DNA triangular prism nanostructure and CRISPR/Cas12a empowered electrochemical biosensor for dual detection of alpha-fetoprotein and microRNA 122

Developing sensitive multi-target-combined detection method is of great significance for the early and accurate diagnosis and prognosis of hepatocellular carcinoma (HCC). Herein, an electrochemical biosensor was proposed for detecting HCC biomarkers miRNA 122 and alpha-fetoprotein (AFP), employing a DNA triangular prism (DTP) nanostructure as an efficient supporting platform integrated with a DNAzyme-hybridization chain reaction (HCR)-CRISPR/Cas12a triple signal amplification strategy. The ssDNA portion at the top of the DTP hybridized with ssDNA (S6) rich in guanine, thereby capturing hemin and forming G-quadruplex/hemin complexes, generating a strong initial electrochemical signal. Target recognition process released Mg²⁺-dependent DNAzyme, which cleaved the substrate hairpin DNA and produced trigger for HCR. The product of HCR activated CRISPR/Cas12a, causing non-specific cleavage of S6 and the ssDNA portion of DTP, which hindered the formation of G-quadruplex/hemin and led to a significant decrease in current signal. The rigid, stable, and size-controllable DTP nano-framework enhanced nucleic acid hybridization and signal molecule binding while minimizing nonspecific adsorption, eliminating masking agent requirements, with its ordered assembly facilitating CRISPR/Cas12a access to ssDNA for improved cleavage efficiency and detection sensitivity. Additionally, the programmable biosensing platform enabled sensitive detection of miRNA 122 and AFP through minimal sequence modifications in target recognition, while maintaining the identical cascade amplification system and DTP framework, demonstrating excellent versatility. Meaningfully, the biosensor sensitively detected miRNA 122 and AFP in real serum samples, and effectively distinguished healthy individuals from HCC patients, indicating its enormous potential for clinical diagnosis.