Lapitan Lorico D S, Zhou Dejian
Department of Chemical Engineering and Advanced Materials Research Laboratory, Research Center for the Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines.
School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
Methods Enzymol. 2020;630:453-480. doi: 10.1016/bs.mie.2019.10.026. Epub 2019 Nov 19.
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 to 10M).
开发了一种用于直接检测超低水平未标记靶DNA的简单磁性纳米颗粒（MNP）-多酶纳米珠夹心测定法。该方法使用与包裹有密集聚乙二醇化聚合物的MNP共价连接的捕获DNA和生物素化的信号DNA来夹心靶DNA。随后进行DNA连接，以在完全匹配和单碱基错配靶DNA之间提供高区分度。只有完全匹配靶的存在才能将生物素化的信号DNA共价连接到MNP表面，以便随后与标记有数千个高活性中性抗生物素蛋白-辣根过氧化物酶（NAV-HRP）拷贝的聚合物纳米珠结合，实现强大的酶信号放大。结合密集MNP表面聚乙二醇化以减少非特异性吸附（测定背景）的优点和多酶纳米珠的强大信号放大功能，该测定法可以直接将靶DNA定量至个位数阿托摩尔，线性动态范围大达5个数量级（从10到10M）。
