Wong Wing Sze, Yung Pun To
Annu Int Conf IEEE Eng Med Biol Soc. 2014;2014:2781-4. doi: 10.1109/EMBC.2014.6944200.
Well organized template for biomolecular conjugation is the foundation for biosensing. Most of the current devices are fabricated using lithographic patterning processes and self-assembly monolayer (SAM) methods. However, the research toward developing a sub-10 nm patterned, self-regenerated template on various types of substrates is limited, mainly due to the limited functional groups of the building material. Bacterial surface layer proteins (S-layer proteins) can self-assemble into ordered lattice with regular pore sizes of 2-8 nm on different material supports and interfaces. The ordered structure can regenerate after extreme variations of solvent conditions. In this work, we developed a nanoscale biomolecular template based on S-layer proteins on gold surface for fabrication of sensing layer in biosensors. S-layer proteins were isolated from Bacillus cereus, Lysinibacillus sphaericus and Geobacillus stearothermophilus. Protein concentrations were measured by Bradford assay. The protein purities were verified by SDS-PAGE, showing molecular weights ranging from 97-135 kDa. The hydrophilicity of the substrate surface was measured after surface treatments of protein recrystallization. Atomic force microscopic (AFM) measurement was performed on substrate surface, indicating a successful immobilization of a monolayer of S-layer protein with 8-9 nm height on gold surface. The template can be applied on various material supports and acts as a self-regenerated sensing layer of biosensors in the future.
生物分子共轭的有序模板是生物传感的基础。当前大多数设备是使用光刻图案化工艺和自组装单分子层（SAM）方法制造的。然而，针对在各种类型的基板上开发亚10纳米图案化、自再生模板的研究有限，主要是由于构建材料的官能团有限。细菌表面层蛋白（S层蛋白）可以在不同的材料载体和界面上自组装成具有2-8纳米规则孔径的有序晶格。在溶剂条件发生极大变化后，这种有序结构可以再生。在这项工作中，我们在金表面开发了一种基于S层蛋白的纳米级生物分子模板，用于制造生物传感器中的传感层。从蜡样芽孢杆菌、球形赖氨酸芽孢杆菌和嗜热栖热菌中分离出S层蛋白。通过Bradford测定法测量蛋白质浓度。通过SDS-PAGE验证蛋白质纯度，显示分子量范围为97-135 kDa。在蛋白质重结晶的表面处理后测量基板表面的亲水性。在基板表面进行原子力显微镜（AFM）测量，表明在金表面成功固定了单层高度为8-9纳米的S层蛋白。该模板可应用于各种材料载体，并在未来作为生物传感器的自再生传感层。
