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

按需诊断：用于多重小分子检测的生物传感器平台

• 批准号: 10720755
• 负责人: Sean Cutler
• 金额: $ 43.6万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10720755
• 关键词: Abscisic Acid; Acceleration; Acids; Address; Affinity; Antibodies; Basic Science; Binding; Biochemical; Biological; Biological Assay; Biomedical Research; Biosensor; Biotechnology; Carrier Proteins; Chemicals; Chemistry; Clinical; Consumption; Deposition; Detection; Development; Diagnostic; Diagnostic Reagent Kits; Diagnostic tests; Dimerization; Directed Molecular Evolution; Engineering; Environmental Monitoring; Enzymes; FDA approved; Genetic; Home; Immobilization; Immunoassay; Kinetics; Legal; Libraries; Ligands; Mediating; Medical; Methods; Mutation; Natural Products; Pharmaceutical Preparations; Plants; Process; Protein Engineering; Proteins; Receptor Activation; Research; Specific qualifier value; Speed; Substance of Abuse; Surface; Technology; Testing; Therapeutic; Time; Work; Workplace; analog; chemical synthesis; clinical diagnostics; computational pipelines; cost; design; detection limit; detection platform; drug testing; empowerment; experience; high throughput screening; home test; immunogenic; improved; interest; meter; multiplex diagnostics; new technology; novel diagnostics; parallelization; point-of-care diagnostics; public repository; receptor; reconstitution; scaffold; sensor; small molecule; tool

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.

小分子检测是许多生物、医学和法律领域的核心， 包括基础研究、临床诊断、环境监测和工作场所药物 测试等应用程序。应用最广泛的诊断是 基于免疫测定，利用针对配体-蛋白质缀合物产生的抗体。这些 易于使用的检测方法可实现多种应用，如现场护理诊断、家庭诊断 测试和现场实时环境监测。虽然分子小 免疫测定很强大，但开发起来既耗时又昂贵，因为类似物 适合与免疫原性载体蛋白缀合的靶分子必须是 化学合成的。能够常规创建小分子的新方法 使用天然分子的传感器将从根本上提高速度并降低成本 需要开发新的诊断测试。拟议的工作通过建立一个 技术将使开发新的小分子生物传感器变得像 开发新的抗体。我们将使用多功能的新型传感支架来实现这一目标 - 植物脱落酸受体 PYR1。该受体参与化学诱导的 与其结合伙伴 HAB1 形成二聚体。我们最近描述了定向进化 用于创建 PYR1/HAB1 二聚 (PAIR) 传感器的管道，并已创建用于 包括 Δ9-THC 在内的 116 种小分子、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、驱动 配体介导的裂解酶重建，并快速创建敏感的诊断测试。 虽然我们的平台功能强大，但命中率、吞吐量和化学物质方面的改进 需要它可以访问的空间来支持高效传感器的开发；达到 为此，我们将结合应变工程、高通量筛选和计算设计。 我们改进的管道将用于对超过 1000 个中等亲和力传感器进行一步隔离 FDA 批准的药物和其他医学相关的小分子。其中 100 个将是 通过后续几轮定向进化进化为高亲和力（nM）传感器。并联， 我们将开发将这些传感器转换为多路诊断的方法。这 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，并将推进生物医学研究和临床和 环境诊断。