Simplified Single Molecule Protein Assays with Unprecedented Sensitivity

项目摘要 准确快速测量极低水平生物分子的能力对于诊断至关重要 监测许多疾病。虽然对于某些生物标志物是足够的，但大多数现有的生物标志物的灵敏度是不确定的。 诊断系统不足以测量存在于容易获得的细胞中的许多蛋白质生物标志物。 浓度低于皮摩尔范围的生物流体。在这个应用程序中，我们建议设计和完善一个 新的超灵敏单分子蛋白质分析平台，将能够常规测量阿托摩尔 蛋白浓度，我们称之为单个计数的分子珠上信号放大（MOSAIC）。 MOSAIC通过珠上信号将单分子测量转换为简化的检测格式 定位，其具有被集成到护理点（POC）设备中的潜力。在马赛克中，一个非- 每个携带靶分子的珠子上都会产生可扩散的信号，从而产生保留的珠子上信号 因此，能够采用替代的检测方案， 不需要将珠限制在微孔或液滴中以定位信号。一个需要解决的关键挑战， 拟议的工作将是确保该MOSAIC平台能够始终优于当前的超灵敏度 蛋白质检测技术在许多蛋白质分析物上的灵敏度提高了一到两个数量级， 这反过来又为将来的工作奠定了基础，将这种增强的分析灵敏度转化为改进的 用于诊断应用的临床灵敏度和特异性。在目标1和2中，我们将优化信号生成 和读出方法的MOSAIC和扩大其多路复用能力。在目标3中，我们将评估 MOSAIC在检测极低丰度的原理验证临床应用中的诊断效用 尿中结核分枝杆菌抗原作为结核病潜在分诊试验。由此产生的生物传感 技术将提供一个超灵敏的诊断平台，将开放蛋白质分析， 难以获得的生物标志物，也可以在研究和临床实验室中方便和经济地使用 使用普通的实验室仪器。

PROJECT SUMMARY The ability to measure extremely low levels of biomolecules accurately and rapidly is essential for diagnosing and monitoring many diseases. While sufficient for certain biomarkers, the sensitivities of most existing diagnostic systems are inadequate for measuring many protein biomarkers that exist in easily accessible biofluids at concentrations below the picomolar range. In this application, we propose to engineer and refine a new ultrasensitive single molecule protein analysis platform that will be able to routinely measure attomolar protein concentrations, which we call Molecular On-bead Signal Amplification for Individual Counting (MOSAIC). MOSAIC transforms single molecule measurements into a simplified assay format via on-bead signal localization, which has the potential to be integrated into a point of care (POC) device. In MOSAIC, a non- diffusible signal is generated on each bead carrying a target molecule, creating an on-bead signal that remains attached for prolonged periods of time, thereby enabling alternative detection schemes to be employed that do not require bead confinement into microwells or droplets to localize signals. A key challenge to be addressed in the proposed work will be to ensure that this MOSAIC platform can consistently outperform current ultrasensitive protein detection technologies in sensitivity by one to two orders of magnitude across many protein analytes, which in turn lays the foundation for future work in translating this enhanced analytical sensitivity to improved clinical sensitivity and specificity for diagnostic applications. In Aims 1 and 2, we will optimize signal generation and readout methods for MOSAIC and expand its multiplexing capabilities. In Aim 3, we will evaluate the diagnostic utility of MOSAIC in a proof-of-principle clinical application to detect very low abundance Mycobacterium tuberculosis antigens in urine as a potential triage test for tuberculosis. The resulting biosensing technology will provide an ultrasensitive diagnostic platform that will open up protein analysis to previously inaccessible biomarkers and also be readily and affordably utilized across both research and clinical laboratories using common laboratory instruments.