Surface Immobilization of Structure-Switching DNA Aptamers on Macroporous Sol−Gel-Derived Films for Solid-Phase Biosensing Applications
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abstract
Structure-switching signaling aptamers (ss-aptamers) are single-stranded DNA molecules that are generated through in vitro selection and have the ability to switch between a duplex composed of a quencher-labeled DNA strand (QDNA) hybridized adjacent to a fluorophore label on the aptamer, and an aptamer-target complex wherein the QDNA strand is released, generating a fluorescence signal. While such species have recently emerged as promising biological recognition and signaling elements, very little has been done to evaluate their potential for solid-phase assays. In this study, we demonstrate that high surface area, sol-gel-derived macroporous silica films are suitable platforms for high-density affinity-based immobilization of functional ss-aptamer molecules, allowing for binding of both large and small target analytes with robust signal development. These films are formed using a poly(ethylene glycol) (PEG)-doped sodium silicate material, and we show that it is possible to control the pore size distribution and surface area of the silica film by varying the amount of PEG. Materials with the highest surface area are shown to be able to immobilize up to 6-fold more ss-aptamer than planar glass surfaces, providing greater detection sensitivity and somewhat improved detection limits as compared to immobilization on conventional glass. The solid-phase assay is performed using two different structure-switching signaling aptamers with high selectivity for adenosine 5'-triphosphate and platelet-derived growth factor, respectively, demonstrating that this immobilization scheme should be suitable for a variety of target ligands.
