Shachaf Catherine M, Elchuri Sailaja V, Koh Ai Leen, Zhu Jing, Nguyen Lienchi N, Mitchell Dennis J, Zhang Jingwu, Swartz Kenneth B, Sun Lei, Chan Selena, Sinclair Robert, Nolan Garry P
Department of Microbiology & Immunology, Stanford University, Stanford, California, United States of America.
PLoS One. 2009;4(4):e5206. doi: 10.1371/journal.pone.0005206. Epub 2009 Apr 15.
Detection 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 FINDINGS: To 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/SIGNIFICANCE: Thus, 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.
三十多年来，单细胞表位检测一直是免疫表型分析的主要手段，主要使用荧光技术进行定量分析。荧光具有广泛的重叠光谱，限制了多重分析能力。
方法/主要发现：为了扩展当前的检测系统，我们开发了一种使用“复合有机-无机纳米颗粒”（COINs）拉曼纳米颗粒在单细胞中进行多色免疫检测的新方法。COINs是表面增强拉曼散射（SERS）纳米颗粒，具有独特的拉曼光谱。为了测量单细胞中的拉曼光谱，我们构建了一种自动化、紧凑、低噪声且灵敏的拉曼显微镜设备（集成拉曼生物分析仪）。利用这项技术，我们检测了人血细胞中区分T细胞的单细胞表面表达的蛋白质。最后，我们测量了Stat1（Y701）和Stat6（Y641）的细胞内磷酸化，结果与流式细胞术相当。
结论/意义：因此，我们证明了应用COIN纳米颗粒测量细胞内磷酸化的实用性，为扩展当前用于单细胞免疫分析的荧光技术提供了新的可能性。
