Dual mode analysis of lead ions in regulation of intestinal flora via split-DNA re-assembly catalyzed colorimetric reaction and trans-cleavage activity of Cas12a/crRNA

Abstract

Chronic exposure to lead ions (Pb²⁺) disrupts intestinal flora homeostasis, underscoring the need for sensitive and practical detection methods. Herein, we report a dual-mode analytical strategy combining split-DNA re-assembly-driven colorimetric reaction with CRISPR/Cas12a trans-cleavage activity for Pb²⁺ analysis. A bifunctional magnetic probe is constructed, where Pb²⁺-specific DNAzyme recognition triggers cleavage and release of crRNA, which subsequently activates Cas12a to generate fluorescence by cleaving a fluorophore-quencher reporter. Concurrently, the remaining magnetic bead-anchored trigger initiates split G-quadruplex re-assembly, catalyzing a visible colorimetric reaction. Under optimized conditions, the fluorescence mode achieves a detection limit of 1.03 fM with a linear range of 5 fM to 100 pM, while the colorimetric mode offers a limit of 62.1 fM from 100 fM to 500 pM. Both modes exhibit excellent specificity against competing metal ions. The platform's clinical feasibility is validated using human serum samples, showing strong correlation with ICP-MS, satisfactory recoveries (97.1–103.4%), and good repeatability (CV = 4.1%). Magnetic separation effectively minimizes matrix interference, making the assay suitable for complex biological specimens such as intestinal contents and fecal samples. This dual-mode design integrates fluorescence for precise quantification and colorimetry for equipment-free visual detection, holding great promise for point-of-care testing, early risk assessment of lead-induced gut microbiota dysbiosis, and environmental monitoring.