Aptamer-biospecies interactions at the single-molecule level
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Nucleic acid aptamers are relatively easy to be produced with low cost, and it maintains the activity with various chemical modifications. The studies on the structure-function relationship of aptamers provide deep understanding of the reaction mechanisms between aptamers and their target molecules. We have investigated the specific single-molecule interactions between aptamers and toxic protein ricin. The ricin molecules were immobilized on Au(111) surface using a series of optimized chemical reactions to specifically expose the active domains of ricin molecule to the aptamers attached to atomic force microscopy (AFM) tips. The AFM topography images of ricin on Au(111) surface showed the morphology of this toxic protein at single-molecule level. The AFM recognition images revealed the binding areas on ricin A chain. It was also observed that the binding sites for aptamers were not interfered by the immobilization reactions for most of ricin molecules. The different locations of the ricin binding sites to aptamer and to antibody were also distinguished by AFM recognition images and interpreted by simulations. The affinity of aptamer to ricin proved to be comparable to the one of antibody. The anti-ricin aptamer with its PCR primers was also used for the studies on the structure-function relationship of aptamer. This aptamer with longer sequence has more complex folding structure and multiple binding sites. A Markov transition model was developed to illustrate the multiple binding sites and multiple unbinding energy barriers of this aptamer-ricin complex. The elastic properties of this aptamer during the unbinding reactions were studied with the dynamic force spectroscopy (DFS). The complex relationships among different unbinding pathways were connected by the mechanism model based on Hook’s law and warm like chain (WLC) theory. The aptamers without primers were further used in the biosensor developments with two label-free platforms: AFM and surface plasmon resonance (SPR). The affinity of aptamers to ricin measured by AFM and SPR were compared. The research findings can be used as a reference for the further development of label-free biosensor for pathogen and toxin detection in food matrices. The Salmonella Typhimurium sample solution was also tested in the AFM and SPR methods by an aptamer with special interaction with the outer membrane proteins of the bacteria, providing the potential application of detecting certain bacteria serotype.