G-triplex molecular beacon‒based fluorescence biosensor for sensitive detection of small molecule-protein interaction via exonuclease III‒assisted recycling amplification

Abstract Small molecule-protein interaction plays a significant role in disease diagnosis and biomedical research. Most of the reported fluorescence strategies for small molecule-protein interaction still involve the use of costly fluorophore-labelled probes. In this work, a label-free fluorescent method for sensitive detection of small molecule-protein interaction is proposed. It is based on G-triplex‒based molecular beacon (G3-MB) and exonuclease III (Exo III)‒aided DNA recycling amplification. Biotin-streptavidin interaction is selected as one model for proof-of-concept. The biotin-linked double-strand DNA (dsDNA) is used as the binding probe. In the presence of streptavidin, Exo III hydrolyzes the binding probe to release the trigger DNA, and the released trigger DNA can hybridize G3-MB to trigger the Exo III cleavage process, accompanied by releasing the trigger DNA and G3 sequences. The trigger DNA can recycle, generating a large number of G3 sequences. The generated G3 sequences bind thioflavin T (ThT) to produce strong fluorescence emission. The interaction between small molecule–protein can be detected by the fluorescence reader. This method exhibits low detection limit (5.6 pg/mL), and owns the merits of the simplicity, high specificity and satisfactory applicability in serum sample.