An Enzyme Self-Amplification System for Ultrasensitive Detection of Biomarkers at the Point of Care

用于在护理点超灵敏检测生物标志物的酶自扩增系统

• 批准号: 10463564
• 负责人: Catherine E. Majors
• 金额: $ 6.5万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10463564
• 关键词: Adenylate Cyclase; Antibodies; Antigens; Automobile Driving; Base Pairing; Binding; Binding Proteins; Biological Assay; Biological Markers; Biology; Chimeric Proteins; Clinical; Communicable Diseases; Communication Research; Complex; Computer Models; Concentration measurement; Critical Thinking; Cyclic AMP; Cyclic AMP Receptor Protein; DNA; Detection; Diagnosis; Diagnostic; Diagnostic Equipment; Differential Equation; Disease; Educational process of instructing; Engineering; Ensure; Enzyme Kinetics; Enzymes; Equipment; Feedback; Future; Generations; Goals; Human; In Vitro; Institutes; Kinetics; Manuscripts; Measures; Mentorship; Methods; Modeling; Pathway interactions; Post-Translational Protein Processing; Postdoctoral Fellow; Preparation; Production; Promoter Regions; Property; Protein Engineering; Proteins; Rapid diagnostics; Research; Research Personnel; Resources; Sampling; Signal Transduction; Signaling Protein; Sirolimus; System; Tacrolimus Binding Protein 1A; Tertiary Protein Structure; Testing; Training; Universities; Work; base; career; career networking; clinically relevant; clinically translatable; design; detection method; detection platform; enzyme reconstitution; global health; in vivo; innovation; lateral flow assay; model design; novel; nucleic acid detection; point of care; prevent; protein complex; reconstitution; response; sensor; skills; small molecule; success; synthetic biology

PROJECT SUMMARY Rapid, inexpensive detection of biomarkers at the point of care is vital for many clinical purposes. However, limitations in current detection platforms have prevented the sensitive detection of many protein and small molecule biomarkers, forcing clinicians to rely either potentially inaccurate empirical diagnosis or expensive lab tests to make critical treatment decisions. Sensitive detection of nucleic acid targets has been readily achieved by exploiting Watson-Crick base pairing to amplify signals (PCR, LAMP, Cas9, etc.), but there has been a lack of innovation for detection of low concentration antigens and small molecules at the point of care. Biology has evolved intricate mechanisms for rapidly amplifying protein signals in vivo via post-translational modification and protein based signaling networks. Towards the goal of developing novel, rapid, ultrasensitive diagnostics, the central hypothesis of this project is that in vitro, protein-based signaling networks incorporating self- amplifying enzymatic pathways will result in biomarker detection platforms with unparalleled sensing capabilities. Specifically, we plan to investigate two mechanisms of protein signaling networks with potential for diagnostics: split enzyme reconstitution and autocatalytic positive feedback loops. First, we will investigate the in vitro use of split adenylate cyclase for small molecule detection. Detection of the analyte will be accomplished by the simultaneously binding two proteins (i.e. a sandwich assay in solution), bringing two halves of adenylate cyclase together and producing cAMP. Second, we will investigate fusions of split adenylate cyclase and cAMP receptor protein to create an autocatalytic feedback loop in vitro. This loop will respond to cAMP by producing more cAMP. Finally, we will develop ordinary differential equation-based models to understand and engineer diagnostic properties. Dynamic models of these protein-signaling networks will be informed by measured experimental parameters. These models will be used to create a combined model for a high sensitivity, fast small molecule sensor as a proof-of-principle for future work. If successful, this system would be broadly applicable for protein and small molecule detection and could be used to detect a wide range of target analytes with known antibody binding domains. As such, this system could be used as a platform for the detection of many protein and small molecule analytes currently unable to be rapidly detected at the point of care. Over the course of the project the fellow will receive technical training in synthetic biology methods, protein engineering, and kinetics computational modeling, in addition to career training in teaching and mentorship best practices, manuscript preparation, grantsmanship, and research communication from the sponsor and co-sponsor and resources available through institutes at Northwestern University. Additionally, the trainee will have the opportunity build a strong professional network of synthetic biologists, diagnostic design experts, and global health clinicians over the course of her training and will continue developing the proposed detection platform into clinically translatable diagnostic devices as an independent researcher.

项目概要 在护理点快速、廉价地检测生物标志物对于许多临床目的至关重要。然而， 当前检测平台的局限性阻碍了许多蛋白质和小分子的灵敏检测 分子生物标志物，迫使临床医生要么依赖可能不准确的经验诊断，要么依赖昂贵的实验室 做出关键治疗决定的测试。轻松实现核酸靶标的灵敏检测 通过利用 Watson-Crick 碱基配对来放大信号（PCR、LAMP、Cas9 等），但一直缺乏 在护理点检测低浓度抗原和小分子的创新。生物学有 进化出通过翻译后修饰快速放大体内蛋白质信号的复杂机制 和基于蛋白质的信号网络。朝着开发新颖、快速、超灵敏诊断方法的目标， 该项目的中心假设是，在体外，基于蛋白质的信号网络结合了自我 放大酶途径将产生具有无与伦比的传感能力的生物标志物检测平台 能力。具体来说，我们计划研究具有潜力的蛋白质信号网络的两种机制 诊断：分裂酶重建和自催化正反馈回路。首先，我们将调查 体外使用分裂腺苷酸环化酶进行小分子检测。分析物的检测将是 通过同时结合两种蛋白质（即溶液中的夹心测定）来完成，使两种蛋白质 一半的腺苷酸环化酶结合在一起并产生cAMP。其次，我们将研究分裂的融合 腺苷酸环化酶和 cAMP 受体蛋白在体外创建自催化反馈回路。这个循环将 通过产生更多的 cAMP 来响应 cAMP。最后，我们将开发基于常微分方程的 模型来理解和设计诊断属性。这些蛋白质信号网络的动态模型 将通过测量的实验参数得知。这些模型将用于创建一个组合的 高灵敏度、快速小分子传感器的模型作为未来工作的原理验证。如果成功的话，这 该系统将广泛适用于蛋白质和小分子检测，并可用于检测 具有已知抗体结合域的多种目标分析物。因此，该系统可以用作 用于检测目前无法快速检测的许多蛋白质和小分子分析物的平台 在护理点。在项目过程中，该研究员将接受合成生物学的技术培训 方法、蛋白质工程和动力学计算模型，以及教学职业培训 和指导最佳实践、手稿准备、资助和研究交流 赞助商和共同赞助商以及通过西北大学研究所提供的资源。此外， 学员将有机会建立强大的合成生物学家、诊断设计专业网络 专家和全球健康临床医生在她的培训过程中将继续制定拟议的 作为独立研究人员将检测平台转化为临床可转化的诊断设备。