Cascade Signal Amplifying Strategy for Ultrasensitive Detection of Tumor Biomarker by Dnazyme Cleaving Mediated Hcr

Abstract

Tumor biomarkers are a kind of important biomolecules for aiding cancer diagnosis and assessing tumor efficacy, among which alpha-fetoprotein (AFP) is a crucial tumor biomarker for the early diagnosis of hepatocellular carcinoma. In this study, cascade signal amplifying strategy was designed for the ultrasensitive detection of AFP by DNAzyme cleaving-mediated HCR. The primary antibody was immobilized on the Au nanoparticles modified glassy carbon electrode, sequentially capturing AFP and the secondary antibody. Then, streptavidin was combined with the biotin-modified secondary antibody and Cu2+-specific DNAzyme. When Cu2+ was present, the substrate strand of the DNAzyme would cleave and produce an ssDNA end. Finally, hybrid chain reaction was triggered to form long double-stranded DNA on the electrode, which would greatly inhibit the charge transfer at the electrode/solution interface. Therefore, the DNAzyme cleaving-mediated HCR strategy can be applied as cascade signal amplification for the ultrasensitive electrochemical determination of tumor biomarker AFP. Under optimal conditions, the immunosensor exhibits a broad linear range of 0.001−100 ng·mL−1 (R2 = 0.990) and a low detection limit of 15.8 fg·mL−1. The proposed sensor displays high sensitivity and selectivity for AFP detection, which can be employed as a universal sensing platform in clinical diagnostics.

Keywords: biomarker, AFP, DNAzyme, Hybridization chain reaction, Immunosensor