适配子修饰的玻璃毛细管基纳米孔用于腺苷的检测

Detection based on nanopore technology is simple, reliable, very sensitive, but it has no specificity; aptamers, as a class of function nucleic acids, are specific and with high affinity. The project intends to sensitive detection of adenosine by combination of the advantages of both nanopore technology and aptamers. First, we cut adenosine aptamer into two subunits and plated gold layer inside the nanopore; the two aptamer subunits bonded to nanopore and gold nanoparticles by gold-sulfur chemistry, respectively; the two aptamer subunits would be linked by the presence of target, adenosine, leading the attachment of AuNPs on the inner wall of nanopore; since these attached AuNPs occupied the ion channels, the declination of ion current would be expected. Quantitative detection of adenosine is realized by recognition of this current change. The same detection strategy can be used for other targets, such as adenosine triphosphate, cocaine. Moreover, the gold layer inside the nanopore promise the possibility of the introduction of other mecapto-functional molecules, which could extend the applications of nanopore technology.

基于纳米孔的检测技术简单、可靠，灵敏度非常高，但其本身没有特异性；适配子具有极高的亲和力和特异性，是近年来最为引人关注的一类功能化核酸。本项目拟将二者的优点结合起来，实现对腺苷的灵敏检测。我们首先在玻璃纳米孔内部镀金;将腺苷适配子切割成两个亚单元,通过金硫键分别与纳米孔内金膜及金纳米粒子键连；当腺苷分子存在时，适配子亚单元与其形成复合体，将金纳米粒子连接在纳米孔内壁，占据了离子通道，导致离子电流降低，利用这一变化定量检测腺苷。该检测策略还可以用来检测三磷酸腺苷、可卡因等其他体系。同时，由于玻璃毛细管基纳米孔内的金膜可以作为巯基功能分子的引入基础，必将极大拓展纳米孔的应用领域。

基于纳米孔的检测技术简单、可靠，灵敏度非常高，但被测物与纳米孔检测区域没有特异性的相互作用，因而基于纳米孔的检测技术本身没有特异性；为了实现特异性检测，需要在纳米孔的检测区域进行特定修饰。而另一方面，金硫键被广泛用于分子界面的剪裁与修饰，可供选择的硫醇分子种类多种多样，也可以对目标的功能分子进行硫醇修饰。但是，将金硫键引入纳米孔的功能界面首先要在纳米孔内壁镀金膜。为此，我们先后采用三种方法在玻璃毛细管纳米孔内部镀金，然后通过金硫键构建特异性的功能检测界面。采用激光拉制法制备的玻璃毛细管纳米孔直径很难降到10纳米以下，这极大限制了在核酸检测方面的应用。为此，我们采用硅酸钠水解的方法，将激光拉制制备的玻璃毛细管纳米孔的孔径从40纳米缩小至5纳米。极大的提高了检测DNA穿孔事件的信噪比和碱基分辨率。