Online Affinity Micro Free Flow Electrophoresis Assays for Continuous Monitoring of Biochemical Messengers

用于连续监测生化信使的在线亲和微自由流电泳分析

基本信息

批准号：
10420769
负责人：
MICHAEL T BOWSER
金额：
$ 32.38万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10420769
关键词：
Address Adipocytes Adsorption Affinity Antibodies Area Benchmarking Binding Biochemical Biological Biological Assay Biological Markers Biological Models Biology Biomedical Research Cell Culture Techniques Cell model Cells Complex Development Electrophoresis Elements Enzyme-Linked Immunosorbent Assay Exhibits Exposure to Goals Home Immune response In Vitro Individual Inflammation Lateral Leptin Ligands Measurement Measures Microfluidics Modeling Modernization Monitor Nature Neurons Obesity Oligonucleotides Optics Pathogen detection Performance Perfusion Play Pregnancy Tests Public Health Reagent Regulation Reproducibility Research Research Personnel Role Signal Transduction Stimulus Surface System TNF gene Time addiction affinity labeling aptamer base biological systems cancer diagnosis design detection limit flexibility improved innovation mast cell neuropeptide Y neurotransmission prevent response sensor

项目摘要

Premise: The combination of selectivity and affinity afforded by biomolecules such as antibodies for their target ligands make them ideal recognition elements for bioassays. While these affinity reagents have enabled the development of many important bioassays, these measurements are almost always performed as static analyses at an individual time point. The slow off rates of affinity reagents makes development of responsive assays that can monitor changes in analyte concentration over time a challenge. Reagent degradation, non-specific surface interactions, and biofouling present additional difficulties. Our goal is to develop an online, flow-through, affinity assay that can continuously monitor the efflux of biochemical messengers from dynamically changing biological systems in real time. Our premise is that microfluidic integration of a perfusion chamber, online mixing of affinity reagents and continuous micro free flow electrophoresis (µFFE) separations will directly address limitations that have restricted the development of time responsive affinity-based assays to date. Innovation: We will use a microfluidic, flow through approach to develop time responsive affinity assays. The biological model (i.e., cell culture) will be housed in a perfusion chamber. Perfusate will be mixed online with a fluorescently labeled affinity reagent (i.e., antibody or aptamer) that selectively binds the target analyte. Online µFFE will then be used to continuously separate the analyte-affinity reagent complex from excess affinity reagent in real time. Online affinity µFFE offers several advantages. Continuous flow removes off rate as a limitation to temporal response. Exposure to the biological matrix is minimal, mitigating reagent degradation. Signal is measured in solution, limiting the effect of non-specific surface interactions and biofouling. µFFE separation enables interference free measurement of the analyte-affinity reagent complex even when the affinity reagent is applied in large excess, improving the LOD of the assay. Approach: Affinity µFFE assays will be developed for representative analytes from three biochemical messenger systems: neuropeptide Y (NPY, neurotransmission), leptin (energy regulation), and tumor necrosis factor α (TNF-α, immune response). Direct comparisons will be made between assays that use antibodies (Aim #1) or aptamers (Aim #2) as the affinity reagent. Figures of merit that will be used to assess assay performance include: LOD, temporal response, minimum detectable change, and long-term stability. Once fully optimized, affinity µFFE assays will be used to continuously monitor both baseline and stimulated efflux from cell models for neurotransmission (neurons), energy regulation (adipocytes) and immune response (mast cells). Benchmarks: We anticipate that affinity µFFE will achieve the following performance metrics: LOD ≤ 1nM; temporal response ≤ 1 s; minimum detectable change ≤ 5%; and long-term stability ≤ 10% over 4 h. Impact: Time responsive µFFE assays will allow researchers to study dynamic changes that occur on a ≤1 s timescale in several critical biochemical messenger systems for the first time.

前提：生物分子（例如抗体）对其靶标提供的选择性和亲和力的结合配体使它们成为生物测定的理想识别元件，而这些亲和试剂使得它们成为可能。许多重要生物测定的发展，这些测量几乎总是以静态分析的形式进行在单个时间点，亲和试剂的缓慢解离速率使得响应测定的开发成为可能。可以监测分析物浓度随时间的变化，挑战试剂降解、非特异性表面。相互作用和生物污垢带来了额外的困难，我们的目标是开发在线、流通、亲和力。可以连续监测动态变化的生物中生化信使的流出的测定我们的前提是灌注室的微流体集成、亲和力的在线混合。试剂和连续微自由流电泳 (µFFE) 分离将直接解决以下限制：迄今为止，限制了基于时间响应的亲和力测定的发展。创新：我们将使用微流体、流通方法来开发时间响应亲和力测定。生物模型（即细胞培养物）将被安置在灌注室中，灌注液将与灌流液在线混合。在线选择性结合目标分析物的荧光标记亲和试剂（即抗体或适体）。然后使用 µFFE 将分析物-亲和试剂复合物与过量的亲和试剂连续分离实时在线亲和 µFFE 具有多种优势，消除了分离速率的限制。与生物基质的接触时间极小，减轻了试剂的降解。在溶液中测量，限制非特异性表面相互作用和 µFFE 分离的影响。即使当亲和试剂处于大量过量应用，提高了测定的 LOD。方法：将为来自三种生化物质的代表性分析物开发亲和 µFFE 测定法信使系统：神经肽Y（NPY，神经传递）、瘦素（能量调节）和肿瘤坏死因子 α（TNF-α，免疫反应）将在使用抗体的测定之间进行直接比较（目的）。 #1) 或适体（目标 #2）作为亲和试剂，用于评估检测性能。包括：LOD、时间响应、最小可检测变化和长期稳定性。亲和 µFFE 测定将用于连续监测细胞模型的基线和刺激流出用于神经传递（神经元）、能量调节（脂肪细胞）和免疫反应（肥大细胞）。基准：我们预计亲和 µFFE 将实现以下性能指标：LOD ≤ 1nM；时间响应 ≤ 1 秒；最小可检测变化 ≤ 5%；4 小时内的长期稳定性 ≤ 10%。影响：时间响应 µFFE 检测将使研究人员能够研究 ≤1 秒内发生的动态变化首次在几个关键生化信使系统中进行时间尺度。