Highly sensitive detection of protein toxins by surface plasmon resonance with biotinylation-based inline atom transfer radical polymerization amplification.
Highly sensitive detection of protein toxins by surface plasmon resonance with biotinylation-based inline atom transfer radical polymerization amplification.
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通过表面等离子体共振对蛋白质毒素的高度敏感检测，其基于生物素化的内联原子转移自由基聚合扩增。
DOI：
10.1021/ac1000114
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发表时间：
2010-05-01
影响因子：
7.4
通讯作者：
Cheng Q
中科院分区：
文献类型：
--
作者：
Liu Y;Dong Y;Jauw J;Linman MJ;Cheng Q
蛋白质的超灵敏检测对蛋白质组学研究具有重要意义。本文报道了一种提高表面等离子体共振（SPR）光谱检测灵敏度的方法，该方法将聚合引发剂耦合到生物特异性相互作用中，并诱导内联原子转移自由基聚合（ATRP）来放大SPR响应。选择细菌霍乱毒素（CT）作为模型蛋白，将其共价固定在表面以演示该原理。特异性识别是通过使用生物素化的抗ct来实现的，它允许带有生物素标签的引发剂通过中性蛋白桥固定在蛋白质结合位点，并通过ATRP机制触发聚甲基丙烯酸羟乙酯（PHEMA）聚合物刷的局部生长。为了进一步增强信号，进行第二次ATRP反应，利用第一步中PHEMA刷的羟基在传感表面形成超支化聚合物。连续的两个ATRP步骤显著提高了SPR的检测，使得无法直接测量的少量CT可以用大信号进行量化。所得聚合物薄膜已通过光学和原子力显微镜进行了表征。在催化剂混合物中加入抗坏血酸（AA）作为脱氧试剂，有效抑制了氧干扰，缩短了反应时间，使该ATRP方法应用于基于流动注射的SPR检测成为可能。建立了基于表面覆盖度的PHEMA放大CT检测标定曲线，其相关关系在8.23 × 10−15 mol/cm2 ~ 3.61 × 10−12 mol/cm2范围内，检测限为6.27 × 10−15 mol/cm2。这里使用的多功能生物素-中性生物素相互作用应该允许ATRP增强适应许多其他系统，包括DNA， RNA，肽和碳水化合物，为流动注射测定和SPR光谱的超灵敏生物分子分析开辟了新的途径。
Ultra-sensitive detection of proteins is of great importance to proteomics studies. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy by coupling a polymerization initiator to a biospecific interaction and inducing inline atom transfer radical polymerization (ATRP) for amplifying SPR response. Bacterial cholera toxin (CT) is chosen as the model protein that has been covalently immobilized on the surface for demonstrating the principle. The specific recognition is achieved by use of biotinylated anti-CT, which allows initiators with a biotin tag to be fixed at the protein binding site through a neutravidin bridge and triggers the localized growth of polymer brushes of poly(hydroxyl-ethyl methacrylate) (PHEMA) via an ATRP mechanism. To further enhance the signal, a second ATRP reaction is conducted that takes advantage of the hydroxyl groups of PHEMA brushes from the first step to form hyper-branched polymers onto the sensing surface. The two consecutive ATRP steps significantly improve SPR detection, allowing low amounts of CT that yield no direct measurement to be quantified with large signals. The resulting polymer film has been characterized by optical and atomic force microscopy. Ascorbic acid (AA) is employed as deoxygen reagent in the catalyst mixture that effectively suppresses oxygen interference, shortening the reaction time and making it possible for applying this ATRP approach to flow injection based SPR detection. A calibration curve of PHEMA amplification for CT detection based on surface coverage has been obtained that displays a correlation in a range from 8.23 × 10−15 mol/cm2 to 3.61 × 10−12 mol/cm2 with a limit of detection of 6.27 × 10−15 mol/cm2. The versatile biotin-neutravidin interaction used here should allow adaptation of ATRP enhancement to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultra-sensitive analysis of biomolecules with flow-injection assay and SPR spectroscopy.
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影响因子：
14.9
作者：
Gao Y;Wolf LK;Georgiadis RM
通讯作者：
Georgiadis RM
影响因子：
3.7
作者：
Jiang, Guoqian;Baba, Akira;Advincula, Rigoberto
通讯作者：
Advincula, Rigoberto
影响因子：
6.2
作者：
Lee, Bong Soo;Chi, Young Shik;Choi, Insung S.
通讯作者：
Choi, Insung S.
影响因子：
7.4
作者：
Lee, Chi-Ying;Nguyen, Phuong-Cac T.;Castner, David G.
通讯作者：
Castner, David G.
影响因子：
2.6
作者：
Kikuchi, J;Furukawa, Y;Hayashi, N
通讯作者：
Hayashi, N