Previous research has primarily focused on recognition of target molecule by characterizing the impedance change of the working electrode. In the present work, we have conducted experiments to understand how the ionic transport (of the electrolyte) across the NAAO membrane is affected due to the functionalization of biomolecules. We have used electrochemical impedance spectroscopy (EIS) to analyze the effect of different biomolecules on the membranes.
As a test example, we have chosen the thrombin binding aptamer as the sensing molecule and human alpha thrombin as the target molecule in the experiments. The NAAO membranes were sputter coated with gold and the effect of sputtering on the pore size was analyzed through scanning electron microscope (SEM). Afterwards the membrane was functionalized with thiolated thrombin binding aptamer (TBA). Then the membrane was placed in a custom made electrochemical cell and a four-electrode system was utilized to measure the impedance change across the membrane. The electrolyte was maintained at a physiological pH value. When the system was treated with thrombin protein, upon binding with the TBA, it produced a change in the impedance, which was characterized by EIS. We have repeated the experiments for different pore diameters, different sputtering time and concentrations of thrombin. A negative control experiment was also carried out to check the effect of other proteins on the system.
Finally, we have modeled the system using a modified Randles circuit to look at the contribution of protein-aptamer binding on the following parameters: resistance due to the pores, capacitance of the membrane and the electrolytic resistance. We have found that there is a positive correlation between these parameters and successful binding of the biomolecules which in turn can be utilized to calibrate the ionic transport across the membrane as well as utilize the system as a sensor for target proteins.