Optimization of SERS tag intensity, binding footprint, and emittance

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.

Figure 1

Fluorescence flow cytometry of microspheres with defined binding capacities. A. Bivariate histogram of FALS vs green fluorescence showing populations of fluorescence-encoded 3.5 um beads (gates B1–B5) and nonfluorescent 5.5 um beads. B. Yellow fluorescence intensity histograms for the indicated populations of neutravidin-coated beads stained with biotin-PE.

Figure 2

Characterization of the plasmonic particles used to prepare SERS tags in this study. A–C. TEM images of large Au nanorods (A, scale bar: 50 nm), Ag@Au nanorods (B, scale bar: 20 nm), and Ag plates (C, scale bar: 50 nm). D. UV/vis extinction spectra of monodisperse (solid line) and aggregated Au rods (dashed line). E. UV/vis extinction spectra of Au rods (solid line) and Ag@Au rods (dashed line). F. UV/vis extinction spectra of Ag plates (solid line) and Ag plate-based SERS tags (dashed line).

Figure 3

SERS flow cytometry of microspheres stained with biotinylated SERS tags. A. SERS spectra of individual SERS-tag stained beads measured on a spectral flow cytometer. B. Average spectra of SERS tag-stained beads. C. SERS intensity histograms of the neutravidin-density multiplex bead set.

Figure 4

Plots of median SERS intensity versus microsphere binding capacity for 3.5 μm (A) and 5.5 μm (B) neutravidin beads stained with biotinylated SERS tags prepared from four different plasmonic nanoparticles: monodisperse Au rods (filled circles), aggregated Au rods (open circles), Ag@Au rods (solid triangles), and Ag plates (open triangles).

Figure 5

SEM of neutravidin microspheres stained with biotinylated SERS tags. 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.

Figure 6

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