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

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，迫使临床医生依赖于可能不准确的经验诊断或昂贵的 实验室测试来做出关键的治疗决定。核酸靶标的灵敏检测已经很容易实现。 通过利用沃森-克里克碱基配对来放大信号（例如，但一直以来， 在护理点检测低浓度抗原和小分子的创新。自然，在 另一方面，已经进化出复杂的机制，通过翻译后途径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，可以适用于生物标志物检测。朝向 该项目的目标是开发新型、快速、超灵敏的诊断方法，其中心假设是， 在体外，结合自放大基序的基于蛋白质的信号网络可以检测临床 生物标志物。具体来说，我们计划将蛋白质信号网络的三种机制与潜在的 用于诊断：裂解酶重建、自催化正反馈回路和小分子 生物传感器我们以前已经证明了在体外使用分裂腺苷酸环化酶的小分子 通过同时结合两种蛋白质进行检测（即溶液中的夹心测定）， 腺苷酸环化酶结合在一起产生cAMP。目标1（K99）将纳入结合结构域， 检测临床相关的生物标志物靶标，并研究人体样本基质中的检测。 我们将测试不同的结合域融合分裂腺苷酸环化酶片段，以检测丙型肝炎核心 抗原（HCVcAg）。为了开发即时诊断，Aim 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 读取器以检测由基于FRET的cAMP生物传感器产生的荧光的偏移。实验台 将使用加入市售合并液中的HCVcAg对所有组分进行验证 血液样本目的3（R 00）探讨裂解腺苷酸环化酶和cAMP受体蛋白的应用 在体外建立一个自我催化的反馈回路这项工作将导致第一个已知的体外蛋白质 信号网络，具有类似开关的传感功能，这是诊断领域的一个重大进步。如果 如果成功，该系统将广泛适用于蛋白质和小分子检测， 用于检测具有已知结合域的广泛的目标分析物。因此，今后的工作包括 结合域的掺入以检测其它高价值蛋白质和小分子分析物 不能在护理点和所述诊断器械的临床转化中快速检测 与国内外临床合作伙伴合作开展试点临床试验。

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.