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Optimization of SERS tag intensity, binding footprint, and emittance.

Nolan JP, Duggan E, Condello D - Bioconjug. Chem. (2014)

Bottom Line: By contrast, SERS tags prepared from smaller gold nanorods coated with a silver shell produce SERS tags that are 2-3 times brighter, on a size-normalized basis, than the Au nanorod-based tags, resulting in labels with improved performance in SERS-based image and flow cytometry assays.SERS tags based on red-resonant Ag plates showed similarly bright signals and small footprint.This approach to evaluating SERS tag brightness is general, uses readily available reagents and instruments, and should be suitable for interlab comparisons of SERS tag brightness.

View Article: PubMed Central - PubMed

Affiliation: La Jolla Bioengineering Institute Suite 210 3535 General Atomics Court San Diego, California 92121, United States.

ABSTRACT
Nanoparticle surface enhanced Raman scattering (SERS) tags have attracted interest as labels for use in a variety of applications, including biomolecular assays. An obstacle to progress in this area is a lack of standardized approaches to compare the brightness of different SERS tags within and between laboratories. Here we present an approach based on binding of SERS tags to beads with known binding capacities that allows evaluation of the average intensity, the relative binding footprint of particles in a SERS tag preparation, and the size-normalized intensity or emittance. We tested this on four different SERS tag compositions and show that aggregated gold nanorods produce SERS tags that are 2-4 times brighter than relatively more monodisperse nanorods, but that the aggregated nanorods are also correspondingly larger, which may negate the intensity if steric hindrance limits the number of tags bound to a target. By contrast, SERS tags prepared from smaller gold nanorods coated with a silver shell produce SERS tags that are 2-3 times brighter, on a size-normalized basis, than the Au nanorod-based tags, resulting in labels with improved performance in SERS-based image and flow cytometry assays. SERS tags based on red-resonant Ag plates showed similarly bright signals and small footprint. This approach to evaluating SERS tag brightness is general, uses readily available reagents and instruments, and should be suitable for interlab comparisons of SERS tag brightness.

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Performanceof SERS tags in spectral flow cytometry. Breast cancercell lines BT474 (HER2+) and MB435 (HER2-) were stained with anti-HER2conjugated primary antibody followed by anti-mouse IgG SERS tags preparedfrom (A) gold nanorods, (B) Ag@Au nanorods, and (C) Ag plates. Leftcolumn: Average spectra from unstained cells (black), BT474 (blue),and MB435 (red), with (solid line) or without (dotted line) primaryantibody. Right column: Intensity histograms from unstained cells(black), BT474 (blue), and MB 435 (red), with (solid fill) or without(no fill) primary antibody.
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fig6: Performanceof SERS tags in spectral flow cytometry. Breast cancercell lines BT474 (HER2+) and MB435 (HER2-) were stained with anti-HER2conjugated primary antibody followed by anti-mouse IgG SERS tags preparedfrom (A) gold nanorods, (B) Ag@Au nanorods, and (C) Ag plates. Leftcolumn: Average spectra from unstained cells (black), BT474 (blue),and MB435 (red), with (solid line) or without (dotted line) primaryantibody. Right column: Intensity histograms from unstained cells(black), BT474 (blue), and MB 435 (red), with (solid fill) or without(no fill) primary antibody.

Mentions: To evaluate how the bright tags performed in cell analysisapplications,we conjugated antibodies to Au rod-, Ag@au rod-, and Ag plate-basedSERS tags (similar to those above, but bearing carboxy groups insteadof biotin) and used these to stain cells for analysis by spectralflow cytometry. We cultured two breast cancer cell lines: BT474, whichexpresses high levels of the cell surface receptor HER2, and MDA-MB-435,which does not. These cells were fixed and stained with an anti-HER2primary antibody, followed by an anti-mouse IgG conjugated SERS tagsecondary label. Presented in Figure 6 areaverage single cell spectra from these cells, as well as spectra fromsecondary-only and unstained cells. The Au rod-based SERS tags allowedresolution of HER2 positive and negative cell lines, but with only∼10-fold difference in the median intensities of the cell populations.As expected from the calibration results summarized in Table 1, cells stained with SERS tags based on Ag@Au rodcores and Ag plates showed brighter staining and better resolutionfrom unstained and secondary-only stained cells, with the Ag plate-basedtags producing a >100-fold separation of the population mean intensities.This performance advantage, combined with the relative ease of preparingAg plate-based SERS tags compared to rod-based SERS tags, makes thesetags attractive for cell analysis and other applications.


