As a crucial biomarker for the early warning and prognosis of liver cancer diseases, elevated levels of alpha-fetoprotein (AFP) are associated with hepatocellular carcinoma and germ cell tumors. Herein, we present a novel signal-on electrochemical aptamer sensor, utilizing AuNPs–MXene composite materials, for sensitive AFP quantitation. The AuNPs–MXene composite was synthesized through a simple one-step method and modified on portable microelectrodes. As signal molecules, AFP aptamers were conjugated with methylene blue (MB) and immobilized on the electrode surface. When interacting with AFP, conformational changes in the aptamer–target complex caused MB to approach the electrode, and the electrochemical signal was enhanced through signal-on mechanisms. The developed sensor demonstrated high sensitivity and selectivity for AFP, with a log-linear relationship defined as 1–300 ng/mL, and the LOD was 0.05 ng/mL (S/N = 3). The method was applied to laboratorial and real clinical samples and presented satisfactory selectivity, reproducibility, and long-term stability. The proposed high-performance sensor highlights the potential of electrochemical aptamer sensors in improving the warning capabilities in disease management.
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