MULTIVALENT APTAMER GENERATION THROUGH ROLLING CIRCLE AMPLIFICATION ON A NANOSTRUCTURED SURFACE FOR BIOSENSING APPLICATIONS

Interest toward multivalent aptamers have been increased due their increased binding strength to target molecules, as they can attach to multiple sites at once, creating more stable interactions.This study introduces a method for creating and modifying multivalent aptamers on a surface using room temperature rolling circle amplification (RCA) technique and chemically modified primers.This technique involves attaching the primers to both flat screen-printed gold electrodes and nano-/microisland (NMI) electrodes where multivalent aptamers are then bio-synthesized and organized.These engineered aptamers were used to detect a specific target which is the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) spike protein (SP) in this study. KEYWORDS: Multimeric aptamers • rolling circle amplification • electrochemical impedance spectroscopy (EIS) • nano-/microislands structures. INTRODUCTION:Multivalency is in a bio-inspired approach which plays a crucial role in various natural interactions.Particularly, in cellular, bacterial, and viral contexts, multivalent interactions contribute significantly to enhanced affinity by clustering receptors on surfaces.Analytical methods targeting low-abundance substances can particularly benefit from multivalent interactions.Among various aptamer-based structures, multimeric aptamers stand out due to their inherent advantages, such as straightforward and cost-effective chemical synthesis, as well as tolerance to diverse chemical modifications 1 .Electrochemical transducers are valued for their simplicity, and portability, offering high sensitivity for point-of-care (POC) applications.They are popular because they are affordable, accurate, require minimal equipment, and being rapid 2 .Notably, label-free affinity-based assays, like multimeric aptamer-based assays, can be easily integrated into these platforms 3 .This study proposes a new biofunctionalization strategy for electrochemical biosensing using gold flat and NMI working electrodes (WEs) and room-temperature RCA.The label-free electrochemical impedance spectroscopy (EIS) multimeric aptasensors can detect SARS-CoV-2 SP in buffer and saliva at femtomolar concentrations within 10 minutes (5 minutes for incubation and 5 minutes for EIS measurement), demonstrating high selectivity and sensitivity.Experimental: Optically transparent ITO SPE from Metrohm, Canada, were used to create gold 3D hierarchical NMIs via electrodeposition.Synthesis of the 3D gold NMIs involved a solution containing HAuCl4 (Sigma Aldrich) at a concentration of 100 mM in an HCl (0.5 M) supporting electrolyte solution, conducted at an applied potential of -600 mV relative to an Ag/AgCl reference electrode (Fig. 1, i).WEs were bio-functionalized with multimeric aptamers by using RCA reaction and modified primers (Fig. 1, ii). RESULTS AND DISCUSSION:In this study we introduced a new approach for generating and biofunctionalizing multimeric aptamers on a surface using RCA.This bioinspired network, featuring multiple capturing domains, demonstrated a high-affinity attachment to the target of interest, leading to higher EIS signals compared to monomeric aptamers.The surface RCA process on both nano-/microislands (NMIs) and flat working electrodes WEs underwent comprehensive characterization using electrochemical, microscopy and gel electrophoresis techniques (Fig. 2).This enabled the determination of the optimal amplification time and surface structure for biosensing applications (Fig. 3, i and ii).The NMI-based multimeric aptasensors demonstrated greater sensitivity within the range of 10 to 10 6 fg/mL of SP in buffer and saliva, with a detection limit of as low as 2 fg/mL (Fig. 3, iii).

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