Affinity-Based Microdialysis Sampling of Cytokines Using Heparin-Immobilized Microspheres

by Duo, Jia; Stenken, Julie Ann

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Microdialysis sampling of cytokines is challenging due to their small diffusion coeffs. Inclusion of affinity agents into the microdialysis perfusion fluid serves to enhance the relative recovery (RR) via the binding reaction with cytokines. This work is to create and use heparin-immobilized microspheres as cytokine-trapping agents during microdialysis sampling. The interaction between heparin and three cytokines (aFGF, MCP-1, and RANTES) was studied by surface plasmon resonance. All cytokines showed high binding affinities to the immobilized heparin on the sensor surface (aFGF, KD: 33.48 ± 6.67 nM; MCP-1, KD: 5.08 ± 1.63 nM; RANTES, KD: 474 ± 110 nM). The heparin-immobilized microspheres were prepd. by covalently coupling heparin onto amine-functionalized microspheres via reductive amination. These microspheres showed broad dynamic ranges for the binding to MCP-1 (0.975-1000 ng/mL) and aFGF (0.5-1000 ng/mL) detected by a flow cytometry-based assay. Enhanced microdialysis RRs of these three cytokines were achieved by including the heparin-immobilized microspheres in the perfusion fluid. Typical control and enhanced RRs at 2.0, 1.0 and 0.5 ±L/min flow rates were as follows (n=3): aFGF, 3.2 ± 2.3% and 4.5 ± 1.8%, 4.9 ± 1.9% and 9.8 ± 0.7%, 8.4 ± 2.3% and 29.2 ± 4.8%; MCP-1, 2.0 ± 0.4% and 8.9 ± 1.2%, 2.8 ± 1.1% and 11.2 ± 1.4%, 6.9 ± 3.0% and 13.8 ± 2.2%; RANTES, 5.3 ± 0.4% and 8.5 ± 1.6%, 7.9 ± 1.2% and 13.0 ± 3.2%, 9.7 ± 1.6% and 26.3 ± 3.3%. NIH EB 001441 funded this work.