Department of Chemistry, University of California-Irvine, Irvine, California 92697, USA.
J Am Chem Soc. 2011 Mar 30;133(12):4271-3. doi: 10.1021/ja2005576. Epub 2011 Mar 10.
DNA microarrays are invaluable tools for the detection and identification of nucleic acids in biosensing applications. The sensitivity and selectivity of multiplexed single-stranded DNA (ssDNA) surface bioaffinity sensing can be greatly enhanced when coupled to a surface enzymatic reaction. Herein we describe a novel method where the specific sequence-dependent adsorption of a target ssDNA template molecule onto an ssDNA-modified gold microarray is followed with the generation of multiple copies of ssRNA via in situ surface transcription by RNA polymerase. The RNA created on this "generator" element is then detected by specific adsorption onto a second adjacent "detector" element of ssDNA that is complementary to one end of the ssRNA transcript. SPR imaging is then used to detect the subsequent hybridization of cDNA-coated gold nanoparticles with the surface-bound RNA. This RNA transcription-based, dual element amplification method is used to detect ssDNA down to a concentration of 1 fM in a volume of 25 μL (25 zeptomoles).
DNA 微阵列是生物传感应用中检测和识别核酸的非常有价值的工具。当与表面酶反应偶联时，多重单链 DNA（ssDNA）表面生物亲和传感的灵敏度和选择性可以大大提高。本文描述了一种新方法，其中目标 ssDNA 模板分子的特定序列依赖性吸附到 ssDNA 修饰的金微阵列上，然后通过 RNA 聚合酶的原位表面转录生成多个 ssRNA 拷贝。在这个“发生器”元件上创建的 RNA 然后通过特异性吸附到与 ssRNA 转录本的一端互补的第二个相邻“检测器”元件上的 ssDNA 上来检测。然后使用 SPR 成像检测与表面结合的 RNA 杂交的 cDNA 涂覆的金纳米粒子。这种基于 RNA 转录的双元件扩增方法可用于在 25μL（25 飞摩尔）体积中检测低至 1 fM 的 ssDNA。
