Engineering genetically encoded FRET-based nanosensors for real time display of arsenic (As3+) dynamics in living cells.

Arsenic poisoning has been a major concern that causes severe toxicological damages. Therefore, intricate and inclusive understanding of arsenic flux rates is required to ascertain the cellular concentration and establish the carcinogenetic mechanism of this toxicant at real time. The lack of sufficiently sensitive sensing systems has hampered research in this area. In this study, we constructed a fluorescent resonance energy transfer (FRET)-based nanosensor, named SenALiB (Sensor for Arsenic Linked Blackfoot disease) which contains a metalloregulatory arsenic-binding protein (ArsR) as the As3+ sensing element inserted between the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus. SenALiB takes advantage of the ratiometic FRET readout which measures arsenic with high specificity and selectivity. SenALiB offers rapid detection response, is stable to pH changes and provides highly accurate, real-time optical readout in cell-based assays. SenALiB-676n with a binding constant (Kd) of 0.676 × 10-6 M is the most efficient affinity mutant and can be a versatile tool for dynamic measurement of arsenic concentration in both prokaryotes and eukaryotes in vivo in a non-invasive manner.

FRET-based nanosensors for monitoring and quantification of alcohols in living cells.

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 µM and covers the physiological range of 500 nM to 12 µM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.

Nigella sativa seed based nanocomposite-MnO2/BC: An antibacterial material for photocatalytic degradation, and adsorptive removal of Methylene blue from water.

Antimicrobial Nigella sativa seed-based nanocomposite, MnO2/BC, was synthesized and utilized for the water purification through adsorption, and the photocatalytic degradation. MnO2/BC was prepared by co-precipitation method, and characterized using FT-IR, XRD, SEM, TEM, TGA, and DSC techniques. The composite was investigated for inhibition of bacterial cells growth. FT-IR spectrum indicated the presence of oxygenous groups on the surface; TGA and DSC showed thermal degradation; and XRD, SEM, and TEM investigations indicated amorphous, and porous nature of MnO2/BC having particle size of 190-220 nm. The nanocomposite inhibited the growth of both Gram-positive and Gram-negative bacteria cells in water. The adsorption of Methylene blue from water was investigated in batch method in terms of amount of MnO2/BC, dye concentration, pH, time, and temperature. 1.0 g L-1 of MnO2/BC removed more than 98% of Methylene blue from aqueous solution having concentration of 10 mg L-1 and pH 7.0 at 27 °C. The maximum Langmuir adsorption capacity of MnO2/BC was 185.185 mg g-1 at 45 °C. The adsorption was an endothermic process which obeyed Freundlich isotherm, and pseudo-second order kinetics. Therefore, the Methylene blue binding onto MnO2/BC surface was site-specific partially through the weak hydrogen bonding, and electrostatic interactions. The photocatalytic activity of MnO2/BC has been investigated by degrading the Methylene blue molecules/ions in water under the sunlight and 85% of degradation was achieved during 120 min irradiation. The dye was desorbed at lower pH and regenerated MnO2/BC was used for second cycle of Methylene blue adsorption. The results obtained for this study are much better than the previous Methylene blue adsorption studies with acid washed Black cumin seeds and MnFe2O4/BC for which the capacities were 73.529 mg g-1 and 10.070 mg g-1 at 27 °C, respectively (J. Mol. liq. 2018a, 264, 275-284; J. Clean. Prod. 2018a, 200, 996-1008).

Role of green fluorescent proteins and their variants in development of FRET-based sensors.

Since the last decade, a lot of advancement has been made to understand biological processes involving complex intracellular pathways. The major challenge faced was monitoring and trafficking of metabolites in real time. Although a range of quantitative and imaging techniques have been developed so far, the discovery of green fluorescent proteins (GFPs) has revolutionized the advancement in the field of metabolomics. GFPs and their variants have enabled researchers to 'paint' a wide range of biological molecules. Fluorescence resonance energy transfer (FRET)-based genetically encoded sensors is a promising technology to decipher the real-time monitoring of the cellular events inside living cells. GFPs and their variants, due to their intrinsic fluorescence properties, are extensively being used nowadays in cell-based assays. This review focuses on structure and function of GFP and its derivatives, mechanism emission and their use in the development of FRET-based sensors for metabolites.

Nanoscale gizmos - the novel fluorescent probes for monitoring protein activity.

Nanobiotechnology has emerged inherently as an interdisciplinary field, with collaborations from researchers belonging to diverse backgrounds like molecular biology, materials science and organic chemistry. Till the current times, researchers have been able to design numerous types of nanoscale fluorescent tool kits for monitoring protein-protein interactions through real time cellular imagery in a fluorescence microscope. It is apparent that supplementing any protein of interest with a fluorescence habit traces its function and regulation within a cell. Our review therefore highlights the application of several fluorescent probes such as molecular organic dyes, quantum dots (QD) and fluorescent proteins (FPs) to determine activity state, expression and localization of proteins in live and fixed cells. The focus is on Fluorescence Resonance Energy Transfer (FRET) based nanosensors that have been developed by researchers to visualize and monitor protein dynamics and quantify metabolites of diverse nature. FRET based toolkits permit the resolution of ambiguities that arise due to the rotation of sensor molecules and flexibility of the probe. Achievements of live cell imaging and efficient spatiotemporal resolution however have been possible only with the advent of fluorescence microscopic technology, equipped with precisely sensitive automated softwares.

Gum ghatti mediated, one pot green synthesis of optimized gold nanoparticles: Investigation of process-variables impact using Box-Behnken based statistical design.

Due to unique inherent catalytic characteristics of different size, shape and surface functionalized gold nanoparticles, their potential applications, are being explored in various fields such as drug delivery, biosensor, diagnosis and theranostics. However conventional process for synthesis of these metallic nanoparticles utilizes toxic reagents as reducing agents, additional capping agent for stability as well as surface functionalization for drug delivery purposes. Hence, in this work suitability of gum Ghatti for reducing, capping and surface functionalization during the synthesis of stable Gold nanoparticles were duly explored. Role and impact of key process variables i.e. volume of chloroauric acid solution, gum solution and temperature at their respective three different levels, as well as mechanism of formation of optimized gold nanoparticles were also investigated using Box- Behnken design. These novel synthesized optimized Gold nanoparticles were further characterized by UV spectrophotometer for its surface plasmon resonance (SPR) at around ∼530nm, dynamic light scattering (DLS) for its hydrodynamic size (112.5nm), PDI (0.222) and zeta potential (-21.3mV) while, transmission electron microscopy (TEM) further revealed surface geometry of these nanoparticles being spherical in shape.