With the incidence of harmful algal blooms (HABs) increasing in recent years, the urgent demand for the detection of domoic acid (DA), an amnesic shellfish toxin mainly produced by red tide algae Pseudonitzschia, has aroused increasing attention. Aptamers, a new molecular recognition element, provide clarity in the monitoring of DA. In this study, aptamers of DA were successfully screened by Capture-SELEX. Through identification and truncation optimization, aptamer C1-d with a high affinity (K_D value, 109 nM) and high specificity for DA was obtained. The binding mechanism between DA and the aptamer was explored by molecular docking and molecular dynamics (MD) simulation, revealing the critical sites for DA–aptamer interaction. Meanwhile, a BLI-based aptasensor was constructed by C1-d, which displayed a linear range from 0.625 to 10 μM and a LOD of 13.7 nM. This aptasensor exhibited high specificity, good precision and repeatability, and high recovery rates for real samples; the process of detection could be completed in 7 min. This study is the first to identify and investigate the binding mechanism of DA–aptamer interaction and constructed a BLI-based aptasensor for DA, which lays a theoretical foundation for the detection and prevention of DA.

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 (K_D) 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.

Azaspiracids are a type of polyether toxin. Currently, the existing detection methods for azaspiracids all have certain drawbacks. Aptamers offer a cost-effective and convenient approach for the detection of azaspiracids. By employing the Capture-SELEX (Systematic evolution of ligands by exponential enrichment) method to screen aptamers specific to azaspiracid-2, a high-affinity aptamer can be identified for toxin detection. The bin ding affinity of the toxin is verified using biolayer interferometry (BLI) technology. Additionally, computer simulations are utilized to explore the binding sites of the aptamer and conduct molecular dynamics simulations to investigate the stability of the aptamer–toxin complex. Further optimization of the obtained aptamers is carried out to enhance their affinity for the toxin. Ultimately, two aptamers, JD2-RM3-27C28T and JD3-RMM1, are obtained, with dissociation constants (K_D) improved by two orders of magnitude (K_D = 8.7 × 10⁻⁸ M and K_D = 6.8 × 10⁻⁸ M, respectively). These aptamers have the advantage of being incorporated into a new AZA2 assay that is more accurate and ethical than biological monitoring methods, and more economical than LC-MS. In the future, this is expected to demonstrate significant advantages in the fields of food safety, environmental toxin monitoring, toxin exposure diagnosis, and public health monitoring.