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

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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.