Ultrasensitive multiplexed mass cytometry immunoassays based on single patch nanoparticles

本文描述了无机纳米颗粒（NPs）的合成，特别是含镧纳米颗粒，以及它们与单克隆抗体的结合研究。np抗体复合物设计用于生物标记物和低拷贝数转录物的定量细胞免疫分析。细胞间低拷贝数生物标志物的分析是细胞生物学的重要研究方向，为癌症等细胞性疾病的诊断提供了重要的新方法。使用np抗体偶联物的生物测定利用了大量细胞术的多路复用能力，这是一项由Richmond Hill ON的DVS Sciences开发的基于原子质谱法和细胞术结合的新技术。目前使用DVS细胞计数仪的方法能够检测到每个细胞中~2000个生物标志物的表达，使用抗体标签携带~200个镧系同位素离子。为了达到检测每个细胞<100拷贝水平的生物标志物所需的高灵敏度，纳米颗粒必须包含至少5000个单一金属同位素原子。这需要直径为10至15纳米的NPs，其大小与抗体本身相当。本研究探索了镧系纳米粒子偶联抗体的新合成策略。对于定量细胞术，每个针对特定生物标志物的抗体都应该由一个单独的NP结合。为了确保抗体不会与抗原交联，我们将探索将反应性官能团限制在NPs表面的小块上的可能性，目的是确保只有少量（1-4）抗体会附着在每个无机纳米颗粒上。金属NP偶联物将通过大量细胞术进行评估，用于定量白血病细胞的高表面和低表面生物标志物表达。然后，我们将量化细胞内抗原，如癌蛋白p53，它在不同水平上表达，显著影响细胞生理。一旦实现，这些np抗体偶联物将用于诊断、监测，并有望找到治疗复杂人类疾病的方法，如癌症、艾滋病等病毒感染、代谢紊乱和心脏病。

This proposal describes research on the synthesis of inorganic nanoparticles (NPs), particularly lanthanide-containing nanoparticles, and their conjugation to monoclonal antibodies. The NP-antibody complexes are designed for quantitative cellular immunoassays of biomarkers and transcripts of low copy number. The analysis of low copy number biomarkers on a cell-by-cell basis is of fundamental interest in cell biology and provides an important new diagnostic for cell based diseases such as cancer. Bioassays with NP-antibody conjugates take advantage of the multiplexing capabilities of mass cytometry, a new technique developed by DVS Sciences, Richmond Hill ON, based upon the union of atomic mass spectrometry and cytometry. Current methods using the DVS mass cytometer are able to detect biomarker expression at the level of ~2000 per cell using antibody tags carrying ~200 ions of a lanthanide isotope. To achieve the high sensitivity needed to detect biomarkers at the level of <100 copies per cell, the nanoparticles must contain atleast 5000 atoms of a single metal isotope. This requires NPs with diameters of 10 to 15 nm, comparable in size to the antibodies themselves. This proposal explores new synthetic strategies for conjugating antibodies to lanthanide nanoparticles. For quantitative mass cytometry, each antibody against a specific biomarker should be bound by a separate NP. To ensure the antibodies do not crosslink the antigens we will explore the potential of confining reactive functional groups to a small patch on the surface of the NPs, with the goal of ensuring that only a small number (1-4) of antibodies will be attached to each inorganic nanoparticle. Metal NP conjugates will be evaluated by mass cytometry for use in quantifying both high and low surface biomarker expression on leukemia cells. Then we will quantify intracellular antigens such as the oncoprotein p53 which is expressed at variable levels dramatically affecting cell physiology. Once realized these NP-antibody conjugates will be useful for the diagnosis, monitoring and hopefully, to find cures for complex human diseases such as cancer, viral infections such as AIDS, metabolic disorders, and heart disease.