State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, 210023, Nanjing, China.
Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, 210023, Nanjing, China.
J Am Chem Soc. 2022 Jan 19;144(2):757-768. doi: 10.1021/jacs.1c09259. Epub 2022 Jan 7.
The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and β-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.
纳米孔正成为一种单分子蛋白质传感的手段。然而，蛋白质表现出不同的电荷特性，这使得设计能够同时感应不同带电蛋白质的传感器变得复杂。在这项工作中，我们引入了一种不对称电解质缓冲液与 porin A（MspA）纳米孔结合，形成电动流（EOF）陷阱。仅在单个血红素上有所不同的脱辅基肌红蛋白和全辅基肌红蛋白可以通过这种方法得到完全区分。MspA EOF 陷阱还可以直接区分溶菌酶、脱辅基/全辅基肌红蛋白和 ACTR/NCBD 蛋白复合物，它们分别是碱性、中性和酸性蛋白质。为了实现事件分类的自动化，提取了多个事件特征来构建机器学习模型，该模型的准确率达到了 99.9%。该方法还应用于直接从乳清蛋白粉中识别α-乳白蛋白和β-乳球蛋白的单分子。这种蛋白质传感策略可用于直接从混合物中识别蛋白质，表明其在快速灵敏地检测生物标志物或实时蛋白质结构分析方面具有广阔的应用前景。
