Reversible Immobilization of Proteins in Sensors and Solid-State Nanopores
Reversible Immobilization of Proteins in Sensors and Solid-State Nanopores
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DOI：
10.1002/smll.201703357
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发表时间：
2018-05-03
期刊：
影响因子：
13.3
通讯作者：
Goerlich, Dirk
中科院分区：
文献类型：
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作者：
Ananth, Adithya;Genua, Maria;Goerlich, Dirk
在生命科学研究和生物医学应用的许多分析方法中，蛋白质表面的受控功能化是至关重要的。在这里，建立了一种硅基表面涂层，它可以稳定和选择性地固定具有控制取向和可调表面密度的蛋白质。该涂层可重复使用，在空气中长期储存仍保持功能，适用于复杂几何形状的表面。在平面上验证了蛋白质锚定方法的有效性，并提出了一种利用氮化硅固体纳米孔实时测量蛋白质锚定过程的方法。为了进行表面附着，利用了重组蛋白中特异位点引入的多组氨酸标签，并以酵母核孔蛋白Nsp1作为模型蛋白。与常用的共价硫醇化学相反，通过多组氨酸标签锚定蛋白质是可逆的，允许将蛋白质取下并用其他蛋白质代替。在单个纳米孔的实验中，这种开关是用离子电导率来实时监测的。最后，研究表明，二氧化硅和金表面可以正交功能化，以容纳二氧化硅上的多组氨酸标记的蛋白质，但防止蛋白质与金结合，这将这种表面功能化方法的适用性扩展到更复杂的传感器设备。
The controlled functionalization of surfaces with proteins is crucial for many analytical methods in life science research and biomedical applications. Here, a coating for silica-based surfaces is established which enables stable and selective immobilization of proteins with controlled orientation and tunable surface density. The coating is reusable, retains functionality upon long-term storage in air, and is applicable to surfaces of complex geometry. The protein anchoring method is validated on planar surfaces, and then a method is developed to measure the anchoring process in real time using silicon nitride solid-state nanopores. For surface attachment, polyhistidine tags that are site specifically introduced into recombinant proteins are exploited, and the yeast nucleoporin Nsp1 is used as model protein. Contrary to the commonly used covalent thiol chemistry, the anchoring of proteins via polyhistidine tag is reversible, permitting to take proteins off and replace them by other ones. Such switching in real time in experiments on individual nanopores is monitored using ion conductivity. Finally, it is demonstrated that silica and gold surfaces can be orthogonally functionalized to accommodate polyhistidine-tagged proteins on silica but prevent protein binding to gold, which extends the applicability of this surface functionalization method to even more complex sensor devices.