Study on aggregation behavior of Cytochrome C–conjugated silver nanoparticles using asymmetrical flow field-flow fractionation

Abstract

In this study, 40nm silver nanoparticles (AgNPs) were synthesized using the citrate reduction method and then the surface of AgNPs was modified by conjugating Cytochrome C (Cyto C) to improve stability and to enhance bioactivity and biocompatibility of AgNPs. It is known that Cyto C may undergo conformational changes under various conditions of pH, temperature, ionic strength, etc., resulting in aggregation of the particles. These parameters also affect the size and size distribution of Cyto C–conjugated AgNPs (Cyto C–AgNP). ζ-potential measurement revealed that the adsorption of Cyto C on the surface of AgNPs is saturated at the molar ratio [Cyto C]/[AgNPs] above about 300. Asymmetrical flow field-flow fractionation (AsFlFFF) analysis showed that hydrodynamic diameter of AgNPs increases by about 4nm when the particle is saturated by Cyto C. The aggregation behavior of Cyto C–AgNP at various conditions of pH, temperature and ionic strength were investigated using AsFlFFF and UV–vis spectroscopy. It was found that the aggregation of Cyto C–AgNP increases with decreasing pH, increasing temperature and ionic strength due to denaturation of Cyto C on AgNPs and reduction in the thickness of electrostatic double layer on the surface of Cyto C–AgNP.