Certain Methods of Electrode Pretreatment Create Misleading Responses in Impedimetric Aptamer Biosensors

Abstract Despite the widespread knowledge of the influence that electrode pretreatments have on electroanalysis, pretreatments of gold electrodes for impedimetric aptasensors remain study-specific. This may be a neglected reason behind the limited consensus in reports focusing on creation and testing of aptasensors. To investigate this, several commonly reported pretreatments were applied to gold electrodes that were subsequently used to fabricate thrombin-specific impedimetric aptasensors, which are widely reported in the literature. Layer-by-layer electrochemical impedance spectroscopy analyses were conducted to determine the effect the pretreatment selection has on impedimetric responses during fabrication and testing of the aptasensor. Pretreatments were evaluated using factors crucial for biosensor deployment: the repeatability of interelectrode measurements and whether biosensors produced statistically significant responses when exposed to 2 μM thrombin. Individual pretreatments were found to impart unique physicochemical properties to electrode surfaces, indicated by variations in measured capacitances (impedimetry) and electrode surface topographies (scanning electron microscopy). Impedimetrically measured resistivity (Rtotal) of electrodes increased during layering of the biosensor across all the investigated pretreatments: from 852 ± 830 Ω (bare electrodes) to 3117 ± 1199 Ω (fabricated aptasensors exposed to thrombin), consistent with prior literature. Crucially, the magnitude and reproducibility of Rtotal values measured during fabrication and testing of the assembled aptasensor were strongly contingent on the pretreatment. Depending on the pretreatment, interelectrode sensor responses exhibited relative standard deviations between 38 and 150%. These findings propose that the choice of gold-surface pretreatment protocols is an overlooked─yet crucial─factor to consider when developing analytically valid impedimetric aptasensors.