Silver sulfide nanoparticles stable in aqueous solutions were obtained in presence of the cells of the bacterium Shewanella oneidensis MR-1 in aqueous solution containing an equimolar mixture of AgNO3 and Na2S2O3. Proteins absorbed on the surface of Ag2S nanoparticles were identified for the first time by MALDITOF/TOF. Among these proteins, multiheme cytochromes MtrC and OmcA, as well as the MtrB membrane porin, which forms a complex on the outer cell membrane, were detected. It was shown that an insoluble precipitate consisting of agglomerated Ag2S nanoparticles with a wide size distribution was formed in the absence of the cells. The role of the detected proteins in the mechanism of the formation and stabilization of the Ag2S nanoparticles in the studied system is discussed.

Проведено исследование электрохимических параметров бактериальных клеток Shewanella oneidensis MR-1. Для регистрации прямого переноса электронов между S. oneidensis MR-1 и электродом клетки предварительно обрабатывали мембраноразрыхляющим веществом – додецилдиметиламмоний бромидом (ДДАБ), что позволило повысить эффективность переноса электронов клеток за счет большей доступности электроактивных белков. Электроанализ бактериальных клеток S. oneidensis MR-1 в анаэробных условиях позволяет зарегистрировать редокс-активные белки и биомолекулы в области восстановительных и окислительных потенциалов: –0.40, –0.16, –0.0 В, что соответствует флавогемопротеинам, производным хинона и цитохромам с-типа внешней мембраны бактериальных клеток S. oneidensis MR-1.

Electrochemical parameters of bacterial cells Shewanella oneidensis MR-1 were investigated. For registration of the direct electron transfer between S. oneidensis MR-1 and electrode, bacterial cells were pretreated with didodecyldimethylammonium bromide (DDAB), a synthetic membrane-like substance of polycationic nature that exhibits membrane-loosening properties. Such pretreatment of S. oneidensis MR-1 allowed increasing the efficiency of extracellular electron transfer by the proteobacterium due to better availability of electroactive proteins for registration of electron transfer processes. The electroanalysis of bacterial cells S. oneidensis MR-1 under anaerobic conditions allows registering redox-active proteins and biomolecules in the range of potentials of–0.40,–0.16, and–0 V, which corresponds to flavohemoproteins, quinone derivatives, and c-type cytochromes of the external membrane of S. oneidensis MR-1 cells.

Представлены результаты исследования применимости современных методов аналитической просвечивающей электронной микроскопии для детекции, идентификации и выявления локализации наночастиц оксидов титана и церия в клетках А549 аденокарциномы легкого человека. Проведен сравнительный анализ особенностей регистрации наночастиц в клетках на основе просвечивающей электронной микроскопии в режиме светлого поля, сканирующей электронной микроскопии в режиме светлого поля, а также микроскопии в режиме темного поля с детекцией электронов, рассеянных на высокие углы. Для идентификации наночастиц в клетках апробированы и сравнены между собой аналитические методы энергодисперсионной рентгеновской спектроскопии и спектроскопии характеристических потерь энергии электронов в режимах получения спектров от отдельных частиц и элементного картирования. Показано, что метод электронной томографии применим для подтверждения того, что наночастицы локализованы в образце, а не занесены на его поверхность путем контаминации. Обсуждаются возможности и области применения различных методов аналитической просвечивающей электронной микроскопии для выявления, визуализации и идентификации наночастиц в биологических образцах.

In the present work it was shown that biosynthesis of silver sulfide nanoparticles from silver nitrate and sodium thiosulfate solutions of millimolar concentration occurs efficiently by living Shewanella oneidensis MR-1 cells, as well as by ultrasonically-disrupted cells and by the membrane fraction of the cells. The size of nanoparticles synthesized in the presence of living cells was 7.8 ± 1.5 nm, while in the presence of ultrasonically-disrupted cells — it was 6.5 ± 2 nm. The shape of nanoparticles in both cases was close to spherical. It was also shown, that synthesis of nanoparticles occurs in a cell-free solution of sodium thiosulfate that has been incubated with cells previously and to which then a silver nitrate solution was added. In this case the nanoparticles were of elongated shape and their size was (11 ± 4) × (24 ± 6) nm. In the control experiment, when only silver nitrate and sodium thiosulfate solutions not incubated with cells were used, the nanoparticles were not detected. It was shown that biosynthesis of nanoparticles occurs both in aerobic and anaerobic conditions. Nanoparticles are not formed by using thermally inactivated cells as it was shown by us previously. The results show the important role of the native structures of cells for the nanoparticles formation.

