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.

项目摘要 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，在这方面， 当前检测平台的局限性已经阻止了对许多蛋白质和小分子的灵敏检测 分子生物标志物，迫使临床医生依赖可能不准确的经验诊断或昂贵的实验室检查， 做出关键的治疗决定。已经容易地实现了核酸靶标的灵敏检测 通过利用沃森-克里克碱基配对来扩增信号（PCR、LAMP、Cas9等），但一直以来 在护理点检测低浓度抗原和小分子的创新。生物学 通过翻译后修饰在体内快速放大蛋白质信号的复杂机制 和基于蛋白质的信号网络。为了实现开发新型、快速、超灵敏诊断的目标， 该项目的中心假设是，在体外，基于蛋白质的信号网络， 放大酶途径将导致具有无与伦比的传感的生物标志物检测平台 能力的具体来说，我们计划研究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 裂解腺苷酸环化酶用于小分子检测体外用途。分析物的检测将 通过同时结合两种蛋白质（即溶液中的夹心测定）， 腺苷酸环化酶的一半在一起，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，以在体外产生自催化反馈环。该循环将 对cAMP的反应是产生更多的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特性。这些蛋白质信号网络的动态模型 将通过测量的实验参数进行通知。这些模型将用于创建一个组合 高灵敏度，快速小分子传感器的模型作为未来工作的原理证明。如果成功，这 系统将广泛地适用于蛋白质和小分子检测， 具有已知抗体结合结构域的广泛的目标分析物。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，研究员将接受合成生物学方面的技术培训 方法，蛋白质工程和动力学计算建模，除了在教学中的职业培训 和指导的最佳做法，手稿准备，赠款，和研究通信从 赞助商和共同赞助商以及西北大学研究所提供的资源。另夕h 培训生将有机会建立一个强大的合成生物学家，诊断设计， 专家和全球卫生临床医生在她的培训过程中，并将继续制定拟议的 作为一名独立研究人员，我们可以将检测平台转化为临床上可翻译的诊断设备。