biosensor for rapid analysis of toxicity of substances by the bioluminescent method, patents & publications

patents & publications
The technology is used for express analysis of the environmental situation by the environment protection bodies of different levels.
Consumer market: EU, USA, Canada, Japan, Australia etc.
The technology is used for express detection of toxic hazards (contamination) of the air medium. Besides, the technology can be used for express contamination control of production sites.
Consumer market: EU, USA, Canada, Japan, Australia etc.
The technology is used for online monitoring of potable water quality in various domains including water supply systems; can be used in residential buildings and all kinds of industrial premises.
Consumer market: Asia, South America, Africa etc.
The technology is used by agricultural and forest management companies for the survey and control of the soil and foliage composition and the conservation of the forest areas.
Consumer market: worldwide.
The technology is used for the express non-invasive monitoring of the athletes' condition during training both in professional and amateur sports and fitness clubs.
Consumer market: EU, USA, Canada, Japan, Australia etc.
The technology is used for the express non-invasive monitoring of stress in people (employees) in companies with the demanding high tech production processes.
Consumer market: USA, Japan, China etc.
The technology is used for the express non-invasive stress monitoring in sport horses and dogs, circus animals, animals resident in zoos and sanctuaries.
Consumer market: worldwide.
The technology is used in environment study practice for school and college students including theory, practice (research) and science project development; the practice is implemented in Russia, and the interest for the program has been confirmed by the EU.
Consumer market: worldwide.
Patents and publications
Monograph (2014)
Bioluminescence: Fundamentals and Applications in Biotechnology
Kratasyuk V. A., Ripp Steven, Sayler Gary, Klumpp Jochen, Loessner Martin J., Frank L. A., Krasitskaya V. V., Oba Yuichi, Schultz Darrin T., Dunlap Paul, Esimbekova Elena, Shimomura Osamu, Xu Tingting, Close Dan, Smartt Abby
This book review series presents current trends in modern biotechnology. The aim is to cover all aspects of this interdisciplinary technology where knowledge, methods and expertise are required from chemistry, biochemistry, microbiology, genetics, chemical engineering and computer science. Volumes are organized topically and provide a comprehensive discussion of developments in the respective field over the past 3-5 years. The series also discusses new discoveries and applications. Special volumes are dedicated to selected topics which focus on new biotechnological products and new processes for their synthesis and purification. In general, special volumes are edited by well-known guest editors. The series editor and publisher will however always be pleased to receive suggestions and supplementary information. Manuscripts are accepted in English.
Articles in the scientific journals (2015-2021)
The Effect of Osmolytes on the Bioluminescent Reaction of Bacteria: Structural and Dynamic Properties
Sukovatyi L. A., Lisitsa A. E., Kratasyuk V. A., Nemtseva E. V.
Journal "Biophysics", article, 2020

The effects of viscous media with glycerol and sucrose (10–40%) on the kinetics of the bacterial bioluminescent reaction have been investigated by stopped-flow technique. Increment of quantum yield in media with 10% of both osmolytes was shown. Higher concentrations of glycerol, up to 30–40%, were found to reduce the efficiency of the reaction, while this effect was not observed in the media with sucrose. The molecular dynamics simulation was used to study the structure of bacterial luciferase surrounded by either water molecules solely or by mixture of water with various numbers of glycerol/sucrose molecules. It was found that both cosolvents at studied concentrations did not cause a significant change in conformation of bacterial luciferase. The calculated root-mean-square fluctuation for Cα-atoms of bacterial luciferase α-subunit indicated that the higher flexibility of the enzyme mobile loop could be responsible for increment of quantum yield in the presence of 10% of both osmolytes. The active site of bacterial luciferase was found to be accessible for glycerol molecules while sucrose did not enter catalytic gorge. Moreover, at 30 and 40% concentration the glycerol molecules were found to locate in the active site of bacterial luciferase throughout the whole simulation time.
Ethylene Diamine Functionalized Citrate-Capped Gold Nanoparticles
for Metal-Enhanced Bioluminescence
Ranjan Rajeev, Kirillova Maria A., Kratasyuk Valentina A.
Journal of Siberian Federal University. Biology, article, 2020
Metal-enhanced bioluminescence (MEB) is a complex photophysical event which manifests itself in manifold luminescence enhancement and depends on many parameters. The parameters include the distance between the luminescent source and the nanomaterial surface, the size and shape of nanoparticles, localized surface plasmon resonance (LSPR) peaks of the nanomaterial and the dielectric constant of the surrounding medium. Studying distance-dependent MEB in the presence of linkers of specified length may provide new insights into luminescence enhancement. In this regard, the present investigation is aimed to understand the role of ethylene diamine as a potential linker in model systems for distance-dependent metal-enhanced bioluminescence (MEB) with gold nanoparticles (AuNPs). Four different types of AuNPs (AuNP1, AuNP2, AuNP3, AuNP4) were synthesized by varying the trisodium citrate (TC) and silver nitrate (AgNO3) concentrations with maximum absorbance values of 0.99, 1.24, 1.21 and 1.38 respectively and the corresponding LSPR peaks (λmax) of 520 nm, 535 nm, 525 nm, and 525 nm. Luminescence enhancement up to 1.44-fold was observed when ethylene diamine (ED) was used as a linker in the presence of 1-N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide (EDC), ATP and AuNP1. The sample consisting of FMN, EDC and AuNP1 showed 1.3-fold luminescence enhancement. It was noted that AuNPs synthesized using AgNO3 as an additional component did not enhance luminescence in all the investigations. The suggested technique of using linkers of predetermined lengths may also prove fairly effective for studying other parameters which can influence MEB and cause sensitivity enhancement of luminescence-based biosensors.
