Detection of Immobilized Protein on Latex Microspheres by IR−Visible Sum Frequency Generation and Scanning Force Microscopy

The molecular-level detection and behavior of bovine serum albumin (BSA) covalently bound to polystyrene microspheres at the air−liquid and air−solid interfaces are reported by infrared−visible sum frequency generation (SFG) vibrational spectroscopy and scanning force microscopy. SFG spectra contain signals corresponding to CH-stretching modes at both the air−liquid and air−solid interfaces, indicating that some amino acid residues in BSA are well-ordered. It is also observed that the orientation and ordering of phenyl rings at the microsphere surface are affected by the presence of BSA. At the air−liquid interface, the interaction of BSA with the substrate alters the orientation of the phenyl groups in the microsphere, relative to bare polystyrene. At the air−solid interface, however, the SFG signal intensity of the CH-stretch mode for the phenyl rings attenuates after BSA adsorption, disrupting the previously ordered phenyl rings in the underlying, bare surface. Scanning force microscope images show that bare microspheres self-assemble into a two-dimensional hexagonal packing, whereas protein-modified microspheres randomly aggregate on the silica surface. Differences in surface tribology were also recorded between interfacial conditions for microspheres with and without BSA attached. Increases in the friction signal of 12 ± 3% and 26 ± 5% over the bare state were found for BSA-modified microspheres at the air−liquid and air−solid interfaces, respectively. The difference in friction values suggests that BSA interacts more strongly with the microsphere at the air−solid interface.