A novel self-autocatalytic DNAzyme and catalytic hairpin assembly molecular circuit for highly sensitive and non-label fluorescence aptamer glypican-3 assay.

Glypican-3 (GPC-3) is universally regarded as an early diagnostic biomarker and plays a crucial role in the sensitive detection and treatment efficacy evaluation of hepatocellular carcinoma (HCC). Here, a novel self-autocatalytic DNAzyme reaction and catalytic hairpin assembly (CHA)-integrated molecular circuit amplification strategy is developed for realizing an aptamer-based, enzyme- and non-label fluorescence assay of GPC-3. The sensing protocol involves GPC-3 analyte binding to the aptamer in aptamer/ssDNA to trigger the liberation of ssDNA for the initiation of CHA formation of duplexes containing both active DNAzyme and G-quadruplex fragments. The DNAzymes further cleave another G-quadruplex- and DNAzyme-caged signal hairpin to free these sequences to trigger the self-autocatalytic circuit process, which leads to the cyclic cutting of signal hairpins to yield large amounts of G-quadruplexes. Subsequent binding of thioflavin T dye to formed G-quadruplexes thus generates significantly intensified fluorescence signals for detecting GPC-3, with a 0.16 pM detection limit between 1 pM and 100 nM. Moreover, the ability of this method to distinguish GPC-3 from non-target proteins and its success in detecting GPC-3 at low levels in diluted human serum is examined, underscoring its promising potential for monitoring disease-specific biomarkers at trace concentrations for early disease diagnosis.