A malachite green light-up aptasensor for the detection of theophylline.

ABSTRACT

Biosensors are of interest for the quantitative detection of small molecules (metabolites, drugs and contaminants for instance). To this end, fluorescence is a widely used technique that is easily associated to aptamers. Light-up aptamers constitute a particular class of oligonucleotides that, specifically induce fluorescence emission when binding to cognate fluorogenic ligands such as malachite green (MG). We engineered a dual aptasensor for theophylline (Th) based on the combination of switching hairpin aptamers specific for MG on the one hand and for Th on the other hand, hence their names malaswitch (Msw) and theoswitch (Thsw). The two aptaswitches form a loop-loop or kissing Msw-Thsw complex only in the presence of theophylline, allowing binding of MG, subsequently generating a fluorescent signal. The combination of the best Msw and Thsw variants, MswG12 and Thsw19.1, results in a 20-fold fluorescence enhancement of MG at saturating theophylline concentration. This aptasensor discriminates between theophylline and its analogues caffeine and theobromine. Kissing aptaswitches derived from light-up aptamers constitute a novel sensing device.