Software for Matching Standard Activity Enzyme Biosensors
for Soil Pollution Analysis
Kratasyuk Valentina A., Kolosova Elizaveta M., Sutormin Oleg S., Lonshakova-Mukina Viktoriya, Baygin Matvey M., Rimatskaya Nadezhda, Sukovataya Irina E., Shpedt Alexander A.
Journal "SENSORS", article, 2021
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
Kratasyuk Valentina A., Stepanova Lyudmila, Ranjan Rajeev, Sutormin Oleg S., Pande Shubhra, Zhukova Galina, Miller Olga M., Maznyak Natalya, Kolenchukova Oksana A.
Journal "LUMINESCENCE", article, 2020
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.
Viscous Media Slow Down the Decay of the Key Intermediate
in Bacterial Bioluminescent Reaction
Lisitsa A. E., Sukovatyi L. A., Kratasyuk V. A.,
Nemtseva E. V.
The effects of medium viscosity on the decay rate of the 4a-hydroperoxyflavin intermediate of the bioluminescent reaction was investigated. It was found that at low concentrations of glycerol or sucrose (viscosity 1.1–1.3 cP) the decay rate rises, whereas a further increase in viscosity to 6.2 cP leads to a decrease in the decay rate following a power function with an exponent of 0.82–0.84. Using molecular dynamics methods, it was shown that the presence of glycerol and sucrose molecules causes a change in the mobility of the amino acid residues in the active center of luciferase, particularly those responsible for binding of flavin. The results obtained are indicative of two opposite effects of viscous media with glycerol and sucrose: (1) destabilization of 4a-hydroperoxyflavin due to a change in the structural and dynamic properties of the protein and (2) stabilization of this intermediate by the decrease in the diffusion rate of its decay products.
Gelatin and Starch: What Better Stabilizes the Enzyme Activity?
Esimbekova E. N., Govorun A. E., Lonshakova-Mukina V. I.,
Kratasyuk V. A.
Journal "GENERAL BIOLOGY", article, 2020
The regularities of the functioning of a number of enzymes in a viscous environment created by natural polymers, starch and gelatin are examined. Based on the analysis of kinetic curves of thermal inactivation, mechanisms of thermal inactivation of enzymes in a viscous microenvironment are proposed. Using the example of butyrylcholinesterase, NAD(P)H:FMN oxidoreductase, and coupled system of the luminous bacteria (NAD(P)H:FMN oxidoreductase + luciferase), the conditions, under which starch and gelatin have a stabilizing effect on enzyme activity during storage and exposure to various physical and chemical environmental factors, were found. A significant increase in the stabilizing effect is achieved by eliminating water during drying the enzyme preparations immobilized in starch and gelatin polymer gels.
Dietary buckwheat enhances sirtuin1 without calorie restriction
Pande Shubhra, Ranjan Rajeev, Shuvaev Anton N., Malinovskaya Natalia A., Ryazanova Maria, Salmina Alla B., Kolenchukova Oksana A.,
Kratasyuk Valentina A.
article, 2020

In the present investigation, the role of dietary intervention in male Wistar rats (n = 8, 3 groups) was studied to observe absolute sirtuin 1 (SIRT1) levels (expressed as ng mg−1 total protein) in serum, stomach, liver, and kidney. Dietary buckwheat at 30% (w/w) level of incorporation in the standard diet (Buckwheat Enriched Diet, BED) improved SIRT1 with values 0.933 ± 0.05, 210 ± 7, 63.26 ± 4, and 69.89 ± 3 in serum, stomach, liver, and kidney respectively when compared to the respective control values of 0.536 ± 0.03, 156 ± 23.3, 31.07 ± 2 and 47.11 ± 4. Moreover, BED though isocaloric to CR diet, led to weight gain (g) by 63.11 ± 3.8, ca. 10%, and 40% higher than control (56.27 ± 5.6) and CR (45.05 ± 4.1) diet groups. A marked rise in Feed Efficiency Ratio (FER) by ca. 37% while a 30% decrease in Feed Conversion Ratio (FCR) was observed for the BED group which supports unexpected weight gain in rats post-dietary intervention. The results justify the superior nutritional profile of buckwheat laden with essential nutrients, essential proteins, and bioactives. In contrast, Calorie Restriction (CR) resulted in a decline of the total protein content in circulation by 19%, while reduction of total protein in stomach, liver, and kidney was estimated to be 95%, 35.2%, and 27% respectively though SIRT1 values were comparatively the highest in all the samples studied. A 30-fold enhancement of SIRT1 in stomach post CR is presumed to counter enhanced stress in gastric tissues. Therefore, mild to moderate expression of SIRT1 may confer beneficial effects such as delayed aging and stress resistance but exceedingly high SIRT1 may evoke increased oxidative stress.
