Simplified Single Molecule Protein Assays with Unprecedented Sensitivity

简化的单分子蛋白质检测，具有前所未有的灵敏度

• 批准号: 10644163
• 负责人: DAVID R. WALT
• 金额: $ 44.75万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-05-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10644163
• 关键词: Accident and Emergency department; Acute; Address; Bacterial Antigens; Bar Codes; Binding; Biological Assay; Biological Markers; Biosensing Techniques; Blood; Clinical; Clinical Sensitivity; Communicable Diseases; Complex; DNA amplification; Detection; Devices; Diagnosis; Diagnostic; Diffuse; Disease; Early treatment; Engineering; Ensure; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Event; Film; Fluorescent Dyes; Fluorescent Probes; Food; Foundations; Future; Generations; Geometry; Human Resources; Image; Individual; Label; Laboratories; Liquid substance; Measurement; Measures; Methods; Microfluidic Microchips; Microfluidics; Microscope; Molecular; Monitor; Neurodegenerative Disorders; Pathogen detection; Plasma; Preparation; Protein Analysis; Proteins; Reagent; Resource-limited setting; Saliva; Sampling; Scheme; Sensitivity and Specificity; Signal Transduction; Slide; Techniques; Technology; Testing; Time; Toxic Environmental Substances; Toxin; Training; Translating; Viral Antigens; Waste Products; Whole Blood; Work; analytical tool; automated image analysis; cancer biomarkers; clinically actionable; cost; cost effective; cross reactivity; design; detection limit; diagnostic platform; digital; disease diagnosis; fluorescence imaging; improved; instrument; instrumentation; medical attention; monolayer; multiplex assay; point of care; portability; protein biomarkers; seal; single molecule

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.

项目概要 准确、快速地测量极低水平的生物分子的能力对于诊断至关重要 并监测许多疾病。虽然对于某些生物标志物来说足够了，但大多数现有的敏感性 诊断系统不足以测量许多容易获得的蛋白质生物标志物 生物流体的浓度远低于其检测限，仍处于皮摩尔范围内。在这个 应用，我们建议开发、设计、工程和精炼一种新的超灵敏单分子蛋白质 分析平台将能够常规测量阿摩尔蛋白质浓度，我们称之为 用于个体计数的分子珠上信号放大 (MOSAIC)。 MOSAIC变身单身 通过珠上信号定位将分子测量转化为简化的测定格式，这具有潜力 集成到护理点 (POC) 设备中。在 MOSAIC 中，每个通道上都会生成一个非扩散信号 珠子携带目标分子，产生珠上信号，该信号可长时间保持附着状态 时间，从而能够采用替代检测方案，不需要将珠加载到 微孔或密封孔来定位信号。拟议工作中要解决的一个关键挑战是 确保该 MOSAIC 平台能够始终优于当前的超灵敏蛋白质检测 技术将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 为未来将这种增强的分析灵敏度转化为改进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，我们将优化信号生成和读出方法 用于 MOSAIC 并扩展其复用能力。在目标 3 中，我们计划将 MOSAIC 集成到一次性 试剂盒和便携式仪器，具有自动样品处理、液体处理、孵化、成像、 和样品分析。由此产生的从样本到答案的生物传感技术将提供超灵敏的 诊断平台可以在紧急情况下在中央临床实验室轻松且经济地使用 部门和资源有限的环境中，实现快速、准确的疾病诊断和早期治疗。