Voltammetric investigation of surface-confined proteins

Development of sensitive and selective methods to detect proteins at trace levels is of great biological importance. Via derivatization with a bifunctional cross-linker 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) and an electrochemical marker 11-ferrocenyl-1-undecanethiol (Fc-SH), voltammetric determination of surface-confined proteins electrostatically adsorbed onto the polyelectrolyte of poly (sodium 4-styrenesulfonate) (PSS) or poly(allylamine hydrochloride) (PAH)-covered surfaces could be realized. The utilization of PSS or PAH was anticipated to reduce the nonspecific adsorption of the proteins on the surface. Two kinds of proteins with no redox activity or exhibiting complex or ill-defined voltammetric peak(s), i.e. the positively charged lysozyme and negatively charged metallothionein (MT) were demonstrated. Due to the incorporation of the bifunctional reagent GMBS and the redox active Fc groups onto the protein-modified electrodes, well-defined voltammetric peaks of high signal intensity were obtained. The anodic peak heights were found to be dependent on the surface density of the proteins electrostatically binded to the polyelectrolyte-coated surface. The present method can measure lysozyme concentration as low as 0.1 nM.