Proof-of-concept electrochemiluminescent assay for the aptamer-based detection of small molecules

This work presents a proof-of-concept assay for the quantification of small molecules based on aptamer recognition and electrochemiluminescence (ECL) readout. The testosterone-binding aptamer (TESS.1) was used to demonstrate the novel methodology. The analytical capability of the ECL array towards testosterone detection was studied using different incubation protocols. Concurrently, and to improve the analytical performance, the optimal concentration of the TESS.1 aptamer was investigated. Subsequently, the selectivity of the proposed array was assessed by performing negative control experiments with a randomized ssDNA sequence and two other steroids, i.e. deoxycholic acid (DCA) and hydrocortisone (HC). In parallel, complementary analytical techniques were employed to confirm the suggested mechanism: i) native nano-electrospray ionization mass spectrometry (native nESI-MS) was used to determine the stoichiometry of binding, and for the characterization of aptamer-target interactions; and, ii) isothermal titration calorimetry (ITC) was carried out to elucidate the dissociation constant (K_d) of TESS.1 for testosterone. The combination of these techniques provided a complete understanding of the aptamer performance, the binding mechanism, affinity and selectivity. This research will pave the way for the development of new aptamer-based biosensor assays coupled with ECL sensing for the detection of relevant small molecules.