Functional divergence between evolutionary-related LuxG and
Fre oxidoreductases of luminous bacteria
Deeva A. A., Nemtseva E. V., Kratasyuk V. A., Zykova E. A.
Journal "Proteins: Structure, Function and Bioinformatics", article, 2019

In luminous bacteria NAD(P)H:flavin-oxidoreductases LuxG and Fre, there are homologous enzymes that could provide a luciferase with reduced flavin. Although Fre functions as a housekeeping enzyme, LuxG appears to be a source of reduced flavin for bioluminescence as it is transcribed together with luciferase. This study is aimed at providing the basic conception of Fre and LuxG evolution and revealing the peculiarities of the active site structure resulted from a functional variation within the oxidoreductase family. A phylogenetic analysis has demonstrated that Fre and LuxG oxidoreductases have evolved separately after the gene duplication event, and consequently, they have acquired changes in the conservation of functionally related sites. Namely, different evolutionary rates have been observed at the site responsible for specificity to flavin substrate (Arg 46). Also, Tyr 72 forming a part of a mobile loop involved in FAD binding has been found to be conserved among Fre in contrast to LuxG oxidoreductases. The conservation of different amino acid types in NAD(P)H binding site has been defined for Fre (arginine) and LuxG (proline) oxidoreductases.
Benefits of biophysical tests of saliva for athletic functionality rating tests
Vyshedko A. M., Stepanova L. V., Kolenchukova O. A., Kratasyuk V. A.
Journal "Teoriya i praktika fizicheskoy kul'tury i sporta
", article, 2019
Modern physical training systems give a high priority to the training process individualizing elements that may be efficient enough only when supported by objective tests to rate the bodily responses to physical workloads. Moreover, success in modern professional sports is impossible unless supported by the newest medical/ biological findings and technologies in the health rating and forecast domains, with a special role played by the modern biophysical research of the cellular- and molecular-level biological processes. The study was designed to assess benefits of biophysical analysis of saliva for the athletic functionality rating tests. Sampled for the purposes of the study were athletes of different skill levels (from Class I to Honorary Masters of Sport) versus the untrained students, with the saliva sampled prior to and after physical trainings by bioluminescence and chemoluminescence test methods. The test data were processed by Statistica 10 (made by StatSoft Inc., the US) toolkit to produce median values (Me) and inter-quartile distribution (C25-C75 percentile) rates. The Mann-Whitney nonparametric U-test of the null hypothesis was used to rate differences of the independent variables, with the statistical difference rated meaningful at p<0.05. The bioluminescence tests found the higher ferment activity of saliva in the athletes' versus untrained students, with the ferment activity in the bioluminescence tests also tested to grow after physical workloads in the highly-skilled athletes and fall in the untrained students. The study found the saliva antioxidant system functional intensity being athletic skills dependent. Therefore, the study has demonstrated benefits of the biophysical (bioluminescence and chemoluminescence) tests of saliva for the athletic functionality rating tests in the physical workload design and management initiatives.
Bioluminescence Analysis of Saliva as Non-Invasive Evaluation Assay
of Physical Exertion in Human
Kratasyuk V. A., Kolenchukova O. A., Litvinova I. S., Tereshchenko S. Yu.
Journal "Allergy", article, 2019
Исследование включает использование бактериальной люциферазы и NADH: FMN-оксидоредуктазы (BLuc-Red) в неинвазивной оценке физических нагрузок у спортсменов.
Образцы слюны, взятые до и после нагрузки у профессиональных спортсменов (борцы вольного стиля, группа 1, n = 25) и студентов (группа 2, n = 25), служили образцом для исследования. В качестве основного метода исследования был использован биолюминесцентный тест, в результате которого был определен процент остаточной люминесценции (Т) биолюминесцентной системы. Для объяснения механизмов работы биферментной системы BLuc-Red под действием слюны во время упражнений изучался ее состав: физические показатели (pH), химические (Ca2 +, Na +, K +, Mg2 +) и биохимические показатели (общий белок , лактат, каталаза) и корреляция проводилась с показателем T.
NAD(P)H:FMN-Oxidoreductase Functioning Under Macromolecular Crowding: InVitro Modeling
Govorun A. E., Esimbekova E. N., Kratasyuk V. A.
The functioning of NAD(P)H:FMN‑oxidoreductase (Red) from Vibrio fischeri under conditions of macromolecular crowding (MMC) simulated in vitro by adding biopolymers (starch and gelatin) was studied. The dissociation rate constants and the activation energies of dissociation of Red to the subunits were calculated, and the process of denaturation of Red was analyzed. It is shown that the functioning of Red both under conditions of MMC and in diluted solutions is the same. This result refutes the common belief that the native conformation of enzymes in vivo is stabilized due to MMC as compared to the in vitro conditions.
