Immunomagnetic Diffractometry for Rapid Biomolecular Detection

描述（由申请人提供）：检测微量生物分子的能力在生物研究和临床中都是至关重要的。定量检测特定样品中的生物分子可以揭示从细胞决策过程到特定疾病状态的重要信息。例如，在美国每年造成50万人死亡的癌症，如果早期诊断，有可能治愈。进行诊断的一种方法是检测体液中的肿瘤标志物。为了成功的早期诊断，检测平台必须是敏感的，并揭示少量目标分子的存在。检测平台还必须是可简化的、快速的、操作相对简单的和便宜的，以实现1）高通量检测和2）在环境（诊所、研究实验室）中足够数量的检测平台。目前存在的大多数检测平台需要使用标记分子，例如荧光和放射性标记。这增加了操作的时间、成本和所需的专业知识，并使得这些基于标记的平台不一定适合于小型化和高通量检测。该提案涵盖了一种生物分子检测平台的开发，该平台是荧光和放射性标记的，易于制造和使用，坚固耐用，同时比许多现有的检测平台更灵敏。所提出的系统是基于免疫磁性分离和衍射，其组合尚未被探索。功能化磁珠用于从溶液中捕获靶分子。在该步骤之后，将珠粒（其表面上含有捕获的分子）简单地暴露于用次级受体分子预官能化的固体表面。功能化以交替的条纹图案进行，使得珠的结合形成固体衍射光栅。结合珠的照射形成衍射模式，其强度揭示珠的存在，并因此揭示捕获的靶分子。检测系统不需要任何额外的扩增步骤或用于信号传导的荧光或放射性标记。该研究有2个PI和3个具体目标。目的1是发展一个数学模型来预测基于微珠的衍射系统的光学行为。该模型将基于蒙特卡罗模拟，并将预测最佳系统参数（例如：珠尺寸、光栅周期性、表面材料、折射率）以获得最大灵敏度。目标2是实际设置的开发，它将与目标1同时开始，但随后将其结果与实验设置相适应。目标2还将在受体和配体浓度以及背景稀释水平方面通过实验优化系统，并探索检测限。目的3是应用该系统检测一种已知的肿瘤标志物：血管内皮生长因子（VEGF）在复杂的混合物，如血清和细胞裂解物。这一目标将探索该系统的“临床实用性”，目标是实现500 fM的检测灵敏度，这是一种将癌症患者与健康人区分开来的浓度变化。

DESCRIPTION (provided by applicant): The ability to detect biomolecules in minute quantities is of paramount importance both in biological research and in clinics. Quantitative detection of biomolecules in a specific sample can reveal significant information ranging from cell decision making processes to state of a specific disease. For example, the disease of cancer, that is responsible for half-million deaths in the U.S. every year, can potentially be cured if diagnosed early. One way to perform the diagnosis is to detect tumor markers in body fluids. For a successful early diagnosis, the detection platform must certainly be sensitive and reveal the presence of small amounts of target molecules. The detection platform must also be miniaturizable, quick, relatively simple to operate and inexpensive to enable 1) high- throughput detection and 2) sufficient number of detection platforms in a setting (clinics, research laboratory). Most detection platforms that currently exist require the use of labeling molecules such as fluorescent and radio labels. This increases time, cost and required expertise for operation and renders these label-based platforms not necessarily suitable for miniaturization and high-throughput detection. This proposal covers the development of a biomolecular detection platform that is fluorescence and radio labelfree, simple to fabricate and use, robust and at the same time more sensitive than many existing detection platforms. The proposed system is based on immunomagnetic separation and diffractometry whose combination has not yet been explored. Functionalized magnetic beads are used to capture target molecules from solutions. After this step, the beads (that contain the captured molecules on their surfaces) are simply exposed to a solid surface that is pre-functionalized with secondary receptor molecules. The functionalization is done in an alternating stripe pattern, so that the binding of the beads forms a solid diffraction grating. The illumination of the bound beads forms diffraction modes whose intensities reveal the presence of the beads, and hence the target molecules captured. The detection system does not require any additional amplification steps or fluorescence or radio labels for signaling. The study has 2 PIs and 3 specific aims. Aim 1 is the development of a mathematical model to predict the optical behavior of a bead-based diffraction system. The model will be based on a Monte Carlo simulation and will predict the optimal system parameters (eg. bead size, grating periodicity, surface material, refractive indices) for maximum sensitivity. Aim 2 is the development of the actual setup which will start simultaneously with Aim 1 but later on adapt its results to the experimental setup. Aim 2 will also optimize the system experimentally in terms of receptor and ligand concentrations and background dilutions levels, and explore the limits of detection. Aim 3 is the application of the system to detection of a known tumor marker: vascular endothelial growth factor (VEGF) in complex mixtures such as sera and cell lysate. This aim will explore the "clinical usefulness" of the system with the goal of achieving 500 fM detection sensitivity, a concentration change that distinguishes cancer patients from healthy people.