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.

Biosensors based on DNA aptamers are increasingly used in diagnostic applications. To improve the sensitivity and specificity of aptasensors, parameters affecting the stability and binding efficiency of the receptor layer need to be identified and studied. For example, the influence of blocking, i.e. the addition of inert molecules to the receptor layer, on sensor performance is well accepted, but its effects on the nano-structure have not been studied in detail. Phenomena such as phase separation into nanodomains have been reported, but their effect on analyte binding remains uncertain. Here, nano-IR spectroscopy is used together with complementary macroscopic spectroscopic methods to study the nano-structural variations during the fabrication of an aptasensor consisting of a mixed self-assembled monolayer (SAM) of sensing aptamers and inert polyethylene glycol. The investigated sensor was recently developed and optimized for the detection of the spike protein of the omega variant of SARS-CoV-2. The initially formed aptamer layer is homogeneous and flat compared to the length of the DNA. In contrast, the film after blocking is much thicker and phase separated into nanodomains consisting of an aptamer-rich and a slightly thicker PEG-rich phase. The unambiguous chemical identification of the nanodomains is achieved by analysis of nano-IR spectra and nano-IR imaging. Furthermore, the analyte bound to the receptor layer was detected at the single molecule level. The detailed analysis of the local nano-IR spectra further revealed chemical properties such as the amorphous state of the PEG molecules within the nanodomains and a strong change in the secondary structure of the analyte. This study significantly advances our understanding of the chemical processes in the receptor layer of biosensors at the nanoscale.

Considering challenges for 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: the removal of the primer binding sites and the inclu-sion of two dithiol phosphoramidite anchor molecules. Thus, reducing fabrication time from 24 to 3 hours for multi-thiol aptasensors compared to a standard aptasensor using single thiols, without 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 pos-sesses a low limit of detection (8.0 fg/mL), the highest sensitivity reported for S protein (209.5 sig-nal per concentration decade), and a wide dynamic detection range (8.0 fg/mL - 38 ng/mL) in na-sopharyngeal 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 In-fluenza. Even more, it showed a 3 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 demonstrated an affordable and variant-selective refined C9t aptasensor that outperformed cur-rent rapid diagnosis tests.