A highly selective sandwich-type FRET assay for ATP detection based on silica coated photon upconverting...

In this paper, we report a highly selective sandwich-type fluorescence resonance energy transfer (FRET) assay for ATP detection by combining the unique optical properties of silica coated photon upconverting NaYF4:Yb(3+), Er(3+) nanoparticles (Si@UCNPs) with the high specific recognition ability of ATP aptamer. In the protocol, a single aptamer of ATP was split into two fragments. One of which was covalently attached to the Si@UCNPs at the 5' end, and the other was labeled with Black Hole Quencher-1 (BHQ1) at the 3' end. In the presence of ATP, the two fragments bound ATP with high affinity to form the sandwich complexes on the surface of Si@UCNPs. ATP induced association of the two fragments, thus bringing the Si@UCNPs and BHQ1 into close proximity. Under the illumination of 980 nm laser, energy transfer took place between the Si@UCNPs as the donor and BHQ1 as the acceptor, creating an optical "sandwich-type" assay for ATP detection. By monitoring the fluorescence change of the Si@UCNPs at 550 nm, the presence of the ATP could be quantitatively detected with a detection limit of 1.70 μM. The linear response range was 2 μM-16 μM. The background of this assay was ignorable because the fluorescence intensity of Si@UCNPs at 550 nm was not changed in the absence of ATP. This assay was also able to discriminate ATP from its analogs.