Tantalum Oxide Nanoparticle-Based Mass Tag for Mass Cytometry.

Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags. Here we report the development of a tantalum oxide nanoparticle (NP)-based mass tag for MC immunoassays. Uniform-sized amine-functionalized tantalum oxide NPs (d ∼ 5.7 nm) were synthesized via a one-pot two-step reverse microemulsion method. These amine-functionalized NPs were further modified with azide groups by reacting with azide-PEG2k succinimidyl carboxymethyl ester (NHS-PEG2k-N3) cross-linkers. The Ab-NP conjugates were prepared by reacting azide-functionalized NPs with dibenzocyclooctyne (DBCO)-functionalized primary or secondary Abs (DBCO-Ab) followed by fast protein size exclusion liquid chromatography (FPLC) purification. Three Ab-NP conjugates (TaO2-PEG2k-goat antimouse, TaO2-PEG2k-CD25, TaO2-PEG2k-CD196) were fabricated and tested in MC immunoassays. For the TaO2-PEG2k-goat antimouse conjugate, we showed that it can effectively detect abundant CD20 biomarkers on Ramos cells. For TaO2-PEG2k-CD25 and TaO2-PEG2k-CD196 conjugates, we demonstrated that these Ab-NP conjugates could be integrated into the commercial Ab staining panels for high-dimensional single-cell immune profiling of human peripheral blood mononuclear cells.

Enabling Indium Channels for Mass Cytometry by Using Reinforced Cyclam-Based Chelating Polylysine.

The synthesis of a polylysine polymer functionalized with the previously reported astonishingly inert [In(cb-te2pa)]+ chelate was performed. A biotin end group allowed the conjugation to biotinylated beads by the intermediary of a fluorescein isothiocyanate/neutravidin receptor. High quality imaging mass cytometry trials, based on 115In detection were performed to highlight the behavior of the material. Anti-CD20 antibody was labeled by the so-obtained In(III)-modified polylysine using the biotin/neutravidin interaction. Ramos (CD20[+]) and HL-60 (CD20[-]) cell lines were costained with the In(III)-modified bioconjugate by finding the best staining conditions. Both immunofluorescence microscopy (IF-M) and mass cytometry analyses confirmed the specific binding of anti-CD20 onto Ramos cells. CyTOF histograms constructed on the 115In detection allowed us to define and to separate, with a good signal-to-noise ratio, two populations (Ramos and HL-60). The inertness of In(III)-MCP-NAv over a three-month storage period was proved by performing new functionality tests involving Jurkat cells (CD20[-]) and multiparametric trials involving the 115In channel. The results ensure a promising future use of the previously announced [In(cb-te2pa)]+ complex-based polymers for mass cytometry.