A pH-Controlled Kit for Total and Direct Bilirubin Built on Mimetic Peroxidase CoFe₂O₄-DOPA-Catalyzed Fluorescence Enhancement

Facile and reliable detection of total bilirubin (Bt, summation of indirect and direct bilirubin) and direct bilirubin (Bd) in human serum is of crucial importance to clinical diagnosis. However, it is still a challenge to explore an ideal recognition system for discriminating Bd and indirect bilirubin (Bi). In this work, a dual-functional sensor for Bt and Bd was first built on pH-controlled and mimetic peroxidase-catalyzed fluorescence enhancement. The fluorescence of nitrogen-doped graphene quantum dots (NGQDs) can be effectively quenched by bilirubin through the IFE process. With the catalysis of dopamine-derived magnetic ferrite nanoparticles (CoFe2O4-DOPA), both Bd and Bi were oxidized by H2O2 to colorless and fluorescent oxidates at pH 8.0. Interestingly, only Bd was oxidized at pH 3.5. The discriminating principle of Bd and Bi relied on their pH-controlled oxidation potentials. A sensitive sensor for Bt and Bd was developed on the enhanced fluorescence of the NGQDs/CoFe2O4-DOPA/H2O2 sensing system after bilirubin oxidation, which was originated from a combination of the fluorescence recovery of NGQDs and newly spawned fluorescence of bilirubin oxidates. The designed probe well quantifies Bt and Bd with the detection limits of 10 and 50 nM, respectively. Moreover, a portable diagnostic kit was fabricated and successfully used for the detection of Bt and Bd in 60 unrelated human serum samples, and the obtained results were almost consistent with those measured by biochemistry analyzer. The present kit exhibits the superiorities of high sensitivity and stability, interference-resistant, and green reagents, making it a promising candidate for bilirubin detection in the clinical diagnosis of jaundice.