Electrochemical impedance based biosensors show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in the impedance of the sensing surface, when a target molecule binds to an immobilized probe. The sensor response is proportional to the number of target molecules bound to the receptor modified surface. In complex matrices such as biological fluids, the sensor response is proportional to both the specific binding of analyte molecules to the receptors (desired signal) as well as non-specific binding of interfering molecules (noise) on the surface. Consequently, reliable detection of the analyte in complex matrices is limited by two parameters: 1) the specific binding affinity between the analyte and receptor molecules; and 2) the sensor sensitivity for analyte-receptor complexes over nonspecifically binding molecules.

We report a novel sensing approach for target detection in solution that can overcome the limitations in specificity due to a lack of sensitive discrimination between specific binding of target analyte and non-specific binding of interfering molecules. The sensing strategy involves functionalizing an array of sensing surfaces with receptor molecules with a reversible bond such that receptor molecules can be released from the surfaces on exposure to the target in solution. Thrombin and its specifically binding aptamer are used as ligand/receptor pair in the sensing experiments. Thrombin aptamers were first immobilized on the sensing surface using a reversible bond and the functionalized surface is exposed to free alpha thrombin in solution. Electrochemical impedance spectroscopy (EIS) is used to monitor the changes in aptamer surface coverage and thus determine the rate of receptor release in presence of thrombin molecules. As a negative control, functionalized surface were also exposed to gamma thrombin as it is structurally similar to alpha thrombin. Experimental results indicate that aptamer release rate is strongly dependent on specifically binding thrombin concentration while it is independent of the non-specifically binding gamma thrombin. In comparison to conventional sensing approaches, this competition sensing shows improvement in dynamic range followed by better sensitivity and specificity.