PFI:AIR-RA: Commercializing of a Molecular Analysis Platform for Plant Genomics in an Industry/University Ecosystem

这个PFI: AIR研究联盟项目的重点是翻译和转移一个集成的基于微流体的筛选和测试分析平台，该平台有可能显著提高育种植物的效率和有效性。在当前的农业生物技术产业中，对植物细胞复杂群落的遗传分析通常以均质化、全群落的方式进行，其输出是关于细胞种群平均状态的全球信息。这可能掩盖了细胞异质性的真实程度，这种异质性是通过平均属性和潜在的丢失有趣的异常值来呈现的。在单细胞水平上的分子分析将有助于更好地开发在各种条件下茁壮成长的植物细胞的增强型菌株。因此，需要一种工具，能够在大群体中提供单细胞分子分析，能够在保持异质性信息的同时实现高通量，并为农业遗传学产业量身定制。该项目的目标是通过应用来自NSF I/UCRC“集成微流体先进设计和制造中心”（CADMIM）的用于分子和单细胞分析的微流控芯片实验室技术来开发这样一种工具，该中心利用了用于细胞和分子处理和检测的微流控设备的设计、微加工和制造方面的专业知识和发现。所提出的技术不仅能够实现单细胞分析，而且与目前的方法相比，它还具有将每个分子测试的成本降低一个数量级的潜力。目前在农业生物技术行业中使用的技术依赖于离散的、笨重的机器人系统，这是时间和劳动密集型的，需要大量的电力、样品和试剂，导致每次测试分析大量样品的成本要高得多。将加强的创新生态系统包括CADMIM、加州大学欧文分校、约翰霍普金斯大学和杜邦先锋。加州大学欧文分校的CADMIM站点带来了微流体细胞操作，裂解和微尺度流体处理专业知识。约翰霍普金斯大学带来了微流控分子处理和检测专业知识。杜邦先锋是全球领先的植物遗传学开发商和供应商，为农民带来了植物基因组专业知识和农业生物技术业务知识。来自两所大学的学生和博士后将通过与杜邦先锋技术团队的频繁互动，获得创业和技术翻译经验，以了解植物基因分型生物技术的当前局限性，跨大陆的国际公司如何解决大规模问题，颠覆性技术取代当前工作技术范式所需的条件。以及跨学科团队如何沟通以开发前沿解决方案。该项目解决了从研究发现到商业应用的几个技术差距。植物细胞基因型筛选需要数百万细胞水平的验证测试，以及随后对特定细胞类型的进一步大规模分子分析。目前的植物细胞处理和分子分析平台由多个笨重且昂贵的细胞处理机器组成，这些机器既耗时又依赖于熟练的实验室工作人员按顺序操作。这极大地限制了被分析细胞的吞吐量，并且使得需要现场植物种子采样和测试的应用成本过高。随着该项目开发的集成微流控技术，多个步骤将集成在一个紧凑且可能便携式的平台上，使用更小的液体体积来获得更快的结果。

This PFI: AIR Research Alliance project focuses on the translation and transfer of an integrated microfluidic based screening and testing analysis platform that has the potential to dramatically improve the efficiency and effectiveness of breeding plants. In the current agriculture biotech industry, genetic analyses of complex communities of plant cells are typically carried out in a homogenized, whole community fashion where the output is global information on the average state of the population of cells. This can mask the true extent of cellular heterogeneity that is present by averaging properties and potentially missing intriguing outliers. Molecular analysis at the single cell level would lead to better capability to develop enhanced strains of plant cells that thrive under various conditions. Thus there is a need for a tool that can provide single cell-molecular analysis in large populations, capable of high throughput while preserving heterogeneity information, and tailored to the agriculture genetics industry. The goal of this project is to develop such a tool by applying microfluidic lab-on-a-chip technologies for molecular and single cell analyses derived from the NSF I/UCRC "Center for Advanced Design and Manufacturing of Integrated Microfluidics" (CADMIM) where expertise and discoveries in the design, microfabrication, and manufacturing of microfluidic devices for cellular and molecular processing and detection are utilized. Not only will the proposed technology enable the single cell analysis, it also has the potential to lower the cost per molecular test by an order of magnitude over current methodologies. Current technology used in the agriculture biotech industry relies on discrete, bulky robotic systems that are time and labor intensive and require larger amounts of power, sample, and reagents resulting in much higher cost per test to analyze large numbers of samples.The innovation ecosystem that will be enhanced includes CADMIM, UC Irvine, Johns Hopkins University, and DuPont Pioneer. The UC Irvine site of CADMIM brings microfluidic cellular manipulation, lysing, and micro-scale fluidic handling expertise. Johns Hopkins University brings microfluidic molecular processing and detection expertise. DuPont Pioneer, a leading developer and supplier of plant genetics to farmers worldwide, brings the plant genomic expertise and agriculture biotech business knowledge. Students and post-doctoral fellows from both universities will gain entrepreneurial and technology translation experience through their frequent interactions with DuPont Pioneer's technical team in understanding the current limitations in plant genotyping biotechnology, how a multi-continent international company works to solve large-scale problems, what is required for disruptive technology to displace current working technology paradigms, and how interdisciplinary teams communicate to develop cutting edge solutions.This project addresses several technology gaps as it translates from research discovery toward commercial application. Plant cell genotype screening requires millions of cell-level verification tests and subsequent further massive molecular analyses of specific cell types. The current plant cell processing and molecular analysis platforms consist of multiple cumbersome and expensive cell processing machines that are time consuming and rely on skilled lab workers operating them in sequence. This greatly limits the throughput of cells being analyzed and makes it cost prohibitive for applications that require field-based plant seed sampling and testing. With the integrated microfluidic technologies developed in this project, the multiple steps will be integrated on a compact and potentially portable platform, using smaller liquid volumes to obtain faster results.