Development of a Novel Split Enzyme Diagnostic Platform for Use at the Point of Care

开发用于护理点的新型裂解酶诊断平台

• 批准号: 10723565
• 负责人: Catherine E. Majors
• 金额: $ 10.79万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10723565
• 关键词: Adenylate Cyclase; Affinity; Antibodies; Antigens; Base Pairing; Binding; Biological Assay; Biological Markers; Biology; Biosensor; Blood; Blood specimen; Clinical; Clinical Trials; Collaborations; Communicable Diseases; Complex; Computer Models; Concentration measurement; Cyclic AMP; Cyclic AMP Receptor Protein; DNA; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Equipment; Disease; Enzymes; Equipment; Feedback; Fluorescence; Fluorescence Resonance Energy Transfer; Freeze Drying; Future; Goals; Hepatitis C; Hepatitis C virus; Human; Immunoglobulin Fragments; In Vitro; Investigation; Modeling; Nature; Output; Pathway interactions; Phase; Post-Translational Protein Processing; Proteins; Rapid diagnostics; Reaction; Reader; Recombinants; Reporter; Sampling; Serum; Signal Transduction; Signaling Protein; Sirolimus; System; Tacrolimus Binding Protein 1A; Tertiary Protein Structure; Testing; Validation; Work; adenylate; antigen binding; clinical biomarkers; clinical translation; clinically relevant; design; detection method; detection platform; diagnostic platform; enzyme reconstitution; in vivo; innovation; lateral flow assay; novel; nucleic acid detection; point of care; point-of-care detection; point-of-care diagnostics; prevent; protein biomarkers; rapid detection; reconstitution; response; scaffold; small molecule; targeted biomarker

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 on 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 (e.g., PCR), but there has been a lack of innovation for detection of low concentration antigens and small molecules at the point of care. Nature, on the other hand, has evolved intricate mechanisms for rapidly amplifying protein signals in vivo via post-translational modification and protein-based signaling networks that could be adapted for biomarker detection. 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 motifs can detect clinical biomarkers. Specifically, we plan to incorporate three mechanisms of protein signaling networks with potential for diagnostics: split enzyme reconstitution, autocatalytic positive feedback loops, and small molecule biosensors. We have previously demonstrated the in vitro use of split adenylate cyclase for small molecule detection via the simultaneous binding of two proteins (i.e. a sandwich assay in solution), bringing two halves of adenylate cyclase together and producing cAMP. Aim 1 (K99) will incorporate binding domains for detection of clinically relevant biomarker targets and investigate detection in human sample matrices. We will test different binding domains fused to split adenylate cyclase fragments to detect Hepatitis C Core Antigen (HCVcAg). Towards the development of a point-of-care diagnostic, Aim 2 (K99) will optimize reaction components for lyophilization and long-term storage and develop a hand-held point-of-care reader to detect the shift in fluorescence produced by the FRET-based cAMP biosensor. Lab-bench validation of all components will be performed using HCVcAg spiked into commercially purchased pooled blood samples. Aim 3 (R00) will investigate the use of split adenylate cyclase and cAMP receptor protein to create an autocatalytic feedback loop in vitro. This work would result in the first known in vitro protein signaling network with bistable switch-like sensing, a significant advancement for the diagnostics field. 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 binding domains. As such, future work includes the incorporation of binding domains to detect other high value protein and small molecule analytes currently unable to be rapidly detected at the point of care and the clinical translation of the described diagnostic device with pilot clinical trials in collaboration with both domestic and foreign clinical partners.

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，迫使临床医生依赖可能不准确的经验诊断或昂贵的 实验室测试以做出关键的治疗决定。核酸靶标的灵敏检测已变得容易 通过利用 Watson-Crick 碱基配对来放大信号（例如 PCR）来实现，但一直缺乏 在护理点检测低浓度抗原和小分子的创新。大自然，在 另一方面，已经进化出复杂的机制，通过翻译后在体内快速放大蛋白质信号 修饰和基于蛋白质的信号网络，可适用于生物标志物检测。迈向 开发新颖、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结合自放大基序可以检测临床 生物标志物。具体来说，我们计划整合蛋白质信号网络的三种机制，具有潜在的潜力 用于诊断：裂解酶重构、自催化正反馈环和小分子 生物传感器。我们之前已经证明了分裂腺苷酸环化酶在小分子中的体外应用 通过同时结合两种蛋白质（即溶液中的夹心测定）进行检测，将两半蛋白质 腺苷酸环化酶一起产生cAMP。目标 1 (K99) 将结合结合域 检测临床相关生物标志物目标并研究人体样本基质中的检测。 我们将测试融合分裂腺苷酸环化酶片段的不同结合域以检测丙型肝炎核心 抗原（HCVcAg）。为了开发即时诊断，目标 2 (K99) 将优化 用于冻干和长期储存的反应组分，并开发手持式护理点 读取器检测基于 FRET 的 cAMP 生物传感器产生的荧光变化。实验台 所有成分的验证将使用掺入商业购买的混合液中的 HCVcAg 进行 血液样本。目标 3 (R00) 将研究分裂腺苷酸环化酶和 cAMP 受体蛋白的用途 在体外创建自催化反馈回路。这项工作将产生第一个已知的体外蛋白质 具有双稳态开关传感功能的信号网络，是诊断领域的重大进步。如果 如果成功的话，该系统将广泛适用于蛋白质和小分子检测，并且可以 用于检测具有已知结合域的各种目标分析物。因此，未来的工作包括 目前结合结合域来检测其他高价值蛋白质和小分子分析物 无法在护理点快速检测以及所述诊断设备的临床转化 与国内外临床合作伙伴合作进行试点临床试验。