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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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SERS flow cytometry ofmicrospheres stained with biotinylated SERStags. A. SERS spectra of individual SERS-tag stained beads measuredon a spectral flow cytometer. B. Average spectra of SERS tag-stainedbeads. C. SERS intensity histograms of the neutravidin-density multiplexbead set.
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fig3: SERS flow cytometry ofmicrospheres stained with biotinylated SERStags. A. SERS spectra of individual SERS-tag stained beads measuredon a spectral flow cytometer. B. Average spectra of SERS tag-stainedbeads. C. SERS intensity histograms of the neutravidin-density multiplexbead set.

Mentions: SERS tags based on gold nanorods are well-established ashavinga readily tunable plasmon resonance and producing strong SERS froma variety of resonant and nonresonant compounds.18−20,23 Given that the use of a resonant compound producesSERS signals that are significantly stronger compared to nonresonantcompounds,3,45 we focused on MGITC, a Raman tag that bindsstrongly to Au and Ag nanoparticles, absorbs near our excitation andnanoparticle resonance wavelength, and produces strong SERS from avariety of nanoparticle types. We stained the calibrated neutravidinmicrospheres with red-excited Au nanorod SERS tags (Figure 2A) and measured the resulting SERS intensity ona custom spectral flow cytometer using excitation at 488 to measurethe green fluorescence from the microsphere encoding dye and excitationat 660 nm to measure the entire microsphere SERS spectra from ∼300to 2000 cm–1 (Figure 3).Presented in Figure 3C are the integrated emissionintensity histograms for the different neutravidin density beads.The intensity axis is scaled to photons detected using the detectorresponse calibration provided by the manufacturer. We also used acommercial flow cytometer (FACSCalibur) to excite the SERS tag stainedbeads at 635 nm and measure the emission between 653 and 669 nm, aband corresponding to a Raman shift of roughly 450–900 cm–1 (Figure 3B), with very similarresults (Supporting Information Figure S1). We calibrated the intensity of the low density bead (∼12Kbinding sites) in units of molecules equivalent soluble fluorochrome(MESF) of allophycocyanin (APC), a red-excited fluorophore, usingcommercially available intensity standard beads and found that thesignal from 12,000 Au rod-based SERS tags was equivalent to ∼25,000MESF of APC.


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

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

SERS flow cytometry ofmicrospheres stained with biotinylated SERStags. A. SERS spectra of individual SERS-tag stained beads measuredon a spectral flow cytometer. B. Average spectra of SERS tag-stainedbeads. C. SERS intensity histograms of the neutravidin-density multiplexbead set.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: SERS flow cytometry ofmicrospheres stained with biotinylated SERStags. A. SERS spectra of individual SERS-tag stained beads measuredon a spectral flow cytometer. B. Average spectra of SERS tag-stainedbeads. C. SERS intensity histograms of the neutravidin-density multiplexbead set.
Mentions: SERS tags based on gold nanorods are well-established ashavinga readily tunable plasmon resonance and producing strong SERS froma variety of resonant and nonresonant compounds.18−20,23 Given that the use of a resonant compound producesSERS signals that are significantly stronger compared to nonresonantcompounds,3,45 we focused on MGITC, a Raman tag that bindsstrongly to Au and Ag nanoparticles, absorbs near our excitation andnanoparticle resonance wavelength, and produces strong SERS from avariety of nanoparticle types. We stained the calibrated neutravidinmicrospheres with red-excited Au nanorod SERS tags (Figure 2A) and measured the resulting SERS intensity ona custom spectral flow cytometer using excitation at 488 to measurethe green fluorescence from the microsphere encoding dye and excitationat 660 nm to measure the entire microsphere SERS spectra from ∼300to 2000 cm–1 (Figure 3).Presented in Figure 3C are the integrated emissionintensity histograms for the different neutravidin density beads.The intensity axis is scaled to photons detected using the detectorresponse calibration provided by the manufacturer. We also used acommercial flow cytometer (FACSCalibur) to excite the SERS tag stainedbeads at 635 nm and measure the emission between 653 and 669 nm, aband corresponding to a Raman shift of roughly 450–900 cm–1 (Figure 3B), with very similarresults (Supporting Information Figure S1). We calibrated the intensity of the low density bead (∼12Kbinding sites) in units of molecules equivalent soluble fluorochrome(MESF) of allophycocyanin (APC), a red-excited fluorophore, usingcommercially available intensity standard beads and found that thesignal from 12,000 Au rod-based SERS tags was equivalent to ∼25,000MESF of APC.

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