Multimeric Aptamers Bio-Generation and Surface Bio-Functionalization through Rolling Circle Amplification Techniques for Biosensing Purposes

Multimeric aptamers have obtained significant attention in recent years due to their relatively higher affinity towards target analytes compared to their monomeric counterparts. These multimeric aptamers can simultaneously bind to different regions of a target molecule, resulting in stronger and more stable interactions. The present work has attempted to propose surface-based generation and bio-functionalization 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 procedure is initiated by immobilizing modified primers onto a working electrode (WE), used as an amplification priming point (capturing primer) for multimeric aptamer generation. The tandemly repeated aptamers are then hybridized to the modified spacer primers to facilitate the positioning of the generated multimeric aptamers in the proximity of the WE surface. These multimeric aptamers can be used as bio-receptors for capturing a specific target, which is the SARS-CoV-2 spike protein in this study. The surface amplification process was validated via electrochemical, microscopy, and spectroscopy techniques, and the optimal amplification time for biosensing purposes was determined. Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (10-fold), as well as higher sensitivity (4-fold) compared to monomeric counterparts. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat WEs and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors proved substantially higher sensitivity within the range of 10 to106 fg/mL of spike protein in buffer and saliva media with the detection limit less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically evaluated with several clinical samples that were validated with the gold standard qPCR technique.