A simple magnetic nanoparticle-poly-enzyme nanobead sandwich assay for direct, ultrasensitive DNA detection

A simple magnetic nanoparticle (MNP)-poly-enzyme nanobead sandwich assay for direct detection of ultralow levels of unlabeled target-DNA is developed. This approach uses a capture-DNA covalently linked to a dense PEGylated polymer encapsulated MNP and a biotinylated signal-DNA to sandwich the target-DNA. A DNA ligation is then followed to offer high discrimination between the perfect-match and single-base mismatch target-DNAs. Only the presence of a perfect-match target can covalently link the biotinylated signal-DNA onto the MNP surface for subsequent binding to a polymer nanobead tagged with thousands of copies of high-activity neutravidin-horseradish peroxidase (NAV-HRP) for great enzymatic signal amplification. Combining the advantages of the dense MNP surface PEGylation to reduce non-specific adsorption (assay background) and the powerful signal amplification of poly-enzyme nanobead, this assay can directly quantify the target-DNA down to single digit attomolar with a large linear dynamic range of 5 orders of magnitude (from 10^-18 to 10^-13M).