Nano‐Phase Separation and Analyte Binding in Aptasensors Investigated by Nano‐IR Spectroscopy

Abstract 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.