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

Shachaf CM, Elchuri SV, Koh AL, Zhu J, Nguyen LN, Mitchell DJ, Zhang J, Swartz KB, Sun L, Chan S, Sinclair R, Nolan GP - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, Stanford University, Stanford, California, United States of America.

ABSTRACT

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.

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.

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Detection of intracellular phosphorylation signaling using COINs.a) Flow analysis of pStat1 and Stat6 phosphorylation following treatment of U937 cells with IFNγ or IL-4 cytokines, compared to non treated (Non stim). b) Raman spectral shift intensity of BFU COIN detecting intracellular pStat1 in IFNγ and c) pStat6 in IL-4, treated and non-treated (Non stim) U937cells. Treated (Stim control) and non treated cells (Non stim control) were stained with non-conjugated BFU COIN. The spectra are representative for five independent experiments. d) Quantitation of change in Raman peak height after IFNγ and e) IL-4 treated cells compared to non-treated cells. The αpStat1 and αStat6 conjugated COINs specifically detected pStat1 and pStat6 respectively on IFNγ and IL-4 treated U937 cells compared to non-treated cells (**p<0.01). e) Fold change ratio of pStat1 and pStat6 phosphorylation in U937 treated cells stained with both BFU and AOH COINs. The changes are the average of five independent experiments.
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pone-0005206-g004: Detection of intracellular phosphorylation signaling using COINs.a) Flow analysis of pStat1 and Stat6 phosphorylation following treatment of U937 cells with IFNγ or IL-4 cytokines, compared to non treated (Non stim). b) Raman spectral shift intensity of BFU COIN detecting intracellular pStat1 in IFNγ and c) pStat6 in IL-4, treated and non-treated (Non stim) U937cells. Treated (Stim control) and non treated cells (Non stim control) were stained with non-conjugated BFU COIN. The spectra are representative for five independent experiments. d) Quantitation of change in Raman peak height after IFNγ and e) IL-4 treated cells compared to non-treated cells. The αpStat1 and αStat6 conjugated COINs specifically detected pStat1 and pStat6 respectively on IFNγ and IL-4 treated U937 cells compared to non-treated cells (**p<0.01). e) Fold change ratio of pStat1 and pStat6 phosphorylation in U937 treated cells stained with both BFU and AOH COINs. The changes are the average of five independent experiments.

Mentions: Next, we tested the potential of COIN nanoparticles for the detection of intracellular phosphorylation events. U937 cells activate intracellular signal transduction pathways when treated with IL-4 and IFNγ. Treatment with IL-4 induces the phosphorylation of Stat6, while treatment with IFNγ induces the phosphorylation of Stat1. We first confirmed the increase in phosphorylation of Stat1 and Stat6 by PhosphoFlow analysis (Figure 4A). We measured a 5.9 fold increase of the phosphorylation of pStat1 following IFNγ treatment and 3.3 fold increase in phosphorylation of pStat6 following IL-4 treatment. BFU and AOH COINs were conjugated to antibodies that recognize the Y701 phosphorylated epitope of the Stat1, and the Y641 epitope of the Stat6 proteins. The cells were then fixed and permeabilized as previously described [15].


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

Shachaf CM, Elchuri SV, Koh AL, Zhu J, Nguyen LN, Mitchell DJ, Zhang J, Swartz KB, Sun L, Chan S, Sinclair R, Nolan GP - PLoS ONE (2009)

Detection of intracellular phosphorylation signaling using COINs.a) Flow analysis of pStat1 and Stat6 phosphorylation following treatment of U937 cells with IFNγ or IL-4 cytokines, compared to non treated (Non stim). b) Raman spectral shift intensity of BFU COIN detecting intracellular pStat1 in IFNγ and c) pStat6 in IL-4, treated and non-treated (Non stim) U937cells. Treated (Stim control) and non treated cells (Non stim control) were stained with non-conjugated BFU COIN. The spectra are representative for five independent experiments. d) Quantitation of change in Raman peak height after IFNγ and e) IL-4 treated cells compared to non-treated cells. The αpStat1 and αStat6 conjugated COINs specifically detected pStat1 and pStat6 respectively on IFNγ and IL-4 treated U937 cells compared to non-treated cells (**p<0.01). e) Fold change ratio of pStat1 and pStat6 phosphorylation in U937 treated cells stained with both BFU and AOH COINs. The changes are the average of five independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666268&req=5

pone-0005206-g004: Detection of intracellular phosphorylation signaling using COINs.a) Flow analysis of pStat1 and Stat6 phosphorylation following treatment of U937 cells with IFNγ or IL-4 cytokines, compared to non treated (Non stim). b) Raman spectral shift intensity of BFU COIN detecting intracellular pStat1 in IFNγ and c) pStat6 in IL-4, treated and non-treated (Non stim) U937cells. Treated (Stim control) and non treated cells (Non stim control) were stained with non-conjugated BFU COIN. The spectra are representative for five independent experiments. d) Quantitation of change in Raman peak height after IFNγ and e) IL-4 treated cells compared to non-treated cells. The αpStat1 and αStat6 conjugated COINs specifically detected pStat1 and pStat6 respectively on IFNγ and IL-4 treated U937 cells compared to non-treated cells (**p<0.01). e) Fold change ratio of pStat1 and pStat6 phosphorylation in U937 treated cells stained with both BFU and AOH COINs. The changes are the average of five independent experiments.
Mentions: Next, we tested the potential of COIN nanoparticles for the detection of intracellular phosphorylation events. U937 cells activate intracellular signal transduction pathways when treated with IL-4 and IFNγ. Treatment with IL-4 induces the phosphorylation of Stat6, while treatment with IFNγ induces the phosphorylation of Stat1. We first confirmed the increase in phosphorylation of Stat1 and Stat6 by PhosphoFlow analysis (Figure 4A). We measured a 5.9 fold increase of the phosphorylation of pStat1 following IFNγ treatment and 3.3 fold increase in phosphorylation of pStat6 following IL-4 treatment. BFU and AOH COINs were conjugated to antibodies that recognize the Y701 phosphorylated epitope of the Stat1, and the Y641 epitope of the Stat6 proteins. The cells were then fixed and permeabilized as previously described [15].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, Stanford University, Stanford, California, United States of America.

ABSTRACT

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.

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.

Show MeSH