Neuropeptide Affinity Enrichment Microdialysis Sampling

描述(由申请人提供)： 在体内，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。在过去的二十年里，尽管传统的微渗析取样的应用空间增加了，但它几乎没有什么改进。显著提高神经肽微透析率的新方法对于研究复杂的神经元化学通讯通路是必不可少的。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设备，以显著提高多肽的微透析RR。我们的假设是，通过将神经肽亲和剂(环糊精或抗体)附着到固体载体上到微透析灌注液中，可以显著改善神经肽微透析RR。这些相同的亲和剂也可用于额外的样品预浓缩。将增强的传质与样品预浓缩相结合，将允许基于分离的检测方法(最终为LC-MS)用于单个小体积(&lt；20ul)透析液样品中神经肽的多重分析。这项工作的成功完成将对脑透析产生重大改进，并将极大地提高对复杂神经元化学通信的整体理解。实验方法。环糊精(CD)和抗体(单抗和多克隆)将被用作阿片肽(强啡肽A、亮氨酸和甲硫氨酸脑啡肽)的增强剂。P物质、促肾上腺皮质激素释放因子(CRF)和神经肽Y的单抗和多克隆抗体将包括在灌注液中。我们将确定与我们的化学分析方法兼容的亲和剂中合适的多肽解离策略。利用表面等离子体共振(SPR)方法得到亲和剂的结合缔合动力学和缔合平衡常数。亲和剂结合动力学和热力学将被纳入用于预测RR增强的质量传输模型中。将在麻醉大鼠身上进行初步的活体研究，以证明增强RR的效果。

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.