Enzyme-mediated individual nanoparticle release assay.
Enzyme-mediated individual nanoparticle release assay.
复制标题
酶介导的单个纳米颗粒释放测定。
DOI：
10.1016/j.ab.2006.03.020
复制
发表时间：
2006
影响因子：
2.9
通讯作者：
Schultz,DavidA
中科院分区：
文献类型：
--
作者：
Glass,JamesR;Dickerson,JanetC;Schultz,DavidA
已经开发了许多方法来测量样品中大分子物质的存在;然而，检测功能活性或该活性的调节剂的方法的数量更有限。为了解决这一限制，开发了一种方法，该方法使用纳米颗粒的光学检测作为酶活性的测量。纳米颗粒越来越多地被用作静态结合试验中的生物标记;在这里，我们描述了它们在释放试验格式中的用途，其中测量单个纳米颗粒从表面的酶介导的释放。DNA的双链片段用作初始系链以将纳米颗粒结合到固体表面。使用暗场光学显微镜和数字图像捕获来确定纳米颗粒的空间分布和数量。DNA系链的位点特异性切割导致纳米颗粒释放。这种方法的方法和验证的酶介导的，个别的DNA切割事件，快速，高特异性，并在实时的描述。这种方法被用来检测和区分非甲基化和甲基化的DNA，并展示了一个新的平台，高通量筛选酶活性的调节剂。
Numerous methods have been developed to measure the presence of macromolecular species in a sample; however, the number of methods that detect functional activity or modulators of that activity is more limited. To address this limitation, an approach was developed that uses the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here, we describe their use in a release assay format, where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double-stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site-specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time are described. This approach was used to detect and discriminate between nonmethylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity.