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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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SEM of neutravidin microspheresstained with biotinylated SERStags. A. Au rod-based SERS tags (small arrows: single nanorods). B.Aggregated Au rod-based SERS tags (large arrows: nanorod aggregates).C. Ag@Au rod-based SERS tags. D. Ag plate-based SERS tags. Magnification:61,472×. Scale bar: 500 nm.
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fig5: SEM of neutravidin microspheresstained with biotinylated SERStags. A. Au rod-based SERS tags (small arrows: single nanorods). B.Aggregated Au rod-based SERS tags (large arrows: nanorod aggregates).C. Ag@Au rod-based SERS tags. D. Ag plate-based SERS tags. Magnification:61,472×. Scale bar: 500 nm.

Mentions: At lowbinding capacities (0–70,000), the SERS intensityon the 3.5 μm beads increases with increasing binding capacity,and then plateaus at the highest capacity beads (Figure 4A, filled circles). We interpret this plateau to result fromsteric hindrance and competition among SERS tags for access to bindingsites on the bead surface. We imaged these beads using SEM. Presentedin Figure S2 are SEM images of high densityavidin beads, showing a high degree of SERS tag surface coverage,low density avidin beads exhibiting subsaturating coverage, and BSA-coatednegative control beads, showing a very low amount of nonspecific bindingof the biotinylated SERS tags. Upon closer inspection of the SERStags bound to the neutravidin beads (Figure 5A), it can be seen that the majority of the SERS tags are singlerods and dimers (small arrows), with relatively few higher order aggregates.Also consistent with the limiting effect of surface density on binding,the larger 5.5 um beads with a higher capacity but comparable Neutravidindensity shows a higher intensity (Figure 4B), reflecting the reduced sterichindrance on the bigger beads. Thus, at low binding site densities,the brightness of a known number of SERS tags can be measured whileat higher surface densities the size of the SERS tags limits binding,providing an indication of the effective “footprint”of the SERS tag. For low binding densities (0–12,000 sites),where intensity is expected to be proportional to binding capacity,the slope of this curve reflects the radiant intensity per SERS tag(Itag). At saturation, intensity (Isat) is limited by the physical size of theSERS tag, and the number of SERS tags bound at saturation (Nsat) can be calculated as Isat/Itag. As the surface areaof the microsphere is known, the average cross-sectional footprintof a SERS tag can be calculated, as can the radiant emittance, orsize-normalized intensity, of each SERS tag. The radiant emittanceis arguably the most important determinant of a SERS tag’sassay performance, and thus is of prime interest in comparing differentSERS tags. We applied this approach to several different SERS tagcompositions based on different plasmonic nanoparticles, but withthe same Raman active compound, MGITC, and the same coating of biotin-functionalizedPEG.


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

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

SEM of neutravidin microspheresstained with biotinylated SERStags. A. Au rod-based SERS tags (small arrows: single nanorods). B.Aggregated Au rod-based SERS tags (large arrows: nanorod aggregates).C. Ag@Au rod-based SERS tags. D. Ag plate-based SERS tags. Magnification:61,472×. Scale bar: 500 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: SEM of neutravidin microspheresstained with biotinylated SERStags. A. Au rod-based SERS tags (small arrows: single nanorods). B.Aggregated Au rod-based SERS tags (large arrows: nanorod aggregates).C. Ag@Au rod-based SERS tags. D. Ag plate-based SERS tags. Magnification:61,472×. Scale bar: 500 nm.
Mentions: At lowbinding capacities (0–70,000), the SERS intensityon the 3.5 μm beads increases with increasing binding capacity,and then plateaus at the highest capacity beads (Figure 4A, filled circles). We interpret this plateau to result fromsteric hindrance and competition among SERS tags for access to bindingsites on the bead surface. We imaged these beads using SEM. Presentedin Figure S2 are SEM images of high densityavidin beads, showing a high degree of SERS tag surface coverage,low density avidin beads exhibiting subsaturating coverage, and BSA-coatednegative control beads, showing a very low amount of nonspecific bindingof the biotinylated SERS tags. Upon closer inspection of the SERStags bound to the neutravidin beads (Figure 5A), it can be seen that the majority of the SERS tags are singlerods and dimers (small arrows), with relatively few higher order aggregates.Also consistent with the limiting effect of surface density on binding,the larger 5.5 um beads with a higher capacity but comparable Neutravidindensity shows a higher intensity (Figure 4B), reflecting the reduced sterichindrance on the bigger beads. Thus, at low binding site densities,the brightness of a known number of SERS tags can be measured whileat higher surface densities the size of the SERS tags limits binding,providing an indication of the effective “footprint”of the SERS tag. For low binding densities (0–12,000 sites),where intensity is expected to be proportional to binding capacity,the slope of this curve reflects the radiant intensity per SERS tag(Itag). At saturation, intensity (Isat) is limited by the physical size of theSERS tag, and the number of SERS tags bound at saturation (Nsat) can be calculated as Isat/Itag. As the surface areaof the microsphere is known, the average cross-sectional footprintof a SERS tag can be calculated, as can the radiant emittance, orsize-normalized intensity, of each SERS tag. The radiant emittanceis arguably the most important determinant of a SERS tag’sassay performance, and thus is of prime interest in comparing differentSERS tags. We applied this approach to several different SERS tagcompositions based on different plasmonic nanoparticles, but withthe same Raman active compound, MGITC, and the same coating of biotin-functionalizedPEG.

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