Matrix conditioning and magnetic bead target transfer for fluorescence aptamer-CRISPR/Cas12a detection of kanamycin in river water

Fluorescence aptamer-CRISPR/Cas12a assays provide efficient signal conversion and amplification for the detection of small molecules. However, their practical application in complex environmental water remains limited. Matrix components can cause signal loss before CRISPR amplification. In this study, a fluorescence aptamer-CRISPR/Cas12a assay was developed by integrating matrix conditioning and magnetic bead target transfer for kanamycin (KAN) detection in complex water matrices. Matrix conditioning reduced interference from humic acid (HA) and inorganic ions, while magnetic bead target transfer captured KAN and transferred it into a small volume of eluate. The transferred KAN induced aptamer release from aptamer/complementary strand duplexes on streptavidin magnetic beads. The released aptamer activated reporter cleavage by Cas12a and generated an increased fluorescence response. In a model matrix containing HA and inorganic ions, the assay produced only weak responses to KAN without matrix conditioning. In contrast, the workflow with matrix conditioning produced a clear increase in response with KAN concentration. In the conditioned model matrix, the assay achieved a linear response over 0.4-4 nM with a limit of detection (LOD) of 0.4 nM. This LOD was below the proposed predicted no-effect concentration (PNEC) for KAN in river water. Recoveries of 90-105% were obtained in spiked real river water samples, supporting its applicability to real water analysis. These results demonstrate that combining matrix conditioning with magnetic bead target transfer improves the matrix compatibility of fluorescence aptamer-CRISPR/Cas12a detection for trace KAN analysis in environmental water.