A split aptamer sensing platform for highly sensitive detection of theophylline based on dual-color fluorescence colocalization and single molecule photobleaching

Abstract A new split aptamer sensing platform is developed for highly sensitive and selective detection of theophylline based on single molecule photobleaching (SMPB) technique. The sensing system contains two probes. One is formed by one streptavidin and four biotinylated RNA fragments labelled with fluorescein isothiocyanate (FITC). Each biotinylated RNA fragment contains two repeating aptamer fragments. The other probe is the complementary aptamer fragment labelled with Cy5 dye. The existence of theophylline can trigger the first probe to bind as many as eight Cy5-labelled probes. The average combined number depends on the theophylline concentration and can be measured by SMPB technique. In the sensing system, the dual-color fluorescence colocalization is performed by the red fluorophore (Cy5) and green fluorophore (FITC), in which the red fluorophore is utilized for quantitative counting of photobleaching steps, while the green fluorophore serves as a counting reference to increase detection efficiency. On basis of the principle, an ultra-sensitive sensing platform of theophylline is created with a low limit of detection (LOD) of 0.092 nM. This work provides not only a highly sensitive method for theophylline detection but also a novel perspective for the applications of SMPB technology to construct biosensors.