Neuropeptide Affinity Enrichment Microdialysis Sampling

点击翻译按钮获取中文摘要

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.