Coupling of NAD(P)H:FMN-oxidoreductase and luciferase from luminous bacteria in a viscous medium: Finding the weakest link in the chain.

The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN-oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5-20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc-Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity.

Bioluminescent-Triple-Enzyme-Based Biosensor with Lactate Dehydrogenase for Non-Invasive Training Load Monitoring.

Saliva is one of the most significant biological liquids for the development of a simple, rapid, and non-invasive biosensor for training load diagnostics. There is an opinion that enzymatic bioassays are more relevant in terms of biology. The present paper is aimed at investigating the effects of saliva samples, upon altering the lactate content, on the activity of a multi-enzyme, namely lactate dehydrogenase + NAD(P)H:FMN-oxidoreductase + luciferase (LDH + Red + Luc). Optimal enzymes and their substrate composition of the proposed multi-enzyme system were chosen. During the tests of the lactate dependence, the enzymatic bioassay showed good linearity to lactate in the range from 0.05 mM to 0.25 mM. The activity of the LDH + Red + Luc enzyme system was tested in the presence of 20 saliva samples taken from students whose lactate levels were compared by the Barker and Summerson colorimetric method. The results showed a good correlation. The proposed LDH + Red + Luc enzyme system could be a useful, competitive, and non-invasive tool for correct and rapid monitoring of lactate in saliva. This enzyme-based bioassay is easy to use, rapid, and has the potential to deliver point-of-care diagnostics in a cost-effective manner.

Bioluminescent-Inhibition-Based Biosensor for Full-Profile Soil Contamination Assessment.

A bioluminescent-enzyme-inhibition-based assay was applied to predict the potential toxicity of the full profile of the following soil samples: agricultural grassland, 10-year fallow land (treated with remediation processes for 10 years) and uncontaminated (virgin) land. This assay specifically detects the influence of aqueous soil extracts from soils on the activity of a coupled enzyme system of luminescent bacteria: NAD(P)H:FMN-oxidoreductase + luciferase (Red + Luc). It was shown that the inhibitory effect of the full-profile soil samples on the Red + Luc system decreased with depth for the 10-year fallow-land and virgin-land samples, which correlated with a decrease in the humic organic matter content in the soils. The inhibitory effect of the agricultural grassland on the Red + Luc enzyme system activity was more complex and involved the presence of the humic organic matter content, as well as the presence of pollutants in the whole-soil profile. However, if the interfering effect of humic organic substances on the Red + Luc system's activity is taken into account during full-profile soil toxicity assessments, it might help to detect pollutant mobility and its leaching into the subsoil layer. Thus, this bioluminescent method, due to the technical simplicity, rapid response time and high sensitivity, has the potential to be developed as a biological part of the inhibition-based assay and/or biosensors for the preventive tracing of potential full-profile soil contamination.

Toxicity of Different Types of Surfactants via Cellular and Enzymatic Assay Systems.

Surfactants have a widespread occurrence, not only as household detergents, but also in their application in industry and medicine. There are numerous bioassays for assessing surfactant toxicity, but investigations of their impact on biological systems at the molecular level are still needed. In this paper, luminous marine bacteria and their coupled NAD(P)H:FMN-oxidoreductase + luciferase (Red + Luc) enzyme system was applied to examine the effects of different types of surfactants, including cationic cetyltrimethylammonium bromide (CTAB), non-ionic polyoxyethylene 20 sorbitan monooleate (Tween 80) and anionic sodium lauryl sulfate (SLS), and to assess whether the Red + Luc enzyme system can be used as a more sensitive indicator of toxicity. It was shown that the greatest inhibitory effect of the surfactants on the activity of luminous bacteria and the Red + Luc enzyme system was in the presence of SLS samples. The calculated IC50 and EC50 values of SLS were 10-5 M and 10-2 M for the enzymatic and cellular assay systems, respectively. The results highlight the benefits of using the enzymatic assay system in ecotoxicology as a tool for revealing surfactant effects on intracellular proteins if the cellular membrane is damaged under a long-term exposure period in the presence of the surfactants. For this purpose, the bioluminescent enzyme-inhibition-based assay could be used as an advanced research tool for the evaluation of surfactant toxicity at the molecular level of living organisms due to its technical simplicity and rapid response time.

Software for Matching Standard Activity Enzyme Biosensors for Soil Pollution Analysis.

This work is dedicated to developing enzyme biosensor software to solve problems regarding soil pollution analysis. An algorithm and specialised software have been developed which stores, analyses and visualises data using JavaScript programming language. The developed software is based on matching data of 51 non-commercial standard soil samples and their inhibitory effects on three enzyme systems of varying complexity. This approach is able to identify the influence of chemical properties soil samples, without toxic agents, on enzyme biosensors. Such software may find wide use in environmental monitoring.

A noninvasive and qualitative bioluminescent assay for express diagnostics of athletes' responses to physical exertion.

Upcoming professional sports authorities seek rapid noninvasive biosensing tools for regular monitoring of athletes' physiological states. The analysis of saliva through luminescence-based biosensors has been perceived as a suitable candidate for such purposes. The present study reports a qualitative bioluminescence assay based on a coupled enzyme system that consists of bacterial luciferase (BLuc) and nicotinamide adenine dinucleotide (NADH):flavin mononucleotide (FMN) oxidoreductase (Red), BLuc-Red, for the express diagnostics of athletes' stress levels before and after physical exertion. The volunteers who participated in the study were grouped as freestyle wrestlers and students who adapted to different levels of physical activities. Under physical exertion modelling conditions, the influence of participant saliva on BLuc-Red catalyzed light emission was investigated. Results showed a significant increase in residual luminescence (Iexp , mean maximum bioluminescence intensity of the experimental measurement (Iexp ); Ic , luminescence intensity in control; Iexp /Ic , %) values for participants in the wrestler group while a decrease in the student group (P < 0.05). Such contrasting residual luminescence values in both groups were found to be dependent on the catalase activity of saliva. The proposed bioluminescence assay can be utilized as a potential nonspecific biosensing tool for determining the physical state of athletes under high loads.

Bioluminescent enzyme inhibition-based assay to predict the potential toxicity of carbon nanomaterials.

A bioluminescent enzyme inhibition-based assay was applied to predict the potential toxicity of carbon nanomaterials (CNM) presented by single- and multi-walled nanotubes (SWCNT and MWCNT) and aqueous solutions of hydrated fullerene С60 (C60HyFn). This assay specifically detects the influence of substances on parameters of the soluble or immobilised coupled enzyme system of luminescent bacteria: NAD(P)Н:FMN-oxidoreductase+luciferase (Red+Luc). A protocol based on the optical properties of CNM for correcting the results of the bioluminescent assay was also developed. It was shown that the inhibitory activity of CNM on Red+Luc decreased in the following order: MWCNT>SWCNT>C60HyFn. The soluble enzyme system Red+Luc had high sensitivity to MWCNT and SWCNT, with values of the inhibition parameter IC50 equal to 0.012 and 0.16mg/L, respectively. The immobilised enzyme system was more vulnerable to C60HyFn than its soluble form, with an IC50 equal to 1.4mg/L. Due to its technical simplicity, rapid response time and high sensitivity, this bioluminescent method has the potential to be developed as a general enzyme inhibition-based assay for a wide variety of nanomaterials.