Apoferritin Nanoparticle-Based Mass Tags: A Novel Metal Tagging Strategy for Mass Cytometry

Mass cytometry (MC), considered the next generation of flow cytometry (FC), uses antibodies tagged with metal isotopes instead of fluorescent molecules for higher-dimensional single cell biomarker assays and can measure more than 50 parameters simultaneously on individual cells. Despite its powerful analytical performance, MC also has limitations in sensitivity and isotope channels using current mass tags. Herein, a new metal tagging strategy was developed to prepare MC mass tags based on apoferritin nanoparticles (AFNPs). For the preparation of AFNPs, rare-earth metals and phosphate ions were sequentially introduced into the cavity of apoferritin by a passive diffusion method to form a metal-phosphate core inside the apoferritin nanocages. The N-hydroxysuccinimide polyethylene glycol (MW = 1000, n = 22) maleimide (NHS-PEG₂₂-MAL) linker was used to link the AFNPs and antibodies to prepare Ab-NP conjugates. The AFNPs have low nonspecific binding to cells, thus resulting in a low background signal for the MC assay. Under optimized conditions, each apoferritin can load an average of 300-800 rare-earth metal atoms, and AFNP tags have more than twice the sensitivity compared with metal-chelating polymer (MCP) tags. Multiparameter assays for the cellular subset analysis of human peripheral blood mononuclear cells (PBMCs) showed good agreement between FC and MC assays by using our AFNP tags. The study presents a new strategy for preparing MC mass tags that are chelator-independent and have multiple staining capabilities, low nonspecific binding, and high sensitivity. The developed AFNP tags are expected to promote the development of MC technology.