A simple fluorescent strategy based on triple-helix molecular switch for sensitive detection of chloramphenicol

A simple fluorescent strategy based on the formation of triple-helix molecular switch (THMS) between a signal transduction probe (STP) and an aptamer (Apt) was constructed for the determination of chloramphenicol (CAP). A weak fluorescence intensity was observed for STP solution due to the proximity of fluorophore and quencher through intramolecular DNA hybridization, causing the fluorescence quenching. The fluorescence intensity of the system was significantly enhanced after the addition of Apt. It was attributed to the formation of THMS between the Apt and STP through the Watson-Crick and Hoogsteen base pairing, resulting in the restoration of fluorescence because of the long distance between the fluorophore and quencher of STP. The fluorescence intensity of the system decreased due to the release of STP caused by the specific binding between Apt and CAP. The quantitative analysis of CAP could be achieved based on the decreased fluorescence intensity. The parameters affecting the performance of THMS including the Apt arm length, pH of buffer solution, Mg²⁺ concentration and the formation time of THMS were investigated in detail. Under the optimal conditions (Apt arm length of 9 bases, pH of 6.5, 2.5 × 10³ μmol L^-1 Mg²⁺, THMS formation time of 30 min), the decreased fluorescence intensity and the concentration of chloramphenicol were linear in the range of 5.0 × 10^-3-2.0 × 10^-1 μmol L^-1 with the correlation coefficient of 0.9963. The limit of detection was 1.2 nmol L^-1. Subsequently, the developed method was applied to the analysis of chloramphenicol in honey sample, and the recovery was between 84.5% and 103.0% with relative standard deviation less than 4.6%.