Enhanced Fluorescence Detection of p53 Gene Using G3 Dimer-Based Palindromic Molecular Beacon for Cancer Diagnosis

• Developed a novel label-free, isothermal amplification for p53 gene detection. • Enhanced specificity using G3 dimers over traditional G3 monomers. • Achieved detection of single-base mutations in complex samples down to 20 pM. • Validated the assay's effectiveness in human serum sample. • Potential broad application in real-time monitoring of nucleic acid biomarkers. This study presents a label-free fluorescence-based assay using a multifunctional palindromic molecular beacon (MPMB) for p53 gene detection, a crucial marker in cancer diagnosis. Compared to PCR or CRISPR-based assays, our external primer-free and isothermal strategy integrates recognition, amplification, and signaling within a single molecular design. Analytical validation revealed that the assay follows a linear response for p53 gene concentrations ranging from 0.5 nM to 200 nM, with a detection limit down to 20 pM. It features a novel application of G-triplex (G3) dimer formation with N-methyl mesoporphyrin IX (NMM), resulting in significantly enhanced fluorescence compared to traditional G3 monomer methods. The system exhibited exceptional specificity, effectively differentiating the target p53 gene from single-base mismatched sequences (MT1-MT4), and maintained robust performance in 50-fold diluted human serum with satisfactory recoveries (97.6%-101.5%). The significant advancements presented by this label-free, external primer-free, and isothermal fluorescence assay signify a major leap in molecular diagnostics. By simplifying genetic testing while maintaining high sensitivity and specificity, this method holds substantial promise for clinical practices and patient management.