Chronoamperometric interrogation of an electrochemical aptamer-based sensor with tetrahedral DNA nanostructure pendulums for continuous biomarker measurements

Tetrahedral DNA nanostructure (TDN) is highly promising in developing electrochemical aptamer-based (E -AB) sensors for biomolecular detection, owing to its inherit programmability, spatial orientation and structural robustness. However, current interrogation strategies applied for TDN-based E -AB sensors, including enzymebased amperometry, voltammetry, and electrochemical impedance spectroscopy, either require complicated probe design or suffer from limited applicability or selectivity. In this study, a TDN pendulum -empowered E -AB sensor interrogated by chronoamperometry for reagent -free and continuous monitoring of a blood clotting enzyme, thrombin, was developed. TDN pendulums with extended aptamer sequences at three vertices were immobilized on a gold electrode via a thiolated double -stranded DNA (dsDNA) at the fourth vertex, and their motion is modulated by the bonding of target thrombin to aptamers. We observed a significantly amplified signalling output on our sensor based on the TDN pendulum compared to E -AB sensors modified with linear pendulums. Moreover, our sensor achieved highly selective and rapidly responsive measurement of thrombin in both PBS and artificial urine, with a wide dynamic range from 1 pM to 10 nM. This study shows chronoamperometry-enabled continuous biomarker monitoring on a sub -second timescale with a drift -free baseline, demonstrating a novel approach to accurately detect molecular dynamics in real time.