Neuropeptide Affinity Enrichment Microdialysis Sampling

描述（由申请人提供）： 体内神经肽脑透析受到分析物扩散特性的严重限制，导致相对回收率（RR）和检测限挑战较差。在过去的二十年中，尽管传统的微透析采样的应用空间增加，但其改进很少。新的方法，大大提高微透析RR的神经肽是必不可少的研究复杂的神经元化学通讯途径。这项工作的目的是创造新的，但简单和直接的方法，可以应用于传统的微透析采样装置，显着增加肽的微透析RR。我们的假设是，神经肽微透析RR可以显着改善通过添加神经肽亲和剂（环糊精或抗体）连接到微透析灌注液的固体支持。这些相同的亲和剂也可用于额外的样品预浓缩。增强的质量传递与样品预浓缩的耦合将允许基于分离的检测方法（最终LC-MS）用于单个低体积（< 20 uL）透析液样品中神经肽的多重分析。这项工作的成功完成将大大改善脑透析，并将大大提高对复杂神经元化学通讯的整体理解。实验方法。环糊精（CD）和抗体（单克隆和多克隆）将被用作阿片肽（强啡肽A和亮脑啡肽和甲硫氨酸脑啡肽）的增强剂。灌注液中将包括P物质、促肾上腺皮质激素释放因子（CRF）和神经肽Y的单克隆和多克隆抗体。将确定与我们的化学分析方法相容的亲和剂的适当肽解离策略。结合缔合动力学和缔合平衡常数将使用表面等离子体共振（SPR）方法获得的亲和剂。亲和剂结合动力学和热力学将被纳入预测RR增强的质量传递模型。将在麻醉大鼠中进行初步体内研究，以证明RR增强功效。

DESCRIPTION (provided by applicant): In vivo neuropeptide brain dialysis is severely limited by analyte diffusion characteristics resulting in poor relative recovery (RR) and detection limit challenges. During the last twenty years there have been few improvements to conventional microdialysis sampling despite its increased application space. New methodologies that considerably improve microdialysis RR of neuropeptides are essential for studying complex neuronal chemical communication pathways. This work aims to create new, yet simple and straightforward, methods that can be applied to conventional microdialysis sampling devices to significantly increase microdialysis RR of peptides. Our hypothesis is that neuropeptide microdialysis RR can be significantly improved by adding neuropeptide affinity agents (cyclodextrins or antibodies) attached to solid supports to the microdialysis perfusion fluid. These same affinity agents can also be used for additional sample preconcentration. The coupling of enhanced mass transport with sample preconcentration will allow separation-based detection methods (ultimately LC-MS) to be used for multiplexed analysis of neuropeptides in a single low volume (< 20 uL) dialysate sample. Successful completion of this work will create significant improvements to brain dialysis and will considerably improve overall understanding of complex neuronal chemical communication. Experimental Approach. Cyclodextrins (CDs) and antibodies (monoclonal and polyclonal) will be used as enhancement agents for opioid peptides (dynorphin A, and leu- and met-enkephalins). Monoclonal and polyclonal antibodies to substance P, corticotropin-releasing factor (CRF) and neuropeptide Y will be included in the perfusion fluid. The appropriate peptide dissociation strategies from the affinity agents compatible with our chemical analysis approaches will be determined. Binding association kinetics and association equilibrium constants will be obtained for the affinity agents using surface plasmon resonance (SPR) methods. The affinity agent binding kinetics and thermodynamics will be incorporated into a mass transport model for predicting RR enhancement. Preliminary in vivo studies in anesthetized rats will be performed to demonstrate RR enhancement efficacy.