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

亲合性：生物分子（如抗体）对其靶点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。虽然这些亲和试剂已经使得 许多重要的生物测定的发展，这些测量几乎总是作为静态分析进行 在一个单独的时间点。亲和试剂的缓慢解离速率使得响应性测定的发展， 可以监测分析物浓度随时间的变化，这是一个挑战。试剂降解，非特异性表面 相互作用和生物结垢带来了额外的困难。我们的目标是发展一种在线的、流动的、亲和的 一种可以连续监测生物化学信使从动态变化的生物化学物质中流出的测定方法， 真实的系统。我们的前提是，微流控集成一个灌注室，在线亲和混合 试剂和连续微量自由流电泳（µFFE）分离将直接解决 限制了基于时间响应的亲和性测定的发展。 创新：我们将使用微流控，流通方法来开发时间响应的亲和测定。的 生物模型（即，细胞培养物）将容纳在灌注室中。Perfusate将与 荧光标记的亲和试剂（即，抗体或适体），其选择性地结合靶分析物。在线 然后使用µFFE连续分离分析物-亲和试剂复合物与过量亲和试剂 在真实的时间里。在线亲和力µFFE提供了几个优势。连续流动消除了作为限制的关闭速率， 时间响应暴露于生物基质的程度最低，可减轻试剂降解。信号 在溶液中测量，限制了非特异性表面相互作用和生物污垢的影响。µFFE分离 使得即使当亲和试剂 大量过量施用，提高了测定的LOD。 方法：将针对三种生化分析物的代表性分析物开发亲和µFFE检测试剂盒。 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（Aim #1）或适体（目标#2）作为亲和试剂。将用于评估检测试剂盒性能的优值 包括：LOD、时间响应、最小可检测变化和长期稳定性。一旦完全优化， 亲和力µFFE测定将用于连续监测细胞模型的基线和刺激外排 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：我们预计亲和力µFFE将达到以下性能指标：LOD ≤ 1 nM; 时间响应≤ 1 s;最小可检测变化≤ 5%; 4 h内长期稳定性≤ 10%。 影响：时间响应型µFFE检测将使研究人员能够研究在≤1 s内发生的动态变化 首次在几个关键的生化信使系统中观察到时间尺度。

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.