In this work, we combined plasmon-enhanced fluorescence and electrochemical (PEF-EC) transduction mechanisms to realize a highly sensitive dual-transducer aptasensor. To implement two traducers in one biosensor, a novel large-scale nanoimprint lithography process was introduced to fabricate gold nanopit arrays (AuNpA) with unique fringe structures. Light transmitting through the AuNpA samples exhibited a surface plasmon polariton peak overlapping with the excitation peak of the C7 aptamer-associated fluorophore methylene blue (MB). We observed a five and seven-times higher average fluorescence intensity over the AuNpA and fringe structure, respectively, in comparison to a plane Au film. Furthermore, the MB fluorophore was simultaneously utilized as a redox probe for electrochemical investigations and is described here as a dual transduction label for the first time. The novel dual transducer system was deployed for the detection of SARS-CoV-2 Spike protein via a C7 aptamer in combination with a strand displacement protocol. The PEF transducer exhibited a detection range from 1 fg/mL to 10 ng/mL with a detection limit of 0.07 fg/mL, while the EC traducer showed an extended dynamic range from 1 fg/mL to 100 ng/mL with a detection limit of 0.15 fg/mL. This work provides insights into an easy-to-perform, large-scale fabrication process for nanostructures enabling plasmon-enhanced fluorescence, and the development of an advanced but universal aptasensor platform.

With the goal of fast and accurate diagnosis of infectious diseases, this study presents a novel electrochemical biosensor that employs a refined aptamer (C9t) for the detection of spike (S) protein SARS-CoV-2 variants in a flexible multielectrode aptasensor array with PoC capabilities. Two aptamer modifications were employed: removing the primer binding sites and including two dithiol phosphoramidite anchor molecules. Thus, reducing fabrication time from 24 to 3 h and increasing the stability and sparseness for multi-thiol aptasensors compared to a standard aptasensor using single thiols, without a reduction in aptamer density. The biosensor fabrication, optimization, and detection were verified in detail by electrochemistry, QCM-D, SPR, and XPS. The analyte–receptor binding was further confirmed spectroscopically at the level of individual molecules by AFM-IR. The aptasensor possesses a low limit of detection (8.0 fg/mL), the highest sensitivity reported for S protein (209.5 signal per concentration decade), and a wide dynamic detection range (8.0 fg/mL–38 ng/mL) in nasopharyngeal samples, covering the clinically relevant range. Furthermore, the C9t aptasensor showed high selectivity for SARS-CoV-2 S proteins over biomarkers for MERS-CoV, RSV, and Influenza. Even more, it showed a three times higher sensitivity for the Omicron in comparison to the Wuhan strain (wild type), alpha, and beta variants of the SARS-CoV-2 virus. Those results demonstrate the creation of an affordable and variant-selective refined C9t aptasensor that outperformed current rapid diagnosis tests.