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

小分子检测在许多生物、医学和法律领域都很重要， 包括基础研究、临床诊断、环境监测和工作场所药物 测试以及其他应用程序。部署最广泛的诊断程序是 以免疫分析为基础，利用针对配体-蛋白质结合物产生的抗体。这些 易于使用的分析支持多种应用程序，从看护点诊断、家庭 测试和现场实时环境监测。虽然小分子 免疫分析是强大的，它们的开发既耗时又昂贵，因为类似的 适合与免疫原性载体蛋白偶联的靶分子必须是 化学合成的。使小分子的常规产生成为可能的新方法 使用天然分子的传感器将从根本上提高速度并降低成本 开发新的诊断测试所需的。拟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像 开发新的抗体。我们将使用一种多功能的新型传感支架来实现这一点- 植物脱落酸受体PYR1。该受体参与化学诱导 与其结合伙伴HAB1的二聚化。我们最近描述了一种定向进化 用于创建PYR1/HAB1二聚(对)传感器的流水线，并已为 116个小分子，包括Δ9-THC，20种FDA批准的药物，以及数十种天然药物 产品。这些传感器可以用来创建配体调节的遗传电路，驱动 配基介导的裂解酶重组，并快速创建敏感的诊断测试。 虽然我们的平台功能强大，但在命中率、吞吐量和化学品方面的改进 需要它可以进入的空间来支持高效传感器的开发；要实现 为此，我们将结合菌株工程、高通量筛选和计算设计。 我们改进的管道将用于一步分离&gt；1000中等亲和力传感器 FDA批准的药物和其他与医学相关的小分子。其中的100个将是 通过随后几轮的定向进化进化成高亲和力(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。这个 所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，并将促进生物医学研究和临床 环境诊断。

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.