Multiplexed Electrochemical Sensing of Single-Nucleotide Polymorphic Microribonucleic Acids in Liver Cancer

Liver cancer remains a highly lethal malignancy, primarily due to the limitations of current biomarkers like α-fetoprotein, which fail to detect many early-stage cases. MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers, particularly those influenced by single-nucleotide polymorphisms (SNPs). However, conventional electrochemical biosensors for miRNA detection often require complex probe modifications to achieve sufficient sensitivity and selectivity. Here, we present a multiplex electrochemical biosensor for the simultaneous detection of SNP-associated miRNAs (SNP-146a and SNP-122) linked to liver cancer. The biosensor leverages amino-rich polyethylenimine gold nanoparticles (PEI-AuNPs) to immobilize capture and signal probes efficiently, enabling robust signal amplification. Using metal ion tags (Pb²⁺ for SNP-146a and Ag⁺ for SNP-122) and differential pulse voltammetry (DPV), we achieved distinct, highly sensitive detection with limits of detection (LODs) of 0.39 and 0.23 pM, respectively, across a broad linear range (1 pM-100 nM). Clinical validation using serum samples yielded statistically significant discrimination (<i>P</i> = 0.0013 for SNP-146a, <i>P</i> = 0.0001 for SNP-122) and outstanding diagnostic performance (ROC-AUC = 0.98 and 0.96, respectively). Meanwhile, the platform's versatility was further demonstrated by detecting miR-181a and miR-155 with exceptional LODs of 0.34 and 0.02 pM. This multiplexed biosensor provides a powerful tool for early liver cancer diagnosis and can be readily adapted for other tumor biomarkers, advancing precision point-of-care diagnostics.