Vladimir I. Ogurtsov

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.

Biosensors on the basis of luminescent oxygen sensor: the use of microporous light-scattering support materials

Abstract New sensor technology and materials for luminescence-based oxygen sensors are described, which utilise microporous light-scattering support in sensor fabrication. Such materials display a number of new features and advantages over conventional oxygen sensor membranes, such as, significantly increased levels of luminescent signals obtainable from the sensor, better mechanical, physical and optical properties and a high capacity for loading biomaterial. The new oxygen sensor technology enables simple and robust procedures of sensor fabrication, as well as saving of the sensing materials, bringing down sensor photobleaching, and improvement in reliability of measurements and signal resolution. Biosensors developed on the basis of the above luminescent oxygen-sensitive materials used as chemical transducers, are described. Direct immobilisation of oxidase enzymes of the oxygen sensor membranes allows simple assays of metabolites. In particular, new batch-type enzyme biosensor for glucose has been investigated, using a new heterogeneous assay format. The latter allows simple, fast and highly reproducible analysis of important metabolites.

Phosphorescent Sensor Approach for Non-Destructive Measurement of Oxygen in Packaged Foods: Optimisation of Disposable Oxygen Sensors and their Characterization Over a Wide Temperature Range

ABSTRACT Phosphorescent oxygen sensors were evaluated for their suitability as a non-destructive method of measuring oxygen in packaged foods. Using phosphorescent phase measurements, characteristics of several types of disposable oxygen sensors were studied in order to optimize sensor chemistry, fabrication technology and performance. The optimal sensor was characterized in both the gas phase and in the liquid phase, over a temperature range of –17°C − +30°C and oxygen concentrations between 0 and 21 kPa. Calibrations, analytical equations and temperature coefficients were obtained, which enabled accurate quantitation of oxygen and correction of optical measurements for sample temperature variations. For disposable sensor elements the resolution of the system at 22°C was about ±0.02 kPa and ±0.5 kPa at 0 and 21 kPa oxygen respectively, and in continuous monitoring mode - ±0.0054 kPa and ±0.081 kPa oxygen, respectively. Results of the use of the oxygen sensors in food packaging applications and practical recommendations are presented.

Modeling of luminescence-based oxygen sensors with non-uniform distribution of excitation and quenching characteristics inside active medium

A generic mathematical model was developed, which describes the integral luminescent signal (intensity and phase shift) of the quenched-luminescence oxygen sensor in the case of non-uniform distribution of the main parameters inside active medium, namely the quenching constant, oxygen and dye concentration and intensity of excitation. This approach, which describes the behaviour of various intensity-based and phase-fluorimetric oxygen sensor systems, was validated by applying it to a set of experimental calibration data obtained with heterogenous microporous sensor membranes on the basis of platinum(II)-octaethylporphine-ketone and polystyrene, and the phase-fluorimetric detector. The cases of one-, two- and three-parametric distribution of the quenching constant were analysed in detail, including discrete single- and double-exponential models and continuous distributions. The latter were represented by: Rayleigh and Maxwell distributions for one-parametric models; Gaussian, Laplace, Cauchy, Extreme Value, Logistic distributions for symmetrical two-parametric models; lognormal, Weibull, gamma and beta distributions for asymmetrical two-parametric models; mixed single-exponential model with one- and two-parametric distribution of the quenching constant-for three-parametric models. Particular models were determined, that provide satisfactory approximation with the same or smaller number of parameters than the classical single and double-exponential models. The best accuracy was achieved with: Maxwell distribution for one-parametric models; lognormal and truncated Gausian (defined only for the quenching constant values larger than distribution mode) for two-parametric models; and discrete double-exponential and mixed discrete with Maxwell distribution for three-parametric models.

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.

