Sensitive fluorescent biosensors based on a cascade signal amplification strategy and a stimuli-responsive hydrogel...

As a key tumor suppressor gene, the mutation and abnormal expression of p53 are closely correlated with tumor occurrence and progression, making p53 an important biomarker for early tumor diagnosis. Herein, a novel fluorescent biosensor based on a Cas12a/alkaline phosphatase (ALP) cascade signal amplification strategy and a stimuli-responsive DNA hydrogel was constructed for sensitive quantification of the p53 gene. Highly stable carbon dot/silica (CD/SiO₂) composites were adopted as excellent fluorescent signal-sensing elements with outstanding optical stability in complex environments. An ALP-encapsulated hyaluronic acid (HA)-DNA hydrogel was prepared as a stimuli-responsive recognition element. Specific binding of the target p53 gene activates the trans-cleavage activity of CRISPR/Cas12a, which cleaves the HA-DNA hydrogel structure and triggers the release of ALP. The released ALP further catalyzes the hydrolysis of the p-nitrophenyl phosphate (PNPP) substrate to generate p-nitrophenol (PNP). The generated PNP quenches the fluorescence of CD/SiO₂ composites through the inner filter effect (IFE), and the fluorescence quenching value is positively correlated with the p53 gene concentration. The biosensor exhibited a favorable linear detection range of 1 pM-100 nM with a low detection limit of 0.074 pM. The proposed strategy eliminates cumbersome pre-amplification procedures for nucleic acids, provides a simple, sensitive and reliable new approach for p53 gene detection in complex biological samples, and exhibits great potential for early tumor auxiliary diagnosis and clinical biological analysis.