A Silica Coating Approach to Enhance Bioconjugation on Metal-Encoded Polystyrene Microbeads for Bead-Based Assays in Mass Cytometry.

Bead-based assays in flow cytometry are multiplexed analytical techniques that allow rapid and simultaneous detection and quantification of a large number of analytes from small volumes of samples. The development of corresponding bead-based assays in mass cytometry (MC) is highly desirable since it could increase the number of analytes detected in a single assay. The microbeads for these assays have to be labeled with metal isotopes for MC detection. One must also be able to functionalize the bead surface with affinity reagents to capture the analytes. Metal-encoded polystyrene microbeads prepared by multi-stage dispersion polymerization can produce effective isotopic signals in MC with relatively small bead-to-bead variations. However, functionalizing this microbead surface with bioaffinity agents remains challenging, possibly due to the interference of the steric-stabilizing PVP corona on the microbead surface. Here, we report a systematic investigation of a silica coating approach to coat Eu-encoded microbeads with thin silica shells, to functionalize the surface with amino groups, and to introduce bioaffinity agents. We examine the effect of silica shell roughness on the bioconjugation capacity and the effect of silica shell thickness on signal quality in MC measurements. To limit non-specific binding, we converted the amino groups on the microbead surface to carboxylic acid groups. Antibodies were effectively attached to microbead by first conjugating NeutrAvidin to the carboxyl-modified bead surface and then attaching biotinylated antibodies to the NeutrAvidin-modified bead surface. The antibody-modified microbeads can specifically capture antigens, which were marked with isotopic labels, and generate strong signals in MC. These are promising results for the development of bead-based assays in MC.

Metal-Encoded Polystyrene Microbeads as a Mass Cytometry Calibration/Normalization Standard Covering Channels from Yttrium (89 amu) to Bismuth (209 amu).

Mass cytometry (MC) measures metal isotope signals from single cells and bead samples. Since large numbers of isotopes can be employed as labels, mass cytometry is a powerful analytical technique for multiparameter cytometric assays. The calibration protocol in MC is a critical algorithm, which employs metal-encoded microbeads as an internal standard to correct the data for instrumental signal drift. The current generation of commercially available beads carries four lanthanide elements (cerium, europium, holmium, and lutetium). However, this is not sufficient to calibrate the full span of detection channels, ranging from yttrium (89 amu) to bismuth (209 amu), which are now available. To address this issue we prepared polystyrene microbeads encoded with seven elements (yttrium, indium, and bismuth in addition to the four lanthanides) by multistage dispersion polymerization for MC calibration and normalization. The bead synthesis conditions were optimized to obtain microbeads that were uniform in size and generated strong MC signal intensities at similar levels for the eight encoded isotopes. Metal ion leaching from the beads under storage and application conditions was also examined. We demonstrated that the precision of normalized MC signals in the MC detection channels was improved by employing seven-element-encoded microbeads as a standard.