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: 10657044
• 负责人: DAVID R. WALT
• 金额: $ 12.8万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-05 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657044
• 关键词: Abbreviations; Address; Antibodies; Antigens; Bacterial Antigens; Bar Codes; Binding; Biological; Biological Assay; Biological Markers; Biological Process; Biosensing Techniques; Blood; Classification; Clinical; Clinical Sensitivity; Complex; DNA amplification; Data; Detection; Devices; Diagnosis; Diagnostic Specificity; Disease; Engineering; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Event; Film; Fingers; Fluorescent Dyes; Fluorescent Probes; Food; Foundations; Future; Generations; Human Resources; Image; Individual; Infrastructure; Label; Laboratories; Licensing; Liquid substance; Measurement; Measures; Methods; Microscope; Modeling; Molecular; Monitor; Mycobacterium tuberculosis antigens; Neurodegenerative Disorders; Pathogen detection; Patients; Performance; Play; Protein Analysis; Proteins; Research; Resource-limited setting; Role; Saliva; Sampling; Scheme; Signal Transduction; Slide; Techniques; Technology; Testing; Time; Toxin; Training; Translating; Triage; Tuberculosis; Urine; Viral Antigens; Work; antigen detection; cancer biomarkers; clinical application; clinically actionable; commercialization; cost; cross reactivity; detection limit; diagnostic platform; diagnostic value; digital; improved; instrument; instrumentation; lipoarabinomannan; monolayer; multiplex assay; multiplex detection; point of care; protein biomarkers; seal; single molecule; urinary

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.

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 浓度低于皮摩尔范围的生物流体。在此应用中，我们建议设计和完善 新型超灵敏单分子蛋白质分析平台将能够常规测量阿托摩尔 蛋白质浓度，我们称之为个体计数分子珠上信号放大 (MOSAIC)。 MOSAIC 通过珠上信号将单分子测量转换为简化的测定格式 本地化，有可能集成到护理点 (POC) 设备中。在 MOSAIC 中，非 每个携带目标分子的珠子上都会产生扩散信号，从而产生一个保留在珠子上的信号 长时间附着，从而能够采用替代检测方案 不需要将珠子限制在微孔或液滴中来定位信号。需要解决的一个关键挑战 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 蛋白质检测技术对许多蛋白质分析物的灵敏度提高一到两个数量级， 这反过来又为未来将这种增强的分析灵敏度转化为改进的工作奠定了基础 诊断应用的临床敏感性和特异性。在目标 1 和 2 中，我们将优化信号生成 和 MOSAIC 的读出方法并扩展其复用功能。在目标 3 中，我们将评估 MOSAIC 在原理验证临床应用中检测极低丰度的诊断效用 尿液中的结核分枝杆菌抗原可作为结核病的潜在分类测试。由此产生的生物传感 技术将提供超灵敏的诊断平台，为以前的蛋白质分析开辟道路 难以获得的生物标志物，并且可以在研究和临床实验室中轻松且经济地使用 使用常见的实验室仪器。