Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore
Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore
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DOI：
10.1038/80295
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发表时间：
2000-10-01
影响因子：
46.9
通讯作者：
Bayley, H
中科院分区：
文献类型：
--
作者：
Movileanu, L;Howorka, S;Bayley, H
在这里，我们描述了一种新型的生物传感器元件，用于检测溶液中的蛋白质在纳摩尔浓度。我们在由葡萄球菌α-溶血素形成的跨膜蛋白孔的内腔内的限定位点处拴系3.4kDa聚乙二醇链。聚合物的自由端共价连接到生物素分子上。在将修饰的孔并入脂质双层中时，生物素基从膜的一侧移动到另一侧，并且通过用突变的链霉亲和素的可逆捕获来检测。捕获事件是通过平面双层中单个孔的离子电流的变化而观察到的。因此，改性孔允许在单分子水平上检测蛋白质分析物，从而通过独特的电流特征促进定量和鉴定。该方法具有较高的时间分辨率相比，其他动力学测量，如表面等离子体共振。
Here we describe a new type of biosensor element for detecting proteins in solution at nanomolar concentrations. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal alpha-hemolysin. The free end of the polymer was covalently attached to a biotin molecule. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are observed as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-molecule level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resolution compared with other kinetic measurements, such as those obtained by surface plasmon resonance.