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

• 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
• 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

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：选择性和亲和力的结合，由生物分子（例如抗体）的靶标提供 配体使其成为生物测定的理想识别元素。尽管这些亲和力试剂使 开发许多重要的生物测定，这些测量值几乎总是作为静态分析进行 在个人时间点。亲和力试剂的速度缓慢使得开发响应迅速的测定法 可以监视分析物浓度随着时间的变化的变化。试剂降解，非特异性表面 相互作用和生物污染物带来了额外的困难。我们的目标是开发在线，流通，亲和力 可以通过动态变化的生物学来不断监测生化使者的外排的测定 实时系统。我们的前提是灌注室的微流体整合，在线混合亲和力 试剂和连续的微自由流电泳（µFFE）分离将直接解决局限性 迄今为止，限制了时间响应基于亲和力的测定的开发。 创新：我们将使用微流体通过方法来开发时间响应亲和力测定。这 生物学模型（即细胞培养）将容纳在灌注室中。灌注液将与 荧光标记的亲和力试剂（即抗体或APATMER）有选择地结合目标分析物。在线的 然后，µFFE将用于连续将分析物 - 亲属试剂复合物与过量亲和力试剂分开 实时。在线亲和力µffe提供了几个优势。连续流量将降低速率作为限制 临时响应。暴露于生物基质的情况下是最小的，减轻试剂降解。信号是 在溶液中测量，限制了非特异性表面相互作用和生物污染的影响。分离 即使亲和力试剂为 施加大量过量，改善了测定法的LOD。 方法：将开发亲和力µFFE分析，用于代表三种生化的分析物 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）。使用抗体的测定法（AIM）将进行直接比较 ＃1）或Aptamers（AIM＃2）作为亲和力试剂。将用于评估评估绩效的功绩数字 包括：LOD，临时响应，最小检测到的变化和长期稳定性。一旦完全优化， 亲和力µFFE测定将用于连续监测基线和刺激的外排从细胞模型中 用于神经传递（神经元），能量调节（脂肪细胞）和免疫响应（肥大细胞）。 基准：我们预计亲和力µFFE将达到以下性能指标：lod≤1NM； 临时响应≤1s;最小可检测的变化≤5％；在4小时内长期稳定性≤10％。 影响：时间响应µFFE分析将使研究人员能够研究在A≤1s上发生的动态变化 第一次在几个关键的生化使者系统中的时间表。