Principles for Construction of Bioluminescent Enzyme Biotests
for Analysis of Complex Media
Kalyabina V. P., Esimbekova E. N., Torgashina I. G., Kopylova K. V.,
Kratasyuk V. A.

In this study, we formulated the principles of designing bioluminescent enzyme tests for assessing the quality of complex media, which consist in providing the maximum sensitivity to potentially toxic chemicals at a minimal impact of uncontaminated complex media. The developed principles served as a basis for designing a new bioluminescent method for an integrated rapid assessment of chemical safety of fruits and vegetables, which is based on using the luminous bacteria enzymes (NAD(P)H:FMN oxidoreductase and luciferase) as a test system.
Functional divergence between evolutionary-related LuxG and Fre oxidoreductases of luminous bacteria
Deeva Anna A., Zykova Evgenia A., Nemtseva Elena V.,
Kratasyuk Valentina A.
Journal "Proteins: Structure, Function and Bioinformatics", article, 2019

In luminous bacteria NAD(P)H:flavin‐oxidoreductases LuxG and Fre, there are homologous enzymes that could provide a luciferase with reduced flavin. Although Fre functions as a housekeeping enzyme, LuxG appears to be a source of reduced flavin for bioluminescence as it is transcribed together with luciferase. This study is aimed at providing the basic conception of Fre and LuxG evolution and revealing the peculiarities of the active site structure resulted from a functional variation within the oxidoreductase family. A phylogenetic analysis has demonstrated that Fre and LuxG oxidoreductases have evolved separately after the gene duplication event, and consequently, they have acquired changes in the conservation of functionally related sites. Namely, different evolutionary rates have been observed at the site responsible for specificity to flavin substrate (Arg 46). Also, Tyr 72 forming a part of a mobile loop involved in FAD binding has been found to be conserved among Fre in contrast to LuxG oxidoreductases. The conservation of different amino acid types in NAD(P)H binding site has been defined for Fre (arginine) and LuxG (proline) oxidoreductases.
Role of Hsp90 and ATP in modulating apyrase activity
and firefly luciferase kinetics
Kirillova Maria A., Ranjan Rajeev, Esimbekova Elena N., Kratasyuk Valentina A.
International Journal of Biological Macromolecules, article, 2019
The present manuscript describes a novel bioassay consisting of apyrase and heat shock protein 90 (Hsp90) without additional co-chaperone supplementation; intended for high-throughput screening of anti-cancer drugs and prognosis of stress. In this regard, Hsp90 and adenosine 5′-triphosphate (ATP) mediated firefly luciferase (FLuc) kinetics was investigated using apyrase and FLuc as client proteins. Bioluminescent assay containing Hsp90, ATP, and apyrase led to complete loss of luminescence at 50 °C which indicates the protective role of Hsp90 against thermal denaturation. Similarly, the assay sample comprising Hsp90, ATP, and FLuc showed 2 fold increments in luminescence than their counterparts. Introduction of bovine serum albumin (BSA) to the pre-incubated assay mixture led to an initial rise in the luminescence (28%) in comparison to the sample containing Hsp90, ATP and FLuc. Therefore, FLuc based HTS assays are not suitable for clinical samples which may contain stabilizing agents. However, thermally denatured FLuc and apyrase could not regain their active conformation even when Hsp90 and ATP were introduced in the assay system. This observation justifies the role of Hsp90 to be protective rather than a reparation agent when acts without co-chaperones.
Is Body Mass Index a potential biomarker for anemia
in obese adolescents?
Pande S., Ranjan R.,
Kratasyuk V. A.
Journal of Nutrition & Intermediary Metabolism, article, 2019
The two paradoxical major health problems namely obesity and anemia are confirmed to affect millions around the world. Hepcidin, a protein synthesized in liver is a negative iron binding regulator. There is an affirmative relation between hepcidin and leptin levels and an inverse co-relation between hepcidin and iron status due to inflammation mediated by obesity in adolescents. So this implicates an alliance between anemia and obesity wherein weight reduction can be a powerful medium to improve iron absorption in obese adolescents. Also the Body Mass Index can serve as a preliminary non-invasive screening tool to identify potential adolescents prone to anemia.
Nutritional biomarkers: Current view and future perspectives
Pande S., Kratasyuk V.A., Medvedeva N.N., Kolenchukova O.A.,
Salmina A.B.
Journal "Food Science and Nutrition", article, 2018
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.
Antioxidant status as the marker of the health in students in the time
of their intensive intellectual activities
Kolenchukova О. А., Dolgushina E. N., Ryupina A. A., Kratasyuk V. A., Medvedeva N. N.