Microelectrochemical Systems on Silicon Chips for the Detection of Pollutants in Seawater

The ever-growing demand for simple, fast and reliable techniques for the detection of pollutants and contaminants in the environment has sparked the development of remote detection and monitoring systems which include application specific sensors, instrumentation and signal processing. We report here the design, fabrication and characterisation of four designs of microelectrochemical systems on silicon chip for the detection of pollutants in artificial seawater. These systems were fabricated by photolithography and incorporate a Pt working microelectrode array (squares or bands), a Pt counter electrode and a Ag|AgCl reference electrode. They have been characterised by cyclic voltammetry of ferricyanide and behaved in good agreement with the theory. These systems were evaluated over 72 hours and showed good stability. Underpotential Deposition – Stripping Voltammetry experiments of Cu2+ in artificial seawater have been carried out at an array of 35 microsquares of 20 µm × 20 µm. The sensitivity achieved was (2.93±0.14) µA cm−2 µM−1, with 1 µM being the lowest Cu2+ concentration measured. These devices provide the basis for the development into sensor systems for remote analysis applications.

Fabrication and Electrochemical Characterization of Micro- and Nanoelectrode Arrays for Sensor Applications

This paper describes the fabrication of microelectrode arrays, with two different geometries: disc (Designs d1 and d2) and band (Designs b1, b2 and b3) using three critical dimensions (100 nm, 1 μm and 10 μm) leading to 5 different designs, fabricated by the combination of UV photolithographic and e-beam lithographic techniques. Three silicon nitride layer thicknesses (200, 300 and 500 nm) were chosen to determine an optimized transducer design and fabrication process. Cyclic voltammetry characterisation using a simple redox probe ion, ferreocenecarboxylic acid in phosphate buffered saline electrolyte solution, demonstrated steady-state voltammetric curves for d1, d2, b1 and b2. A good agreement between experimental and theoretical data is found for devices d1, d2, b1 and b2. The experimental current for b3, on the other hand, is much lower compared to the calculated one- perhaps due to the overlapping of the diffusion layers of neighbouring microelectrodes in the array.

Enzymatic flow-injection analysis of metabolites using new type of oxygen sensor membranes and phosphorescence phase measurements

ABSTRACT A simple system for enzymatic flow-injection analysis of metabolites is described, which is based on the phosphorescence lifetime based detection of molecular oxygen using phase-modulation techniques and a simple instrument - phosphorescence phase detector equipped with a fibre-optic probe. The phase detector is connected to the oxygen sensor membrane and allows real-time continuous monitoring of the phosphorescence phase shift. This parameter is related to the phosphorescence lifetime of the oxygen probe, therefore giving a measure of the dissolved oxygen concentration, and its changes as a result of the enzymatic oxidative reaction with the substrate. The sensor membrane is positioned in a compact integrated flow-through cell and exposed to the flow stream. Using glucose as a test analyte and glucose oxidase enzyme, two different sensor setups were tested: 1) the membrane type biosensor in which the enzyme is immobilized directly on the oxygen sensor membrane; 2) the microcolumn type biosensor ...

Development of an Integrated Electrochemical Sensing System to Monitor Port Water Quality Using Autonomous Robotic Fish

The development of autonomous systems for environmental monitoring is a great challenge posed to scientists. Robots might offer an interesting solution for monitoring where access is difficult such as oceans and seas. Here, we describe the approach envisaged for pollution monitoring in port waters based on robot fish equipped with chemical sensors. We will briefly review the state of the art of environmental sensors before describing the electrochemical sensors and instrumentation developed to be integrated inside a robot fish. Finally, we will present laboratory and real sample tests obtained with the developed electrochemical microsystems.

Ultra-Sensitive determination of pesticides via cholinesterase-based sensors for environmental analysis.

Publisher Summary The detection of many pesticides at extremely low levels can be best achieved not by direct detection of the pesticide itself but rather by detection of its inhibitory effects on enzyme reactions. The detection of organophosphate and other pesticides based on the inhibition of the enzyme acetylcholinesterase by these compounds has received considerable attention primarily because of high specificity and sensitivity. Other techniques such as use of multiple electrodes, pattern recognition software and flow-injection techniques have enabled the subtraction of matrix effects such as heavy metals from the system as well as the determination of pesticides in systems containing more than one compound. The signal processing algorithms allow automation of the pesticide quantification enabling use of the instrumentation by unskilled personal, thereby removing this sensing platform from specialized laboratories and making it available to the end-users. Thus this application could conceivably be utilized in the field as well as under laboratory conditions. The relative low cost of electrochemical technology compared with many of the other technologies used makes it an attractive alternative, especially if the enzyme electrodes can be inexpensively mass-produced using screen-printing to allow single-shot use.