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，