Cytokines are small proteins that are secreted by macrophages, dendritic, and T-cells allowing these cells to drive immunological responses. An imbalance in cytokine concentrations can be an indication of inflammation and/or autoimmune response. Cytokines can be collected using microdialysis, which is a diffusion-based sampling technique that allows collection and quantification of unbound solutes without additional sample clean-up. Since it is difficult to collect cytokines due to their low concentrations and poor diffusivity, affinity agents are added to the perfusion fluid to bind cytokines and improve relative recovery. Cibacron blue (3G-A) triazine dye is used due to the dye’s chemical stability, commercial availability, and ability to bind many different proteins. The dye can be coupled to various solid supports. Our goal is to improve relative recovery of cytokines using affinity agents during microdialysis sampling. All microdialysis experiments were done using a CMA 20 probe (10 mm membrane length, 0.5 mm diameter) with a 100 kDa molecular weight cutoff (MWCO). Each sample was perfused at 1 µL/min using phosphate buffered saline (PBS). Poly (<i>N</i>-isopropylacrylamide) (PNIPAM) nanoparticles, about 450 nm in diameter, were able to bind approximately 1 mmol/g cibacron blue via covalent bond between the amine group of the nanoparticles with the chlorine atom in the cibacron blue dye. The small diameter of the particles prevents them from being trapped in the microdialysis tubing and from settling in solution. PNIPAM-Cibacron blue particles were able to capture approximately twice as much lysozyme as PNIPAM nanoparticle with no cibacron blue. Future work will include the incorporation of other affinity agents during microdialysis of cytokines and samples will be analyzed by Enzyme Linked Immunosorbent Assay (ELISA).

Improving relative recovery of cytokines using cibacron blue

255th National Meeting (2018)

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