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.

中文翻译：

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基于氧化钽纳米粒子的质量标记，用于细胞计数。

质谱分析（MC）是一种生物分析技术，它使用金属标签抗体（Abs）进行高维单细胞免疫测定。当前，该技术可以使用基于金属螯合聚合物（MCP）的试剂同时在单个电池上测量40多个参数。然而，随着新的元素质量标签的发展，MC原则上可以检测多达135个参数。在这里，我们报告为MC免疫测定基于钽氧化物纳米粒子（NP）的质量标签的发展。通过一锅两步反相微乳液法合成了均一尺寸的胺官能化氧化钽纳米颗粒（d〜5.7 nm）。通过与叠氮化物-PEG2k琥珀酰亚胺基羧甲基酯（NHS-PEG2k-N3）交联剂反应，将这些胺官能化的NP进一步用叠氮基进行修饰。通过叠氮化物官能化的NP与二苯并环辛炔（DBCO）官能化的一级或二级Abs（DBCO-Ab）反应，然后进行快速蛋白质大小排阻液相色谱（FPLC）纯化，制备Ab-NP共轭物。制备了三种Ab-NP偶联物（TaO2-PEG2k-山羊抗小鼠，TaO2-PEG2k-CD25，TaO2-PEG2k-CD196），并在MC免疫分析中进行了测试。对于TaO2-PEG2k-山羊抗小鼠偶联物，我们表明它可以有效地检测Ramos细胞上丰富的CD20生物标记。对于TaO2-PEG2k-CD25和TaO2-PEG2k-CD196缀合物，我们证明了这些Ab-NP缀合物可以整合到商业Ab染色板中，用于人外周血单核细胞的高维单细胞免疫谱分析。