Monitoring protein binding to phospholipid monolayers using electrochemical impedance spectroscopy

Biotin was incorporated into dioleoyl phosphatidylcholine (DOPC) monolayers as dioleoyl phosphatidylethanolamine (DOPE)–biotin, DOPE–caproyl–biotin and DOPE–polyethylene glycol (PEG)–biotin. Experiments were carried out in electrolyte KCl (0.1 mol dm−3) buffered with 0.001 mol dm−3 phosphate buffer to pH 6.9. Bovine serum albumin (BSA), avidin, biotinylated anti-haemoglobin (IgG) and haemoglobin were added to the solution. The frequency dependence of the complex impedance of the coated electrode surfaces was estimated between 65,000 and 0.1 Hz at potentials −0.4 V versus Ag/AgCl 3.5 mol dm−3 KCl. The capacitance of the monolayers was measured at 75 Hz between potentials −0.4 and −1.1 V. Both non-specific and specific binding of the soluble proteins to DOPC gave rise to the occurrence of low frequency relaxations in the impedance data. The non-specific binding of BSA to DOPC can be suppressed by the incorporation of DOPE–PEG into the DOPC monolayer. The nature of the effect of specific binding of neutravidin to biotin on the impedance data depended on the positioning of the biotin group in relation to the DOPC monolayer surface. Successive binding of proteins to the biotin and then to each other gave rise to an increase in the significance of low frequency relaxations in the impedance data respectively.