The response of an Electrochemical Impedance Spectroscopy (EIS) sensor using DNA aptamers is affected by many factors such as DNA density, charge and conformational changes upon DNA-target binding and buffer conditions. We report here for the first time on the optimisation of an EIS aptamerbased sensor by using Quartz Crystal Microbalance with Dissipation mode (QCM-D). As a case study we employed a DNA aptamer against Prostate Specific Antigen (PSA). PSA detection was achieved by functionalizing the gold sensor surface via thiol chemistry with different ratios of thiolated-DNA aptamer and 6-mercapto-1-hexanol (MCH) used as spacer molecules. PSA binding efficiency can be monitored by measuring QCM-D signals which not only provides information about the mass of PSA bound on the sensor surface but also crucial information about the aptamer conformation and layer hydration.Data generated through QCM-D analysis provided the optimal conditions in terms of aptamer/MCH ratio to maximize the PSA binding. The ratio of 1:200 for DNA aptamer/spacer molecule was found to be optimal for ensuring maximum PSA binding. However, this study showed how a maximum analyte binding does not necessarily correspond to a maximum EIS response, which revealed to be enhanced if a ratio of 1:100 for DNA aptamer/spacer molecule was used. Moreover, by monitoring the QCM-D signal, for the first time a value of the dissociation constant (K d ), equal to 37 nM, was found for the PSA DNA aptamer towards its target. The combination of QCM-D with EIS techniques provide further insight into the effects of mass loading and charge effects that govern the response of an EIS aptasensor, serving as a valuable support for future EIS aptamer-based applications.

Adaptation of conventional lateral flow tests: demonstration of a lateral flow assay in which gold nanoparticle conjugates, when bound to aptamers, serve as a reporting system to screen novel aptamers for the ability to bind to target proteins.

The nonspecific uptake of aptamers by dead cells is an often-overlooked factor during the study of aptamer binding to their targets in vivo. Accounting for this can aid in the identification of aptamers with high affinity and specificity.

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