Ultrasensitive multiplexed mass cytometry immunoassays based on single patch nanoparticles

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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.