Comparison of the three optical platforms for measurement of cellular respiration.

We compared three optical platforms for measurement of cellular respiration: absolute oxygen consumption rates (OCRs) in hermetically sealed microcuvettes, relative OCRs measured in a 96-well plate with oil seal, and steady-state oxygenation of cells in an open 96-well plate. Using mouse embryonic fibroblasts cell line, the phosphorescent intracellular O2 probe MitoXpress-Intra, and time-resolved fluorescence reader, we determined algorithms for conversion of relative OCRs and cell oxygenation into absolute OCRs, thereby allowing simple high-throughput measurement of absolute OCR values.

On-chip electrochemical microsystems for measurements of copper and conductivity in artificial seawater.

The fabrication and characterisation of microelectrochemical sensors for Cu(2+) and conductivity suitable for operation in the marine environment are presented. The impact of the designs on sensor performance and their adequacy to operate in real conditions are discussed. The sensors, tailored to voltammetric and impedimetric measurements, are fabricated on silicon using photolithographic and thin film deposition techniques. The impedimetric sensor is made of Pt interdigitated electrodes which are used for the measurement of conductivity. The voltammetric sensors are based on a three electrode electrochemical cell with on-chip Ag|AgCl reference and Pt counter and working electrodes, used for detection of copper by underpotential deposition-stripping voltammetry at microelectrode array. The sensors operated in the Cu(2+) concentrations ranging from 0.48 to 3.97 µM with a limit of detection of 0.115 µM. The impact of the temperature, the pH and the salinity of the artificial seawater on the sensitivity for Cu(2+) detection are also considered. Measurements of copper concentration and conductivity are validated using certified reference materials and standard solutions.

Characterization of the electrochemical behavior of gastrointestinal fluids using a multielectrode sensor probe.

A characterization of gastrointestinal fluids has been performed by means of an electrochemical sensor that has potential for clinical in vivo and in vitro monitoring applications. The sensor comprised a three-electrode cell with a counter, reference, and four working electrodes, Au, Pt, Ir, and Rh. Cyclic voltammetry was used to obtain chemical information from faecal water (in vitro) and gut model (in vivo) fluids. Stable voltammetric responses were obtained for both fluids at these noble metal working electrodes. The responses differed in shape that demonstrated the discrimination capability and the potential for practical use as a tool for gastrointestinal fluid investigation. The analysis of the stability profiles in faecal water over a 14-h duration has indicated a possible adsorption mechanism with the formation of a biolayer on the sensor surface. The stability in gut model fluids over a 42-h duration has demonstrated a more stable profile, but the mechanisms involved are more complicated to determine.

Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique.

Quenched-phosphorescence oxygen (O(2)) sensing technique allows non-invasive, real-time monitoring of both intra- and extracellular O(2) concentration in respiring samples. Using this technique we investigated O(2) gradients in populations of neurosecretory PC12 cells cultured in 96-well plates and exposed to graded hypoxia at rest and upon metabolic stimulation. Under high atmospheric O(2) (10-21%) the respiration of resting cells dictated that local O(2) was moderately reduced, and at a certain threshold (6% in galactose medium) cell layer became practically anoxic. Furthermore, cell stimulation triggered a major redistribution of O(2) and a prominent 'hypoxic overshoot' mediated by diffusion. The deep, prolonged cell deoxygenation upon stimulation was matched by an increase in nuclear HIF-1alpha levels. In the presence of nitric oxide the hypoxic overshoot was truncated and HIF-1alpha stabilization inhibited. Thus, the main determinants which impact upon cellular O(2) levels and oxygen-sensitive signaling pathways are the atmospheric O(2), sample geometry, cell density, respiration rate and its dynamics. Changes in any of these parameters can significantly alter the O(2) levels experienced by the cells and the subsequently activated signaling pathways. This technique, which provides simple and reliable monitoring of cell oxygenation, is therefore important for hypoxia research, metabolic studies and experiments with respiring cells.

Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes.

Mitochondrial dysfunction is a common mechanism of drug-induced toxicity. Early identification of new chemical entities (NCEs) that perturb mitochondrial function is of significant importance to avoid attrition in later stages of drug development. One of the most informative ways of assessing mitochondrial dysfunction is by measuring mitochondrial oxygen consumption. However, the conventional polarographic method of measuring oxygen consumption is not amenable to high sample throughput or automation. We present an alternative, low-bulk, high-throughput approach to the analysis of isolated-mitochondrial oxygen consumption using luminescent oxygen-sensitive probes. These probes are dispensable and are analyzed in standard microtitre plates on a fluorescence plate reader. Respiratory substrate and adenosine diphosphate (ADP) dependencies of mitochondrial oxygen consumption were assessed using the fluorescence-based method, and results compared favourably to conventional polarographic analysis. To assess assay performance, the method was then applied to the analysis of a panel of classical modulators of oxidative phosphorylation. The effect of uncoupler concentration was analyzed in detail to identify factors which would be important in applying this method to large scale NCE screening and mechanistic investigations. Results demonstrate that the 96-well format can accommodate up to approximately 200 compounds/day at a single concentration or alternatively IC(50) values can be generated for approximately 25 compounds. Throughput may be increased by moving to a 384-well plate format.

Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes.

Mitochondrial dysfunction has been associated with a variety of currently marketed therapeutics and has also been implicated in many disease states. Alterations in the rate of oxygen consumption are an informative indicator of mitochondrial dysfunction, but the use of such assays has been limited by the constraints of traditional measurement approaches. Here, we present a high-throughput, fluorescence-based methodology for the analysis of mitochondrial oxygen consumption using a phosphorescent oxygen-sensitive probe, standard microtitre plates and plate reader detection. The protocol describes the isolation of mitochondria from animal tissue, initial establishment and optimization of the oxygen consumption assay, subsequent screening of compounds for mitochondrial toxicity (uncoupling and inhibition), data analysis and generation of dose-response curves. It allows dozens of compounds (or hundreds of assay points) to be analyzed in a single day, and can be further up-scaled, automated and adapted for other enzyme- and cell-based screening applications.