dioxygenases, e.g. 2-aminoethanethiol dioxygenase (ADO), and similar principles can be applied to determine kinetic characteristics of other oxygenase enzymes towards O2.

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Since their initial discovery some 30 years ago, heme-based O2 sensors have been extensively studied. Among many other lessons, we have learned that they have adapted a wide variety of folds to bind heme for O2 sensing, and they can couple those sensory domains to transducer domains with many different activities. There is no question that we have learned a great deal about those systems by solving X-ray structures of the truncated pieces of larger multi-domain proteins. All of the studies have, for example, hinted at the importance of protein residues, which were further investigated, usually by site-directed mutagenesis of the full-length proteins together with physico-chemical measurements and enzymatic studies. The biochemistry has suggested that the sensing functions of heme-based O2 sensors involve not only the entire proteins but also, and quite often, their associated regulatory partners and targets. Here we critically examine the state of knowledge for some well-studied sensors and discuss outstanding questions regarding their structures. For the near future, we may foresee many large complexes with sensor proteins being solved by cryo-EM, to enhance our understanding of their mechanisms.

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Oxygen (O2) uptake by cells and tissues is a critical indicator of metabolic demand, changes in microenvironment, and pathophysiology. O2 uptake from the atmosphere accounts for virtually all the O2 consumption in the avascular cornea; however, a detailed spatiotemporal profile of corneal O2 uptake (COU) remains undetermined. Here, we used a non-invasive self-referencing optical fiber O2 sensor-the scanning micro-optrode technique (SMOT)-to report the O2 partial pressure and flux variations at the ocular surface of rodents and non-human primates. In vivo spatial mapping in mice revealed a distinct COU, characterized by a centripetal gradient with a significantly higher O2 influx at the limbus and conjunctiva regions than at the center of the cornea. This regional COU profile was reproduced ex vivo in freshly enucleated eyes. The centripetal gradient was conserved across the following species analyzed: mice, rats, and rhesus monkeys. In vivo temporal mapping in mice showed a significant increase in the O2 flux in the limbus in the evening compared to other times. Altogether, the data unveiled a conserved centripetal COU profile, which may be associated with the limbal epithelial stem cells residing at the intersection of the limbus and conjunctiva. These physiological observations will serve as a useful baseline for comparative studies with contact lens wear, ocular disease, diabetes, etc. Moreover, the sensor may be applied to understand the responses of the cornea and other tissues to various insults, drugs, or changes in the environment.

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Previous studies have shown that the pollutants in exhaust gas can cause performance deterioration in air-fuel oxygen sensors. Although the content of Pb in fuel oil is as low as 5 mg/L, the effect of long-term Pb accumulation on TiO2 oxygen sensors is still unclear. In this paper, the influence mechanism of Pb-containing additives in automobile exhaust gas on the response characteristics of TiO2 oxygen sensors was simulated and studied by depositing Pb-containing pollutants on the surface of a TiO2 sensitive film. It was found that the accumulation of Pb changed the surface gas adsorption state and reduced the activation energy of TiO2, thus affecting the steady-state response voltage and response speed of the TiO2-based oxygen sensor.

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A highly sensitive, biocompatible, and scalable phosphorescent oxygen sensor formulation is designed and evaluated for use in continuous metabolite sensors for biological systems. Ethyl cellulose (EC) and polystyrene (PS) nanoparticles (NPs) stabilized with Pluronic F68 (PF 68), Polydimethylsiloxane-b-polyethyleneglycol methyl ether (PDMS-PEG), sodium dodecylsulfate (SDS), and cetyltimethylammonium bromide (CTAB) were prepared and studied. The resulting NPs with eight different surfactant−polymer matrix combinations were evaluated for physical properties, oxygen sensitivity, effect of changes in dispersion matrix, and cytotoxicity. The EC NPs exhibited a narrower size distribution and 40% higher sensitivity than PS, with Stern−Volmer constants (Ksv) 0.041−0.052 µM−1 for EC, compared to 0.029−0.034 µM−1 for PS. Notably, ethyl cellulose NPs protected with PF68 were selected as the preferred formulation, as they were not cytotoxic towards 3T3 fibroblasts and exhibited a wide phosphorescence lifetime response of >211.1 µs over 258−0 µM and ~100 µs over 2.58−0 µM oxygen, with a limit of detection (LoD) of oxygen in aqueous phase of 0.0016 µM. The EC-PF68 NPs were then efficiently encapsulated in alginate microparticles along with glucose oxidase (GOx) and catalase (CAT) to form phosphorescent nanoparticles-in-microparticle (NIMs) glucose sensing microdomains. The fabricated glucose sensors showed a sensitivity of 0.40 µs dL mg−1 with a dynamic phosphorescence lifetime range of 46.6−197.1 µs over 0−150 mg dL−1 glucose, with a glucose LoD of 18.3 mg dL−1 and maximum distinguishable concentration of 111.1 mg dL−1. Similarly, lactate sensors were prepared with NIMs microdomains containing lactate oxidase (LOx) and found to have a detection range of 0−14 mg dL−1 with LoD of 1.8 mg dL−1 and maximum concentration of 13.7 mg dL−1 with lactate sensitivity of 10.7 µs dL mg−1. Owing to its versatility, the proposed NIMs-based design can be extended to a wide range of metabolites and different oxygen-sensing dyes with different excitation wavelengths based on specific application.

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Accurate monitoring of dissolved oxygen (DO) is vital for aerobic fermentation process control. This work presents an autoclavable Micro-Dissolved oxygen Sensor (MDS) that can monitor real time DO. The proposed sensor is much cheaper to be manufactured (< $35) and can be adapted to varying measurement environments. An ultra-micropore matrix was created using femtosecond laser processing technology to reduce flow dependency of probe signals. The validity of the proposed DO sensor was verified by testing it under different DO levels. The result revealed consistency between the new designed sensor and a commercial DO sensor. The obtained sensitivity was- 7.93 µA·L·mg-1 (MDS with ultra-micropore matrix). Moreover, the MDS can function without an oxygen-permeable membrane and a solid electrolyte was used which reduced the response time (4.6 s). For real-time monitoring, the stability of the MDS was validated during a yeast batch fermentation carried out until 18 h.

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