An electrochemical/colorimetric sensor for 5-methyltetrahydrofolate based on a screened specific aptamer and DNA walker-driven CRISPR/Cas12a cascade amplification.

5-Methyltetrahydrofolate (5-MTHF), the primary bioactive form of folate (vitamin B9), played a vital role in human metabolism. In this work, an electrochemical/colorimetric dual-mode aptasensor for 5-MTHF was constructed by combining a DNA Walker-driven CRISPR-Cas12a trans-cleavage system. A 5-MTHF aptamer D1a was obtained through Capture-SELEX with subsequent trimming of non-binding regions, which exhibiting high affinity and specificity. CuMOF@CuO@RuO@IrO, serving as a multifunctional indicator with high conductivity and peroxidase-like activity, was immobilized on a gold electrode (AuE) via ssDNA. The DNA Walker and nicking enzyme (Nt.BbvCI) continuously released Cas12a activators, triggering Cas12a-mediated cleavage of ssDNA on the AuE, leading to reduced electrochemical signals. The released materials were collected for colorimetric detection. The detection limits were determined to be 0.044 ng/mL (electrochemical) and 0.114 ng/mL (colorimetric). This dual-mode aptasensor demonstrated high selectivity, sensitivity, and stability for 5-MTHF detection, establishing a novel sensing platform for 5-MTHF analysis in food samples. The proposed aptasensor exhibited excellent sensitivity, selectivity, and reproducibility. This strategy offered a promising platform for 5-MTHF analysis in nutritional evaluation and dietary supplementation guidance.