Enzyme immunosensing of pepsinogens 1 and 2 by scanning electrochemical microscopy.
Enzyme immunosensing of pepsinogens 1 and 2 by scanning electrochemical microscopy.
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DOI：
10.1016/j.bios.2007.01.015
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发表时间：
2007-06
影响因子：
12.6
通讯作者：
T. Yasukawa;Y. Hirano;Naomi Motochi;H. Shiku;T. Matsue
中科院分区：
文献类型：
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作者：
T. Yasukawa;Y. Hirano;Naomi Motochi;H. Shiku;T. Matsue
将扫描电化学显微镜 (SECM) 应用到双酶免疫分析中检测胃蛋白酶原 1 (PG1) 和胃蛋白酶原 2 (PG2)。在由抗 PG1 IgG 抗体和抗 PG2 IgG 抗体组成的微点上构建了辣根过氧化物酶 (HRP) 标记的三明治型免疫复合物。这些微点是使用毛细管微点技术在疏水性玻璃基板上制造的。在 H2O2 和二茂铁甲醇（FcOH；用作电子介体）存在下，标记的 HRP 催化 H2O2 对 FcOH 的氧化，在对应于含有两种免疫复合物的微点的局部区域生成氧化形式的 FcOH (Fc+OH)。酶促生成的 Fc+OH 被还原，并用 SECM 探针（0.05V 相对于 Ag/AgCl）进行检测，并对基质表面进行绘图以生成 PG1 和 PG2 点的 SECM 图像。在 1.6–60.3ng/ml 蛋白质范围内获得了 SECM 图像中的还原电流与 PG1 和 PG2 浓度之间的关系。使用含有 PG1 和 PG2 免疫复合物的微点实现 PG1 和 PG2 的双重成像，微点由基底上的 200μm 物理屏障隔开。利用硅片上具有 100μm×100μm 开口的金字塔孔阵列，利用聚二甲基硅氧烷 (PDMS) 膜上的抗体来制造斑点。与从使用毛细管方法制造的点获得的响应相比，从用金字塔孔制造的微点获得的电流响应明显更尖锐。
Scanning electrochemical microscopy (SECM) was applied to a dual enzyme immunoassay for the detection of pepsinogen 1 (PG1) and pepsinogen 2 (PG2). Sandwich-type immunocomplexes labeled with horseradish peroxidase (HRP) were constructed on microspots consisting of anti-PG1 IgG antibody and anti-PG2 IgG antibody. These microspots were fabricated on a hydrophobic glass substrate using a capillary microspotting technique. In the presence of H2O2and ferrocenemethanol (FcOH; used as an electron mediator), the labeled HRP catalyzed the oxidation of FcOH by H2O2to generate the oxidized form of FcOH (Fc+OH) at localized areas corresponding to microspots containing both immunocomplexes. The enzymatically generated Fc+OH was reduced and detected with a SECM probe (0.05V versus Ag/AgCl), and the substrate surface was mapped to generate SECM images of the PG1 and PG2 spots. Relationships between the reduction current in the SECM images and the concentrations of PG1 and PG2 were obtained in the range 1.6–60.3ng/ml protein. Dual imaging of PG1 and PG2 was achieved using microspots containing PG1 and PG2 immunocomplexes separated by a 200μm physical barrier on the substrate. Pyramidal hole arrays with 100μm×100μm openings on the silicon wafer were utilized to fabricate spots using antibodies on poly(dimethylsiloxane) (PDMS) membranes. Current responses obtained from microspots fabricated with pyramidal holes are significantly sharper compared to the responses obtained from spots fabricated using the capillary method.