Abstract
Electrochemical impedance spectroscopy (EIS), an extremely sensitive analytical technique, is a widely used signal transduction method for the electrochemical detection of target analytes in a broad range of applications. The use of nucleic acids (aptamers) for sequence-specific or molecular detection in electrochemical biosensor development has been extensive, and the field continues to grow. Although nucleic acid-based sensors using EIS offer exceptional sensitivity, signal fidelity is often linked to the physical and chemical properties of the electrode-solution interface. Little emphasis has been placed on the stability of nucleic acid self-assembled monolayers (SAMs) over repeated voltammetric and impedimetric analyses. We have studied the stability and performance of electrochemical biosensors with mixed SAMs of varying length thiolated nucleic acids and short mercapto alcohols on gold surfaces under repeated electrochemical interrogation. This systematic study demonstrates that signal fidelity is linked to the stability of the SAM layer and nucleic acid structure and the packing density of the nucleic acid on the surface. A decrease in packing density and structural changes of nucleic acids significantly influence the signal change observed with EIS after routine voltammetric analysis. The goal of this article is to improve our understanding of the effect of multiple factors on EIS signal response and to optimize the experimental conditions for development of sensitive and reproducible sensors. Our data demonstrate a need for rigorous control experiments to ensure that the measured change in impedance is unequivocally a result of a specific interaction between the target analyte and nucleic recognition element.