Renewable surface fluorescence sandwich immunoassay biosensor for rapid sensitive botulinum toxin detection in an automated fluidic format
Renewable surface fluorescence sandwich immunoassay biosensor for rapid sensitive botulinum toxin detection in an automated fluidic format
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DOI：
10.1039/b900794f
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发表时间：
2009-01-01
期刊：
影响因子：
4.2
通讯作者：
Bruckner-Lea, Cynthia J.
中科院分区：
文献类型：
--
作者：
Grate, Jay W.;Warner, Marvin G.;Bruckner-Lea, Cynthia J.
一种可再生的表面生物传感器，用于快速检测肉毒杆菌神经毒素血清型A的荧光夹心免疫分析的流体自动化的基础上，使用重组蛋白片段的毒素重链（类似于50 kDa）作为结构上有效的模拟。单克隆抗体AR 4和RAZ 1结合完整肉毒杆菌全毒素的单独的非重叠表位（类似于150 kDa）。两个靶向表位均位于重组片段上。AR 4抗体与琼脂糖凝胶珠共价结合并用作捕获抗体。使用旋转杆流动池来捕获通过顺序注射流动系统以悬浮液形式递送的这些珠粒，产生3.6 μ L柱。在用样品灌注珠柱并洗去基质后，用Alexa 647染料标记的RAZ1抗体作为报告物灌注柱。以彼此成90度角耦合到旋转杆流动池的光纤使用一根光纤递送来自HeNe激光器（633nm）的激发光，并使用另一根光纤收集荧光发射光用于检测。每次测量后，释放使用过的琼脂糖凝胶珠，并用新鲜珠替换。在样品分析的快速筛选方法中，使用该系统在不到20分钟内检测到毒素模拟物浓度为10 pM。
A renewable surface biosensor for rapid detection of botulinum neurotoxin serotype A is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant protein fragment of the toxin heavy chain (similar to 50 kDa) as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate non-overlapping epitopes of the full botulinum holotoxin (similar to 150 kDa). Both of the targeted epitopes are located on the recombinant fragment. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by a sequential injection flow system, creating a 3.6 mu L column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degrees angle to one another delivered excitation light from a HeNe laser (633 nm) using one fiber and collected fluorescent emission light for detection with the other. After each measurement, the used Sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes using this system.