Ultrahigh Resolution Optical Barcode

描述（由申请人提供）：随着对高通量和低成本反应的需求的增加，多路复用技术在生物学和生物化学领域越来越受欢迎。光复用包括将已知的生物探针连接到具有已知光码的头上，该头允许通过读取其唯一的光码来对池样中的每个探针进行光学识别。在随后的步骤中，染料标记目标的发射与探针身份相关联，以确定所讨论的反应的程度。目前用于光复用的两类材料是荧光有机染料和量子点，但目前只有不到100种光学代码可用于商业（例如来自Luminex）。对于第一阶段的工作，我们假设，如果有机染料的宽发射（在FWHM时高达30-60 nm）被窄发射体（如稀土元素，通常显示2-10 nm范围内的发射峰宽度）所取代，则有可能获得更高分辨率的光学编码。在第二阶段，我们证明了在理想条件下，通过使用已知数量的稀土基平行材料（可发射多达六种颜色的光复用），可以解析多达10亿个光代码。与当前基于流式细胞仪的系统相比，该系统的优点包括：每个发射器的光谱离散发射，通过使用可变发射器浓度实现非常高水平的多路复用，高量子效率，优异的光稳定性，可变粒径，低成本，自动化并行合成。非常重要的是，我们还开发了一种完全便携式，电池供电的原型头部读取器（多路分析读取系统，或“MARS”），具有超亮led和基于非常便宜的商用单反相机的成像系统。为响应NHGRI主持下的复杂仪器竞争性更新应用的具体项目公告PA-08-115， Parallel Synthesis提出基于Parallume平台构建一个完整的后期原型，该平台由Parallume微管、MARS硬件和软件组成。具体来说，我们建议(1)为Parallume珠开发一种自动化的并行合成方法，以满足编码集的要求；(2)创建两个自动化的第二代MARS原型，并将它们交付给合作者进行beta测试，从而获得反馈，以创建原型的最终版本；(3)创建用于MARS的低成本珠定位幻灯片（BLS），以实现高密度的珠图像。(4)完成一个软件包来控制MARS并分析MARS的数据输出，也将与MARS一起进行beta测试；(5)通过将我们的恰加斯病诊断方法与使用Luminex的已知诊断方法进行比较，测试完整的功能系统。在三年的开发期结束时，Parallel Synthesis将拥有一个功能齐全的G3 MARS，具有用户友好的软件以及BLS和编码的parallelme珠作为消耗品。该平台为多重分析开发和复杂生物成像和可视化领域提供了一种全新的技术，将面向潜在的战略合作伙伴、被许可方或直接销售给最终用户。这种低成本的系统非常适合低资源设置的应用，如发展中国家。公共卫生相关性：如果能够同时进行多种反应，在发达国家和资源匮乏环境中使用的检测方法都将大大受益于成本降低和产量增加。为了一次处理许多汇集的样本，必须有一种区分单个样本的方法。这个提议描述了一种方法，通过这种方法，每个头在激光激发时发出独特的光学特征。每个珠的光学签名允许珠的含量和测定期间的反应程度的测定。用这种方法可以解析成千上万的光码。通过便携式检测器/头读卡器读取光学特征，可以廉价地执行和并行分析数千种分析。

DESCRIPTION (provided by applicant): Multiplexing technologies have steadily gained in popularity in the fields of biology and biochemistry as the desire to perform higher-throughput and lower-cost reactions has increased. Optical multiplexing consists of attaching a known biological probe to a bead with a known optical code which allows the optical identification of each probe in the pooled sample by reading its unique optical code. In a subsequent step, the emission of a dye-labeled target is associated with the probe identity to determine the extent of the reaction in question. The two classes of materials currently used for optical multiplexing are fluorescent organic dyes and Quantum Dots but only <100 optical codes are currently available commercially (e.g. from Luminex). For the Phase I effort we hypothesized that it would be possible to obtain far more resolvable optical codes if the broad emission from organic dyes (up to 30-60 nm at FWHM) were replaced with narrow emitters such as the rare earth elements which often display emission peak widths in the 2-10 nm range. In Phase II we demonstrated that it was possible to resolve up to a billion optical codes under ideal conditions by using known amounts of rare-earth-based Parallume materials which emit up to six colors for optical multiplexing. The advantages of this system over the current flow cytometer-based systems include: spectrally discrete emission from each of the emitters, a very high level of multiplexing available through the use of variable emitter concentrations, high quantum efficiency, excellent photostability, variable particle size, and a low cost, automated parallel synthesis. Very importantly, we have also developed a completely portable, battery-powered prototype bead reader (Multiplexed Assay Reader System, or "MARS") with on superbright LEDs with an imaging system based on a very inexpensive commercial DSLR camera. In response to the specific Program Announcement, PA-08-115, for Competing Renewal applications for Complex Instrumentation under the auspices of the NHGRI, Parallel Synthesis proposes to build a complete late-stage prototype based on the Parallume platform consisting of the Parallume beads, the MARS hardware and software. Specifically, we propose to (1) develop an automated, parallel synthesis methodology for the Parallume beads to fulfill encoded set requirements, (2) create two automated, second-generation MARS prototypes and deliver them to collaborators for beta- testing resulting in feedback to create a final version of the prototype, (3) create a low-cost bead localization slide (BLS) for use in the MARS to enable high-density bead images, (4) complete a software package to control the MARS and analyze the data output from the MARS, also to be beta-tested along with the MARS, and (5) test the completely functional system by comparing our diagnostic assay for Chagas disease against a known diagnostic assay using Luminex. At the end of the three-year development period, Parallel Synthesis will have a fully functional G3 MARS with user- friendly software along with the BLS and encoded Parallume beads as consumables. This platform, offering a completely new technology for the fields of multiplex assay development and complex biological imaging and visualization, will be available for potential strategic partners, licensees, or for direct sale to end-users. This low cost system is ideally suited for low resource setting applications such as developing countries. PUBLIC HEALTH RELEVANCE: Assays used in both the developed countries and low resource settings would greatly benefit from reduced costs and increased throughput if it were possible to perform many reactions simultaneously. In order to process many pooled samples at once it is necessary to have a means of distinguishing the individual samples. This proposal describes a method by which each bead emits a unique optical signature upon excitation with a laser. The optical signature of each bead allows the determination of the bead's content and the extent of reaction during the assay. It is possible to resolve many thousands of optical codes by this method. Reading of the optical signatures by a portable detector/bead reader allows thousands of assays to be inexpensively performed and analyzed in parallel.