Ultrahigh Resolution Optical Barcode

描述(申请人提供)：随着对更高产量和更低成本反应的需求的增加，多路复用技术在生物和生物化学领域得到了稳步的普及。光学多路传输包括将已知的生物探针连接到具有已知光学代码的珠子上，该已知光学代码允许通过读取其唯一的光学代码来光学识别汇集样本中的每个探针。在接下来的步骤中，染料标记目标的发射与探针特性相关联，以确定所讨论的反应的程度。目前用于光学多路复用的两类材料是荧光有机染料和量子点，但目前只有&lt；100光学编码在商业上可用(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(在半高宽时高达30-60 nm)被窄发射体取代，如稀土元素，其发射峰宽通常在2-10 nm范围内，那么将有可能获得更可分辨的光码。在第二阶段，我们展示了在理想的条件下，通过使用已知数量的稀土基Parallume材料来解析高达10亿个光码是可能的，这些材料发射多达六种颜色用于光学多路复用。与当前基于流式细胞仪的系统相比，该系统的优势包括：每个发射器的光谱离散发射，通过使用可变发射器浓度可获得非常高水平的多路复用，高量子效率，出色的光稳定性，可变的颗粒大小，以及低成本的自动并行合成。非常重要的是，我们还开发了一款完全便携、电池供电的样机磁珠阅读器(Multiplexed Assay Reader System，简称“MARS”)，带有超高亮度LED和基于非常便宜的商用单反相机的成像系统。为了回应具体的计划公告，PA-08-115，在NHGRI的支持下，针对复杂仪器的竞争更新应用，并行合成建议基于Parallume平台构建一个完整的后期原型，该平台由Parallume珠子、MARS硬件和软件组成。具体地说，我们建议(1)为Parallume珠子开发一种自动化的并行合成方法以满足编码集要求，(2)创建两个自动化的第二代火星原型并将它们交付给合作者进行Beta测试，从而产生反馈以创建原型的最终版本，(3)创建用于火星上的低成本珠子定位幻灯片(BLS)以实现高密度珠子图像，(4)完成一个软件包来控制火星并分析来自火星的数据输出，也将与火星一起进行Beta测试，以及(5)通过将我们的恰加斯病诊断试剂盒与已知的Luminex诊断试剂盒进行比较，测试完整的功能系统。在三年的开发期结束时，并行合成将拥有一个功能齐全的G3火星，带有用户友好的软件，以及BLS和编码的Parallume珠子作为消耗品。这一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，将向潜在的战略合作伙伴、被许可方提供，或直接销售给最终用户。这种低成本的系统非常适合发展中国家等低资源环境的应用。公共卫生相关性：如果可以同时进行许多反应，发达国家和低资源环境中使用的分析将从降低成本和增加产量中受益匪浅。为了一次处理许多混合样品，有必要有一种方法来区分单独的样品。该方案描述了一种方法，通过该方法，每个珠子在用激光激励时发出唯一的光学签名。每个珠子的光学标记允许在分析过程中确定珠子的含量和反应程度。通过这种方法可以解析数以千计的光码。通过便携式检测器/珠粒读取器读取光学特征允许以低成本并行执行和分析数千次分析。

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.