Colorimetric aptamer-based assays for the detection of small molecules, proteins, and cells are of great interest. The unique properties of gold nanoparticles (AuNPs) and aptamers are utilized for the detection of targets with the unaided eye. The intricate interactions of DNA-aptamers and AuNPs, especially in the presence of target molecules, are not well understood. For these assays, aptamers are absorbed onto the surface of AuNPs. In turn, the aptamers stabilize the AuNP solution by increasing the surface charge, which keeps the nanoparticles from aggregating. Aptamer-target binding alters the stability of the AuNPs resulting in a color change. We have investigated the riboflavin binding aptamer (RBA) used in the design of a colorimetric assay involving AuNPs and the DNA-aptamer. RBA consists of a total of 29 DNA bases—15 bases in the loop and a 7 base pair stem in the absence of riboflavin (target). It is believed that the RBA forms a G-quartet structure in the presence of the target. For these investigations, the RBA was chosen for its simple structure—a stem and loop (or hairpin)—and known activity as a recognition element in the design of colorimetric assays. Initial nuclear magnetic resonance (NMR) investigations indicated that the stem and loop structure was too stable and prevented the formation of the G-quartet with the aptamer’s native 29 base structure. The G-quartet was able to form only when the buffer used was altered to include potassium ions, essential for G-quartet formation, and the temperature was raised 15 °C above room temperature. We then altered the native RBA structure by weakening the stem strength—either shortening the stem length or mismatching the stem pairs. The aptamer variants were then investigated for their activity in colorimetric assays. Further NMR investigations provided essential DNA structural details of the aptamer variants. These investigations provide crucial information into the mechanism and function that governs AuNP-aptamer based colorimetric assays.

Aptamer-gold nanoparticle colorimetric assay evaluation of the role of DNA, small molecule targets, and gold nanoparticle interactions via NMR

253rd National Meeting (2017)

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