Web hybrid chain reaction enhanced fluorescent magnetic bead array for digital nucleic acid detection

The detection of biomarkers at low concentrations is important in clinical diagnostic analyses and has attracted continuous research. In this work, absolute quantification of hepatitis B virus (HBV) DNA was achieved using magnetic beads with isothermal, enzyme-free DNA nanostructure for fluorescence amplification. Firstly, the DNA-functionalized bead captured the target nucleic acid in the form of sandwich hybridization, and the individual target lighted up the entire bead by isothermal web hybridization chain reaction (wHCR). After the microarray scanning, the target nucleic acids can be digitally quantified based on the Poisson statistics. Therefore, the fluorescent bead assay enabled precise detection of HBV DNA down to 5 fM level without external calibration curves. Moreover, this method not only specifically distinguished single-base mismatched sequences, but also obtained the quantitative detection of HBV DNA in serum samples. Unlike routine digital detection usually combined with complex compartment partitioning operations, the amplification structure immobilized on beads can be conducted in microcentrifuge tubes with a volume of microliter scale. This work expands the application of magnetic beads in the digital quantitative detection via enzyme-free and isothermal method.