Nanocaged Metal Tags in Massively Multiplexed Leukemia Bioassay and Beyond

大规模多重白血病生物测定及其他领域中的纳米笼金属标签

• 批准号: 8035589
• 负责人: Janice Paige Buchanan
• 金额: $ 17.5万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035589
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Address; Advanced Development; Affinity; Alkenes; Antibodies; Antigens; Binding; Biological Assay; Biological Markers; Carbon; Cells; Characteristics; Classification; Colorado; Coupled; Detection; Diagnosis; Disease; Elements; Feedback; Flow Cytometry; Fullerenes; Gene Expression; Gene Proteins; Genes; Goals; Growth Factor; Healthcare; Human; Immunoassay; Investigation; Life; Link; Mass Spectrum Analysis; Measurement; Medical; Metals; Microspheres; Mississippi; Oligonucleotides; Patients; Plasma; Polymers; Preparation; Proteins; Relative (related person); Reporter; Robotics; Sampling; Serum; Surface; System; Techniques; Technology; Testing; Time; Transcript; Universities; Validation; Work; analytical method; base; chemical synthesis; chemokine; cytokine; functional group; instrumentation; leukemia; light scattering; mass spectrometer; monomer; novel; outcome forecast; polymerization; receptor; small molecule

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. PUBLIC HEALTH RELEVANCE: The goals of the proposed investigation are the development of an advanced metal encoding system for bead-based assays. This novel system uses combinations of trimetallic endometallofullerenes embedded in polymeric microbeads to encode hundreds of thousands of unique carriers which together with time-resolved multi- element detection by flow-cytometry inductively-coupled-plasma mass spectrometry (FC-ICP-MS) will enable quantitative gene expression analysis and massively multiplexed immunoassays. Unambiguous disease identification and patient-specific diagnosis and prognosis will all be enabled by the simultaneous, rapid, and quantitative determination of many biomarkers in a patient's sample.

描述(由申请人提供)：急性白血病(AML)细胞的特征需要测量生物标记物，如蛋白质、基因和小分子。一个明确的生物标志物特征不能仅通过测定几个蛋白质来获得。对一系列蛋白质、小分子和基因转录本进行定量、大规模多重生物检测(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记物，将能够实现“个性化保健”、明确的疾病识别和患者特定的诊断/预后。我们将开发一种基于功能化聚合物微珠(PMB)的新的、健壮的多金属标记系统，该系统通过成熟的高灵敏度、时间分辨和元素特定的检测技术流式细胞仪电感耦合等离子体质谱(FC-ICP-MS)进行分析。我们由公认的专家组成的跨学科小组将应对这一重大挑战。史蒂文森教授(大学)密西西比州南部，USM)将优化7种不同的M3n@C80内金属富勒烯(EMF)的合成，这些EMF使用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(科罗拉多州立大学，CSU)将优化电动势化学衍生物的合成，确定实现均一性的最佳官能团类型和数量。优选的衍生品将被发送给J.P.Phillips教授(USM)，他将在EMF衍生品上连接可聚合基团，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(所有这些都将永久隔离在其碳纳米笼中，因此不会随着时间的推移而渗出)。大学。由V.I.Baranov教授领导的多伦多大学(UofT)团队将开发针对特定目的的分析方法，将机器人进样和FC-ICP-MS仪器相结合，实现对数千种具有元素标记报告亲和力分子的多金属编码珠的大规模多路复用检测和分类。他们将估计标记的多重性，并显示标记的PMB的完整性和几乎无限的保质期，并且当这项技术被简化为实践时，与数千个不同标记的PMB进行大规模多路复用是可能的。O·I·奥纳茨基博士(UofT)将对编码微珠、免疫分析和寡核苷酸杂交进行分析物选择、测试和验证，并将一系列抗体(Abs)共价连接到标记的PMB的表面，这些抗体将在夹心检测中与连接到报告标签的次级Abs进行测试。我们将证明我们的标签系统具有用于大规模多重生物检测的潜力，在这种生物检测中，许多疾病和疾病的数千个抗原、基因转录本和小分子细胞标记物可以在一个样本中同时确定。我们将在急性髓系白血病(AML)细胞上测试我们的大规模多路生物分析平台。我们将展示FC-ICPMS分析包被捕获抗体的多金属标记PMB针对人体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优势。 公共卫生相关性：拟议调查的目标是开发一种先进的金属编码系统，用于基于珠子的分析。这一新的系统使用嵌入在聚合物微球中的三金属内金属富勒烯的组合来编码数十万个独特的载体，这些载体与流式细胞仪电感耦合等离子体质谱(FC-ICP-MS)的时间分辨多元素检测一起将使定量基因表达分析和大规模多重免疫分析成为可能。同时、快速和定量地测定患者样本中的许多生物标志物，将使明确的疾病识别和患者特定的诊断和预后成为可能。