Показано, что биосинтез наночастиц сульфида серебра в миллимолярном растворе солей тиосульфата натрия и азотнокислого серебра эффективно происходит при использовании как живых, так и разрушенных ультразвуком клеток Shewanella oneidensis MR-1, а также мембранной фракции этих клеток. Наночастицы, синтезированные в присутствии живых клеток, имеют средний размер 7,8 ± 1,5 нм, в присутствии разрушенных ультразвуком клеток - 6,5 ± 2,0 нм. Форма наночастиц в обоих случаях близка к сферической. Также обнаружено, что синтез наночастиц происходит в бесклеточной фракции раствора тиосульфата натрия, ранее инкубированного с клетками S. oneidensis MR-1 и затем соединенного с раствором азотнокислого серебра. В данном случае частицы имеют в основном удлиненную форму, их размеры составляют (11 ± 4) х (24 ± 6) нм. В контрольном эксперименте при использовании растворов солей сульфида натрия и азотнокислого серебра без инкубации с клетками наночастицы обнаружены не были. Установлено, что биосинтез наночастиц осуществляется при проведении реакции биосинтеза как в аэробных, так и в анаэробных условиях. Наночастицы не образуются при использовании клеток, инактивированных повышенной температурой, как было показано нами ранее. Полученные данные указывают на важную роль нативных структур клетки в образовании наночастиц.

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy (TEM) for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549, human lung adenocarcinoma cell line. Comparative analysis was performed for images of the nanoparticles in cells obtained in the bright-field mode of TEM, bright-field scanning TEM, and high-angle annular dark field scanning TEM. For identification of nanoparticles in the cells, the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the modes of obtaining energy spectra from different particles and of element mapping. It was shown that electron tomography is applicable to confirm that nanoparticles are localized in the sample rather than brought in by contamination. The possibilities and fields of using different techniques of analytical TEM for detection, visualization and identification of nanoparticles in biological samples are discussed.

The metal-reducing bacterium Shewanella oneidensis MR-1 has been employed to obtain Ag 2 S nanoparticles from an aqueous solution of AgNO 3 and Na 2 S 2 O 3 at an ordinary temperature and pressure. The nanoparticles vary in size within 2–16 nm, and the fraction 6 to 12 nm in size constitutes about 70%. The maximum yield of nanoparticles in silver equivalent is 53%. Being visualized by transmission electron microscopy, the particles look like spheres with average diameters varying from 7 ± 2 nm to 9 ± 2 nm. The elemental composition of synthesized nanoparticles has been analyzed by energy-dispersive X-ray spectroscopy, and the estimated silver to sulfur atomic ratio is 2: 1. The presence of living bacterial cells is mandatory for the formation of Ag 2 S nanoparticles in the aqueous salt solution. Changes in the reaction conditions (reagent concentrations, temperature, and cell-incubation time in the reaction mixture) influence the yield of nanoparticles dramatically, but have little influence on their size.

We report a classification of the crystallographic structures of bovine and squid rhodopsins corresponding to different stages of their photocycles. Using the resource Protein (Structure) Comparison, Knowledge, Similarity, and Information server (ProCKSI, http://www.procksi.net/), selected spatial structures were compared on the basis of classification schemes (dendrograms). To compare the spatial structures of transmembrane proteins, optimal consensus was developed from methods implemented in ProCKSI. Structures were also clustered using principal component analysis, resulting in good agreement with the classification based on the ProCKSI consensus method. Analysis of the results revealed the basic movements of individual transmembrane domains of these proteins that we were able to relate to different stages of the photoactivation of rhodopsin. A combination of methods identified in this study can be used as an up-to-date analytical tool to study the conformational dynamics of membrane receptors.

The virus particles of live mumps virus vaccine widely used for vaccination in Russia have been detected and visualized by the atomic force microscopy. For quantitative estimation of the number of observed virus particles the special method has been developed. The presence of the vaccine virus protein component was tested by ELISA and dot-blot analysis. Using a quantitative real-time PCR assay the number of copies of viral RNA was estimated. The results of the quantitative estimation obtained by real-time PCR corresponded to the atomic force microscopy data.