Nano-phase separation and analyte binding in a SARS-CoV-2 biosensor investigated by nano-IR spectroscopy

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.