Analysis of activity and inhibition of oxygen-dependent enzymes by optical respirometry on the LightCycler system.

There is currently a need for a method capable of measuring the activity and inhibition of biologically relevant oxygenases in a format that enables the convenient, fast, and cost-efficient generation of dose-response information. Here we describe a low-volume luminescence-based assay for the measurement of such oxygen-dependent enzymes. The assay employs a photoluminescent oxygen-sensitive probe and glass capillary microcuvettes measured on the Roche LightCycler detection platform. Three discrete types of oxygen probe were evaluated for this application: (i) solid-state coatings, (ii) soluble macromolecular MitoXpress probe, both phosphorescent porphyrin-based, and (iii) a luminescent Ir(III)-based nanoparticle probe. Measurement parameters were optimised and subsequently applied to the analysis of three biologically relevant oxygenases, namely cytochrome P450 (CYP), monoamine oxygenase (MAO), and cyclooxygenase (COX). CYP enzymes are central players in drug detoxification while specific inhibitors of MAO and COX are important for therapeutic intervention and treatment of neurological and inflammatory diseases, respectively. To determine assay utility, oxygen consumption catalysed by all three enzyme types was measured and the effect of specific inhibitors determined. The panel included the MAO-A/B inhibitors clorgyline, toloxatone, deprenyl, and the COX-1/2 inhibitors niflumic acid, nimesulide, SC-560, ketoprofen, and phenylbutazone. IC(50) values were then compared with literature values. The measurement methodology described allows the low-volume analysis of biologically relevant oxygenases and displays the requisite sensitivity and throughput to facilitate routine analysis. It is also applicable to other O(2)-dependent enzymes and enzymatic systems.

Toxicological profiling of chemical and environmental samples using panels of test organisms and optical oxygen respirometry.

A simple and versatile methodology for high throughput toxicological assessment of chemical and environmental samples is presented. It uses panels of test organisms ranging from prokaryotic (E. coli, V. fischeri) and eukaryotic (Jurkat) cells to invertebrate (Artemia salina) and vertebrate (Danio rerio) organisms, to analyze alterations in their oxygen consumption by optical oxygen respirometry. All the assays are carried out in a convenient microtiter plate format using commercial reagents (phosphorescent oxygen probe, microplates) and detection on a standard fluorescent plate reader. Simple experimental set-up and mix-and-measure procedure allow parallel assessment of up to 96 samples (or assay points) in 2 h, easy generation of dose- and time-dependent responses, and EC(50) values. The methodology was demonstrated with several different classes of chemicals including heavy metal ions, PAHs, pesticides, their mixtures, and also validated with complex environmental samples such as wastewater from a wastewater treatment plant. It has been shown to provide high sensitivity, sample throughput and information content, flexibility and general robustness. It allows ranking and profiling of samples, compares favorably with alternative methods such as MicroTox and mortality tests with animal models, and is well suited for large-scale monitoring programs such as CWA and WFD.

Toxicological assessment of chemicals using Caenorhabditis elegans and optical oxygen respirometry.

Oxygen consumption is indicative of an organism's metabolic state, whereby alterations in respiration rate can result from the presence of different stimuli. Here, we develop a novel approach based on quenched fluorescence oxygen sensing and respirometry method for toxicity screening assays using the nematode Caenorhabditis elegans. Previously, C. elegans was established as a useful model in soil and aquatic toxicology studies. For existing toxicology screening approaches with C. elegans, however, the endpoint is lethality. In addition, the assay time frame for the existing approaches is considerably longer than that for the approach described here. We present a sensitive, robust, high-throughput platform using standard laboratory equipment for toxicological studies by measuring respiration rate in C. elegans animals using a phosphorescent probe.

Investigating oligonucleotide hybridization at subnanomolar level by surface plasmon resonance biosensor method.

We have optimized surface plasmon resonance (SPR) biosensor technology for a rapid, direct, and low-consumption label-free multianalyte screening of synthetic oligonucleotides (ONs) with modified internucleotide linkages potentially applicable in antisense therapy. Monitoring of the ONs hybridization is based on the formation of complex between the natural oligonucleotide probe immobilized on the sensor surface and the ON in solution in contact with the sensor surface. An immobilization chemistry utilizing the streptavidin-biotin interaction was employed to obtain desired ligand density and high hybridization efficiency. It was demonstrated that the sensor is capable of detecting complementary 23-mer ONs in concentrations as low as 0.1 nM with high specificity and reproducibility.