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Sensitive mutant DNA biomarker detection based on magnetic nanoparticles and nicking endonuclease assisted fluorescence signal amplification

机译：基于磁性纳米粒子和切口核酸内切酶辅助的荧光信号放大的敏感突变DNA生物标志物检测

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摘要

Based on the nicking endonuclease (NEase)-assisted target recycling and magnetic nanoparticle (MNPs) separation process via the streptavidin-biotin system, we developed a new, signal amplified and ultrasensitive fluorescent biosensor for the detection of mutant human p53 gene. The target mutant DNA hybridizes with the loop portion of a hairpin probe (HP) modified with biotin and a fluorescein isothiocyanate dye (FITC) at its 5' and 3' ends separately and forms a nicking site for NEase, which cleaves the HP and releases the target DNA. The released target DNA again hybridizes with the intact HP and initiates the DNA recycling process with the assistance of NEase, leading to the cleavage of a large number of HPs and detachment of the biotin labeled part with the FITC tagged signal portion. Only these cleaved fragments corresponding to target DNAs could remain in solution and function as a signaling flare, while the biotin labeled sequences including intact hairpin probes could be trapped and removed by the streptavidin coated MNPs. The developed method exhibits a detection limit as low as 198 fM and high discrimination efficiency toward a single-base mismatched sequence. Therefore, the novel NEase-amplified magnetic nanoparticle assay has great potential for sensitive and accurate detection of trace amounts of DNA in clinical diagnosis and biomedical research.
机译：基于切口内切核酸酶（NEase）辅助的靶标回收和通过链霉亲和素-生物素系统的磁性纳米颗粒（MNPs）分离过程，我们开发了一种新型的信号放大和超灵敏荧光生物传感器，用于检测突变型人p53基因。目标突变体DNA与经过生物素修饰的发夹探针（HP）的环部分杂交，并在其5'和3'末端分别与异硫氰酸荧光素染料（FITC）杂交，并形成NEase的切刻位点，从而裂解HP并释放靶DNA。释放的靶DNA再次与完整的HP杂交，并在NEase的帮助下启动DNA回收过程，导致大量HP的裂解以及带有FITC标记信号部分的生物素标记部分的分离。只有对应于目标DNA的这些裂解片段可以保留在溶液中，并起信号弹的作用，而包括完整发夹探针的生物素标记序列可以被链霉亲和素包被的MNP捕获和去除。所开发的方法展现出低至198 fM的检测限和对单碱基错配序列的高识别效率。因此，新型的NEase扩增磁性纳米颗粒测定法在临床诊断和生物医学研究中具有灵敏而准确地检测痕量DNA的巨大潜力。

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