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。 生物流体的浓度远低于其检测限，检测限保持在皮摩尔范围内。在这 应用，我们建议开发，设计，工程师，并完善一个新的超灵敏的单分子蛋白质 分析平台，将能够常规测量阿摩尔蛋白浓度，我们称之为 用于单个计数的分子珠上信号放大（MOSAIC）。MOSAIC转换单个 通过珠上信号定位将分子测量转化为简化的测定形式，这具有潜在的 以集成到护理点（POC）设备中。在MOSAIC中，在每个信号上产生不可扩散的信号。 携带靶分子的珠粒，产生珠粒上信号，该信号在长时间内保持附着， 时间，从而使得能够采用不需要将珠加载到微球中的替代检测方案。 微孔或孔密封以定位信号。拟议工作中需要解决的一个关键挑战是 确保该MOSAIC平台始终优于当前的超灵敏蛋白质检测 技术在许多蛋白质分析物上的灵敏度提高了两个数量级，这反过来又奠定了 为将来将这种增强的分析灵敏度转化为提高的临床灵敏度的工作奠定了基础， 诊断应用中的特异性。在目标1和2中，我们将优化信号产生和读出方法 并扩展其多路复用功能。在目标3中，我们计划将MOSAIC集成到一次性 盒和具有自动化样品处理、液体处理、孵育、成像 和样品分析。由此产生的样本到答案的生物传感技术将提供一个超灵敏的 在紧急情况下，中心临床实验室可以方便且经济地使用的诊断平台 在资源有限的情况下，能够快速、准确地诊断疾病和早期治疗。

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 much lower than their detection limits, which remain in the picomolar range. In this application, we propose to develop, design, 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 loading into microwells or well sealing 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 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 in 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 plan to integrate MOSAIC into a disposable cartridge and a portable instrument with automated sample processing, liquid handling, incubation, imaging, and sample analysis. The resulting sample-to-answer biosensing technology will provide an ultrasensitive diagnostic platform that can be readily and affordably utilized in the central clinical laboratory, in emergency departments, and in resource-limited settings, enabling rapid, accurate disease diagnosis and early treatments.