Surface-Based Multimeric Aptamer Generation and Bio-Functionalization for Electrochemical Biosensing Applications

Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface-based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio-receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS-CoV-2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10-fold), as well as higher sensitivity (almost 4-fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.