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

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

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

Metal enhanced luminescence: Current trend and future Perspectives – A review

Текст статьи не публикуется в открытом доступе в соответствии с политикой журнала

Metal enhanced luminescence: Current trend and future Perspectives – A review

Текст статьи не публикуется в открытом доступе в соответствии с политикой журнала

Покрытые цитратной оболочкой и функционализированные этилендиамином наночастицы золота для усиления биолюминесценции металлом

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 biosensorsМ еталл – у силенная б иолюминесценция ( MEB) п редставляет с обой с ложное фотофизическое событие, проявляющееся в виде многократного усиления люминесценции и зависящее от множества параметров. К этим параметрам относится расстояние между источником люминесценции и поверхностью наноматериала, размер и форма наночастиц, а также пики плазмонного резонанса локализованной поверхности (LSPR) наноматериала и диэлектрическая проницаемость окружающей среды. Изучение расстояния между источником люминесценции и поверхностью наноматериала с использованием связующих агентов (линкеров) заданной длины может дать новое понимание эффекта усиления люминесценции. Поэтому целью настоящего исследования была проверка возможности использования этилендиамина в качестве потенциального линкера для дистанционно- зависимой MEB с наночастицами золота (AuNPs). Путем варьирования концентраций тринатрийцитрата (TC) и нитрата серебра (AgNO3) были синтезированы четыре типа наночастиц золота (AuNP1, AuNP2, AuNP3, AuNP4) с максимумами поглощений 0,99; 1,24; 1,21 и 1,38 при λmax 5 20, 5 35, 5 25 и 5 25 н м. У силение л юминесценции в 1 ,44 р аза наблюдалось при использовании этилендиамина (ED) в качестве линкера в присутствии 1-этил-3-(3-диметиламинопропил)карбодиимида (EDC), ATP и AuNP1. В образце, состоящем из флавинмононуклеотида (FMN), EDC и AuNP1, было зафиксировано усиление люминесценции в 1,3 раза. Использование AgNO3 в качестве дополнительного компонента при синтезе AuNPs не приводит к усилению уровня люминесценции. Предложенный подход с применением линкеров заданной длины может быть полезным для изучения других параметров, влияющих на MEB и приводящих к увеличению чувствительности люминесцентных биосенсоро

Antimicrobial activity of new silver (thio)semicarbazone derivatives

Background: Among the bioactive metals, silver exhibits the highest toxicity to bacteria, viruses, and other eukaryotic microorganisms. A large number of bioactive Ag(I) coordination compounds with different ligand environment have been reported and, in particular, the derivatives containing weak Ag−O and/or Ag−N bonds can exhibit a high bioactivity, lower light stability, and inferior solubility in water than the compounds with rather strong Ag−S or Ag−P bonds. Herein we describe the synthesis and antimicrobial activity of two new silver(I) compounds that feature the {AgNO} or {AgSO} environments and were derived from semicarbazone and thiosemicarbazone type ligands.This study was supported by FCT and Portugal 2020 (projects PTDC/QUI-QIN/29697/2017, LISBOA-01 -0145-FEDER-029697, UID/QUI/00100/2013, and REM2013), and the RUDN University Program 5 -100. TAF acknowledges the FCT for BPD grant SFRH/BPD/119980/2016.info:eu-repo/semantics/publishedVersio

Биолюминесцентная система светящихся бактерий для анализа микробного загрязнения

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 FMNДля анализа микробного загрязнения широко используется биолюминесцентный метод, основанный на определении аденозин-5’-трифосфата (АТР) с помощью люциферин- люциферазной системы светляков. Существует принципиальная возможность оценки степени бактериальной обсемененности различных объектов исходя из количественного анализа других нуклеотидов. В работе исследована возможность применения биолюминесцентной системы светящихся бактерий NADH:FMN-оксидоредуктаза (Red) и люцифераза (BLuc) для количественного анализа бактериальных клеток путем определения количества никотинамидадениндинуклеотида (NADH) и флавинмононуклеотида (FMN) в образце. Для увеличения чувствительности биолюминесцентной системы к FMN и NADH осуществлена оптимизация условий проведения анализа путем подбора концентраций ферментов и субстратов в реакционной смеси. Максимальная чувствительность биферментной системы Red + BLuc составила 1,2 нМ FMN и 0,1 пМ NADH. Для проведения количественного анализа бактериальных клеток использовали модельный бактериальный образец – культуру Escherichia coli. Экстрагирование FMN и NADH проводили путем разрушения клеточной мембраны ультразвуковым дезинтегратором. Клеточную суспензию добавляли в реакционную смесь вместо раствора FMN или NADH, при этом интенсивность свечения биолюминесцентной системы зависела от количества бактериальных клеток в реакционной смеси. Введение в пробоподготовку дополнительной процедуры центрифугирования образца, подвергнутого ультразвуковой обработке, не привело к увеличению чувствительности метода. Таким образом, было показано, что чувствительности биферментной системы Red + BLuc достаточно для определения не менее 800 тыс бактериальных клеток в миллилитре путем экстрагирования NADH из разрушенных клеток. При анализе, основанном на определении концентрации FMN в бактериальном образце, чувствительность метода составила 3,9 млн клеток на миллилит