A base-stacking-driven ratiometric electrochemical biosensor using dsDNA-mediated MB-and-cholesterol co-immobilization: A model of hydrophobic versatile platform for biosensing.

It remains a huge challenge to integrate the stability, reproducibility, and sensitivity of electrochemical DNA biosensors (E-sensors) for practical applications in a simplistic yet cost-effective way. In this work, we present a versatile and inclusive hexanethiol self-assembled monolayer (HT SAM) platform that strategically recruits cholesterol and methylene blue (MB) through double-stranded DNA (dsDNA) coordination, incorporating immobilization and reference functionalities onto the HT SAM. Systematically augmented anchoring sites substantially enhanced interfacial DNA probe immobilization stability and efficiency. Additionally, co-immobilized MB functions as an intrinsic reference signal, effectively mitigating the precision limitations arising from reproducibility issues inherent in conventional E-sensors. The upright dsDNA and the coaxial base-stacking promote the target-probe interactions and improve both hybridization efficiency and rate for the interface DNA probes. The tightly packed hydrophobic HT SAM facilitates [Fe(CN)]-mediated cascade electrocatalytic amplification, further increasing E-sensor sensitivity. As a proof-of-concept, the designed base-stacking-driven ratiometric E-sensor using dsDNA-mediated MB-and-cholesterol co-immobilization successfully detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene-related fragments, demonstrates a wide dynamic range (10 fM to 10 nM) with a low detection limit of 1.32 fM, exhibiting excellent reproducibility and selectivity. With its high detection performance, ease of operation and low cost, this E-sensor is well-suited for point-of-care testing in large-scale disease screening. Above all, the hydrophobic HT SAM as a versatile and inclusive platform combined with the ease of modification of DNA structures to recruit functional molecules and maximize their contributions is key to synergistically enhancing the overall performance of E-sensors.