Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics.
Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics.
复制标题
多孔聚合物整体中的触手探针夹心测定提高了特异性、灵敏度和动力学。
DOI：
10.1093/nar/gkn564
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发表时间：
2008
影响因子：
14.9
通讯作者：
West,JayAA
中科院分区：
文献类型：
--
作者：
Satterfield,BrentC;Caplan,MichaelR;West,JayAA
核酸夹心法通过添加捕获探针和特异性标签，提高了低密度阵列分析的特异性和灵敏度。在这里，我们采用光激发多孔聚合物单体（PPM）作为三明治分析的高表面积底物。PPMs对2μl样品的萃取效率提高了20倍。我们进一步比较了标记线性探针、量子点标记探针、分子信标（MBs）和触手探针（TPs）的性能。将每种探针技术与传统的标记样品杂交方法进行了比较和对比。所有探针都表现出与传统杂交技术相似的灵敏度和更高的特异性。由于MBs和tp的“开-关”信号机制，它们能够绕过洗涤步骤。TPs的反应动力学比MBs快37.6倍，最快检测时间为5分钟。我们的数据进一步表明，在这些实验中所有被测探针中，tp具有最灵敏的检测限（<1 nM）和最高的特异性（>1×104improvement）。通过将PPM的提高萃取效率与tp的选择性相匹配，我们创建了一种改进的夹层分析格式。
Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2μl of sample. We further compare the performance of labeled linear probes, quantum dot labeled probes, molecular beacons (MBs) and tentacle probes (TPs). Each probe technology was compared and contrasted with traditional hybridization methods using labeled sample. All probes demonstrated similar sensitivity and greater specificity than traditional hybridization techniques. MBs and TPs were able to bypass a wash step due to their ‘on–off’ signaling mechanism. TPs demonstrated reaction kinetics 37.6 times faster than MBs, resulting in the fastest assay time of 5 min. Our data further indicate TPs had the most sensitive detection limit (<1 nM) as well as the highest specificity (>1×104improvement) among all tested probes in these experiments. By matching the enhanced extraction efficiencies of PPM with the selectivity of TPs, we have created a format for improved sandwich assays.