Electrochemical Biosensor Based on a Tetrahedral DNA Nanostructure and an “AND” Logic Gate-Regulated Cascade Amplification System for Parallel Detection of Dual Disease Biomarkers

Hepatocellular carcinoma (HCC) is the most common form of liver cancer globally, and early diagnosis is of great significance for improving the survival rate of patients. The combined detection of important biomarkers of HCC, alpha-fetoprotein (AFP) and microRNA-122 (miRNA-122), has significant value for improving the accuracy of early diagnosis of HCC. Herein, we constructed an electrochemical biosensor based on a tetrahedral DNA nanostructure and an "AND" logic gate-regulated cascade amplification system for the detection of AFP and miRNA-122. Specifically, the product of the catalytic hairpin assembly (CHA) triggered by miRNA-122 converted the signal probe on the tetrahedral DNA nanostructure (TDN) from the double-stranded structure to the single-stranded structure. The product of the hybridization chain reaction (HCR) triggered by AFP activated the Cas12a/crRNA system. The two amplification circuits occurred in parallel on the electrode surface and in the solution, respectively. The activated Cas12a/crRNA nonspecifically cleaved the single-stranded signal probe on the TDN, resulting in significant signal changes. Since the single-stranded signal probe on the TDN can only be cleaved when both AFP and miRNA-122 are present, an "AND" logic gate with a single-signal probe output was constructed, enabling the highly sensitive combined detection of AFP and miRNA-122. The limits of detection (LOD) are as low as 4.05 fg/mL and 34.84 aM (S/N = 3), respectively. Meaningfully, this sensing platform was successfully applied to detect AFP and miRNA-122 in real human serum samples and effectively distinguished between healthy individuals and HCC patients, demonstrating its great potential in the accurate diagnosis of HCC.