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: 10836196
• 负责人: Hsien Wei YEH
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10836196
• 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; Clinic; Clinical; Collection; Color; Colorimetry; Consumption; Coupled; Coupling; Creativeness; Darkness; 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; Incubated; 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; 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; 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 impact; pathogen; personalized medicine; point of care; point of care testing; point-of-care diagnostics; precision medicine; protein biomarkers; ratiometric; receptor; receptor binding; seasonal influenza; 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.

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，或在家里，这可能会减轻医院的负担，例如，在新冠肺炎期间， 19大流行病。精准化和个性化医疗革命也需要POC检测提供便捷的 为临床医生提供生物标志物信息。这份职业发展计划的目标是快速创造， 廉价、灵敏和可靠的分子诊断技术，以应对21世纪的医疗保健挑战。 核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的蛋白质。 变构蛋白质开关，称为LOCKR（闭锁正交笼键蛋白），使快速 以及相互作用时可逆的构象变化。作为原则的证明，我们证明LOCKR- 基于的生物传感器可以被配置为在添加临床目标时产生生物发光（例如， 肉毒杆菌毒素，心肌肌钙蛋白I，HER 2受体，Fc结构域，抗HBV mAb，抗SARS-CoV 2抗体， 和SARS-CoV 2受体结合结构域/刺突蛋白，图1和2）在均质“全溶液”中 分析。由于LOCKR传感器平台的模块化和从头装订设计的进步， 任意蛋白质靶点，我们提出将这两个特征整合作为开发的通用策略 为用户定义的目标定制生物传感器。独立阶段的主要具体目标是 迭代地扩展基于LOCKR的诊断，具有以下协同作用：（1）从头蛋白结合剂设计， 检测各种疾病蛋白质生物标志物，和（2）间接检测与所述蛋白质生物标志物竞争的抗体。 设计的接口，如POC设备;和（3）重新利用原始发光信号与其他 通过交换报告器模块来实现兼容的读数。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， 制造白细胞介素-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COVID-19血清学检测为例，展示生物传感器。凭借我在生物传感器方面的专业知识 工程，我试图在目标3开发一个比率生物发光共振能量转移（BRET） 分析HBV抗体的生物传感器和测量人心肌肌钙蛋白I的比色生物传感器 水平最终，我预计这种新的传感器平台对开发鲁棒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兼容的读数，为未来 诊断