Control of Metal Content in Polystyrene Microbeads Prepared with Metal Complexes of DTPA Derivatives

Abstract

Mass cytometry (MC) is an emerging analytical technique in which an inductively coupled plasma time-of-flight mass spectrometer is employed to analyze the signals of isotopic labels on cell and microbead samples. Bead-based applications in mass cytometry require one to label the microbeads with various metal ions that can be individually identified by MC. This paper describes a novel approach to encoding microbeads with controlled levels of metal ions by introducing polymerizable metal complexes of the structure M­(DTPA-VBAm2) in two-stage dispersion polymerization reactions. Using this method, the incorporation of various metal ions is effective and consistent in the investigated concentration range. As a result, this method provides a convenient path for synthesizing microbeads with controlled metal content for mass cytometry applications. As a proof-of-concept experiment, a sample of europium-labeled microbeads was surface-functionalized with a goat anti-mouse IgG antibody and tested by mass cytometry for its ability to detect a Lu-labeled mouse IgG. Surface functionalization involved a thin silica shell to which neutravidin was covalently attached. This experiment demonstrates the potential use of these microbeads as classifier beads for bead-based assays in MC.

Development of Multiplexed Bead-Based Immunoassays for Profiling Soluble Cytokines and CD163 Using Mass Cytometry

“…The cerium isotopes 140 Ce and 142 Ce can serve as a microbead identifier for the convenience of gating classifier signals in the MC results. As a demonstration of the design strategy, a set of 11 binary-metal-encoded 3 μm polystyrene (PS) microbeads for bead-based assays were synthesized by a series of two-stage dispersion polymerization (DisP) by following the bead synthesis protocol reported in our previous publications …”

“…As a demonstration of the design strategy, a set of 11 binary-metal-encoded 3 μm polystyrene (PS) microbeads for bead-based assays were synthesized by a series of two-stage dispersion polymerization (DisP) by following the bead synthesis protocol reported in our previous publications. 30 Key details of the bead synthesis and characterization are described in the Supporting Information. A scanning electron microscopy image of the bead sample C-1 (see Table S2 and S3 for synthesis details) is presented in panel (a) of Figure S1 in the Supporting Information.…”

“…We have shown that we can label the beads with three different levels of lanthanide content with baseline resolution. 30 For this example, with K = 4 and M = 36, a very large variety of multiplexing (n ∼ 10 21 ) can theoretically be created.…”

Reagents for Mass Cytometry

“…DTPA binds metal ions rapidly and the binding is sufficiently rapid that it can be characterized by isothermal titration calorimetry . In the Winnik group, DTPA is most commonly used for mass cytometry applications, − whereas DOTA has been used for radioimmunotherapy applications …”

“…Nothing comparable is currently commercially available for mass cytometry. The metal-loading strategies used to synthesize the polystyrene calibration beads described above do not provide a sufficient level of metal ion control to enable reproducible synthesis of 68 This approach provides uniform microbeads with control over lanthanide ion content. The dispersion polymerization method employed produces microbeads with a surface corona of poly(N-vinylpyrrolidone).…”