基于纳米金探针和基因芯片的DNA检测新方法

A Novel DNA Detection Method Based on Gold Nanoparticle Probes and Gene Chips

运用荧光纳米金探针和基因芯片杂交建立一种新的DNA检测方法.荧光纳米金探针表面标记有两种DNA探针:一种为带有Cy5荧光分子的信号探针BP1,起信号放大作用;另一种为与靶DNA一部分互补的检测探针P532,两种探针比例为5∶1.当靶DNA存在时,芯片上捕捉探针(与靶DNA的另一部分互补)通过碱基互补配对结合靶DNA,将靶DNA固定于芯片上;荧光纳米金探针通过检测探针与靶DNA及芯片结合,在芯片上形成"三明治"复合结构,最后通过检测信号探针上荧光分子的信号强度来确定靶DNA的量.新方法检测灵敏度高,可以检测浓度为1 pmol/L的靶DNA,操作简单,检测时间短.通过改进纳米金探针的标记和优化杂交条件,可进一步提高核酸检测的灵敏度,这将在核酸检测方面具有重要的应用价值.

A new DNA detection method based on fluorescent gold nanoparticle （AuNP） probes and DNA chips was introduced here. Firstly, chips were modified with amine-oligonucleotide probe complementary to target DNA （capture probe）. Gold nanoparticles were modified with two types of probes, one was another thiolated-oligonucleotide probe complementary to target DNA （detecting probe） and the other was thiolated-oligonucleotide probe without homologous sequence to detected samples （signal probe）. The end of signal probe opposite the gold nanoparticles was modified with Cy5. The ratio of them is 1 ： 5. Through hybridization, the sandwich structures （DNA chip-target DNA-AuNP probes） were formed. Excess samples and AuNP probes were removed by washing for several times. And the amount of target DNA was determined by detecting the fluorescent signal of the AuNP probes. Low to 1 pmol/L target DNA could be detected with this method. Because the signal DNA probe with fluorescence rather than the target DNA was identified in this approach, the different proportions of detecting probe and signal probe could affect the detection signal significantly. Effect of different proportions of probes on detecting signal would be studied at a ratio of 1 ： 30, 1 ： 60, 1 ： 100 and higher sensitivity would be achieved. So, this will be one of the most important DNA detection methods.