Integration of exonuclease III-assisted recycling amplification and multi-site enzyme polymerization labeling for sensitive detection of p53 gene

Abstract

• A sensitive biosensor for p53 gene detection was proposed and characterized. • The parameters of ExoIII amplification were optimized to ameliorate the non-specific reaction. • sTDN provided accurate enzyme loading and well-organized spatial arrangement for multi-site enzymatic labeling. • Compared with exogenous nanomaterials, sTDN possessed higher yield, better biocompatibility and stability. • The integration of the ExoIII amplification with multi-site enzymatic labeling enhanced the biosensor's sensitivity. • With low detection limit, the modified biosensor exhibited acceptable stability and reproducibility. Protein p53 induces cell growth arrest, differentiation, apoptosis and DNA repair. More than 50% of malignant tumors produce mutations in the p53 gene. Hence, p53 sequence-specific analysis helps in early cancer diagnosis, increasing the treatment success rate. The single nucleic acid amplification method limits sensitivity, so we innovatively integrated exonuclease III (ExoIII)-assisted target recycling amplification with multi-site enzyme polymerization labeling. The integration dramatically improved the biosensor’s sensitivity. Given the nonspecific reaction of ExoIII, we optimized the ExoIII amplification system to reduce false positives and background current. The single-strand DNA produced in the ExoIII system served as the bridge between the electrode and signal DNA tetrahedron (sTDN). Moreover, sTDN provided accurate enzyme loading and well-organized spatial arrangement for multi-site enzymatic labeling. Compared with single-stranded signal probes, sTDN modified by terminal transferase provided higher catalytic efficiency. Under the optimal experimental conditions, the cathodic current exhibited a logarithmic relation over 1.0–5.0 × 10 6 fM p53, with a detection limit of 0.47 fM. Applying this biosensor has successfully detected p53 in serum samples, providing a potential tool for cancer diagnosis and treatment.