Covalent attachment of proteins on self-assembled monolayer electrodes as characterized by electrochemical impedance measurements and FT-IR spectroscopy

A gold substrate surface was modified with a self-assembled monolayer (SAM) from ω-carboxyalkanethiol of N-hydroxysuccinimide (NHS) ester form, followed by a reaction with a series of proteins with different molecular weights to create a protein-SAM/gold structure. The resulting substrates were first characterized by electrochemical measurements. A frequency-response analysis showed that the magnitude of the impedance of the substrate increased with increasing molecular weight of the immobilized protein. Reflection-absorption Fourier-transform infrared spectroscopy (FT-IRRAS) was employed for a detailed study of the protein-SAM/gold structure. A quantitative analysis of FT-IRRAS spectra showed that both the surface concentration of the protein and the apparent thickness of the protein adlayer correlated to the molecular weight of the immobilized protein. It was also suggested that the magnitude of the impedance of the substrate is linked to the apparent thickness of the protein adlayer, since its characteristic molecular-weight dependence was consistent with that for the protein adlayer thickness. This result seems to be important for chemical sensor applications of the present system based on an impedimetric principle.