Biotechnology Resource Center of Biomodular Multi scale Systems CBM2 for Precision Molecular Diagnostics

摘要 构建完全可操作的混合规模系统，用于从循环中进行精确分子分析 标记物对于许多疾病状态的有效管理具有巨大的吸引力。混合尺度系统（mm → nm）为疾病诊断/预后提供了一些有吸引力的机会，例如， 从血液中提取必要的标记物（毫升; mL），然后使用数字计数提取分子信息 技术（亚飞升; fL），用于以高度复用的方式提供定量数据。比如说， 分离携带mRNA货物的疾病特异性生物细胞或外来体可用于提供 可用于辅助脑损伤的诊断和预后的物质，如mRNA， 中风，这表明mRNA表达差异，在某些白细胞亚型造成的组织 脑损伤然而，要建立这样的系统，可以跨越各种规模，差距， 必须解决体积处理中的问题;从1 mL血液输入中分离循环标志物需要 体积减少到1 fL，用于有效的分子规模分析，体积减少1012倍。综合混合- 提出了一种规模模块化流体系统来解决这些挑战;一种通用的分子处理系统， 系统- uMPS。uMPS被配置为类似于个人计算机的配置，其中流体母板 在用户定义的配置中，使用装配到主板上的任务特定模块进行填充。的 模块由热塑性塑料制成，并包含适合提供有效操作的结构 模块的预期应用。这些模块可以以低成本的高规模生产模式生产， 成本和高保真度，适用于体外诊断，使用热塑性基底。uMPS 需要适当的封装和组装技术来互连必要的处理模块， 例如用于模块和母板之间互连的有效密封策略， 用于输送流体通过系统的防漏、对准结构、阀门和泵送单元，以及 支持外围设备，以识别由指示 用于提供临床数据的特定生物标志物的存在。所提出的uMPS的独特之处在于能够 直接从全血（≥1 mL）中分离出多种循环标志物类型， 将分子标记物测量到sub-nL体积，并以sub-femtoliter体积读出分子标记物的存在。那里 为uMPS设想了许多配置格式，但将提供一个工作演示， 来自血液传播的生物细胞和/或亚纳米囊泡（外来体）的表达谱mRNA。的 系统将直接处理全血，亲和选择相关循环标志物，收获mRNA 形成循环标记物并对某些 成绩单相关转录本的表达将通过spLDR的分子计数来推断 使用靶的分子依赖性特征的产品;电泳飞行时间从 spLDR产物通过长度为50 x 50 nm和100 µm的纳米飞行管的运输。

Abstract Building fully operational mixed-scale systems for performing precision molecular analysis from circulating markers has great appeal for the effective management of many disease states. Mixed-scale systems (mm → nm) provides some attractive opportunities for disease diagnosis/prognosis, for example securing the necessary markers from blood (milliliters; mL), and then extract molecular information using digital counting techniques (sub-femtoliters; fL) for supplying quantitative data in a highly multiplexed fashion. For example, isolating disease-specific biological cells or exosomes that carry mRNA cargos can be used for supplying material, such as mRNA, that can be used to assist in both the diagnosis and prognosis of brain damage, such as stroke, which shows mRNA expression differences in certain leukocyte sub-types resulting from tissue damage in the brain. However, to build such systems that can cut across a diverse range of scales, disparities in volume processing must be addressed; isolating circulating markers from a 1 mL blood input requires a volume reduction to 1 fL for efficient molecular-scale analyses, a 1012-fold volume reduction. Integrated mixed- scale modular fluidic systems are proposed to address these challenges; a universal Molecular Processing System – uMPS. The uMPS is configured similar to that of a personal computer in which a fluidic motherboard is populated with task-specific modules assembled to the motherboard in a user-defined configuration. The modules are fabricated in thermoplastics and contain structures appropriate to provide efficient operation of the module for the intended application. The modules can be produced in a high-scale production mode at low- cost and with high fidelity, appropriate for in vitro diagnostics, using thermoplastic substrates. The uMPS requires appropriate packaging and assembly technologies to interconnect the necessary processing modules, such as efficient sealing strategies for interconnections between the module and the motherboard that are leak-proof, alignment structures, valving and pumping units for transporting fluids through the system and support peripherals to transduce the necessary signals arising from single molecule signatures indicative of the presence of a particular biomarker used to provide clinical data. Unique to the proposed uMPS is the ability to isolate a number of circulating marker types directly from whole blood (≥1 mL), concentrate the relevant markers to sub-nL volumes and readout the presence of molecular markers in sub-femtoliter volumes. There are many configurational formats envisioned for the uMPS, but a working demonstration will be offered to expression profile mRNA from blood-borne biological cells and/or sub-nanometer vesicles (exosomes). The system will process whole blood directly, affinity select the relevant circulating markers, harvest the mRNA form the circulating markers and perform solid-phase ligase detection reactions (spLDRs) on certain transcripts. The expression of the relevant transcripts will be deduce from molecular counting of spLDR products using a molecular-dependent signature of the target; the electrophoretic flight time deduce from the transport of the spLDR product through a nanometer flight tube measuring 50 x 50 nm and 100 µm in length.