Rational design of ratiometric fluorescent aptasensor for patulin in traditional Chinese medicine through the studies of interaction mechanism between DNA aptamer and target molecule

Patulin (PAT) is a kind of mycotoxin which must be monitored for the sake of quality and safety in traditional Chinese medicine (TCM) owing to its harm to human health. On this account, a rationally designed ratiometric fluorescent aptasensor was developed based on the study of the interaction mechanism between PAT and its aptamer (PAT-APT). Firstly, CD spectroscopy, molecular docking, and molecular dynamic simulation were applied to investigate the details on how PAT-APT bind with its target molecule. The results indicated that the structure of PAT-APT changed to a certain extent and was stabilized after binding with PAT. C-11, C-37 and C-38 were the key sites for the recognition and interaction between PAT-APT and its target. Secondly, based on these results, a ratiometric aptasensor made use of fluorescence resonance energy transfer (FRET) and synchronous fluorescence spectroscopy was designed. The complementary sequence (cDNA) to the aptamer with appropriate length and hybridization position was obtained through rationally design and optimization. Both PAT-APT and cDNA were labeled by a pair of fluorophores, which could generate FRET when the two single-stranded oligonucleotides hybridized. The accurate detection of PAT could be realized according to the change ratio of fluorescence intensity at the corresponding wavelength of the two fluorophores before and after assay. The aptasensor achieved an ultralow limit of detection 0.16 nM, perfect selectivity, and satisfactory practibility in complex TCM sample. To our knowledge, this is the first aptasensor for PAT designed through the interaction mechanism between aptamer and target molecule. Moreover, the assay for PAT is cost-effective, does not need complicated pretreatment and only takes less than an hour. Summarily, this study makes contribution to safety control of TCM, and provides a thinking mode from mechanism to rational design to conquer the problem of sensitive aptasensing of one component in a complex system.