Zhao, M., Li, H., Guo, Z., Liu, H., Peng, J., Hu, Y., Fu, B., Li, B., Guo, L., Fu, R. and Lu, Y., 2025. Rapid Single‐Cell Proteomics Using Nanoconfined Enzyme Reactors on a Microscale Digital Microfluidics Platform. Advanced Functional Materials, p.e02142.

Rapid Single-Cell Proteomics Using Nanoconfined Enzyme Reactors on a Microscale Digital Microfluidics Platform

Abstract

Multicellular organisms exhibit cellular heterogeneity, crucial for understanding physiological and pathological processes. Single-cell proteomics (SCP) enables exploration of this diversity but faces challenges such as sample loss due to nonspecific adsorption and relies on free protease solutions for enzymatic digestion. Here, a microfluidic platform is reported that enhances proteomic analysis of single cells by integrating nanoconfined enzyme reactors with digital microfluidics (DMF). Trypsin immobilized on NHS-activated magnetic beads via click chemistry (Try@Fe3O4) shows improved stability and enzyme loading, reducing autolysis risks. Using DMF-Try@Fe3O4, it achieves over twice the sequence coverage and four times the peptide matches for standard proteins in 10 min compared to conventional 10-h methods. The densely packed enzymes in the nanoscale microenvironment enhance reaction rates. This system identifies 3,916 and 1,849 protein groups from 50 HeLa cells and single cells, respectively, showing 27% and 201% increases over tube digestion. The platform also classifies leukocyte subtypes (HL-60, Jurkat, and Raji, with N = 20 for each) with SCP and identifies key upregulated proteins. Proteomic analysis of gemcitabine-treated PANC-1 cells reveal alterations consistent with known drug mechanisms. This approach enhances protein digestion efficiency and identification rates, offering a rapid, automated SCP solution for high-throughput applications and broader biological investigations.

摘要
多细胞生物存在细胞异质性，这种异质性对于理解生理和病理过程至关重要。单细胞蛋白质组学（Single-cell Proteomics, SCP）为探究这种异质性提供了可能，但该技术面临非特异性吸附导致的样本损失、依赖游离蛋白酶溶液进行酶解等挑战。本研究报道了一种微流控平台，该平台通过将纳米受限酶反应器与数字微流控（Digital Microfluidics, DMF）技术相结合，可提升单细胞的蛋白质组学分析效果。
研究人员通过点击化学技术，将胰蛋白酶固定在 N - 羟基琥珀酰亚胺（NHS）活化的磁珠表面（记为 Try@Fe₃O₄），这种固定化胰蛋白酶不仅稳定性提升、酶负载量增加，还降低了自溶风险。利用 DMF-Try@Fe₃O₄系统分析标准蛋白质时，仅需 10 分钟即可实现超过传统 10 小时酶解方法 2 倍以上的序列覆盖率和 4 倍以上的肽段匹配数。纳米级微环境中密集分布的酶可显著提高反应速率。
该系统对 50 个 HeLa 细胞和单个 HeLa 细胞分别鉴定出 3916 个和 1849 个蛋白组，相较于试管酶解方法，鉴定数量分别增加 27% 和 201%。此外，该平台还可通过单细胞蛋白质组学对白细胞亚型（HL-60 细胞、Jurkat 细胞、Raji 细胞，每种细胞样本量 N=20）进行分类，并识别出关键的上调蛋白。对吉西他滨处理后的 PANC-1 细胞进行蛋白质组学分析，发现其蛋白表达变化与已知药物作用机制一致。
该方法显著提升了蛋白质酶解效率和鉴定率，为高通量应用及更广泛的生物学研究提供了一种快速、自动化的单细胞蛋白质组学解决方案。

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