Protein Adsorption on Carboxy-Functionalized Microparticles Revealed by Zeta Potential and Absorption Spectroscopy Measurements

Addition of micro/nanoparticles to a protein solution leads to the formation of a protein layer on the particle surface, called a protein corona. We investigate here the adsorption behavior of myoglobin, hemoglobin, cytochrome-c, and lysozyme on carboxy-functionalized polystyrene microparticles using zeta potential and absorption spectroscopy measurements. The observed adsorption behavior differs according to the method of detection: monolayer for zeta potential and multilayer for absorption spectroscopy. Langmuir-type monolayer adsorption is observed with zeta potential measurements, because zeta potential (ζ) responds only to the charge density at the outermost protein layer. Multilayer adsorption is observed by absorption spectroscopy. Spectroscopic results were analyzed by the Guggenheim-Anderson-de Boer (GAB) model, which comprises a hard corona formed by strong interaction between the protein and the particle surface and a soft corona formed by weak interaction between adsorbed and bulk-solution proteins. The extent of hard and soft corona formation depends on pH. When a protein monolayer is prepared by covalent modification of the particle surface, the number of layers in the protein corona decreases relative to the case of protein adsorption on bare particles. This result demonstrates that electrostatic interactions between the protein and particle surface play a key role in the formation of a protein corona.