Bioluminescent System of Luminous Bacteria for Detection of Microbial Contamination

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 FM

Bioluminescent enzyme inhibition based assay of metal nanoparticles

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Анализ параметров активной фазированной антенной решетки радиотелескопа ГУРТ

Представлены методика расчета и результаты численного анализа параметров активной фазированной антенной решетки (АФАР) Гигантского украинского радиотелескопа (ГУРТ) декаметрового и метрового диапазонов волн, который сооружается в настоящее время вблизи г. Харькова на территории Радиоастрономической обсерватории им. С. Я. Брауде Радиоастрономического института Национальной академии наук Украины. Методика базируется на матричной теории антенных решеток, сочетающей в себе электродинамический подход к анализу решетки излучателей с методами теории многополюсников СВЧ для описания фидерной схемы АФАР. Приведены и проанализированы результаты численного расчета эффективной площади АФАР и коэффициента передачи, который в случае пассивной ФАР ассоциируется с КПД, в широком секторе сканирования луча в диапазоне частот 10- 80 МГц.Надаються методика розрахунку та результати числового аналізу параметрів активної фазованої антенної решітки (АФАР) Гігантського українського радіотелескопу (ГУРТ) декаметрового та метрового діапазонів хвиль, що наразі споруджується поблизу м. Харкова на території Радіоастрономічної обсерваторії ім. С. Я. Брауде Радіоастрономічного інституту Національної академії наук України. Методика базується на матричній теорії антенних решіток, що поєднує електродинамічний підхід до аналізу решітки випромінювачів з методами теорії багатополюсників НВЧ для опису фідерної схеми АФАР. Наведені та проаналізовані результати числового розрахунку ефективної площі АФАР та коефіцієнту передачі, що в разі пасивної ФАР асоціюється з ККД, у широкому секторі сканування променя в діапазоні частот 10 - 80 МГц.The calculation technique results of numerical analysis of parameters of active phased antenna array (APAA) of the Giant Ukrainian Radio Telescope (GURT) of decameter and meter wavelengths which is being built now nearby Kharkiv at the area of S. Ya. Braude Radio Astronomy Observatory of the Institute of Radio Astronomy of the National Academy of Sciences of Ukraine are presented. The technique is based on the matrix theory of antenna arrays which combines an electromagnetic approach to analysis of radiators array with the methods of microwave multiport theory for the APAA feed network description. The results of numerical calculation of the APAA effective area and its gain, which in case of passive array is associated with its efficiency, are given and analyzed for a wide scan range within 10 to 80 MHz

Immobilization of Firefly Bioluminescent System: Development and Application of Reagents

The present study describes the method of preparing reagents containing firefly luciferase (FLuc) and its substrate, D-luciferin, immobilized into gelatin gel separately or together. The addition of stabilizers dithiothreitol (DTT) and bovine serum albumin (BSA) to the reagent is a factor in achieving higher activity of reagents and their stability during storage. The use of immobilized reagents substantially simplifies the procedure of assay for microbial contamination. The mechanism of action of the reagents is based on the relationship between the intensity of the bioluminescent signal and the level of ATP contained in the solution of the lysed bacterial cells. The highest sensitivity to ATP is achieved by using immobilized FLuc or reagents containing separately immobilized FLuc and D-luciferase. The limit of detection of ATP by the developed reagents is 0.3 pM, which corresponds to 20,000 cells·mL−1. The linear response range is between 0.3 pM and 3 nM ATP. The multicomponent reagent, containing co-immobilized FLuc and D-luciferin, shows insignificantly lower sensitivity to ATP—0.6 pM. Moreover, the proposed method of producing an immobilized firefly luciferin-luciferase system holds considerable promise for the development of bioluminescent biosensors intended for the analysis of microbial contamination

