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

Abstract

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.

Revisiting carboxylic group functionalization of silica sol–gel materials

“…122 There exists an ample variety of materials modified with this procedure: silica-coated Eu-encoded polystyrene beads, polystyrene core/SiO 2 shell particles, SiO 2 -coated Fe 3 O 4 particles and SiO 2 NPs doped with Ru(bpy) 3 Cl 2 , among others. [123][124][125][126][127][128][129][130][131][132][133][134][135][136] Alternatively, it is possible to synthesize anhydrides from polycarboxylic acids such as EDTA as shown in Scheme 15. 137 Finally, Amesh et al reported the synthesis of a COOH silane precursor from the reaction between APTES and succinic anhydride in chloroform and using an anhydrous CaCl 2 moisture trap tube.…”

“…There exists an ample variety of materials modified with this procedure: silica-coated Eu-encoded polystyrene beads, polystyrene core/SiO 2 shell particles, SiO 2 -coated Fe 3 O 4 particles and SiO 2 NPs doped with Ru(bpy) 3 Cl 2 , among others. 123–136…”

Generalized Analysis Approach of the Profile Roughness by Electron Microscopy with the Example of Hierarchically Grown Polystyrene–Iron Oxide–Silica Core–Shell–Shell Particles

“…[ 10–14 ] A very promising material combination for single particle‐based assays is achieved by merging the benefits of polystyrene (PS) and silica (SiO 2 ) particles in a core–shell particle format. [ 12,15,16 ]…”

“…[10][11][12][13][14] A very promising material combination for single particle-based assays is achieved by merging the benefits of polystyrene (PS) and silica (SiO 2 ) particles in a core-shell particle format. [12,15,16] Besides equipping CS particles with optical or electrochemical features and grafting (bio)chemical units to their outer surface that drive many of the developments in the field, two less explicit aspects gained increasing attention recently, surface morphology and its impact on functional performance [10,17,18] as well as magnetic properties, strongly facilitating handling and processibility [19,20] especially in view of automated workflows. [21,22] Examples of CS particles with magnetic features and controlled surface morphology, however, are scarce.…”

Exploiting Wavelength Orthogonality in Photoinitiated RAFT Dispersion Polymerization and Photografting for Monodisperse Surface-Functional Polymeric Microspheres

“…Monodisperse polymeric microspheres with various funcional groups on the surface have attracted widespread attention for a variety of applications such as molecular imprinting technology, charomatography, calibration standards, templates for organic/inorganic composites, and bead-based biomedical assays. − Polymeric microspheres with a micrometer-dimension can be prepared by seeded emulsion polymerization, activated swelling suspension polymerization, or dispersion polymerization . In particular, dispersion polymerization is an attractive one-pot method that can prepare monodisperse polymeric microspheres with diameters in the range of 0.5–15 μm …”