Journal "Helicobacter", article, 2018
Currently, the implementation of innovative non-invasive methods for controlling the state of the human organism under stress conditions is highly actual. Long-time intellectual burdens lead to the chronic stress and may result in the exhaustion of antioxidant system. The aim of the research is to study the characteristics of the antioxidant status in saliva under the conditions of intensive intellectual activities. In order to carry out the experiment, we collected the cohort of 123students aged from 20 to 22 years of the second and third years of the V.F.Voino-Yasenetsky Krasnoyarsk State Medical University. The examined materials, namely saliva, had been taken by direct spitting into test-tubes. Saliva was collected twice: firstly background sampling had been taken during the course of regular classes when the students were supposed to be in the state of emotional balance; secondly, the test sampling had been made in the time of examination session characterized by considerable intellectual burdens. We carried out questionnaire survey for the students using Spielberger-Hanin and Nemchin-Taylor tests. With the help of these tests, we determined the situational and personal anxiety as well as the predisposition to stress development. Antioxidant status was evaluated by Н2О2-luminol-dependent chemiluminescence technique. Chemiluminescent tests were performed by tablet luminometer TriStar LB 941, (made by Berthold). We had proved the existence of the association between the antioxidant status of saliva and the state of intellectual tension. We have noted that during a session in a group with the high level of uneasiness indices have grown, in a group with the low level of uneasiness they have decreased. We can suppose that in the time of examination sessions there is an increase in the common level of anxiety under the lowering of antioxidant activity. In intellectual stress, the antioxidant system performs the interception of radicals less intensively, because the speed of active forms of oxygen (AFO) neutralization is decreased. So, under the conditions of intensive intellectual activities, the degradation of antioxidant protection is observed. It is caused, supposedly, by the lowering of the activity of the enzymes of peroxidase protection. The pro-oxidant system also works less efficiently. This is confirmed by the decline of such markers of CL-fluorescence as maximum intensity, amplitude and light sum, which showed AFO number.
Effect of viscosity on efficiency of enzyme catalysis
of bacterial luciferase coupled with lactate dehydrogenase
and NAD(P)H:FMN-Oxidoreductase
Sutormin Oleg S., Sukovataya Irina E., Pande Shubhra,
Kratasyuk Valentina A.
Journal "Molecular Catalysis", article, 2018
One of the current trends of the modern biology figures out cellular enzyme behaviour. Numerous researches look more closely at the chemical composition of creating in vivo simulated media conditions. The aim of this work was to find out a thermodynamic cooperativity of enzymes in a triple-enzyme chain (lactate dehydrogenase + NAD(P)H: FMN-oxidoreductase + bacterial luciferase) under in vivo simulated condition. The thermodynamic cooperativity effects were found out based on the influence of the viscogens (glycerol and sucrose) on the thermal stability of the triple-enzyme system. The results showed that the viscogens do not lead to an increase in the thermal stability of the triple-enzyme system. In addition, organic solvents (sucrose and glycerol) added as viscous agents to the reaction medium altered the kinetics of this triple-enzyme chain, including changing the light emission decay constant (kdec) and quantum yield of luminescence (Q). Plus, sucrose was found to be more efficient in limiting the flexibility of enzymes than glycerol. The high sensitivity of the triple-enzyme system to the viscogens may be connected with a fact that lactate dehydrogenase does not bound with couple enzyme system NAD(P)H: FMN-oxidoreductase + bacterial luciferase inside the real cell. Since this approach may be used as a method to understand the real connection between enzymes in cellular multi-enzyme metabolic chains inside the luminous bacteria cell.
Bioluminescent System of Luminous Bacteria
for Detection of Microbial Contamination
Kirillova M.A., Esimbekova E.N., Ranjan R., Torgashina I.G.,
Kratasyuk V.A.
Journal of Siberian Federal University. Biology,
article, 2018
Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection of adenosine-5'-triphosphate (ATP). There is an opportunity to assess the bacterial contamination of various objects based on a quantitative analysis of other nucleotides. In the present study, a bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase (BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions was performed by varying enzymes and substrates concentrations. The lowest limits of detection were 1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension was added into the reaction mixture instead of FMN and NADH, and light intensity depended on number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional step of sample preparation did not improve the sensitivity of method. The experimental results showed that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by determining concentration of FMN.
Stabilization of Butyrylcholinesterase by the Entrapment
into the Natural Polymer-Based Gels
Lonshakova-Mukina V. I., Esimbekova E. N.,
Kratasyuk V. A.

A new method for obtaining stable butyrylcholinesterase (BuChE) samples based on the enzyme immobilization in starch and gelatin gels followed by drying is proposed. Coimmobilization of BuChE with the thiol group indicator 5,5'-dithiobis(2-nitrobenzoic) acid did not reduce the activity of BuChE, which allowed us to simplify the procedure and reduce the time of analysis of organophosphorus pesticides. The resulting immobilized samples retained activity for at least 300 days. BuChE samples based on the starch gel showed a greater sensitivity in the determination of pesticides as compared to the samples based on the gelatin gel.