APPLICATION OF BIOLUMINESCENT ENZYMATIC TESTS IN ECOTOXICOLOGY

This paper examines the general principles of bioluminescent enzymatic toxicity bioassays and describes the applications of these methods and the implementation in commercial biosensors. Bioluminescent Enzyme System Technology (BEST) has been proposed in the bacterial coupled enzyme system, wherein NADH:FMN-oxidoreductase-luciferase substitutes for living organisms. BEST was introduced to facilitate and accelerate the development of cost-competitive enzymatic systems for use in biosensors for medical, environmental, and industrial applications. For wide-spread use of BEST, the multi-component reagent ‘Enzymolum’ has been developed, which contains the bacterial luciferase, NADH:FMN-oxidoreductase and their substrates, co-immobilized in starch or gelatin gel. ‘Enzymolum’ can be integrated as a biological module into the portable biodetector-biosensor originally constructed for personal use. Based on the example of ‘Enzymolum’ and the algorithm of creating new enzyme biotests with tailored characteristics, a new approach was demonstrated in biotechnological design and construction. The examples of biotechnological design of various bioluminescent methods for ecological monitoring were provided. The advantages of enzymatic assays are their rapidity (the period of time required does not exceed 3-5 minutes), high sensitivity, simplicity and safety of procedure, possibility of automation of ecological monitoring; required luminometer is easily available

Биолюминесцентный ферментативный ингибиторный анализ наночастиц на основе металлов

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 nanomaterialsПредложен метод оценки биотоксичности наноматериалов, основанный на использовании в качестве объекта воздействия растворимой и иммобилизованной биолюминесцентной биферментной системы: НАД(Ф)·Н:ФМН-оксидоредуктаза и люцифераза. Принцип метода состоит в обнаружении токсических свойств тестируемых веществ по их влиянию на параметры биолюминесценции используемой биферментной системы. Проведено тестирование коммерчески доступных наночастиц на основе металлов (МНЧ), в том числе наночастиц серебра (Ag), и различающихся по размеру наночастиц диоксидов кремния (SiO2) и титана (TiO2). Эти МНЧ оказывают ингибирующий эффект на активность биферментной системы, причем растворимые ферменты в большей степени подвержены ингибирующему воздействию МНЧ по сравнению с иммобилизованными. Степень ингибирующего воздействия уменьшается в ряду Ag > TiO2 > SiO2, что согласуется с результатами других биологических методов. Биолюминесцентный ферментативный метод анализа занимает 2-3 мин, отличается высокой чувствительностью, технической простотой и может использоваться для оценки безопасности различных классов наноматериало

Alternative Enzyme Inhibition Assay for Safety Evaluation of Food Preservatives

While food additives are widely used in the modern food industry and generally are important in maintaining the ability to provide food for the increasing world population, the progress occurring in this field is much ahead of the evaluation of their possible consequences for human health. The present study suggests a set of single- and multi-enzyme assay systems for revealing toxic effects of the most widely spread food preservatives, such as sorbic acid (E200), potassium sorbate (E202), and sodium benzoate (E211) at the primary molecular level of their interaction with enzymes. The assay is based on the inhibition of enzyme activity by toxic substances proportional to the amount of the toxicants in the sample. The single-enzyme assay system based on NAD(P)H:FMN oxidoreductase (Red) proved to be most sensitive to the impact of food additives, with the IC50 values being 29, 14, and 0.02 mg/L for sodium benzoate, potassium sorbate, and sorbic acid, respectively, which is considerably lower than their acceptable daily intake (ADI). No reliable change in the degree of inhibition of the enzyme assay systems by food preservatives was observed upon elongating the series of coupled redox reactions. However, the inhibition of activity of the multi-enzyme systems by 50% was found at a preservative concentration below the maximum permissible level for food. The inhibition effect of food preservatives on the activity of butyrylcholinesterase (BChE), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH) was either absent or found in the presence of food preservatives at concentrations significantly exceeding their ADI. Among the preservatives under study, sodium benzoate is considered to be the safest in terms of the inhibiting effect on the enzyme activity. The results show that the negative effect of the food preservatives at the molecular level of organization of living things is highly pronounced, while at the organismal level it may not be obvious

Role of Hsp90 and ATP in modulating apyrase activity and firefly luciferase kinetic

Текст статьи не публикуется в открытом доступе в соответствии с политикой журнала.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 50ºC 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