Optimization of SERS Tag Intensity, Binding Footprint, and Emittance

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

Methodology for evaluating and comparing flow cytometers: A multisite study of 23 instruments

We demonstrate improved methods for making valid and accurate comparisons of fluorescence measurement capabilities among instruments tested at different sites and times. We designed a suite of measurements and automated data processing methods to obtain consistent objective results and applied them to a selection of 23 instruments at nine sites to provide a range of instruments as well as multiple instances of similar instruments. As far as we know, this study represents the most accurate methods and results so far demonstrated for this purpose. The first component of the study reporting improved methods for photoelectron scale (Spe) evaluations, which was published previously (Parks, El Khettabi, Chase, Hoffman, Perfetto, Spidlen, Wood, Moore, and Brinkman: Cytometry A 91 (2017) 232-249). Those results which were within themselves are not sufficient for instrument comparisons, so here, we use the Spe scale results for the 23 cytometers and combine them with additional information from the analysis suite to obtain the metrics actually needed for instrument evaluations and comparisons. We adopted what we call the 2+2SD limit of resolution as a maximally informative metric, for evaluating and comparing dye measurement sensitivity among different instruments and measurement channels. Our results demonstrate substantial differences among different classes of instruments in both dye response and detection sensitivity and some surprisingly large differences among similar instruments, even among instruments with nominally identical configurations. On some instruments, we detected defective measurement channels needing service. The system can be applied in shared resource laboratories and other facilities as an aspect of quality assurance, and accurate instrument comparisons can be valuable for selecting instruments for particular purposes and for making informed instrument acquisition decisions. An institutionally supported program could serve the cytometry community by facilitating access to materials, and analysis and maintaining an archive of results. © 2018 International Society for Advancement of Cytometry