Agglomeration behavior of lipid-capped gold nanoparticles
Ranjan Rajeev, Kirillova M.A., Esimbekova E.N., Zharkov S.M.,
Kratasyuk Valentina A.
Journal of Nanoparticle Research
article, 2018
The current investigation deciphers aggregation pattern of gold nanoparticles (AuNPs) and lipid-treated AuNPs when subjected to aqueous sodium chloride solution with increasing ionic strengths (100–400 nM). AuNPs were synthesized using 0.29 mM chloroauric acid and by varying the concentrations of trisodium citrate (AuNP1 1.55 mM, AuNP2 3.1 mM) and silver nitrate (AuNP3 5.3 μM, AuNP4 10.6 μM) with characteristic LSPR peaks in the range of 525–533 nm. TEM analysis revealed AuNPs to be predominantly faceted nanocrystals with the average size of AuNP1 to be 35 ± 5 nm, AuNP2 15 ± 5 nm, AuNP3 30 ± 5 nm, and AuNP4 30 ± 5 nm and the zeta-average for AuNPs were calculated to be 31.23, 63.80, 26.08, and 28 nm respectively. Induced aggregation was observed within 10 s in all synthesized AuNPs while lipid-treated AuNP2 (AuNP2-L) was found to withstand ionic interferences at all concentration levels. However, lipid-treated AuNPs synthesized using silver nitrate and 1.55 mM trisodium citrate (AuNP3, AuNP4) showed much lower stability. The zeta potential values of lipid-treated AuNPs (AuNP1-L-1x/200, − 17.93 ± 1.02 mV; AuNP2-L-1x/200, − 21.63 ± 0.70; AuNP3-L-1x/200, − 14.54 ± 0.90; AuNP3-L-1x/200 − 13.77 ± 0.83) justified these observations. To summarize, AuNP1 and AuNP2 treated with lipid mixture 1 equals or above 1x/200 or 1x/1000 respectively showed strong resistance against ionic interferences (up to 400 mM NaCl). Use of lipid mixture 1 for obtaining highly stable AuNPs also provided functional arms of various lengths which can be used for covalent coupling.
Physiological properties of fluid circulation in the crystalline lens
in animals with regard to a phase of accommodation
Stepanova L. V., Sychev G. M., Kratasiuk V. A., Svetlova O. V.
Journal "Oftalmologicheskii Zhurnal", article, 2017
Traditional ideas about the fluid circulation in the lens assume its movement through the lens capsule inside-out and its surface distribution in the lens bulk. It is alleged that "fresh" intraocular fluid directionally diffuses from the posterior chambera into the lens toward its center through both its the anterior capsul epithelium and the posterior lens capsule wall. Then intraocular fluid moves along the lamillar structures of the lens towards the equator, where, likely, the Na+, K+-pump activity is maximal. Removal of "waste" of fluid from the lens bag goes in all directions: either through the anterior and posterior capsule, or through the equatorial part. However, these views do not take into account the physiological characteristics of the transport properties of the anterior capsule epithelium that is capable, due to an existing therein ion exchange system, to provide only the one-way fluid flow: from outside to inside. The possible changes in the level of pressure within the lens in different accommodation phases, which may affect the intensity or direction of water exchange, are also not taken into account. The aim of the Research. To identify the main mechanisms of the water-exchange process in the lenses of animals, taking into account a phase of accommodation. Materials and Methods. Lenses of rabbits and cattle were studied. The fluid transport processes in the lenses were examined in vitro by changing their weight when placed in incubatory solutions with or without an inhibitor of Na+, K+ -ATPase. In the first part of the in vitro studies, the lens partially (some lenses with the anterior surface, others with posterior surface) were immersed in solutions representing the lens surrounding medium. In the second part of the in vitro study, the lenses were completely immersed in a solution similar in ionic composition to aqueous humor, with different osmotic pressures. The direction of movement of intraocular fluid was studied in vivo according to dye distribution using biomicroscopy and a stopped diffusion method. Results. The epithelium of the anterior lens capsule of rabbits and cattle supports the water transport from the posterior chamber of the eye into the lens through the work of Na+, K+ -ATPase. This active ion transport system facilitates the directed movement of the "fresh" fluid, rich in metabolites, through the anterior capsular epithelium from the anterior surface of the lens capsule to the posterior one. For the first time we found that at the completely "near" accommodation phase the maximal pressure inside the lens capsule is 6 mm Hg. This state is a dynamic balance for the lens, i.e. the value of the osmotic pressure of 6 mm Hg balances in the lens the level of the mechanical pressure of the capsule. The lens weight right after removing was close to its original weight with the osmotic pressure of 6 mm Hg. When looking completely "far", the lens bag is stretched with a ciliary zonule and minimally compresses the masses inside the lens (minimal intracranial pressure). This contributes to the intensive inflow into the lens of "fresh" fluid. The anterior capsular epithelium of the lens was found to support the water transport from the posterior chamber of the eye into the lens by the active ion transport system Na+, K+ -ATPase. It is important to note that the fluid circulation inside the lens occurs along the osmotic gradient and is always unidirectional from its anterior epithelium to the posterior wall of the capsule. Diffusive fluid circulation inside the lens does not occur through its nucleus, but along the fibers inside the lens, followed by diffusion of the "waste" fluid outward through the posterior surface of the lens into the vitreous chamber. This mechanism of micro changes in the volume and/or fluid replacement in the lens can be thought of as a mechanism of "fluctuations" of the lens volume. Conclusion. The theory of "lens volume fluctuations" at the "near-far" accommodation phases is presented; the theory is confirmed in animal experiments in vivo and in vitro. Understanding this physiological process makes it possible to selectively choose the type of rational correction for more effective inhibition and prevention of the cataract or presbyopic process.
