Atul Bharde

Fungus-mediated biosynthesis of silica and titania particles

The synthesis of inorganic materials by biological systems is characterized by processes that occur at close to ambient temperatures, pressures and neutral pH. This is exemplified by biosilicification in marine organisms such as diatoms while laboratory-based synthesis of silica involves extreme temperature and pH conditions. We show here that silica and titania particles may be produced by challenging the fungus Fusarium oxysporum with aqueous anionic complexes SiF62− and TiF62− respectively. Extra-cellular protein-mediated hydrolysis of the anionic complexes results in the facile room temperature synthesis of crystalline titania particles while calcination at 300 °C is required for crystallization of silica.

Bacteria-mediated precursor-dependent biosynthesis of superparamagnetic iron oxide and iron sulfide nanoparticles.

The bacterium Actinobacter sp. has been shown to be capable of extracellularly synthesizing iron based magnetic nanoparticles, namely maghemite (gamma-Fe2O3) and greigite (Fe3S4) under ambient conditions depending on the nature of precursors used. More precisely, the bacterium synthesized maghemite when reacted with ferric chloride and iron sulfide when exposed to the aqueous solution of ferric chloride-ferrous sulfate. Challenging the bacterium with different metal ions resulted in induction of different proteins, which bring about the specific biochemical transformations in each case leading to the observed products. Maghemite and iron sulfide nanoparticles show superparamagnetic characteristics as expected. Compared to the earlier reports of magnetite and greigite synthesis by magnetotactic bacteria and iron reducing bacteria, which take place strictly under anaerobic conditions, the present procedure offers significant advancement since the reaction occurs under aerobic condition. Moreover, reaction end products can be tuned by the choice of precursors used.

On-chip magnetic separation and encapsulation of cells in droplets

Single cell study is gaining importance because of the cell-to-cell variation that exists within cell population, even after significant initial sorting. Analysis of such variation at the gene expression level could impact single cell functional genomics, cancer, stem-cell research, and drug screening. The on-chip monitoring of individual cells in an isolated environment would prevent cross-contamination, provide high recovery yield, and enable study of biological traits at a single cell level. These advantages of on-chip biological experiments is a significant improvement for a myriad of cell analyses methods, compared to conventional methods, which require bulk samples and provide only averaged information on cell structure and function. We report on a device that integrates a mobile magnetic trap array with microfluidic technology to provide the possibility of separation of immunomagnetically labeled cells and their encapsulation with reagents into picoliter droplets for single cell analysis. The simultaneous reagent delivery and compartmentalization of the cells immediately following sorting are all performed seamlessly within the same chip. These steps offer unique advantages such as the ability to capture cell traits as originated from its native environment, reduced chance of contamination, minimal use of the reagents, and tunable encapsulation characteristics independent of the input flow. Preliminary assay on cell viability demonstrates the potential for the device to be integrated with other up- or downstream on-chip modules to become a powerful single-cell analysis tool.

Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

Background Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. Methodology/Principal Findings The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. Conclusions/Significance We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.

Protein manipulation by stimuli-responsive polymers encapsulated in erythrocyte ghosts.

Stimuli-responsive polymers exhibit dramatic, reversible alterations of their physical properties in response to minor environmental changes. Such alterations can be controlled systematically and are exploited in a variety of biologically relevant applications. We describe several properties of the temperature-responsive polymer, poly(N-isopropylacrylamide), encapsulated within erythrocyte ghosts. The manipulation of target proteins inside the cells was demonstrated with polymer–biomolecule conjugates and fusion of differentially labeled erythrocyte ghosts. Harnessing the properties of such materials to actively alter the functionality of proteins provides unique modes of interference with cellular events.

Bacterial Synthesis of Photocatalytically Active and Biocompatible TiO2 and ZnO Nanoparticles

TiO2 and ZnO nanoparticles are well known for their photocatalytic and ultraviolet (UV)-absorbent properties and have successfully entered into the market as consumer products. Large volume production of these nanoparticles by industries require environmentally friendly processes of synthesis. In this article, we show that when challenged with an appropriate precursor, the bacterium Actinobacter spp. can lead to the extracellular synthesis of ZnO and TiO2 nanoparticles. The biological way of synthesizing these materials probably leads to the in situ doping of elements like C, F, and N into their crystal lattices, which is evidenced by a red shift in the absorption edge. Because the doping is expected to affect the photocatalytic activity of these materials under different light conditions, such investigations have also been undertaken here. Further, the biocompatibility of these nanomaterials (cytotoxicity and genotoxicity) has also been investigated in detail.