Phillips K Scott, Han Jong-Ho, Martinez Marilyn, Wang Zhuangzhi, Carter David, Cheng Quan
Department of Chemistry, University of California, Riverside, California 92521, USA.
Anal Chem. 2006 Jan 15;78(2):596-603. doi: 10.1021/ac051644y.
Surface plasmon resonance (SPR) spectroscopy, a powerful tool for biosensing and protein interaction analysis, is currently confined to gold substrates and the relevant surface chemistries involving dextran and functional thiols. Drawbacks of using self-assembled monolayers (SAMs) for SPR-related surface modification include limited stability, pinhole defects, bioincompatibility, and nonspecific protein adsorption. Here we report the development of stable nanometer-scale glass (silicate) layers on gold substrates for SPR analysis of protein toxins. The nanoscale silicate layers were built up with layer-by-layer deposition of poly(allylamine hydrochloride) and sodium silicate, followed by calcination at high temperature. The resulting silicate films have a thickness ranging from 2 to 15 nm and demonstrate outstanding stability in flow cell conditions. The use of these surfaces as a platform to construct supported bilayer membranes (SBMs) is demonstrated, and improved performance against protein adsorption on SBM-coated surfaces is quantified by SPR measurements. SBMs can be formed reproducibly on the silicate surface via vesicle fusion and quantitatively removed using injection of 5% Triton X-100 solution, generating a fresh surface for each test. Membrane properties such as lateral diffusion of the SBMs on the silicate films are characterized with photobleaching methods. Studies of protein binding with biotin/avidin and ganglioside/cholera toxin systems show detection limits lower than 1 microg/mL (i.e., nanomolar range), and the response reproducibility is better than 7% RSD. The method reported here allows many assay techniques developed for glass surfaces to be transferred to label-free SPR analysis without the need for adaptation of protocols and time-consuming synthetic development of thiol-based materials and opens new avenues for developing novel bioanalytical technologies for protein analysis.
表面等离子体共振（SPR）光谱技术是一种用于生物传感和蛋白质相互作用分析的强大工具，目前仅限于金基底以及涉及葡聚糖和功能性硫醇的相关表面化学。使用自组装单分子层（SAMs）进行与SPR相关的表面修饰存在稳定性有限、针孔缺陷、生物不相容性和非特异性蛋白质吸附等缺点。在此，我们报告了在金基底上开发用于蛋白质毒素SPR分析的稳定纳米级玻璃（硅酸盐）层。通过聚（烯丙胺盐酸盐）和硅酸钠的逐层沉积构建纳米级硅酸盐层，然后在高温下煅烧。所得硅酸盐薄膜的厚度范围为2至15纳米，并在流动池条件下表现出出色的稳定性。展示了将这些表面用作构建支撑双层膜（SBMs）平台的用途，并通过SPR测量对SBM涂层表面上蛋白质吸附的改进性能进行了量化。SBMs可以通过囊泡融合在硅酸盐表面可重复地形成，并使用5% Triton X - 100溶液注入定量去除，为每次测试生成新表面。使用光漂白方法表征了SBMs在硅酸盐薄膜上的膜性质，如横向扩散。对蛋白质与生物素/抗生物素蛋白和神经节苷脂/霍乱毒素系统结合的研究表明，检测限低于1微克/毫升（即纳摩尔范围），响应重现性优于7%相对标准偏差（RSD）。本文报道的方法允许许多为玻璃表面开发的检测技术转移到无标记SPR分析，而无需调整方案和耗时的基于硫醇材料的合成开发，并为开发用于蛋白质分析的新型生物分析技术开辟了新途径。