Optimization of SERS tag intensity, binding footprint, and emittance.

Nolan JP, Duggan E, Condello D - Bioconjug. Chem. (2014)

Performanceof SERS tags in spectral flow cytometry. Breast cancercell lines BT474 (HER2+) and MB435 (HER2-) were stained with anti-HER2conjugated primary antibody followed by anti-mouse IgG SERS tags preparedfrom (A) gold nanorods, (B) Ag@Au nanorods, and (C) Ag plates. Leftcolumn: Average spectra from unstained cells (black), BT474 (blue),and MB435 (red), with (solid line) or without (dotted line) primaryantibody. Right column: Intensity histograms from unstained cells(black), BT474 (blue), and MB 435 (red), with (solid fill) or without(no fill) primary antibody.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215889&req=5

fig6: Performanceof SERS tags in spectral flow cytometry. Breast cancercell lines BT474 (HER2+) and MB435 (HER2-) were stained with anti-HER2conjugated primary antibody followed by anti-mouse IgG SERS tags preparedfrom (A) gold nanorods, (B) Ag@Au nanorods, and (C) Ag plates. Leftcolumn: Average spectra from unstained cells (black), BT474 (blue),and MB435 (red), with (solid line) or without (dotted line) primaryantibody. Right column: Intensity histograms from unstained cells(black), BT474 (blue), and MB 435 (red), with (solid fill) or without(no fill) primary antibody.
Mentions: To evaluate how the bright tags performed in cell analysisapplications,we conjugated antibodies to Au rod-, Ag@au rod-, and Ag plate-basedSERS tags (similar to those above, but bearing carboxy groups insteadof biotin) and used these to stain cells for analysis by spectralflow cytometry. We cultured two breast cancer cell lines: BT474, whichexpresses high levels of the cell surface receptor HER2, and MDA-MB-435,which does not. These cells were fixed and stained with an anti-HER2primary antibody, followed by an anti-mouse IgG conjugated SERS tagsecondary label. Presented in Figure 6 areaverage single cell spectra from these cells, as well as spectra fromsecondary-only and unstained cells. The Au rod-based SERS tags allowedresolution of HER2 positive and negative cell lines, but with only∼10-fold difference in the median intensities of the cell populations.As expected from the calibration results summarized in Table 1, cells stained with SERS tags based on Ag@Au rodcores and Ag plates showed brighter staining and better resolutionfrom unstained and secondary-only stained cells, with the Ag plate-basedtags producing a >100-fold separation of the population mean intensities.This performance advantage, combined with the relative ease of preparingAg plate-based SERS tags compared to rod-based SERS tags, makes thesetags attractive for cell analysis and other applications.

Bottom Line: By contrast, SERS tags prepared from smaller gold nanorods coated with a silver shell produce SERS tags that are 2-3 times brighter, on a size-normalized basis, than the Au nanorod-based tags, resulting in labels with improved performance in SERS-based image and flow cytometry assays.SERS tags based on red-resonant Ag plates showed similarly bright signals and small footprint.This approach to evaluating SERS tag brightness is general, uses readily available reagents and instruments, and should be suitable for interlab comparisons of SERS tag brightness.

View Article: PubMed Central - PubMed

Affiliation: La Jolla Bioengineering Institute Suite 210 3535 General Atomics Court San Diego, California 92121, United States.

ABSTRACT
Nanoparticle surface enhanced Raman scattering (SERS) tags have attracted interest as labels for use in a variety of applications, including biomolecular assays. An obstacle to progress in this area is a lack of standardized approaches to compare the brightness of different SERS tags within and between laboratories. Here we present an approach based on binding of SERS tags to beads with known binding capacities that allows evaluation of the average intensity, the relative binding footprint of particles in a SERS tag preparation, and the size-normalized intensity or emittance. We tested this on four different SERS tag compositions and show that aggregated gold nanorods produce SERS tags that are 2-4 times brighter than relatively more monodisperse nanorods, but that the aggregated nanorods are also correspondingly larger, which may negate the intensity if steric hindrance limits the number of tags bound to a target. By contrast, SERS tags prepared from smaller gold nanorods coated with a silver shell produce SERS tags that are 2-3 times brighter, on a size-normalized basis, than the Au nanorod-based tags, resulting in labels with improved performance in SERS-based image and flow cytometry assays. SERS tags based on red-resonant Ag plates showed similarly bright signals and small footprint. This approach to evaluating SERS tag brightness is general, uses readily available reagents and instruments, and should be suitable for interlab comparisons of SERS tag brightness.

Show MeSH
Related in: MedlinePlus