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Sensitive and selective detection of the p53 gene based on a triple-helix magnetic probe coupled to a fluorescent liposome hybridization assembly via rolling circle amplification
Analyst ( IF 3.6 ) Pub Date : 2017-08-23 00:00:00 , DOI: 10.1039/c7an01255a Xia Li 1, 2, 3, 4, 5 , Juan Song 1, 2, 3, 4 , Qingwang Xue 1, 2, 3, 4 , Haiyan Zhao 5, 6, 7, 8, 9 , Min Liu 1, 2, 3, 4 , Baoli Chen 1, 2, 3, 4 , Yun Liu 1, 2, 3, 4 , Wei Jiang 5, 6, 7, 8, 9 , Chen-zhong Li 1, 2, 3, 4, 10
Analyst ( IF 3.6 ) Pub Date : 2017-08-23 00:00:00 , DOI: 10.1039/c7an01255a Xia Li 1, 2, 3, 4, 5 , Juan Song 1, 2, 3, 4 , Qingwang Xue 1, 2, 3, 4 , Haiyan Zhao 5, 6, 7, 8, 9 , Min Liu 1, 2, 3, 4 , Baoli Chen 1, 2, 3, 4 , Yun Liu 1, 2, 3, 4 , Wei Jiang 5, 6, 7, 8, 9 , Chen-zhong Li 1, 2, 3, 4, 10
Affiliation
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Developing a sensitive and selective sensing platform for the p53 gene and its mutation analysis is essential and may aid in early cancer screening and assessment of prognosis. Here, we developed a highly sensitive and selective p53 gene assay based on the coupling of a triple-helix magnetic probe (THMP) to a fluorescent liposome hybridization assembly, a process initiated by rolling circle amplification (RCA). In the presence of p53, the THMP unfolds and activates an enzymatic cleavage reaction, thus releasing the RCA primer and initiating the RCA product-assisted fluorescent liposome hybridization assembly. The resultant double-stranded DNA structures bind the intercalating SG dye from the fluorescent liposomes, thus dramatically enhancing the fluorescence signal. In the absence of p53, the THMP remains intact and blocks the trigger release and fluorescent liposome assembly, thus resulting in a low background signal. The THMPs were designed with integrated target recognition by Watson–Crick base-pairing, site-specific cleavage by an endonuclease and background signal elimination by magnetic isolation, thus avoiding the need to design multiple probes. Moreover, the use of fluorescent liposome assembly and magnetic isolation helps in avoiding sample matrix interference and nonspecific staining. Through cooperative amplification coupling with enzyme cleavage recycling, the RCA-assisted fluorescent liposome assembly and magnetic isolation improved the sensitivity, with a detection limit of 0.07 fM. The excellent capacity of the THMP to specifically detect the involved targets and the precise site-specific endonuclease cleavage ensured remarkable selectivity for p53 against single-base mismatches. This proposed approach worked well in biological samples, thus demonstrating great potential for biomedical and clinical diagnosis applications.
中文翻译：
基于三螺旋磁性探针通过滚环扩增与荧光脂质体杂交组件偶联的p53基因的灵敏和选择性检测
为p53基因及其突变分析开发灵敏且选择性的传感平台至关重要，并且可能有助于早期癌症筛查和评估预后。在这里，我们基于三螺旋磁探针（THMP）与荧光脂质体杂交组件的结合，开发了一种高度灵敏且选择性的p53基因测定法，该过程由滚环扩增（RCA）启动。在p53存在下，THMP会展开并激活酶促裂解反应，从而释放RCA引物并启动RCA产品辅助的荧光脂质体杂交装配。所得的双链DNA结构结合了来自荧光脂质体的插入型SG染料，从而显着增强了荧光信号。在没有p53的情况下，THMP保持完整并阻止触发释放和荧光脂质体装配，因此导致低背景信号。THMPs的设计具有整合的靶标，可通过Watson-Crick碱基配对，通过核酸内切酶进行位点特异性切割以及通过磁隔离消除背景信号，从而避免了设计多个探针的需要。此外，使用荧光脂质体组装和磁隔离有助于避免样品基质干扰和非特异性染色。通过协同扩增与酶裂解循环的结合，RCA辅助的荧光脂质体组装和磁分离提高了灵敏度，检测极限为0.07 fM。THMP具有出色的特异性检测相关靶标的能力和精确的位点特异性内切核酸酶裂解能力，确保了p53对单碱基错配的显着选择性。该提议的方法在生物样品中效果很好，因此证明了在生物医学和临床诊断应用中的巨大潜力。
更新日期：2017-09-25
中文翻译：
基于三螺旋磁性探针通过滚环扩增与荧光脂质体杂交组件偶联的p53基因的灵敏和选择性检测
为p53基因及其突变分析开发灵敏且选择性的传感平台至关重要，并且可能有助于早期癌症筛查和评估预后。在这里，我们基于三螺旋磁探针（THMP）与荧光脂质体杂交组件的结合，开发了一种高度灵敏且选择性的p53基因测定法，该过程由滚环扩增（RCA）启动。在p53存在下，THMP会展开并激活酶促裂解反应，从而释放RCA引物并启动RCA产品辅助的荧光脂质体杂交装配。所得的双链DNA结构结合了来自荧光脂质体的插入型SG染料，从而显着增强了荧光信号。在没有p53的情况下，THMP保持完整并阻止触发释放和荧光脂质体装配，因此导致低背景信号。THMPs的设计具有整合的靶标，可通过Watson-Crick碱基配对，通过核酸内切酶进行位点特异性切割以及通过磁隔离消除背景信号，从而避免了设计多个探针的需要。此外，使用荧光脂质体组装和磁隔离有助于避免样品基质干扰和非特异性染色。通过协同扩增与酶裂解循环的结合，RCA辅助的荧光脂质体组装和磁分离提高了灵敏度，检测极限为0.07 fM。THMP具有出色的特异性检测相关靶标的能力和精确的位点特异性内切核酸酶裂解能力，确保了p53对单碱基错配的显着选择性。该提议的方法在生物样品中效果很好，因此证明了在生物医学和临床诊断应用中的巨大潜力。
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