Nanocaged Metal Tags in Massively Multiplexed Leukemia Bioassay and Beyond

描述（由申请人提供）：急性白血病（AML）细胞的表征需要测量生物标志物，如蛋白质、基因和小分子。仅通过测定少数蛋白质无法获得明确的生物标志物特征。一个定量的，大规模多路生物测定（MMB）的蛋白质，小分子和基因转录的星座将是一个重大的医学突破。通过同时定量测定患者样品中的许多生物标志物，将能够实现“个性化医疗保健”、明确的疾病鉴定和患者特异性诊断/预后。我们将开发一种基于功能化聚合物微珠（PMB）的新型、稳健的多金属标记系统，该系统通过流式细胞仪电感耦合等离子体质谱（FC-ICP-MS）的成熟、高灵敏度、时间分辨和元素特异性检测技术进行分析。我们的跨学科专家小组将应对这一重大挑战。S.史蒂文森（南密西西比大学，USM）将优化7种不同的M3N@C80内突富勒烯（EMF）的合成，其中无干扰金属在生物医学样品中不是内源性的。S. H.施特劳斯和O. V. Boltalina（科罗拉多州立大学，CSU）将优化EMF的化学衍生物的合成，确定最佳官能团的类型和数量以实现均匀性。优选的衍生物将被送到J. P.菲利普斯教授（USM），他将把可聚合基团连接到EMF衍生物上，优化含有七种金属的许多特定混合物的窄尺寸分布PMB的制备（所有这些金属都将永久地隔离在碳纳米笼中，因此不会随着时间的推移而浸出）。多伦多大学（UofT）的研究小组，由V.I. Baranov将开发特定目的的分析方法，该方法将联合收割机机器人样品引入和FC-ICP-MS仪器相结合，以允许对数千个具有元素标记的报告亲和分子的多金属编码珠进行大规模多重检测和分类。他们将估计标记的复用性，并显示标记的PMB的完整性和几乎无限的保质期，以及当该技术减少到实践中时，数千个不同标记的PMB的大规模复用是可能的。欧博士I. Ornatsky（UofT）将进行分析物选择、编码珠的检测和验证、免疫测定和寡核苷酸杂交，并将一系列抗体（Ab）共价连接到标记的PMB表面，将在夹心试验中使用连接到报告标签的二级Ab进行检测。我们将证明我们的标记系统有潜力用于大规模多路生物测定，其中在单个样品中同时测定许多疾病和病症的数千种抗原，基因转录物和小分子细胞标记物。我们将在急性髓性白血病（AML）细胞上测试我们的大规模多重生物分析平台。我们将展示FC-ICP-MS分析包被有针对人血清中存在的细胞因子、趋化因子、生长因子和可溶性受体的捕获Ab的多金属标记的PMB的优势。

DESCRIPTION (provided by applicant): Characterization of an acute leukemia (AML) cell requires measurement of biomarkers such as proteins, genes and small molecules. An unambiguous biomarker signature cannot be obtained by determining only a few proteins. A quantitative, massively multiplexed bioassay (MMB) for a constellation of proteins, small molecules, and gene transcripts would be a significant medical breakthrough. "Personalized health care", unambiguous disease identification, and patient- specific diagnosis/prognosis will be enabled by the simultaneous quantitative determination of many biomarkers in a patient's sample. We will develop a new, robust, multimetallic tagging system based on functionalized polymer microbeads (PMBs) analyzed by the proven, high-sensitivity, time-resolved, and element-specific detection technique of flow-cytometry inductively- coupled-plasma mass spectrometry (FC-ICP-MS). Our interdisciplinary group of recognized experts will address this significant challenge. Prof. S. Stevenson (Univ. of Southern Mississippi, USM) will optimize the synthesis of 7 different M3N@C80 endometallofullerenes (EMFs) with interference-free metals that are not endogenous in biomedical samples. Prof. S. H. Strauss and Dr. O. V. Boltalina (Colorado State Univ., CSU) will optimize the synthesis of chemical derivatives of the EMFs, determine the optimum type and number of functional groups to achieve uniformity. Preferred derivatives will be sent to Prof. J. P. Phillips (USM), who will attach polymerizable groups to EMF derivatives, optimize preparation of narrow-size-distribution PMBs containing many specific mixtures of the seven metals (all of which will be permanently sequestered in their carbon nanocages and therefore cannot leach out over time). The Univ. of Toronto (UofT) group, led by Prof. V. I. Baranov, will develop purpose-specific analytical methods that combine robotic sample introduction and FC-ICP-MS instrumentation to allow massively multiplexed detection and classification of thousands of multimetallic encoded beads with element-tagged reporter affinity molecules. They will estimate the tagging multiplexity and show the integrity and virtually infinite shelf-life of the tagged PMBs, and that massive multiplexing with thousands of differently tagged PMBs is possible when this technology is reduced to practice. Dr. O. I. Ornatsky (UofT) will perform analyte selection, testing and validation of the encoded beads, immunoassays, and oligonucleotide hybridization, and will covalently link a range of antibodies (Abs) to the surfaces of the tagged PMBs, which will be tested in sandwich assays with secondary Abs linked to a reporter tag. We will prove that our tagging system has the potential to be used for massively multiplexed bioassays in which thousands of antigens, gene transcripts, and small-molecule cell markers for many diseases and conditions are determined simultaneously in a single sample. We will test our massively multiplexed bioanalytical platform on acute myeloid leukemia (AML) cells. We will show the advantages of FC-ICP-MS analysis of multi-metal-tagged PMBs coated with capture Abs against cytokines, chemokines, growth factors, and soluble receptors present in human serum.