Simplified Single Molecule Protein Assays with Unprecedented Sensitivity

项目总结 准确而快速地测量极低水平的生物分子的能力对于诊断是必不可少的 并监测许多疾病。虽然对某些生物标志物来说足够了，但大多数现有的 诊断系统不足以测量存在于易于获取的许多蛋白质生物标志物 生物体液的浓度远低于其检测限值，保持在皮摩尔范围内。在这 应用，我们计划开发、设计、工程和提纯一种新的超灵敏的单分子蛋白质。 一个分析平台，将能够常规测量大气分子蛋白浓度，我们称之为 用于个体计数的分子在珠信号放大(马赛克)。马赛克变换单个 通过珠上信号定位将分子测量转化为简化的分析格式，这具有潜在的 集成到护理点(POC)设备中。在马赛克中，每个图像上都生成一个不可扩散信号 携带目标分子的珠子，产生在珠子上的信号，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子加载到 微孔或井口密封以定位信号。拟议工作中要解决的一个关键挑战将是 确保这个马赛克平台能够持续超越当前的超灵敏蛋白质检测 技术对许多蛋白质分析物的灵敏度提高了两个数量级，这反过来又奠定了 为今后的工作奠定基础，将这种增强的分析灵敏度转化为改进的临床灵敏度 诊断应用中的特异性。在目标1和目标2中，我们将优化信号产生和读出方法 用于马赛克，并扩展其多路复用能力。在目标3中，我们计划将马赛克整合到一次性产品中 色谱柱和便携式仪器，具有自动样品处理、液体处理、培养、成像、 和样本分析。由此产生的样本到答案生物传感技术将提供一种超灵敏的 在紧急情况下，可在中心临床实验室方便且经济实惠地使用的诊断平台 在资源有限的情况下，能够进行快速、准确的疾病诊断和早期治疗。

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.