Electrochemical Monitoring of Respiratory Infectious Disease on Nanostructured Surface Functionalized with Biosynthetic Multimer Aptamers

Multivalent aptamers have gained substantial interest owing to their enhanced affinity towards target analytes compared to their monomeric counterparts due to their capability to bind simultaneously to diverse regions of a target molecule, fostering more robust and enduring interactions. This study focuses on engineering the surface-based production and modification of multivalent aptamers through room temperature rolling circle amplification technique and chemically modified primers. This process starts with immobilizing modified primers onto an electrode to generate and arrange biosynthesised multivalent aptamers. These bio-engineered multivalent aptamers are utilized as bio-receptors for capturing a specific target (in this case, the SARS-CoV-2 spike protein). The entire surface amplification procedure is extensively characterized using electrochemical, microscopy, and spectroscopy methods, determining the optimal amplification duration for biosensing applications. Impressively, multivalent aptasensors shows substantially increased response signals and affinity compared to monomeric aptasensors. Moreover, the influence of surface structures on response signals is explored by employing both flat screen-printed gold electrodes and nano-/microisland (NMI) electrodes. NMIs-based multimeric aptasensors demonstrate notably heightened sensitivity in detecting SARS-CoV-2 spike protein within the range of 10 to 106 fg/mL in saliva and buffer media. Finally, the signal of the proposed aptasensor has been successfully assessed using several patient samples.