Zhou Zi-Qi, Liu Shao-Chuang, Wang Jia, Chen Ke-Le, Xie Bao-Kang, Ying Yi-Lun, Long Yi-Tao
Molecular Sensing and Imaging Center, School of Chemistry and Chemical Engineering, Nanjing University Nanjing 210023 P. R. China
Chemistry and Biomedicine Innovation Center, Nanjing University Nanjing 210023 P. R. China.
Chem Sci. 2025 Apr 7;16(19):8501-8508. doi: 10.1039/d5sc01764e. eCollection 2025 May 14.
Direct protein analysis from complex cellular samples is crucial for understanding cellular diversity and disease mechanisms. Here, we explored the potential of SiN solid-state nanopores for single-molecule protein analysis from complex cellular samples. Using the LOV2 protein as a model, we designed a nanopore electrophoretic driver protein and fused it with LOV2, thereby enhancing the capture efficiency of the target protein. Then, we performed single-cell protein analysis by directly extracting the contents of individual cells using glass nanopipette-based single-cell extraction and successfully identified and monitored the conformational changes of the LOV2 protein from single-cell extracts using SiN nanopores. Our results reveal significant differences between proteins measured directly from single cells and those obtained from purified samples. This work demonstrates the potential of solid-state nanopores as a powerful tool for single-cell, single-molecule protein analysis, opening avenues for investigating protein dynamics and interactions at the cellular level.
直接对复杂细胞样本进行蛋白质分析对于理解细胞多样性和疾病机制至关重要。在此，我们探索了氮化硅（SiN）固态纳米孔用于对复杂细胞样本进行单分子蛋白质分析的潜力。以光氧电压感受域2（LOV2）蛋白为模型，我们设计了一种纳米孔电泳驱动蛋白并将其与LOV2融合，从而提高了目标蛋白的捕获效率。然后，我们通过基于玻璃纳米吸管的单细胞提取直接提取单个细胞的内容物来进行单细胞蛋白质分析，并使用氮化硅纳米孔成功地从单细胞提取物中识别和监测了LOV2蛋白的构象变化。我们的结果揭示了直接从单个细胞测量的蛋白质与从纯化样本中获得的蛋白质之间存在显著差异。这项工作证明了固态纳米孔作为单细胞、单分子蛋白质分析强大工具的潜力，为在细胞水平研究蛋白质动力学和相互作用开辟了道路。
