Ratiometric Fluorescence Sensor Based on 3-Hydroxyphenylboronic Acid and Gold Nanoclusters for Ultrasensitive Detection of Xanthine and Hypoxanthine

Developing a sensitive and reliable method for detecting xanthine (XT) and hypoxanthine (HX) is crucial for the early diagnosis of diseases such as hyperuricemia. Herein, we developed a ratiometric fluorescence sensor based on a target-triggered cascade reaction by integrating 3-hydroxyphenylboronic acid (3-HBA) with lysozyme-functionalized gold nanoclusters (MTL@AuNCs), enabling ultrasensitive and highly accurate detection of XT and HX. In the sensing system, hydrogen peroxide (H₂O₂) was generated through the catalytic oxidation of XT/HX by xanthine oxidase (XOD) in the presence of O₂. The unique nucleophilic reaction between H₂O₂ and the boric acid group resulted in the conversion of 3-HBA into resorcinol (Res), which then reacted with dopamine (DA) to form blue fluorescent azamonardine (λ_em = 465 nm) compounds. This Res-DA system acts as the fluorescence-responsive unit, producing a progressively intensified emission, while the red-emitting MTL@AuNCs (λ_em = 660 nm) serve as an invariant reference signal. The proposed fluorescence sensor exhibited a linear response range of 0.005-300 μM for XT and 0.01-300 μM for HX, with ultralow detection limits of 1.7 nM and 3.4 nM, respectively. This method was successfully applied to detect XT and HX in real serum and urine samples, yielding satisfactory results. Furthermore, this study provides valuable insights for designing biosensors with enhanced stability and sensitivity by incorporating a cascade reaction and internal reference fluorescence.