Glass James R, Dickerson Janet C, Schultz David A
Seashell Technology, LLC, La Jolla, CA 92037, USA.
Anal Biochem. 2006 Jun 15;353(2):209-16. doi: 10.1016/j.ab.2006.03.020. Epub 2006 Mar 31.
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.
已经开发出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调节剂的方法数量更为有限。为了解决这一局限性，开发了一种方法，该方法利用纳米颗粒的光学检测作为酶活性的一种度量。纳米颗粒越来越多地被用作静态结合测定中的生物标记物；在此，我们描述了它们在释放测定形式中的应用，其中测量酶介导的单个纳米颗粒从表面的释放。一段双链DNA片段用作初始系链，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。描述了这种用于快速、高特异性和实时测量酶介导的单个DNA切割事件的方法及其验证。该方法用于检测和区分未甲基化和甲基化的DNA，并展示了一个用于酶活性调节剂高通量筛选的新型平台。
