An Ultrasensitive Label-Free Aptasensor for Insulin Detection Assisted by Exonuclease III and 2-Aminopurine

We designed a label-free fluorescent aptasensor assisted by exonuclease III (Exo III) for sensitive insulin (Ins) detection. The method has high sensitivity, anti-interference properties and repeatability. Additionally, the label-free fluorescent aptasensor assisted by Exo III used to detect Ins has not been reported on yet. In this study, we connected a modified DNA sequence to the 5′ end of an aptamer, modifying it into a hairpin structure and exposing 11 nucleotides at the 3′ end containing the base adenine (A). The A was substituted with base 2-aminopurine (2AP) to provide a label-free stable hairpin fluorescent probe (2AP-hairpin probe). This strategy took advantage of the high binding affinity of the Ins aptamer and the susceptibility of 2AP to the local base stacking environment. When Ins is added to the detection system, the 2AP-hairpin probe binds to Ins, adopts a folded state, and blocks Exo III’s access to the binding site for cutting DNA. 2AP cannot be released, and the fluorescence of the 2AP-hairpin probe/cDNA/Ins/Exo III system cannot be restored. Ins detection is achieved by comparing changes in the fluorescent intensity before and after adding Ins to the detection system. The detection limit of the aptasensor is as low as 1.62 nM with a linear range of 3–130 nM. Furthermore, it is able to selectively and directly detect Ins in biological fluids, demonstrating significant clinical application value and research significance.