Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6–15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4–7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.

细胞异质性是普遍存在的，并且在生物学中至关重要。单细胞分析技术对于了解细胞的异质性和功能是必不可少的。低拷贝数蛋白质（每个细胞少于 1,000 个分子）执行多种关键功能，例如基因表达、细胞代谢和细胞信号传导。单个细胞低拷贝数蛋白的表达水平为深入了解生物过程和疾病提供了关键信息。然而，单细胞中低拷贝数蛋白质的定量分析仍然具有挑战性。为了克服这个问题，我们开发了一种称为单细胞等离子体免疫夹心法 (scPISA) 的方法，用于定量测量单个活细胞中的低拷贝数蛋白质。scPISA 结合了用于从细胞中特异性富集靶蛋白的体内微萃取和称为等离子增强拉曼散射的最先进技术，用于超灵敏地检测低拷贝数蛋白质。等离子体增强拉曼散射检测依赖于银基等离子体纳米标签和金基提取微探针之间的等离子体耦合效应（热点），显着增强纳米标签表面增强拉曼散射的信号强度，从而使单分子水平的灵敏度。scPISA 是一种简单且微创的技术，仅需约 6-15 分钟（从体内提取到拉曼光谱读数）。它通常适用于所有自由漂浮的细胞内蛋白，只要有适当的抗体或替代品（例如，分子印迹聚合物或适体）是可用的。整个协议需要约 4-7 天才能完成，包括材料制造、单细胞操作、蛋白质标记、信号采集和数据分析。