Aptamer based fluorescent cocaine assay based on the use of graphene oxide and exonuclease III-assisted signal amplification

The article reports an aptamer based assay for cocaine by employing graphene oxide and exonuclease III-assisted signal amplification. It is based on the following scheme and experimental steps: (1) Exo III can digest dsDNA with blunt or recessed 3-terminus, but it has limited activity to ssDNA or dsDNA with protruding 3-terminus; (2) GO can absorb the FAM-labeled ssDNA probe and quench the fluorescence of probe, while the affinity between FAM-labeled mononucleotide and GO is negligible; (3) Cocaine aptamer can be split into two flexible ssDNA pieces (Probe 1 and Probe 2) without significant perturbation of cocaine-binding abilities; (4) The triple complex consisting of Probe 1, Probe 2 and cocaine can be digested by Exo III with the similar efficiency as normal dsDNA. Cocaine aptamer is split into two flexible ssDNA pieces (Probe 2 and 3′-FAM-labeled Probe 1). Cocaine can mediate the cocaine aptamer fragments forming a triplex. The triple complex has unique characteristic with 3′-FAM-labeled blunt end at the Probe 1 and 3′-overhang end at Probe 2. If exonuclease III is added, it will catalyze the stepwise removal of fluorescein (FAM) labeled mononucleotides from the 3-hydroxy termini of the special triplex complex, resulting in liberation of cocaine. The cocaine released in this step can produce a new cleavage cycle, thereby leading to target recycling. Through such a cyclic bound-hydrolysis process, small amounts of cocaine can induce the cleavage of a large number of FAM-labeled probe 1. The cleaved FAM-labeled mononucleotides are not adsorbed on the surface of graphene oxide (GO), so a strong fluorescence signal enhancement is observed as the cocaine triggers enzymatic digestion. Under optimized conditions, the assay allows cocaine to be detected in the 1 to 500 nM concentration range with a detection limit of 0.1 nM. The method was applied to the determination of cocaine in spiked human plasma, with recoveries ranging from 92.0 to 111.8 % and RSD of <12.8 %.