Homogeneous, nanoparticle-based quantitative colorimetric detection of oligonucleotides
Homogeneous, nanoparticle-based quantitative colorimetric detection of oligonucleotides
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DOI：
10.1021/ja000133k
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发表时间：
2000-04-19
影响因子：
15
通讯作者：
Letsinger, RL
中科院分区：
文献类型：
--
作者：
Reynolds, RA;Mirkin, CA;Letsinger, RL
寡核苷酸的同源检测对于监测和量化聚合酶链式反应产生的产物的数量是必不可少的。1一般来说，均相分析是有吸引力的检测形式，因为它们提供了实时监测的可能性，易于自动化，降低了污染的风险，并消除了耗时的洗涤步骤。2、3目前，大多数同源寡核苷酸检测格式依赖于荧光、4、5，虽然灵敏度很高，但它们需要相对昂贵的探针和监测设备。最近，本实验室开发了一种基于烷基硫醇功能化寡核苷酸修饰的13 nm金探针的多相比色寡核苷酸检测系统。6在该体系中，分散的寡核苷酸修饰的金纳米颗粒通过与互补的目标寡核苷酸的杂交事件组装成聚集的聚合物网络，并随着温度的变化而斑点在反相硅胶载体上。寡核苷酸诱导的聚集伴随着从红到蓝的颜色变化，很容易在载体上看到。这种现场测试的特点是：(1)超高选择性；(2)肉眼可监测；(3)成本低。关于使用类似的策略对溶液中的寡核苷酸进行均相检测，该系统的缺点是：(1)检测下限为1 nm，这要求它在聚合酶链式反应后的非定量形式中使用；(2)纳米颗粒网络的沉降特性，这排除了获得可接受的终点的可能性。在这里，我们描述了一种新的策略，该策略使用50 nm金颗粒探针和通过非单位探针(方案1)比率产生更小的聚集结构来展示具有确定终点的寡核苷酸的定量比色均相分析的概念。
The homogeneous detection of oligonucleotides is essential for monitoring and quantifying the amount of product generated by PCR. 1 In general, homogeneous assays are attractive detection formats since they offer the potential to be monitored in real time, are amenable to automation, reduce the risk of contamination, and eliminate time-consuming washing steps. 2, 3 Currently, most homogeneous oligonucleotide detection formats rely on fluorescence, 4, 5 and although highly sensitive, they require relatively expensive probes and monitoring equipment. Recently, a heterogeneous colorimetric oligonucleotide detection system based on alkylthiol-functionalized oligonucleotide-modified 13 nm gold probes was developed in this laboratory. 6 In this system, dispersed oligonucleotide-modified gold nanoparticles are assembled into aggregated polymeric networks via hybridization events with complementary target oligonucleotides and spotted onto a reverse phase silica gel support as a function of temperature. The oligonucleotide-induced aggregation occurs with a concomitant red-to-blue color change that is easily visualized on the support. The attributes of this spot test are:(1) ultrahigh selectivity,(2) ability to be monitored by the naked eye, and (3) low cost. With respect to using a similar strategy for the homogeneous detection of oligonucleotides in solution, the drawbacks of the system are:(1) a detection limit of 1 nM, which requires it to be used in a post PCR non-quantitatiVe format, and (2) the sedimentation properties of the nanoparticle networks, which precludes the possibility of obtaining acceptable endpoints. Herein, we describe a new strategy that employs the use of 50 nm gold particle probes and the generation of smaller aggregate structures through nonunity probe (Scheme 1) ratios to demonstrate the concept of a quantitative colorimetric homogeneous assay for oligonucleotides with definitive endpoints.