Carbon Nanospikes: Synthesis, characterization and application for high resolution AFM.

We present a detailed study of synthesis, structural characterization of sharp, spike-like carbon structures and its application for high-resolution atomic-force microscopy (AFM) measurements of biological molecules. The probes are obtained by chemical vapor deposition of spike-like carbon structures on the apexes of common AFM silicon probes. The deposition process is carried out in carbonaceous gas mixture activated by a direct-current discharge. It was revealed by electron microscopy and Raman spectroscopy that the having dimensions at their ends of few nanometers the structures consist of amorphous carbon. The carbon spikes demonstrate high efficiency and resolutions in AFM studies of biological objects. Sub-molecular resolution is demonstrated on the samples of DNA and streptavidin molecules in AFM measurements with the ultra-sharp carbon tips.

Protein nanoparticles with ligand-binding and enzymatic activities.

PURPOSE: To develop a general method for NP fabrication from various proteins with maintenance of biological activity. METHODS: A novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay. RESULTS: Water-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20-250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls. CONCLUSION: Thus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities.