Electrochemical impedance spectroscopy as a platform for reagentless bioaffinity sensing

Simple reagentless immunosensor formats have been difficult to achieve, particularly for electrochemical devices, since antigen/hapten recognition by an antibody does not directly lead to a reaction cascade. A direct reading electrochemical immunosensor would have major advantages with respect to speed, de-skilled analysis and the development of multi-analyte sensors. Electrically conducting polymers, such as poly(pyrrole), allow for the intimate association between a biological recognition element and a potential reporter polymeric chain. We have polymerised poly(pyrrole), loaded with avidin or antibody to luteinising hormone (LH), on gold interdigitated electrodes (IDE) and employed two-electrode electrochemical impedance spectroscopy (EIS) to "visualise" charge transfer through the polymer as the basis for a reagentless protocol. We investigated bulk redox processes in the poly(pyrrole) films by cyclic voltammetry in order to ascertain the redox state of the film prior to EIS. The redox process of the immobilised protein molecule was identified and allowed the focusing of the EIS studies on polymer associated with the immobilised bioaffinity molecule. The polymer displayed both polaronic and electronic charge transfer during EIS studies. A possible binding-dependent response, observed as a decrease in peak polaronic phase angle, occurred when a redox cycle was performed on the film following exposure to the appropriate analyte. The response of poly(pyrrole) films loaded with avidin to D-biotin and two derivatives was assessed, which was shown to be sensitive to electrode pre-treatment. Poly(pyrrole) films loaded with antibody to LH allowed a calibration for LH to be constructed between I and 800 IU/l. Importantly, the films were responsive to LH within the clinically relevant range of 1-10 IU/l. (C) 2001 Elsevier Science B.V. All rights reserved.