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: 10641748
• 负责人: MICHAEL T BOWSER
• 金额: $ 30.78万
• 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/10641748
• 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; 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; biological systems; cancer diagnosis; design; detection limit; flexibility; improved; innovation; lateral flow assay; 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：生物分子如抗体对其靶标的选择性和亲和力的结合