Valentina A. Kratasyuk

Development of the Bioluminescent Bioindicators for Analyses of Pollutions

Biotests are currently used for monitoring of ecological systems. Bioluminescent bacterial systems are applied instead of plants, fishes and other living organisms as test-objects [1-2]. The main principle of bioluminescent biotests is the correlation between the toxicity of the medium under study and its effect on the light intensity. Applying of the bioluminescent assays develops in two directions: the test-systems of BL bacteria (in vivo) and bioluminescent enzymic systems (in vitro). Reagents based on the test-organisms (e.g. lyophilized luminous bacteria) and on the enzymic systems (e.g. bacterial luciferase systems) are created. Luciferase biotesting is based on the inhibition of the enzymes’ activity by the components of a medium under study.

Rapid biosensing tools for cancer biomarkers

The present review critically discusses the latest developments in the field of smart diagnostic systems for cancer biomarkers. A wide coverage of recent biosensing approaches involving aptamers, enzymes, DNA probes, fluorescent probes, interacting proteins and antibodies in vicinity to transducers such as electrochemical, optical and piezoelectric is presented. Recent advanced developments in biosensing approaches for cancer biomarker owes much credit to functionalized nanomaterials due to their unique opto-electronic properties and enhanced surface to volume ratio. Biosensing methods for a plenty of cancer biomarkers has been summarized emphasizing the key principles involved.

Bioluminescent enzymatic rapid assay of water integral toxicity

A bioluminescent rapid method was developed to estimate the integral toxicity of natural and wastewater. This method is based on registering the effect of the polluted water sample on the parameters of the bioluminescent reaction catalyzed by the multi-component reagent containing NADH:FMN oxidoreductase, luciferase, and their substrates co-immobilized in a starch carrier. Several ways to increase the methods sensitivity to toxic substances were suggested; conditions were selected to make it possible to determine, with maximum efficiency, the content of toxic substances corresponding to a certain maximum permissible concentration. The sensitivity of soluble and immobilized coupled enzymatic systems to a series of organic pollutants (phenols, quinones, and salts of heavy metals) was compared. It was shown that the reagent is the most sensitive to the effect of phenols and quinones. The method was tested during analysis of the wastewater from a pulp and paper plant and can be used for biotesting in both laboratory and field conditions.

Comparative study of immobilized and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system.

The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in Km app was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.

Estimation of energy of the upper electron-excited states of the bacterial bioluminescent emitter

The hypothesis of activity of the upper electron-excited states of the bacterial bioluminescent emitter was verified using dye molecules as foreign energy acceptors. Six compounds were selected having fluorescent state energies ranging from 25,700 to 32,000 cm(-1) (anthracene, pyrene, 1.4-bis(5-phenyloxasol-2-yl)benzene (POPOP), p-bis(o-methylstyryl)benzene (MSB), 2-methoxy-naphtalene, p-terphenyl), exceeding that of the bioluminescent emitter (22,000 cm(-1)). Their absorption spectra do not overlap with the bioluminescence spectrum; the trivial light absorption and the intermolecular resonance S-S energy transfer were excluded. Bacterial bioluminescent spectra of the coupled enzyme system NADH:FMN-oxidoreductase-luciferase in the presence of MSB were presented as an example. The weak sensitized fluorescence of MSB was registered. The results obtained have confirmed the activity of the energetic precursor in the bacterial bioluminescence. Its energy can be located in the interval of 26,000-27,000 cm(-1).

