A novel method for detection of phosphorylation in single cells by surface enhanced Raman scattering (SERS) using composite organic-inorganic nanoparticles (COINs).
BACKGROUND
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.


RESULTS
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
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.
Department of Microbiology & Immunology, Stanford University, Stanford, California, United States of America.
Catherine M
CM
Shachaf
Sailaja V
SV
Elchuri
Ai Leen
AL
Koh
Jing
J
Zhu
Lienchi N
LN
Nguyen
Dennis J
DJ
Mitchell
Jingwu
J
Zhang
Kenneth B
KB
Swartz
Lei
L
Sun
Selena
S
Chan
Robert
R
Sinclair
Garry P
GP
Nolan
D000954:Antigens, Surface / Q000032:analysis*; D002460:Cell Line; D002477:Cells / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D053768:Metal Nanoparticles; D053758:Nanoparticles; D010766:Phosphorylation; D050794:STAT1 Transcription Factor / Q000378:metabolism; D050800:STAT6 Transcription Factor / Q000378:metabolism; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013601:T-Lymphocytes
D000954:Antigens, Surface; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human