A Broadly Generalizable Dye for Colorimetric Aptamer-Based Sensors

Aptamer-based dye-displacement assays offer a simple, rapid, and robust approach for detecting analytes via a visible color change. These assays utilize a dye molecule that binds to aptamers in a monomeric or dimeric form; target binding to the aptamer subsequently displaces the dye, which aggregates in solution and undergoes a marked shift in absorbance. However, the generalizability of these assays remains an open question, and it is unclear whether existing dyes are broadly capable of binding to and undergoing target-induced displacement from diverse aptamers of varying sequence and structure. Here, we evaluate the compatibility of a panel of 105 diverse small-molecule-binding DNA aptamers with dye-displacement assays based on two commonly used cyanine dyes: 3,3'-diethylthiatricarbocyanine (Cy7) and 3,3'-di-(3-sulfopropyl)-4,5,4',5'-dibenzo-9-methyl-thiacarbocyanine (MTC). We found that roughly half of the aptamers tested were incompatible with these dyes. Through extensive evaluation of a large dye library, we identified X-732-91Ban asymmetric cyanine dye that displays unprecedented generalizability in dye-displacement assays. This dye worked with 95% of our 105-member panel of aptamer-target pairs, producing a measurable signal within seconds. This discovery could enable 'plug-and-play' development of aptamer-based colorimetric sensors for a wide range of targets in applications including medical diagnostics, environmental monitoring, quality control, and forensics.