Metal-enhanced luminescence: Current trend and future perspectives- A review

Optically enhanced biosensing strategies are prerequisites for developing miniature and highly sensitive multiplexed analytical platforms. Such smart biosensing systems are highly promising for use in the fields of biomedicine and environmental monitoring. Optical signal enhancement during bioassays is attributed to the complex opto-electronic interactions of incoming photonic signals at the nanomaterial interface. Research on the use of metals other than gold and silver for such purposes tends to extend the spectral window to observe luminescence enhancement effects. Such manifold increase in luminescence may be explained by the principles of plasmon coupling, directional emission led high collection efficiency, Rayleigh scattering and related opto-electronic events. The present review begins with a mechanistic description of important phenomena associated with metal-induced luminescence enhancement, particularly focusing on the origin of metal-enhanced luminescence. This review further analyses the hybrid nanostructure capabilities responsible for maintaining unique opto-electronic properties during bio-functionalisation. Current research trends in this area, future scope of this field for designing useful bioassays and concluding remarks are then discussed.

Bioluminescence characteristics of Lake Shira water

Bioluminescence bioassays based on luminous bacteria (Photobacterium phosphopreum) and coupled enzyme system NADH-FMN-oxidoreductase-luciferase were adapted for monitoring the saline-water conditions of Lake Shira (Khakasia, Siberia). The differences in bioluminescence responses have been found to be related to the salt composition and the oxidation-reduction properties of water. Bioluminescent kinetics parameters, which are mostly sensitive to pollution under conditions of saline water, have been observed. The enzymatic system in the presence of 1,4-benzoquinone are shown to be more sensitive to redox characteristics of the salt water than this in the absence of 1,4-benzoquinone. 1,4-benzoquinone should be applied for the preparation of a model solution for the monitoring of redox properties of the salt water. Using this technique, the results of bioluminescence analysis are used to construct a heterogeneity map that characterizes the spatial and temporal water quality of lake Shira. A partial map was based on the bioluminescence characteristics of water samples taken along the shoreline, sampling stations in the different places and in different depths of the lake. It has been demonstrated that the bioluminescence assay measurements must be done within two hours after the sampling time.

Bioluminescent water quality monitoring of salt lake Shira

The coupled bioluminescent enzyme system luciferase-NADH:FMN-oxidoreductase was used as a biotest in ecological monitoring of the health resort salt lake Shira (South Siberia, Russia). The technique was adapted to saltwater conditions. Bioluminescence kinetic parameters sensitive to pollutants were determined. Conditions for the use of bacterial bioluminescence biotests in salty environmental media were established.

Nutritional biomarkers: Current view and future perspectives

ABSTRACT There is a poor relationship between nutrient intake and existing nutritional biomarkers due to variety of factors affecting their sensitivity and specificity. To explore the impact of nutrients at molecular level and devising a sensitive biomarker, proteomics is a central technology with sirtuins as one of the most promising nutritional biomarker. Sirtuins (seven mammalian sirtuins reported so far) have been reported to perform protein deacetylases and ADP-ribosyltransferases activity. It is distributed in different cellular compartments thereby controlling several metabolic processes. Sirtuins are oxidized nicotinamide adenine dinucleotide dependent, which implicates a direct effect of the metabolic state of the cell on its activity. Calorie restriction upregulates the mammalian sirtuin protein levels in variety of tissues and organs where it acts upon both histone and nonhistone substrates. Sirtuin senses nutrient availability and impacts gluconeogenesis, glycolysis, and insulin sensitivity. It deacetylates and inhibits the nuclear receptor that activates fat synthesis and adipogenesis in the body, leading to fat loss and bringing favorable cellular and health changes. Sirtuins mediates intracellular response that promotes cell survival, DNA damage repair thereby increasing the cell longitivity. The activation of sirtuins brings a wide spectrum of other health benefits and its activity levels are indicative of nutritional status as well as disease progression in cancer, inflammation, obesity, cardiovascular diseases, and viral infections. There are several foods that activate sirtuin activity and offer significant health benefits by their consumption.

Design of multicomponent reagents for enzymatic assays

102 Currently, kits containing several lyophilized reagents, which are dissolved and dosed during the analytical procedure, are widely used in biochemical tests. The disadvantages of such reagents are the short shelf life of preparations after dissolution, the neces sity of dosing many solutions during analysis, the necessity to maintain the pH, temperature, and other conditions for preserving the high activity of enzymes, etc.