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 aptamer receptors 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 blocking step, i.e., the addition of inert molecules to the receptor layer, can improve sensor performance, but can also cause phase separation into nanodomains of unknown composition and structure. Here, nano‐IR spectroscopy is used together with complementary macroscopic spectroscopic methods to study the nano‐structural variations during the fabrication of a recently developed SARS‐CoV‐2 aptasensor. The blocking step by polyethylene glycol (PEG) causes a significant thickening of the receptor layer and a phase separation 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 images. Furthermore, bound analyte (spike protein of SARS‐CoV‐2) is detected at the single molecule level. Detailed analysis of the local nano‐IR spectra revealed structural 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 the understanding of nanoscale chemical processes in the receptor layer of aptasensors.

Self-assembled monolayers (SAMs) of oligoethylene glycol (OEG)-terminated thiols are often used in the design of protein-repellent interfaces. It is well known that their repellency depends on the crystallinity and conformation of the EG chains, and it is thought to be a consequence of interface water interacting with the OEG chains. Here, we provide novel insight into the structure and molecular conformation of OEG SAMs at a nanoscale level, focusing on the impact of immersion time (t_im) during preparation and relative humidity. Macroscopically, an increase in t_im leads to SAM densification and an increase in thickness, as well as a transition from amorphous OEG chains to predominantly helical crystalline chains. At the nanoscale, the morphology showed nano-domains of size dependent on t_im, and nano IR spectroscopy revealed direct spectroscopic evidence for different conformations prevalent in these domains. Hydration experiments conducted in an environment with increased humidity resulted in the nano-domains being hydrated to different extents. Water uptake led to an irreversible conformational transition of the helical domains. Additionally, nano IR spectroscopy demonstrated the absorbance of two differently hydrogen-bound water populations. These results shed new light on the nanophase behaviour of OEG SAMs and help to explain how nano-structure influences their protein repellency.

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.