Bioluminescent enzyme inhibition-based assay to predict the potential toxicity of carbon nanomaterials
Esimbekova E.N., Nemtseva E.V., Bezrukikh A.E., Jukova G.V., Lisitsa A.E., Lonshakova-Mukina V.I., Rimatskaya N.V., Sutormin O.S.,
Kratasyuk V.A.
Juornal "Toxicology in Vitro", article, 2017
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 0.16 mg/L, respectively. The immobilised enzyme system was more vulnerable to C60HyFn than its soluble form, with an IC50 equal to 1.4 mg/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.
Bioluminescent Enzyme Inhibition Based Assay
of Metal Nanoparticles
Fritsler Yana V., Esimbekova Elena N., Kratasyuk Valentina A.
Journal of Siberian Federal University. Biology
, article, 2017
The bioluminescent enzymatic bioassays for assessment of nanomaterial biotoxicity using the soluble or immobilized coupled enzyme system of luminous bacteria NAD(P)Н:FMN-oxidoreductase + luciferase (Red + Luc) as a test system were employed in this study. This method specifically detects the toxic properties of substances based on their effect on the parameters of the bioluminescent enzyme reactions. The commercially available metal nanoparticles (MNPs), including silver nanoparticles (Ag), nanoparticles of silicon dioxide (SiO2), and titanium dioxide (TiO2), of different sizes were tested in the study. The inhibitory effects of MNPs on the bioluminescent Red + Luc enzyme system were measured. Results indicated that the soluble Red + Luc coupled enzyme system was more sensitive to the inhibition effect of MNPs than its immobilized form. The inhibitory activity of MNPs decreased in the following order: Ag TiO2 SiO2. That correlated well with results of other biological methods. Due to substantial advantages such as technical simplicity, short response time and high sensitivity to analysis, this bioluminescent enzymatic bioassay has the potential to be developed as a general bioassay for safety assessment of a wide variety of nanomaterials.
Bioluminescent assay for toxicological assessment of nanomaterials
Esimbekova E. N., Nemtseva E. V., Kirillova M. A., Asanova A. A.,
Kratasyuk V. A.
Journal "Biochemistry, Biophysics, and Molecular Biology", article, 2017
A new method for assessing biotoxicity of nanomaterials, based on the use of soluble bioluminescent coupled enzyme system NAD(P)⋅H:FMN oxidoreductase and luciferase, is proposed. The results of this study indicate a significant adverse biological effect exerted by nanoparticles at the molecular level. It was found that the most toxic nanoparticles the nanoparticles are based on copper and copper oxide, as well as single-walled carbon nanotubes and multi-walled carbon nanofibers, which are referred to hazard class II.
Design of multicomponent reagents for enzymatic assays
Esimbekova E. N., Kratasyuk V. A., Lonshakova-Mukina V. I., Bezrukikh A. E.
Journal " Biochemistry, Biophysics and Molecular Biology", article, 2015
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 necessity of dosing many solutions during analysis, the necessity to maintain the pH, temperature, and other conditions for preserving the high activity of enzymes, etc.
In our study, the method for creating multicomponent enzyme preparations is exemplified using the reagents for bioluminescent analysis (in particular, bioluminescent enzyme bioassay methods). When such methods are used, the toxicity of test substances and mixtures is determined by their effect onthe bioluminescence parameters of a chain of coupled reactions catalyzed by the enzyme system NADPH:FMN-oxidoreductase + luciferase. Bioluminescent methods of analysis are characterized by a high sensitivity, speed, and ease of analysis. The development of highly stable immobilized preparations will make it possible to solve the problems associated with the use of soluble enzymes and extend the range of application of bioluminescent methods of analysis in ecology, medicine, agriculture,and other fields.
Rapid biosensing tools for cancer biomarkers
Ranjan Rajeev, Esimbekova Elena N.,
Kratasyuk Valentina A.
Journal "Biosensors and Bioelectronics", article, 2017
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.
