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.