Barcoded Hydrogel Microparticles and Scanner for Multiplexed Biomolecule Assays

描述(由申请人提供)：多重筛查是一种在药物发现、基因分型、医疗诊断和输血安全的应用中广泛使用的工具，在未来的“个性化”医学领域将是至关重要的。这两种商业上可用的筛选技术要么提供高“密度”的被测分析物(即平面微阵列)，要么提供高样品吞吐量(即。基于珠的系统)，但不能两者兼而有之。这一应用提出了一种基于多功能编码颗粒的新筛选技术的基础发展，该技术可以提供微阵列的密度和基于微珠的系统的吞吐量。初步结果表明，由海绵状水凝胶材料组成的颗粒，一半写着穿孔代码条形码，另一半写着捕捉目标的条纹，可以用来同时对单个生物样本中的目标进行量化，编码能力超过100万。在概念验证演示的基础上，假设(1)增加水凝胶结构中的孔大小将允许靶标在颗粒中结合，从而提高每次检测的灵敏度，同时减少所需的孵育时间；(2)可以调整杂交条件，以实现与现有技术的性能竞争；以及(3)可以使用基于微流控和光电倍增技术的穿透系统来快速扫描颗粒(即读取代码和量化靶标)。该项目的具体目标是：(1)通过探索化学变化和工艺条件来增强颗粒合成，以产生足够多孔和机械坚固的颗粒。颗粒将通过显微镜进行检查，并用FITC标记的右旋糖苷进行探测。(2)优化DNA杂交检测的物理和化学条件，以最大限度地提高灵敏度、特异性和重复性。(3)开发了一套基于微流控流动的快速扫描系统，该系统集成了流动聚焦微流控装置、光电倍增管辅助荧光检测和采集信号的解码软件。该项目的最终目标是拥有一个能够量化每个样本2500个核酸目标的系统，能够以比商业上可用的系统更高的灵敏度用单碱基对分辨来探测目标，并以每分钟500个粒子的速度扫描5000个粒子。该项目与公共卫生的相关性在于开发了一种从疾病诊断到药物发现的基因组医学变革性技术。与公共卫生相关：该项目将开发一种新技术，可用于同时检测溶液中的数千种生物分子。这项新技术将在疾病诊断/治疗、提高输血安全性的血液分型和药物开发方面找到潜在的用途。

DESCRIPTION (provided by applicant): Multiplexed screening is a tool that finds broad use in applications such as drug discovery, genotyping, medical diagnostics, and blood typing for transfusion safety, and will be of utmost importance in the up-and-coming field of "personalized" medicine. The two commercially available screening technologies offer either a high "density" of analytes measured (i.e. planar microarrays) or high sample throughput (ie. bead-based systems), but not both. This application proposes the fundamental development of a new screening technology, based on multi-functional encoded particles, which could provide the density of microarrays and throughput of bead-based systems. Preliminary results show that particles composed of a spongy hydrogel material, with a punch-code barcode written on one half and a stripe for target capture on the other, can be used to simultaneously quantify targets in a single biological sample, with coding capabilities of over one million. In building upon a proof-of-concept demonstration, it is hypothesized that (1) increasing the size of pores in the hydrogel structure will allow targets to bind throughout the particle, increasing the sensitivity of each assay while decreasing required incubation times; (2) that hybridization conditions can be tuned to achieve performance competitive with existing technologies; and that (3) a flow-through system based on microfluidics and photomultiplier technologies can be used to rapidly scan particles (i.e. read codes and quantify targets). The specific aims of the project are: (1) Enhance particle synthesis by exploring chemical variations and processing conditions to generate particles that are sufficiently porous and mechanically robust. Particles will be examined via microscopy and probed with FITC-conjugated dextrans. (2) Optimize the physical and chemical conditions of DNA hybridization assays to maximize sensitivity, specificity, and reproducibility. (3) Develop a microfluidic flow-based system for rapid scanning that integrates a flow-focusing microfluidic device, photomultiplier-aided fluorescence detection, and software to decode the acquired signal. The end goal of this project is to have a system capable of quantifying 2,500 nucleic acid targets per sample, detecting targets with single base-pair discrimination at a better sensitivity than commercially available systems, and scanning 5,000 particles at a rate of 500 particles per minute. The relevance of this project to public health is the development of a transformative technology for genomic medicine, ranging from disease diagnosis to drug discovery. PUBLIC HEALTH RELEVANCE: This project will develop a new technology that can be used to simultaneously detect thousands of biomolecules in a solution. This new technology will find potential use in disease diagnosis/treatment, blood typing for increasing the safety of transfusions and drug development.