Date of Graduation

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Julie Stenken

Committee Member

Ranil Wickramasinghe

Second Committee Member

Dan Davis

Third Committee Member

David Paul

Fourth Committee Member

Ingrid Fritsch

Fifth Committee Member

Wayne Kuenzel

Keywords

Pure sciences, Affinity agent, Microdialysis, Nanoparticle agglomeration, Thermoresponsive nanoparticles

Abstract

Affinity agents increase microdialysis protein relative recovery, yet they have not seen widespread use within the microdialysis community due to their additional instrumentation requirements and prohibitive cost. This dissertation describes new affinity agents for microdialysis that require no additional instrumentation to use, have nearly 100% particle recovery, are 7 times more cost efficient than alternatives, and have low specificity enabling their use for a wide variety of proteins. Initially gold nanoparticles were chosen as an affinity ligand support due to their high surface area/volume ratio and colloidal stability. Poly (N-isopropylacrylamide) was immobilized to the gold nanoparticles, which served to sterically stabilize the particles and to act as a generic, reversible protein capture agent. A method was developed to reproducibly vary and quantify poly (N-isopropylacrylamide) graft density from 0.09 to 0.40 ligands/nm2 on gold nanoparticles. During characterization of the polymer coated gold nanoparticles, irreversible particle agglomeration was observed at low polymer graft density in ionic solutions, which prevented further development as a protein capture agent. Poly (N-isopropylacrylamide) nanogels, which have low nonspecific adsorption, low interparticle attractive forces owing to the low curvature of the particle, and a low Hamaker constant, were synthesized to overcome the agglomeration problem. A generic protein affinity ligand cibacron blue, was immobilized to the nanogels, which enabled rapid determination of particle recovery. The perfusion of the nanogels through a microdialysis probe was optimized yielding ~ 100% particle recovery using a combination of a syringe and peristaltic pump. The microdialysis collection efficiency of CCL2, a physiologically relevant cytokine, was increased 3-fold with addition of the nanogel to the microdialysis perfusion fluid. The reduction in instrumentation requirements, low cost, and low specificity obtained with the new affinity agents will lead to increased affinity agent use for microdiaylsis protein sampling.

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