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

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.