Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

Magnesium aminoclays were synthesized and used to transform non-competent Escherichia coli JM109 using the exogenous plasmid pUC19. The structure determined for the Mg aminoclays is analogous to 2:1 trioctahedral smectites such as talc, with an approximate composition R8Si8Mg6O16(OH)4, where R = CH2CH2NH2, morphologically arranged in layered sheets. Mg aminoclays were employed as a cationic vehicle that enabled the passage of plasmids across the cell envelope and led to genetic modification of the host. A stock solution of 10 mg/mL of Mg aminoclays was prepared, mixed with E. coli JM109 and pUC19 plasmid, and spread over Petri dishes containing lysogeny broth (LB), isopropyl ?-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (14%). The transformation efficiency obtained was higher for 1% and 2% agar even though transformation also occurred at agar concentrations of 3% and 4%. The optical density of E. coli JM109 and spreading time were also adjusted, favoring transformation when cells were used in their exponential growth phase (OD600 = 1.0) and spread for 90 s. Transformation was confirmed by the growth of blue colonies in LB/IPTG/X-gal/agar Petri dishes containing ampicillin, by regrowth of biomass in liquid media containing ampicillin and by agarose gel electrophoresis of the linearized pUC19 plasmid that followed plasmidic DNA extraction from 4 blue colonies. The maximum transformation efficiency achieved was 7.0 × 103 CFU/?g pUC19. This transformation approach proved to be suitable for a convenient, cost-effective, room-temperature, risk-free and rapid transformation of non-competent E. coli JM109.This study was supported by the Portuguese Foundation for Science and Technology (FCT) and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects FCOMP-01- 0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/2013, UID/FIS/04650/2020, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioEnv - Biotech nology and Bioengineering for a sustainable world”. The authors acknowledge the fellowship SFRH/BD/71661/2010 awarded to Gabriel Mendes under the scope of the MIT-Portugal Program. The authors also thank Paul Brown and Takuya Harada for the help in obtaining TEM images.info:eu-repo/semantics/publishedVersio

Effective Peroxidase-Like Activity of Co-Aminoclay [CoAC] and Its Application for Glucose Detection

In this study, we describe a novel peroxidase-like activity of Co-aminoclay [CoAC] present at pH ~5.0 and its application to fluorescent biosensor for the determination of H2O2 and glucose. It is synthesized with aminoclays (ACs) entrapping cationic metals such as Fe, Cu, Al, Co., Ce, Ni, Mn, and Zn to find enzyme mimicking ACs by sol–gel ambient conditions. Through the screening of catalytic activities by the typical colorimetric reaction employing 2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)diammonium salt (ABTS) as a substrate with or without H2O2, Fe, Cu, and CoACs are found to exhibit peroxidase-like activity, as well as oxidase-like activity was observed from Ce and MnACs. Among them, CoAC shows exceptionally high peroxidase-like activity, presumably due to its ability to induce electron transfer between substrates and H2O2. CoAC is then used to catalyze the oxidation of Amplex® UltraRed (AUR) into a fluorescent end product, which enables a sensitive fluorescent detection of H2O2. Moreover, a highly sensitive and selective glucose biosensing strategy is developed, based on enzyme cascade reaction between glucose oxidase (GOx) and CoAC. Using this strategy, a highly linear fluorescence enhancement is verified when the concentration of glucose is increased in a wide range from 10 μM to 1 mM with a lower detection limit of 5 μM. The practical diagnostic capability of the assay system is also verified by its use to detect glucose in human blood serum. Based on these results, it is anticipated that CoAC can serve as potent peroxidase mimetics for the detection of clinically important target molecules

Effective Peroxidase-Like Activity of Co-Aminoclay [CoAC] and Its Application for Glucose Detection

