Surface plasmon resonance detection of endocrine disruptors using immunoprobes based on self-assembled monolayers
Surface plasmon resonance detection of endocrine disruptors using immunoprobes based on self-assembled monolayers
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DOI：
10.1016/j.snb.2004.11.094
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发表时间：
2005-07-22
影响因子：
8.4
通讯作者：
Miura, N
中科院分区：
文献类型：
--
作者：
Gobi, KV;Kataoka, C;Miura, N
本文报道了一种可再生的表面等离子体共振（SPR）免疫传感器的制备和表征。混合单层用作免疫探针制造中的基底层，以确保对免疫探针的稳定性和对非选择性半抗原和蛋白质的惰性。用SPR角探询法研究了BaP单克隆抗体（BaP-Ab）与单层膜的界面作用。随着BaP头部基团暴露在自组装单层之外，BaP-Ab与免疫探针发生选择性免疫反应并牢固结合。高浓度的牛血清白蛋白（BSA）的流动显示出可忽略的响应，表明传感器表面对蛋白质的非选择性吸附的高电阻率。采用间接抑制免疫测定法分析传感器对BaP的响应。在该方法中，BaP-Ab与游离分析物一起通过使用流动系统的传感器芯片。随着BaP一起存在于分析物溶液中，溶液中的BaP分子与传感器芯片上存在的BaP官能团之间发生与BaP-Ab结合的竞争，因此发现BaP-Ab与传感器表面的结合被抑制。在测定BaP浓度时，遵循BaP存在对免疫反应的抑制程度。获得了对应于50 ppt（pg/ml）至100 ppb（ng/ml）BaP范围内的分析物浓度的校准曲线。该免疫传感器的最低检测限为50 ppt（pg/ml），响应时间约为10 min。在用于BaP的测定之后，通过用胃蛋白酶溶液处理使结合的抗体从传感器芯片解离，再次产生活性传感器表面以重复使用。同一传感器芯片可重复使用，用于BaP浓度的测定，重复不少于20个循环，而传感器活性没有显著降低。(c)2004 Elsevier B. V.保留所有权利。
This paper describes the formation and characterization of a regenerable surface plasmon resonance (SPR) immunosensor for the detection of benzo(a)pyrene (BaP) by employing a self-assembled mixed monolayer presenting BaP head groups as the sensor immumoprobe. The mixed monolayer was used as the base layer in the fabrication of immunoprobe to assure stability to the immunoprobe and inertness to nonselective haptens and proteins. Interfacial interaction of a monoclonal anti-BaP antibody (BaP-Ab) with the monolayer is studied by SPR angle interrogation method. With BaP head groups exposing out of the self-assembled monolayer, BaP-Ab undergoes selective immunoreaction with the immumoprobe and binds firmly. Flow of high concentrations of bovine serum albumin (BSA) showed negligible response indicating high resistivity of the sensor surface to non-selective adsorption of proteins. An indirect inhibition immunoassay method is employed for analyzing sensor responses to BaP. In this method, BaP-Ab is passed together with the free analyte over the sensor chip using a flow system. With the presence of BaP together in the analyte solution, competition for binding to BaP-Ab occurs between BaP molecules in solution and the BaP functional groups present on the sensor chip and thus the binding of BaP-Ab with the sensor surface is found inhibited. Extent of the inhibition of immunoreaction by the presence of BaP is followed in determining the concentration of BaP. Calibration curves corresponding to analyte concentrations ranging from 50 ppt (pg/ml) to 100 ppb (ng/ml) BaP are obtained. The lowest detection limit of the present SPR-based immunosensor is 50 ppt (pg/ml) with a response time of ca. 20 min. After the use in determination of BaP, active sensor surface is generated again for repeated use by dissociating the bound antibody from the sensor chip with the treatment of a pepsin solution. A same sensor chip is reusable in the determination of BaP concentration repeatedly for not less than 20 cycles without significant decrease in the sensor activity. (c) 2004 Elsevier B.V. All rights reserved.