Highly Selective Protein Patterning on Gold−Silicon Substrates for Biosensor Applications

Abstract

Proteins were precisely patterned on 2D sensor surfaces using photolithography and chemical selectivity. Microarrays of gold squares were fabricated on silicon substrates. The gold regions were modified with mixed COOH-terminated self-assembled monolayers (SAMs) to have a high affinity for the desired proteins or peptides. The silicon regions were modified with polyethylene glycol (PEG) by silanization to provide a high resistivity to protein adsorption. Protein surface coverage was visualized by fluorescence microscopy and atomic force microscopy (AFM). AFM was also used for studying protein morphology to understand the interaction of proteins with SAMs at the molecular level. Proteins and peptides immobilized on SAMs were examined by Fourier transform infrared (FTIR) spectroscopy. Contact angle measurements for surface wettability were conducted to confirm the success of the surface modification reactions. Protein resistance by the PEGs immobilized on bare silicon substrates and on the silicon regions of gold-patterned silicon substrates was compared, and it was found that the latter has a higher resistivity to protein adsorption. Both fluorescence and high-resolution AFM images indicated that bovine serum albumin (BSA) and fibronectin molecules formed a densely packed layer on the gold regions of the patterned substrates, while the immunoglobulin's (IgG) coverage was low. Specific antigen−antibody binding (BSA−anti-BSA) was studied using the surface plasmon resonance (SPR) technique for characterizing the bioactivity of the antigen attached to the gold substrates. The SPR results showed that the BSA proteins bound covalently to the gold surfaces have a much better bioactivity than those bound physically. This study suggests that protein or peptide, molecular structures, and the immobilization technique influence the coverage, morphology, and bioactivity of the attached proteins on the substrates which is crucial to the operational behavior of biosensors.