Limits...
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.

Show MeSH
Raman microscopy.a) Image of the “Integrated Raman BioAnalyzer” - IRBA. The arrow indicates the placement of the chamber with the sample prior to insertion into the apparatus. b) Generic configuration of Raman microscopic setup. c) Optimization of scans using IRBA. Wells containing COINs were scanned with a laser beam of 1 µm using a matrices of 5×5, 10×10, 15×15, 17×17 and 20×20 at 100 µm distances. The spectra are indicated (left) and the calculated peak heights are represented as histograms (right). The experiments were performed 3 times in duplicates. The peak heights for the 15×15 and 17×17 are significantly different from the 5×5, 10×10 and 20×20; **p<0.01. d) Raman intensity of spectra from cells stained with different concentrations of αCD54-AOH-COIN (red - 0.5 mM, blue – 0.25 mM and yellow – 0.1 mM) and AOH-COIN (purple - 0.5 mM, green – 0.25 mM and orange – 0.1 mM), scanned by IRBA (left). Quantitation of the Raman peak height from the spectra observed illustrated as histograms *p<0.05 and **<0.01. The experiment was performed three times in duplicates.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2666268&req=5

pone-0005206-g002: Raman microscopy.a) Image of the “Integrated Raman BioAnalyzer” - IRBA. The arrow indicates the placement of the chamber with the sample prior to insertion into the apparatus. b) Generic configuration of Raman microscopic setup. c) Optimization of scans using IRBA. Wells containing COINs were scanned with a laser beam of 1 µm using a matrices of 5×5, 10×10, 15×15, 17×17 and 20×20 at 100 µm distances. The spectra are indicated (left) and the calculated peak heights are represented as histograms (right). The experiments were performed 3 times in duplicates. The peak heights for the 15×15 and 17×17 are significantly different from the 5×5, 10×10 and 20×20; **p<0.01. d) Raman intensity of spectra from cells stained with different concentrations of αCD54-AOH-COIN (red - 0.5 mM, blue – 0.25 mM and yellow – 0.1 mM) and AOH-COIN (purple - 0.5 mM, green – 0.25 mM and orange – 0.1 mM), scanned by IRBA (left). Quantitation of the Raman peak height from the spectra observed illustrated as histograms *p<0.05 and **<0.01. The experiment was performed three times in duplicates.

Mentions: To reliably detect the Raman signal in a format appropriate for cellular analyses, we developed a automated Raman scanner (Intel Raman BioAnalyser – IRBA) that is suitable for detecting Raman signals (Figure 2A). The schema for the IRBA is illustrated in (Figure 2B). The key components of the microscope are the dichroic filter and notch filter. The dichroic filter allows the laser light to reach the sample, and reflect all other wavelengths. The notch filter blocks the laser light, and transmits all other light wavelengths. The Raman scattering is measured as spectral shifts as little as 30 nm from the excitation laser-light source, hence the slope of the notch filter is high (∼90 degrees).


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)

Raman microscopy.a) Image of the “Integrated Raman BioAnalyzer” - IRBA. The arrow indicates the placement of the chamber with the sample prior to insertion into the apparatus. b) Generic configuration of Raman microscopic setup. c) Optimization of scans using IRBA. Wells containing COINs were scanned with a laser beam of 1 µm using a matrices of 5×5, 10×10, 15×15, 17×17 and 20×20 at 100 µm distances. The spectra are indicated (left) and the calculated peak heights are represented as histograms (right). The experiments were performed 3 times in duplicates. The peak heights for the 15×15 and 17×17 are significantly different from the 5×5, 10×10 and 20×20; **p<0.01. d) Raman intensity of spectra from cells stained with different concentrations of αCD54-AOH-COIN (red - 0.5 mM, blue – 0.25 mM and yellow – 0.1 mM) and AOH-COIN (purple - 0.5 mM, green – 0.25 mM and orange – 0.1 mM), scanned by IRBA (left). Quantitation of the Raman peak height from the spectra observed illustrated as histograms *p<0.05 and **<0.01. The experiment was performed three times in duplicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005206-g002: Raman microscopy.a) Image of the “Integrated Raman BioAnalyzer” - IRBA. The arrow indicates the placement of the chamber with the sample prior to insertion into the apparatus. b) Generic configuration of Raman microscopic setup. c) Optimization of scans using IRBA. Wells containing COINs were scanned with a laser beam of 1 µm using a matrices of 5×5, 10×10, 15×15, 17×17 and 20×20 at 100 µm distances. The spectra are indicated (left) and the calculated peak heights are represented as histograms (right). The experiments were performed 3 times in duplicates. The peak heights for the 15×15 and 17×17 are significantly different from the 5×5, 10×10 and 20×20; **p<0.01. d) Raman intensity of spectra from cells stained with different concentrations of αCD54-AOH-COIN (red - 0.5 mM, blue – 0.25 mM and yellow – 0.1 mM) and AOH-COIN (purple - 0.5 mM, green – 0.25 mM and orange – 0.1 mM), scanned by IRBA (left). Quantitation of the Raman peak height from the spectra observed illustrated as histograms *p<0.05 and **<0.01. The experiment was performed three times in duplicates.
Mentions: To reliably detect the Raman signal in a format appropriate for cellular analyses, we developed a automated Raman scanner (Intel Raman BioAnalyser – IRBA) that is suitable for detecting Raman signals (Figure 2A). The schema for the IRBA is illustrated in (Figure 2B). The key components of the microscope are the dichroic filter and notch filter. The dichroic filter allows the laser light to reach the sample, and reflect all other wavelengths. The notch filter blocks the laser light, and transmits all other light wavelengths. The Raman scattering is measured as spectral shifts as little as 30 nm from the excitation laser-light source, hence the slope of the notch filter is high (∼90 degrees).

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