Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
J Nanobiotechnology. 2011 Jun 20;9:26. doi: 10.1186/1477-3155-9-26.
The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system.
Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S) bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function.
Water contact angle (65-70°), as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate) and the other (SH/S-S) at the ω-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity ~100 times that expected for the same amount of protein in solution.
To the best of our knowledge, immobilization of a protein by the method presented here, with the resulting high enzymatic activity, has never been reported. There are many potential applications for selective localization of active proteins at patterned surfaces, for example, bioMEMS (MEMS--Micro-Electro-Mechanical Systems. Due to the success of the method, presented here, it was decided to continue a research project of a biosensor by transferring it to a high aspect ratio platform--nanotubes.
将生态清洁、高效的酶引入现代工业的兴趣一直在稳步增长。然而，控制其取向并保持其选择性和反应性仍然是一个重大障碍。我们已经开发出精确的生物分子固定化方法，同时保留其天然功能，并报告了一种新的、快速、简单、可靠的蛋白质固定化方法，该方法使用腺苷酸激酶作为模型系统。
在金表面形成了十六烷-1,6-二硫醇的自组装单层。通过接触角测量、Elman 试剂反应、QCM 和 XPS 对单层进行了表征。使用经过专门设计的、突变的腺苷酸激酶，其中在 75 位插入半胱氨酸，并且 77 位的半胱氨酸被丝氨酸取代，通过自发形成的二硫键（S-S）键附着在 SAM 表面上。使用 QCM 和 XPS 对固定化蛋白层进行了表征。XPS 测量中的曲线拟合使用了高斯-洛伦兹函数。
水接触角（65-70°）以及使用的所有表征技术都证实了具有表面 SH 基团的自组装单层的形成。X 射线光电子能谱清楚地显示了两种类型的硫原子，一种附着在金上（三醇），另一种（SH/S-S）位于十六烷-1,6-二硫醇 SAM 的ω 位。使用 XPS 和 QCM 确认了蛋白质单层的形成，其中 QCM 确定的表面上的蛋白质量与考虑单个蛋白质分子表面积的模型一致。固定化蛋白质的酶活性测试证实酶功能没有变化，并揭示了比溶液中相同量蛋白质预期的活性高 100 倍。
据我们所知，通过本文提出的方法固定蛋白质并获得高酶活性的方法从未有过报道。在图案化表面上选择性定位活性蛋白质有许多潜在的应用，例如生物 MEMS（MEMS-微机电系统）。由于本文提出的方法取得了成功，因此决定将其转移到高纵横比平台-纳米管上，继续进行生物传感器的研究项目。
