基于纳米金复合探针的基因芯片膜转印检测法

Membrane transfer-based colorimetric DNA detection using enzyme modified gold nanoparticles

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针(T10,T40),其中检测探针与靶DNA5′端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1(与靶DNA分子3′端互补)通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA5′端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成"三明治"结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9∶1时可以检测1pmol/L合成靶DNA分子或0.23pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。

We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle （AuNP） probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3＇ end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5＇ end of target DNA, and two biotin-labeled signal probes （T10 and T40） with different lengths. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a ＂sandwich＂ format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9：1：1 （T10：T40：P2）. The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.