• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Analytica chimica acta >On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay
【24h】

On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay

机译：用于乙酰胆碱酯酶测定的片上集成水解，荧光标记和电泳分离

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

A sensitive on-chip acetylcholinesterase (AChE) assay that serves as a basis for the development of a fully integrated on-chip AChE-inhibitor detection assay is presented. The sequential steps required for the on-chip analysis process were integrated into a microchip. Transport and mixing of the reagents occurred by a combination of electroosmosis and electrophoresis using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was determined by on-chip reaction of thiocholine with eosinmaleimide, and the resulting thioether was electrophoretically separated and detected by laser-induced fluorescence (LIF). Enzyme-substrate mixing and reaction by confluent flow of reagents was compared with electrophoretically mediated microanalysis (EMMA), based on injection of an enzyme plug, and the utilization of differences in electrophoretic mobility as a driving force for efficient mixing and reaction. Both methods yielded similar results, however the EMMA-plug technique is preferable. The EMMA-plug technique was optimized for length and pushing time of enzyme plug, length of dyes mixture plug, acetylthiocholine concentration, and detector location. Detection of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and paraoxon, two AChE inhibitors, was demonstrated by off-chip mixing of the inhibitor and AChE, followed by the on-chip AChE assay. Limit of detection of VX for 5.5 min incubation and of paraoxon for 8 min incubation was 4 x 10(-10) and 4 x 10(-7) M, respectively. Utilization of the AChE microchip assay for inhibition kinetics was demonstrated also by evaluation of the inhibitor-enzyme bimolecular reaction constant (k(i)). The evaluated k(i) values for VX and paraoxon for AChE from the electric eel were 3.5 x 10(7) and 1.7 x 10(5) M-1 min(-1), respectively, conforming well to reported values obtained by bulk methods. (c) 2006 Elsevier B.V. All rights reserved.
机译：提出了一种敏感的片上乙酰胆碱酯酶（AChE）检测方法，该方法可作为开发完全集成的片上AChE抑制剂检测方法的基础。片上分析过程所需的顺序步骤已集成到微芯片中。试剂的传输和混合是通过电渗和电泳结合使用计算机控制的电动传输进行的。通过乙酰胆碱与曙红马来酰亚胺的片上反应来测定乙酰胆碱酯基催化的乙酰基硫代胆碱水解为硫代胆碱，然后电泳分离所得硫醚，并通过激光诱导荧光（LIF）进行检测。基于注入的酶塞，并通过电泳介导的微量分析与电泳介导的微量分析（EMMA）进行了比较，并基于电泳迁移率的差异作为有效混合和反应的驱动力。两种方法均产生相似的结果，但是EMMA-plug技术是可取的。 EMMA塞技术针对酶塞的长度和推入时间，染料混合物塞的长度，乙酰胆碱浓度和检测器位置进行了优化。两种AChE抑制剂O-乙基S- [2-（二异丙基氨基）乙基]甲基硫代磷酸酯（VX）和对氧磷的检测通过抑制剂和AChE的芯片外混合，然后进行芯片上的AChE分析来证明。孵育5.5分钟时对VX的检测限和孵育8分钟时对氧磷的检测限分别为4 x 10（-10）和4 x 10（-7）M。还通过评估抑制剂-酶双分子反应常数（k（i））证明了AChE微芯片测定法用于抑制动力学。来自鳗鱼的VCh和对氧磷对AChE的对氧磷的评估k（i）值分别为3.5 x 10（7）和1.7 x 10（5）M-1 min（-1），与批量获得的报告值非常吻合方法。 （c）2006 Elsevier B.V.保留所有权利。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号