Influence of Molecular Crowding on Aptamers and Application in Lateral Flow Assays

It is generally believed that the practical application of aptamers in food safety monitoring is often hindered by their attenuated performance in complex, crowded food matrices. As a fundamental characteristic of realistic sample matrices, how molecular crowding influences aptamer functionality was investigated. Using non-pretreated milk as a model crowded environment, it was found that molecular crowding significantly enhanced the performance of an Escherichia coli O157:H7-specific aptamer, which is contrary to common sense. Compared to a dilute buffer, the crowding condition increased binding affinity by 25%, boosted the maximum binding capacity by 95.1%, and elevated the melting temperature by 31.7%, while also enhancing nuclease resistance. Mechanistic studies revealed that the crowding agent polyethylene glycol with a molecular weight of 200 Da (PEG200) induced the formation of densely structured aptamer condensates, accelerated binding kinetics, and shifted the binding thermodynamics from entropy-driven to enthalpy-driven. Capitalizing on this effect, molecular crowding agents were integrated into an aptamer-based lateral flow assay. Systematic screening identified 5% (w/v) polyvinylpyrrolidone (PVP58) as the optimal formulation, which intensified the colorimetric signal and reduced the visual detection limit by an order of magnitude without inducing false positives. This work elucidates the mechanism of crowding-enhanced aptamer binding and provides a simple yet powerful strategy to advance the practical application of aptamers in food safety monitoring.