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。一个明确的生物标记不能通过仅确定少数蛋白质来获得。对蛋白质、小分子和基因转录物进行定量、大规模多重生物测定（MMB）将是一项重大的医学突破。通过同时定量测定患者样本中的许多生物标志物，“个性化医疗”、明确的疾病识别和患者特异性诊断/预后将成为可能。我们将开发一种新的、强大的、基于功能化聚合物微珠（PMBs）的多金属标记系统，通过成熟的、高灵敏度、时间分辨和元素特异性的流式细胞术电感耦合等离子体质谱（FC-ICP-MS）检测技术进行分析。我们由知名专家组成的跨学科小组将应对这一重大挑战。S. Stevenson教授（university of Southern Mississippi， USM）将优化7种不同的M3N@C80子宫内膜富勒烯（EMFs）的合成，这些emf是生物医学样品中非内源性的无干扰金属。S. H. Strauss教授和O. V. Boltalina博士（Colorado State university， CSU）将优化电磁场化学衍生物的合成，确定最佳的官能团类型和数量，以达到均匀性。优选的衍生物将被发送给j.p. Phillips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PMBs的制备（所有这些都将永久地隔离在碳纳米笼中，因此不会随着时间的推移浸出）。多伦多大学（UofT）小组，由V. I. Baranov教授领导，将开发特定目的的分析方法，结合机器人样品导入和FC-ICP-MS仪器，允许大规模多路检测和分类数千个多金属编码珠与元素标记的报告亲和分子。他们将估计标记的多路性，并显示标记的pmb的完整性和几乎无限的保质期，并且当该技术减少到实践中时，数千种不同标记的pmb的大规模多路复用是可能的。O. I. Ornatsky博士（UofT）将进行分析物选择、编码小珠的测试和验证、免疫测定和寡核苷酸杂交，并将一系列抗体（Abs）共价连接到标记的PMBs表面，这些抗体将在夹心分析中与连接到报告标记的次级抗体进行测试。我们将证明我们的标记系统具有用于大规模多重生物测定的潜力，其中数千种抗原、基因转录物和许多疾病和病症的小分子细胞标记物可以在单个样品中同时测定。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分析多金属标记的PMBs的优势，这些PMBs包被捕获的抗体，可以对抗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。

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.