In this study, we describe a novel peroxidase-like activity of Co-aminoclay [CoAC] present at pH ~5.0 and its application to fluorescent biosensor for the determination of H2O2 and glucose. It is synthesized with aminoclays (ACs) entrapping cationic metals such as Fe, Cu, Al, Co., Ce, Ni, Mn, and Zn to find enzyme mimicking ACs by sol–gel ambient conditions. Through the screening of catalytic activities by the typical colorimetric reaction employing 2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)diammonium salt (ABTS) as a substrate with or without H2O2, Fe, Cu, and CoACs are found to exhibit peroxidase-like activity, as well as oxidase-like activity was observed from Ce and MnACs. Among them, CoAC shows exceptionally high peroxidase-like activity, presumably due to its ability to induce electron transfer between substrates and H2O2. CoAC is then used to catalyze the oxidation of Amplex® UltraRed (AUR) into a fluorescent end product, which enables a sensitive fluorescent detection of H2O2. Moreover, a highly sensitive and selective glucose biosensing strategy is developed, based on enzyme cascade reaction between glucose oxidase (GOx) and CoAC. Using this strategy, a highly linear fluorescence enhancement is verified when the concentration of glucose is increased in a wide range from 10 μM to 1 mM with a lower detection limit of 5 μM. The practical diagnostic capability of the assay system is also verified by its use to detect glucose in human blood serum. Based on these results, it is anticipated that CoAC can serve as potent peroxidase mimetics for the detection of clinically important target molecules

Síntesis y caracterización de materiales nanoestructurados aplicados al diseño de sensores y biosensores electroquímicos

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2019En este trabajo de Tesis Doctoral se sintetizaron y caracterizaron nanoestructuras de magnetita, níquel y oro, las cuales se inmovilizaron en matrices de grafito o sustratos de oro con el objetivo de diseñar plataformas (bio)analíticas para el desarrollo de (bio)sensores electroquímicos. En el Capítulo 1 se presenta una introducción general acerca de la importancia de la utilización de sensores y biosensores en la química analítica y el diseño de nuevas plataformas mediante la incorporación de nanomateriales y biomoléculas. Se describen las características principales de las biomoléculas utilizadas en esta Tesis, enzimas y aptámeros. Además, se presentan distintas metodologías de inmovilización de biomoléculas en la construcción de biosensores. En el Capítulo 2 se describen las técnicas utilizadas y se discuten los fundamentos y conceptos necesarios para la compresión de los resultados presentados. También se detallan los materiales y metodologías utilizadas en este trabajo de Tesis. En el Capítulo 3 se reporta el desarrollo de un biosensor destinado a la cuantificación de glucosa basado en el uso de nanopartículas de magnetita recubiertas con carbono amorfo. Se discuten las propiedades electroquímicas de dichas nanopartículas, las ventajas de su incorporación junto a la enzima glucosa oxidasa en un electrodo compósito para el desarrollo de un biosensor para glucosa. Asimismo, se presenta la optimización de la plataforma a fin de obtener los parámetros analíticos óptimos para el mejor desempeño del biosensor. En el Capítulo 4 se presenta el desarrollo de una plataforma electroquímica basada en el empleo de nanohilos de níquel. Esta plataforma fue aplicada para la cuantificación de etanol, tomando ventaja de las propiedades electrocatalíticas del níquel hacia compuestos orgánicos en medio alcalino. Asimismo, se presentan los parámetros analíticos del sensor obtenido. El Capítulo 5 detalla la construcción de una superficie de oro modificada basada en la inmovilización de 4,4'-bifenilditiol y el posterior anclaje de nanopartículas anisotrópicas de oro o nanorods. La plataforma resultante se utilizó como punto de partida para la inmovilización de un aptámero específico para la detección de α-trombina humana. Adicionalmente, se presenta la optimización en cada etapa de la construcción de la plataforma diseñada para el mejor rendimiento analítico del aptasensor. En el Capítulo 6 se exponen las conclusiones generales del presente trabajo de Tesis Doctoral.202