Cancer molecular analysis using quantum dots

 描述(申请人提供)：单个细胞的全面分子特征有助于了解细胞的病理生理学和我们诊断的能力 并控制复杂病理过程的进展，如癌症。原则上，用独特的报告探针标记每个生物分子，并在亚细胞分辨率上高灵敏度地检测其定位，可以实现这一目标。然而，没有一种传统的生物医学技术能够经受住挑战，因为可以同时分析的分子靶点数量有限，提供的单细胞信息有限，而且往往利用定性而不是定量分析。将基于纳米颗粒的新型成像技术转化为生物医学研究和临床诊断学有望为解决肿瘤的表型异质性提供强大的工具，打开研究通常被批处理掩盖或完全擦除的低丰度事件的大门，并阐明癌症生物标记物的特征以进行准确诊断和靶向治疗。为了实现这一承诺，我们最近发明了一种多色多周期分子图谱技术，该技术基于具有光学成像分辨率(NAT)的单个细胞中数十到数百个肿瘤生物标记物的循环成像。交警。2013年&NAT。协议2013)。在这项工作和我们在DNA链介导的置换用于快速和多周期分析方面的另一项进展(JACS2011)的基础上，我们建议探索和开发具有快速但温和脱色条件的新一代多色多周期蛋白质组技术。我们的方法利用简单的生物偶联和自组装机制来直接制备探针，利用抗体-DNA杂交探针来编码分子靶标，利用量子点独特的光学特性来进行定量和多色成像，以及DNA脚趾介导的置换的温和和快速动力学。这些功能的独特组合(得到我们最近的初步结果的支持)产生了简单但强大的技术，该技术应该能够在原位对单个癌细胞进行分子表征，而不需要专门的技术技能或对常见的染色方法和成像仪器进行重大修改。因此，如果成功，建议的QD技术应该可以很容易地应用于广泛的应用，特别是检测癌细胞中的分子信号通路，评估治疗反应，以及开发多重诊断和预后小组，从而有望在癌症研究和临床实践中产生重大影响。

 DESCRIPTION (provided by applicant): Comprehensive molecular characterization of individual cells is instrumental for understanding cell pathophysiology and our ability to diagnose and control the progression of complex pathological processes, such as cancer. In principle, tagging each biomolecule with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution can achieve this goal. Yet, none of the conventional biomedical techniques can stand up to the challenge, suffering from a limitation in the number of molecular targets that can be analyzed simultaneously, providing limited single cell information, and often utilizing qualitative rather than quantitative analysis. Translation of novel nanoparticl-based imaging technologies into biomedical research and clinical diagnostics promises to provide powerful tools for addressing phenotypic heterogeneity of tumors, opening access to studying low-abundance events often masked or completely erased by batch processing, and elucidating cancer biomarker signatures for accurate diagnosis and targeted therapy. To deliver on this promise, we recently invented a multicolor multicycle molecular profiling technology based on cyclic imaging of tens to hundreds of tumor biomarkers in single cells with optical imaging resolution (Nat. Commun. 2013 & Nat. Protocols 2013). Built on this work and another advance we made on DNA strand mediated displacement for fast and multicycle analysis (JACS 2011), we propose to explore and develop a new generation of multicolor multicycle proteomic technology with fast yet mild destaining condition. Our approach takes advantage of simple bioconjugation and self-assembly mechanisms for straightforward probe preparation, hybrid antibody-DNA probes for encoding of molecular targets, the unique optical properties of quantum dots for quantitative and multicolor imaging, and the mildness and fast kinetics of DNA toehold- mediated displacement. Unique combination of these features (which are supported by our recent preliminary results) yields simple, but powerful technology that should enable molecular characterization of individual cancer cells in situ, while requiring no specialized technical skills or significant modifications to common staining methodology and imaging instrumentation. As a result, if successful, the proposed QD technology should be readily applicable for a wide range of applications, in particular examination of molecular signaling pathways in cancer cells, evaluation of response to therapy, and development of multiplexed diagnostic and prognostic panels, thus promising to produce a substantial impact in cancer research and clinical practice.