Kratasyuk Valentina Aleksandrovna (RU), Zhukova Galina Viktorovna (RU), Kolenchukova Oksana Aleksandrovna (RU), Stepanova Lyudmila Vasilevna (RU), Sutormin Oleg Sergeevich (RU), Esimbekova Elena Nikolaevna (RU), Gulnov Dmitrij Valerevich (RU). 2017
Invention relates to medicine, in particular, it is possible to use in healthcare, medical and sports diagnostics. Method for determining the level of human stress resistance includes determining the maximum intensity of luminescence of samples with saliva I0 and the magnitude of the maximum luminescence intensity of control samples without saliva Ik, the calculation of the luciferase index LI0 samples with saliva and luciferase index LIk a control sample without saliva, calculation of the luciferase stress index by the formula LIstress=LI0-LIk, while LIstress 18–30 % corresponds to the average level of stress resistance; LIstress more than 30 % – high; LIstress less than 18 % – low level of stress resistance.
Kirillova Mariya Aleksandrovna (RU), Esimbekova Elena Nikolaevna (RU), Kratasyuk Valentina Aleksandrovna (RU). 2017
Invention relates to the field of biochemistry. Disclosed is a complex of reagents for the adenosine-5'-triphosphate (ATP) quantitative analysis. Complex includes luciferin and an enzyme preparation. Enzyme preparation contains firefly luciferase, a buffer solution, dithiothriotol and bovine serum albumin stabilizing additives. At that, the dose of each of the reagents for one quantitative analysis is immobilized separately. Thus, the enzyme preparation is immobilized in the gelatin gel with a quantitative ratio of firefly luciferase and gelatin from 1:50 to 1:90, and luciferin is immobilized in a gelatin gel in a quantitative ratio of 1:15 to 1:50 or in a starch gel at a quantitative ratio of 1:40 to 1:150. Invention provides an increase in the stability of the reagents for determining ATP during storage and use, as well as simplifying the reagent preparing procedure.
Esimbekova Elena Nikolaevna (RU), Kratasjuk Valentina Aleksandrovna (RU), Lonshakova-Mukina Viktorija Ivanovna (RU). 2015
Invention proposes a preparation method of an enzymatic agent based on immobilised butyrylcholin esterase and an enzymatic agent for determination of carbamates and organic phosphorus compounds. Starch or gelatine gel is prepared in a phosphate buffer pH 7.9-8.0. The gel is cooled down to 31-35°C. Then, it is mixed with the buffer solution of butyrylcholin esterase and a buffer solution of an indicator of thiolic groups of 5,5'-dithio-bis(2-nitrobenzoic acid) with a concentration of 0.56 M. The obtained mixture is dosed to a hydrophobic substrate and dried at the temperature of 8°C. The inventions allow simplifying an enzyme immobilisation procedure, increasing storage time of an enzymatic agent without any loss of its activity during more than one year. Agent has high sensitivity to action of organic phosphorus compounds and carbamates.
Kratasjuk Valentina Aleksandrovna (RU), Esimbekova Elena Nikolaevna (RU). 2011
Biomodule contains the following, immobilised into gel: nicotinamide adenine dinucleotide (NADN): flavine mononucleotide (FMN)-oxidoreductase, myristic aldehyde, NADN, enzyme stabiliser - dithiothreitol in concentration of 1·10-4 M, a FMN solution and a substrate. The method of preparing the biomodule involves preparation of 3-5% gel by boiling a starch suspension in a phosphate buffer or heating a gelatin suspension in a phosphate buffer to 60-80°C, cooling the gel to 24°-30°C, mixing the buffer solution of a bienzymatic system of luminescent bacteria NADN:FMN-oxidoreductase - luciferase, solutions of myristic aldehyde, reduced nicotinamide adenine dinucleotide, enzyme stabiliser - dithiothreitol in concentration of 1·10-4 M with gel, depositing onto the substrate and drying at 4-10°C. The biomodule is activated with a solution of flavin mononucleotide. Invention increases activity output and maximum luminous intensity of the biomodule by 1,3-3 times, increases the time for constant illumination level of the biomodule to 2-20 minutes, increases the storage time of the biomodule without loss of activity by 2 times, increases thermal stability and resistance of the biomodule to chemical factors of the environment.
Kratasjuk Valentina Aleksandrovna (RU), Esimbekova Elena Nikolaevna (RU). 2011
Rapid method involves preparation of working and control bioluminescent samples based on enzymes and measurement of bioluminescence kinetics. The control sample is prepared using a bioluminescent biomodule which contains co-immobilised enzymes and substrates of a bienzymatic system of luminescent bacteria with addition of distilled water or a phosphate buffer. The working sample is prepared using a bioluminescent biomodule which contains co-immobilised enzymes and substrates of a bienzymatic system of luminescent bacteria with addition of the analysed water sample. Further, both samples are held for 30-300 seconds; a bioluminescent reaction is initiated in both samples using an FMN solution, the volume ratio of the FMN solution in concentration of 5×10-4 M to the control or analysed solution lying between 1:10 and 1:50; and bioluminescence intensity of both samples is measured. The water toxicity criterion is the 20% or more fall or 20% or more increase in the value of the maximum bioluminescence intensity measured in the working sample compared to these parameters in the control sample. Invention increases sensitivity of the rapid method for biotesting natural and waste water and aqueous solutions.