Jin Furui, Liu Min, Xu Danke
State Key Laboratory of Analytical Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, No 163, Xianlin Avenue, Nanjing, 210023, China.
State Key Laboratory of Analytical Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, No 163, Xianlin Avenue, Nanjing, 210023, China.
Talanta. 2023 Feb 1;253:123968. doi: 10.1016/j.talanta.2022.123968. Epub 2022 Sep 27.
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.
低浓度生物标志物的检测在临床诊断分析中具有重要意义，并一直吸引着持续的研究。在这项工作中，使用具有等温、无酶DNA纳米结构用于荧光放大的磁珠实现了乙型肝炎病毒（HBV）DNA的绝对定量。首先，DNA功能化磁珠以夹心杂交的形式捕获目标核酸，单个目标通过等温网状杂交链反应（wHCR）点亮整个磁珠。在微阵列扫描后，可基于泊松统计对目标核酸进行数字定量。因此，荧光磁珠检测能够在无需外部校准曲线的情况下精确检测低至5 fM水平的HBV DNA。此外，该方法不仅能特异性区分单碱基错配序列，还能对血清样本中的HBV DNA进行定量检测。与通常结合复杂隔室划分操作的常规数字检测不同，固定在磁珠上的扩增结构可在微升规模体积的微量离心管中进行。这项工作通过无酶和等温方法扩展了磁珠在数字定量检测中的应用。
