Conotoxins (CTXs) are a variety of mixed polypeptide toxins, among which α-conotoxin MI (CTX-MI) is the most toxic. Serious toxic symptoms, a lack of counteracting drugs, and cumbersome detection processes have made CTX-MI a hidden danger for humans. One of the obstacles to resolving this problem is the absence of specific recognition elements. Aptamers have shown great advantages in the fields of molecule detection, drug development, etc. In this study, we screened and characterized aptamers for CTX-MI through a programmed process. MBMI-01c, the isolated aptamer, showed great affinity, with an affinity constant (KD) of 0.524 μM, and it formed an antiparallel G-quadruplet (GQ) structure for the specific recognition of CTX-MI. Additionally, an aptasensor based on the biolayer interferometry (BLI) platform was developed and displayed high precision, specificity, and repeatability with a limit of detection (LOD) of 0.26 μM. This aptasensor provides a potential tool for the rapid detection of CTX-MI in 10 min. The aptamer can be further developed for the enrichment, detoxification, and biological studies of CTX-MI. Additionally, the programmed process is applicable to screening and characterizing aptamers for other CTXs.
