Organic films for the immobilization of modified proteins for biosensor applications

The effective immobilization of proteins to surfaces is a key step in the design of many bioassays and sensing devices. The modification of the protein capture agent with a tag provides a pathway for immobilization with controlled orientation. In this work, surface plasmon resonance imaging (SPRi) was used to measure the binding of four hexahistidine-tagged (His 6) proteins, with molecular masses of 48 to 480 kDa, to alkyl thiol monolayers of nitrilotriacetic acid (NTA) via coordination with Ni 2+. Protein attachment to a pure NTA monolayer was found to be primarily controlled by the ability of the proteins to pack on the surface. NTA and -OH terminated monolayers proved effective in reducing non-specific adsorption by the larger proteins. Lower molecular weight proteins tended to non-specifically adsorb to a greater extent because less of their surface area is exposed to the OH groups.;An alternative surface chemistry to that of alkyl thiol monolayers was also explored in the form of electrografted aryl films derived from diazonium salts. Para-benzoic acid aryl layers were generated on gold from the diazonium salt by sweeping across a reductive potential at five different scanning rates. Peak currents obtained from cyclic voltammetry increased with voltammetric scanning rates (nu) in a manner consistent with a quasi-reversible, diffusion-limited process. Incomplete film formation at higher values of nu was evident from observable current on the second cycles. Further characterization was carried out with infrared reflectance absorbance spectroscopy (IRRAS). Changes in the wavenumber and absorbance of the carbonyl stretch with varying voltammetric sweep rates suggests nu to partially control the aryl film's structure. The binding of His6-tagged cAMP receptor protein (CRP) to these aryl layers was measured using SPRi following EDC/NHS coupling to produce NTA terminal groups on the surface. Binding curves in nickel-loaded buffer confirmed the IRRAS results, showing nu to determine the extent of CRP attachment. Control experiments demonstrated attachment to the NTA-aryl layers occurring both nonspecifically and via NTA-Ni2+-His6 coordination.