A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs)

BackgroundDetection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities.
Methodology/Principal FindingsTo expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using “Composite Organic-Inorganic Nanoparticles” (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry.
Conclusions/SignificanceThus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

背景 三十余年来，单细胞表位检测一直是免疫分型（immunophenotyping）的核心技术手段，其定量分析主要依赖荧光技术。但荧光光谱存在宽幅重叠的固有缺陷，极大限制了多重检测能力。
方法/主要发现 为突破现有检测系统的局限，我们开发了一种基于"Composite Organic-Inorganic Nanoparticles"（COINs）拉曼纳米颗粒的新型单细胞多色免疫检测方法。该复合有机-无机纳米颗粒即表面增强拉曼散射（"Surface-Enhanced Raman Scattering"，SERS）纳米颗粒，具备独特的拉曼光谱特征。为实现单细胞拉曼光谱检测，我们构建了一套自动化、小型化、低噪声且高灵敏度的拉曼显微镜装置——集成式拉曼生物分析仪（"Integrated Raman BioAnalyzer"）。利用该技术，我们在单细胞表面成功检测到可区分人血细胞中T细胞的表达蛋白；最终，我们还完成了Stat1（Y701）与Stat6（Y641）的细胞内磷酸化水平检测，所得结果与流式细胞术（flow cytometry）的检测结果相当。
结论与意义 综上，本研究证实了COIN纳米颗粒用于细胞内磷酸化水平检测的实用性，为拓展当前单细胞免疫检测所用的荧光技术体系提供了全新的可能性。