De novo design of a generalizable protein biosensor platform for point-of-care testing

用于即时测试的通用蛋白质生物传感器平台的从头设计

• 批准号: 10448046
• 负责人: Hsien Wei YEH
• 金额: $ 8.02万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10448046
• 关键词: 2019-nCoV; Address; Affinity; Algorithms; Antibodies; Area; Basic Science; Binding; Binding Proteins; Biological Assay; Biological Markers; Bioluminescence; Biosensor; Body Fluids; Botulinum Toxins; Bypass; COVID-19; COVID-19 pandemic; Cardiac; Caring; Centers for Disease Control and Prevention (U.S.); Clinic; Clinical; Collection; Color; Colorimetry; Consumption; Coupled; Coupling; Creativeness; Detection; Development; Development Plans; Devices; Diagnostic; Diagnostic Reagent; Diagnostic Services; Directed Molecular Evolution; Disease; Drops; ERBB2 gene; Economics; Energy Transfer; Engineering; Enzyme-Linked Immunosorbent Assay; Equilibrium; Event; Fc domain; Future; Gene Order; Goals; Health Priorities; Healthcare; Hepatitis B Antibodies; Hepatitis B Virus; Home; Hospitals; Human; Interleukin-6; Light; Luciferases; Measurable; Measures; Medical; Mentors; Mentorship; Methods; Mission; Modality; Molecular; Molecular Conformation; Monitor; Monoclonal Antibodies; Names; Oligonucleotide Microarrays; Patients; Peptides; Phase; Protein Engineering; Proteins; Published Comment; Reporter; Reporting; Research; Research Personnel; SARS-CoV-2 antibody; Sampling; Seasons; Serology test; Signal Transduction; Specimen; Surface; System; Technology; Therapeutic; Thermodynamics; Time; Treatment outcome; Troponin I; United States National Institutes of Health; Work; Yeasts; antibody detection; base; career; career development; clinically relevant; complement system; design; disability; emergency settings; flu; health care availability; health care quality; health care service; improved; instrument; interest; luminescence; molecular diagnostics; neutralizing antibody; novel; pandemic disease; pathogen; personalized medicine; point of care; point of care testing; point-of-care diagnostics; precision medicine; protein biomarkers; ratiometric; receptor; receptor binding; sensor; synergism; systemic inflammatory response; tv watching

úú PROJECT SUMMARY/ABSTRACT Delivering diagnostic services at the point-of-care (POC) can improve the quality of healthcare in clinics, in emergency settings, or at home, which can potentially ease hospitals’ burden, for instance, during the COVID- 19 pandemic. Precision and personalized medicine revolution also require POC testing to provide readily available biomarker information to clinicians. The goal of this career development proposal is to create fast, inexpensive, sensitive, and reliable molecular diagnostics to address the 21st-century healthcare challenges. The central hypothesis is that we can efficiently utilize computational protein design to create modular allosteric protein switches, named LOCKR (Latching Orthogonal Cage–Key pRotein), that enable the rapid and reversible conformational changes upon interaction. As a proof of principle, we demonstrate that LOCKR- based biosensors can be configured to produce bioluminescence upon the addition of clinical targets (e.g., botulinum toxin, cardiac troponin I, HER2 receptor, Fc domain, anti-HBV mAb, anti-SARS-CoV2 antibodies, and SARS-CoV2 receptor-binding domain/spike protein, Fig 1 and 2) in homogeneous “all-in-solution” assays. Due to the modularity of LOCKR sensor platform and the advance in de novo binder design for arbitrary protein targets, we proposed the integration of both features as the universal strategy to develop tailored biosensors for user-defined targets. The main specific aims for the independent phase are to iteratively expand LOCKR-based diagnostics with the synergy of (1) de novo protein binder design to directly detect various disease protein biomarkers, and (2) indirectly detect the antibodies that compete with the designed interface, as POC devices; and (3) to repurpose the original luminescence signal with other compatible readouts by exchanging the reporter modules. For more specific proof-of-concept projects during the mentored phase, I describe in Aim 1 the use of state-of-the-art computational protein design methods to create an interleukin-6 binder and biosensor. In Aim 2, I propose a general way to develop antibody biosensors by demonstrating COVID-19 serological tests as an example. With my expertise in biosensor engineering, I attempt in Aim 3 to develop a ratiometric bioluminescence resonance energy transfer (BRET) biosensor to analyze the HBV antibody and a colorimetric biosensor to measure human cardiac troponin I level. Ultimately, I anticipate this new sensor platform is significant for the development of robust protein sensors that will be broadly applicable to arbitrary targets and enabling its POC compatible readouts for future diagnostics.

úú 项目概要/摘要 在护理点 (POC) 提供诊断服务可以提高诊所的医疗保健质量 紧急情况下或在家中，这可能会减轻医院的负担，例如在新冠疫情期间 19 流行病。精准化、个性化医疗革命也需要POC测试能够轻松提供 向临床医生提供生物标志物信息。该职业发展提案的目标是快速创造、 廉价、灵敏且可靠的分子诊断技术可应对 21 世纪的医疗保健挑战。 中心假设是我们可以有效地利用计算蛋白质设计来创建模块化 变构蛋白开关，称为 LOCKR（闩锁正交笼 - 关键蛋白），能够快速 以及相互作用时可逆的构象变化。作为原理证明，我们证明 LOCKR- 基于生物传感器的配置可以在添加临床目标（例如， 肉毒杆菌毒素、心肌肌钙蛋白 I、HER2 受体、Fc 结构域、抗 HBV mAb、抗 SARS-CoV2 抗体、 和 SARS-CoV2 受体结合结构域/刺突蛋白，图 1 和 2）均质“全溶液” 化验。由于 LOCKR 传感器平台的模块化以及从头绑定设计的进步， 任意蛋白质目标，我们提出将这两种特征的整合作为开发的通用策略 针对用户定义的目标量身定制的生物传感器。独立阶段的主要具体目标是 通过 (1) 从头蛋白结合剂设计的协同作用迭代扩展基于 LOCKR 的诊断，以直接 检测各种疾病蛋白生物标志物，(2)间接检测与疾病蛋白竞争的抗体 设计接口，如POC设备； (3)用其他信号重新利用原始发光信号 通过交换报告模块实现兼容读数。对于更具体的概念验证项目 在指导阶段，我在目标 1 中描述了使用最先进的计算蛋白质设计方法来 创建 IL-6 结合剂和生物传感器。在目标 2 中，我提出了一种开发抗体的通用方法 以展示 COVID-19 血清学测试为例。凭借我在生物传感器方面的专业知识 工程方面，我尝试在目标 3 中开发比例生物发光共振能量转移 (BRET) 用于分析 HBV 抗体的生物传感器和用于测量人心肌肌钙蛋白 I 的比色生物传感器 等级。最终，我预计这个新的传感器平台对于开发强大的蛋白质具有重要意义 传感器将广泛适用于任意目标，并使其 POC 兼容读数可供未来使用 诊断。