Abstract: This study aimed to enable rapid detection of hypoxanthine (Hx) in marine fish by developing a colorimetric method integrating liquid-liquid extraction with enzyme cascade catalysis. An iron-based metal-organic framework (Fe-MOF) nanozyme was employed to replace natural peroxidase and combined with xanthine oxidase (XOD) to construct a cascade reaction system for the sensitive detection of Hx. Meanwhile, ether liquid-liquid extraction was introduced to optimize the trichloroacetic acid (TCA)-based pretreatment procedure and eliminate interference in color development. The results showed that the XOD/Fe-MOF detection system exhibited a linear response toward Hx in the concentration range of 0.005–0.1 mmol/L (R2 = 0.99) with a detection limit of 1.69 μmol/L. Three cycles of ether extraction effectively removed the inhibitory effect of TCA on the chromogenic reaction, significantly improving the sensitivity and accuracy. The spiked recoveries for real samples were in the range of 96.26%–108.68%. The results of sea bass samples stored for different durations measured by this method demonstrated a good linear correlation with those measured by high performance liquid chromatography (HPLC) (R2 = 0.96). From the perspective of synergistic regulation of nanozyme performance optimization and sample pretreatment, this study proposed a strategy to improve the performance of biosensors for Hx detection, providing a new approach for quality evaluation of aquatic products.

Key words: hypoxanthine; metal-organic framework; nanozyme; liquid-liquid extraction; marine fish