Fluorescent aptaswitch for chloramphenicol detection - Quantification enabled by immobilization of aptamer

Chloramphenicol is a potent drug used to treat a variety of bacterial infections. However, due to its toxic effects on human health, use of chloramphenicol is banned in food-producing animals, emphasizing the necessity for its ultrasensitive detection. A molecular beacon-based fluorescent sensing method was developed for the purpose. Fluorophore-and quencher-tagged oligonucleotides complementary to aptamer recognising chloramphenicol were hybridized to construct an aptaswitch sensor complex. On analyte binding the fluorescence was turned on, due to desorption of quencher-oligonucleotide from the complex. With optimized concentrations, the linear range of detection was observed to be from 10 pg mL(-1) to 107 pg mL(-1) of CAP and LOD in buffer was estimated to be 0.987 pg mL(-1). In a modified fluorescence turn-off approach, the aptamers were immobilized through maleimide chemistry on microplates to improve detection limit and resolution. In this format, only fluorophore-oligonucleotide was hybridized to the aptamer which could be detached in presence of chloramphenicol, turning the aptaswitch off. This oligonucleotide was removed to avoid background interference leading to higher resolution and sensitive detection (detection limit 0.039 pg mL(-1)), with a linear range from 1 pg mL(-1) to 104 pg mL(-1) and regression coefficient 0.99. The performance of the aptaswitch sensor was tested in real honey samples where it showed excellent reproducibility, selectivity, accuracy and a detection limit of 0.285 pg mL(-1), which is considerably lower than previously reported biosensors.