Immunomagnetic Diffractometry for Rapid Biomolecular Detection

描述(申请人提供)：检测微量生物分子的能力在生物学研究和临床上都是极其重要的。对特定样本中生物分子的定量检测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，每年在美国造成50万人死亡的癌症，如果及早诊断，可能是可以治愈的。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，检测平台当然必须灵敏，并揭示少量目标分子的存在。检测平台还必须是可小型化的、快速的、操作相对简单和廉价的，以实现1)高通量检测和2)在一个环境(诊所、研究实验室)中有足够数量的检测平台。目前存在的大多数检测平台都需要使用标记分子，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，并使得这些基于标签的平台不一定适合小型化和高通量检测。这项建议包括开发一种生物分子检测平台，该平台无需荧光和放射性标记，制造和使用简单，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分离和衍射法，它们的组合还没有被探索。功能化磁珠用于从溶液中捕获目标分子。在这一步之后，珠子(在它们的表面包含捕获的分子)简单地暴露在一个固体表面，该固体表面预先用二级受体分子进行了功能化。功能化是以交替的条纹图案完成的，因此珠子的结合形成了固体衍射光栅。结合的珠子的照射形成了衍射模，其强度揭示了珠子的存在，从而捕获了目标分子。该检测系统不需要任何额外的放大步骤或荧光或无线电标记来发送信号。这项研究有2个绩效指标和3个具体目标。目标1是开发一种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，并将预测最优系统参数(例如，磁珠大小、光栅周期、表面材料、折射率)，以实现最高灵敏度。目标2是实际设置的开发，它将与目标1同时开始，但稍后将其结果应用于实验设置。Aim 2还将在受体和配体浓度以及背景稀释度方面对系统进行实验优化，并探索检测限。目的3应用该系统检测血清和细胞裂解物等复杂混合物中已知的肿瘤标志物：血管内皮生长因子(VEGF)。这一目标将探索该系统的“临床实用性”，目标是达到500 FM检测灵敏度，这是一种区分癌症患者和健康人的浓度变化。

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.