Competitive FRET-Aptamer-Based detection of methylphosphonic acid, a common nerve agent metabolite
Competitive FRET-Aptamer-Based detection of methylphosphonic acid, a common nerve agent metabolite
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DOI：
10.1007/s10895-008-0316-3
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发表时间：
2008-09-01
影响因子：
2.7
通讯作者：
Hanson, Douglas
中科院分区：
文献类型：
--
作者：
Bruno, John G.;Carrillo, Maria P.;Hanson, Douglas
描述了竞争荧光共振能量转移(FRET)-基于适体的分析格式，用于一步检测甲基膦酸(MPA；几种有机磷(OP)神经毒剂的代谢物)。将氨基甲酸甲酯连接到对甲苯磺酰磁珠上，用于DNA适配子的选择，从中产生一个优势适配子序列。尝试了两种不同的FRET方法。在一种方法中，互补DNA序列用作模板，用于通过不对称PCR用Alexa Fluor 546(AF 546)-14-dUTP标记适配子。在对适配子-MPA复合体进行三维分子模拟后，合成了一系列标记在最佳结合口袋中的50、51和52位的荧光适配子。在两种FRET形式中，氨基MPA连接到黑洞Quencher(BHQ-1或BHQ-2)-琥珀酰亚胺，并允许与荧光素或AF546标记的MPA适配子结合。在凝胶过滤纯化标记的MPA适配子-BHQ-氨基MPA FRET络合物后，在滴定和交叉反应研究中，这些络合物与不同浓度的未标记MPA、MPA衍生物和无关化合物竞争。这两种方法都产生了低微克/毫升的MPA检测下限，与无关化合物的交叉反应水平通常较低。然而，数据显示了一种可能影响焦虑症的方向(开灯或关灯)和强度的特征模式。
Competitive fluorescence resonance energy transfer (FRET)-aptamer-based assay formats are described for one-step detection of methylphosphonic acid (MPA; a metabolite of several organophosphorus (OP) nerve agents). AminoMPA was attached to tosyl-magnetic beads and used for DNA aptamer selection from which one dominant aptamer sequence emerged. Two different FRET approaches were attempted. In one approach, the complementary DNA sequence was used as a template for labeling the aptamer with Alexa Fluor 546 (AF 546)-14-dUTP by asymmetric PCR. Following 3-dimensional (3-D), molecular modeling of the aptamer-MPA complex, a series of three fluoresceinated aptamers labeled at positions 50, 51, and 52 in the putative optimal binding pocket were synthesized. In both FRET formats, aminoMPA was linked to Black Hole Quencher (BHQ-1 or BHQ-2)-succinimides and allowed to bind the fluorescein or AF 546-labeled MPA aptamer. Following gel filtration to purify the labeled MPA aptamer-BHQ-aminoMPA FRET complexes, the complexes were competed against various concentrations of unlabeled MPA, MPA derivatives, and unrelated compounds in titration and cross-reactivity studies. Both approaches yielded low microgram per milliliter detection limits for MPA with generally low levels of cross-reactivity for unrelated compounds. However, the data suggest a pattern of traits that may effect the direction (lights on or off) and intensity of the FRET.