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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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Fluorescence flow cytometryof microspheres with defined bindingcapacities. A. Bivariate histogram of FALS vs green fluorescence showingpopulations of fluorescence-encoded 3.5 um beads (gates B1–B5)and nonfluorescent 5.5 um beads. B. Yellow fluorescence intensityhistograms for the indicated populations of neutravidin-coated beadsstained with biotin-PE.
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fig1: Fluorescence flow cytometryof microspheres with defined bindingcapacities. A. Bivariate histogram of FALS vs green fluorescence showingpopulations of fluorescence-encoded 3.5 um beads (gates B1–B5)and nonfluorescent 5.5 um beads. B. Yellow fluorescence intensityhistograms for the indicated populations of neutravidin-coated beadsstained with biotin-PE.

Mentions: Our SERS calibration approach is based onsurfaces with a knownnumber of SERS tag binding sites. Here, these are polymer microspheresfunctionalized with different amounts of neutravidin that can be quantifiedusing conventional fluorescence flow cytometry. To create microsphereswith defined numbers of binding sites and surface densities, we mixedneutravidin with BSA in different ratios and coupled these to microspheres.The microspheres were fluorescently encoded with different intensitiesof a green fluorochrome and by different diameters so that they canbe identified in a mixture. Staining this multiplexed bead set allowsus to assess capture protein capacity and density simultaneously inone tube. As presented in Figure 1A, 3.5-μm-diameterbeads bearing different amounts of capture protein could be identifiedby their green fluorescence (gates B1–B5), while 5.5 μmbeads could be distinguished by their increased light scatter. Gatingindividual populations in the side scatter vs green fluorescence histogramallows us to measure the fluorescence or SERS from each bead. To determinethe bead binding capacity, we stained these beads with a fluorescentligand, biotinylated phycoerythrin (biotin-PE), and at saturation,and measured them by flow cytometry. After appropriate calibrationof the fluorescence signals, we estimated the binding capacity ofeach bead population (Figure 1B), which rangedfrom 0 to ∼200,000 molecules of biotin-PE.


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

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

Fluorescence flow cytometryof microspheres with defined bindingcapacities. A. Bivariate histogram of FALS vs green fluorescence showingpopulations of fluorescence-encoded 3.5 um beads (gates B1–B5)and nonfluorescent 5.5 um beads. B. Yellow fluorescence intensityhistograms for the indicated populations of neutravidin-coated beadsstained with biotin-PE.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Fluorescence flow cytometryof microspheres with defined bindingcapacities. A. Bivariate histogram of FALS vs green fluorescence showingpopulations of fluorescence-encoded 3.5 um beads (gates B1–B5)and nonfluorescent 5.5 um beads. B. Yellow fluorescence intensityhistograms for the indicated populations of neutravidin-coated beadsstained with biotin-PE.
Mentions: Our SERS calibration approach is based onsurfaces with a knownnumber of SERS tag binding sites. Here, these are polymer microspheresfunctionalized with different amounts of neutravidin that can be quantifiedusing conventional fluorescence flow cytometry. To create microsphereswith defined numbers of binding sites and surface densities, we mixedneutravidin with BSA in different ratios and coupled these to microspheres.The microspheres were fluorescently encoded with different intensitiesof a green fluorochrome and by different diameters so that they canbe identified in a mixture. Staining this multiplexed bead set allowsus to assess capture protein capacity and density simultaneously inone tube. As presented in Figure 1A, 3.5-μm-diameterbeads bearing different amounts of capture protein could be identifiedby their green fluorescence (gates B1–B5), while 5.5 μmbeads could be distinguished by their increased light scatter. Gatingindividual populations in the side scatter vs green fluorescence histogramallows us to measure the fluorescence or SERS from each bead. To determinethe bead binding capacity, we stained these beads with a fluorescentligand, biotinylated phycoerythrin (biotin-PE), and at saturation,and measured them by flow cytometry. After appropriate calibrationof the fluorescence signals, we estimated the binding capacity ofeach bead population (Figure 1B), which rangedfrom 0 to ∼200,000 molecules of biotin-PE.

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