Diagnostics on demand: a biosensor platform for multiplexed small molecule detection

小分子检测在许多生物学、医学和法律的领域中是核心， 包括基础研究、临床诊断、环境监测和工作场所用药 测试，以及其他应用。部署最广泛的诊断程序包括 基于免疫测定，其利用针对配体-蛋白质缀合物产生的抗体。这些 易于使用的检测试剂盒可实现多种应用，如即时诊断、家庭 测试和现场实时环境监测。虽然小分子 免疫测定是强大的，它们是耗时和昂贵的开发，因为类似物 适合与免疫原性载体蛋白缀合的靶分子的量必须 化学合成的新方法，使小分子的常规创造 使用天然分子的传感器将从根本上提高速度并降低成本 需要开发新的诊断测试。建议的工作通过构建一个 这项技术将使开发新的小分子生物传感器变得简单可靠， 开发新的抗体我们会用一个多功能的新型传感支架来完成- 植物脱落酸受体PYR 1。该受体参与化学诱导的 与其结合配偶体HAB 1二聚化。我们最近描述了一种定向进化 用于创建PYR 1/HAB 1二聚化（PAIR）传感器的管道，并且已经创建了用于 116种小分子，包括Δ9-THC，20种FDA批准的药物，以及数十种天然药物。 产品.这些传感器可用于创建配体调节的遗传电路， 配体介导的分裂酶的重建，并迅速创建灵敏的诊断测试。 虽然我们的平台功能强大，但命中率，吞吐量和化学成分的改进 它可以访问的空间需要授权高效传感器开发;实现 为此，我们将结合联合收割机应变工程，高通量筛选和计算设计。 我们改进的管道将用于1步分离>1000个中等亲和力的传感器， FDA批准的药物和其他医学相关的小分子。其中100个将是 通过随后的定向进化轮进化为高亲和力（nM）传感器。同时， 我们将开发将这些传感器转换为多路诊断的方法。的 开发的技术将为开发传感器提供新的工具和方法， 用户指定的分子，并打开用户指定的化学调节过程的大门， 将具有广泛的生物医学相关性，并将推动生物医学研究和临床， 环境诊断学

Small molecule detection is central in many biological, medical, and legal domains, including basic research, clinical diagnostics, environmental monitoring, and workplace drug testing, among other applications. The most widely deployed diagnostics are immunoassay-based that utilize antibodies raised against ligand-protein conjugates. These easy-to-use assays enable applications as diverse as point-of-care diagnostics, in-home testing, and real-time environmental monitoring in the field. Although small molecule immunoassays are powerful, they are time-consuming and costly to develop because analogs of target molecules suitable for conjugation to immunogenic carrier proteins must be chemically synthesized. New methods that enable the routine creation of small molecule sensors using native molecules would radically increase the speed and decrease the costs required to develop new diagnostic tests. The proposed work addresses this by building a technology that will make developing new small molecule biosensors as easy and reliable as developing new antibodies. We will accomplish this using a versatile new sensing scaffold – the plant abscisic acid receptor PYR1. This receptor participates in chemical-induced dimerization with its binding partner, HAB1. We recently described a directed evolution pipeline for creating PYR1/HAB1 dimerization (PAIR) sensors and have created sensors for 116 small molecules, including Δ9-THC, 20 FDA-approved drugs, and dozens of natural products. These sensors can be used to create ligand-regulated genetic circuits, drive ligand-mediated reconstitution of split enzymes, and rapidly create sensitive diagnostic tests. While our platform is powerful, improvements in hit rate, throughput, and the chemical space it can access are needed to empower high-efficiency sensor development; to achieve this, we will combine strain engineering, high-throughput screening, and computational design. Our improved pipeline will be used for 1-step isolation of >1000 moderate-affinity sensors of FDA-approved drugs and other medically-relevant small molecules. 100 of these will be evolved to high-affinity (nM) sensors by subsequent rounds of directed evolution. In parallel, we will develop methods for converting these sensors into multiplexable diagnostics. The technology developed will deliver new tools and methods for developing sensors of user-specified molecules and open the door to user-specified chemical-regulated processes, will have broad biomedical relevance and will advance biomedical research and clinical and environmental diagnostics.