Department of Chemical Engineering, University of Washington, Seattle, WA 98195-1750, USA.
Biosens Bioelectron. 2010 Jun 15;25(10):2276-82. doi: 10.1016/j.bios.2010.03.012. Epub 2010 Mar 12.
Non-specific protein binding from human plasma and serum has severely hindered the full capabilities of biosensors concerned with cancer biomarker detection. Currently, there is a strong desire for developing new materials which allow for the convenient attachment of an ultra-low fouling and functionalizable surface coating which can be used for highly sensitive and label-free detection of target analytes directly from complex media. In this work, a short 20 min in situ "graft to" protocol using Tris pH 8.5 buffer was developed for zwitterionic carboxybetaine methacrylate (CBMA) polymer conjugates containing the adhesive biomimetic moiety, 3,4-dihydroxy-L-phenylalanine (DOPA), on SiO(2) substrates. Using a surface plasmon resonance (SPR) biosensor, different buffers, pH values, salt concentrations, and temperatures were investigated for determining the "graft to" conditions that yield dense polymer films which both minimize non-specific protein adsorption and maximize antibody immobilization. The optimized surface coatings were shown to be highly protein resistant to 100% human blood plasma and serum. Subsequent antibody functionalized surfaces without any blocking agents enabled the specific detection of the cancer biomarker ALCAM directly from undiluted human serum down to 64 ng/mL. The successful use of this zwitterionic surface coating for detection from complex media on SiO(2) surfaces indicates its potential for broad impacts in the development of implantable medical devices, in vivo diagnostics, and nano-scale biosensors.
来自人血浆和血清的非特异性蛋白质结合严重阻碍了与癌症生物标志物检测相关的生物传感器的全部功能。目前,人们强烈希望开发新的材料,这些材料允许方便地连接超低污染和功能化的表面涂层,可用于直接从复杂介质中对目标分析物进行高灵敏度和无标记检测。在这项工作中,开发了一种使用 Tris pH 8.5 缓冲液的 20 分钟原位“接枝到”方案,用于在 SiO2 基底上含有粘附仿生部分 3,4-二羟基-L-苯丙氨酸 (DOPA) 的两性离子羧基甜菜碱甲基丙烯酸酯 (CBMA) 聚合物缀合物。使用表面等离子体共振 (SPR) 生物传感器,研究了不同的缓冲液、pH 值、盐浓度和温度,以确定产生既能最大程度地减少非特异性蛋白质吸附又能最大程度地固定抗体的致密聚合物膜的“接枝到”条件。优化的表面涂层对 100%人血浆和血清具有高度的蛋白质抗性。随后,无需任何封闭剂的抗体功能化表面能够直接从未稀释的人血清中特异性检测到癌症标志物 ALCAM,检测下限低至 64ng/mL。这种两性离子表面涂层在 SiO2 表面上从复杂介质中进行检测的成功应用表明,它在植入式医疗设备、体内诊断和纳米级生物传感器的开发方面具有广泛的应用潜力。