Date of Graduation

5-2012

Document Type

Thesis

Degree Name

Master of Science in Chemistry (MS)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Stenken, Julie A.

Committee Member

Fritsch, Ingrid

Second Committee Member

Hestekin, Christa N.

Third Committee Member

Heyes, Colin D.

Fourth Committee Member

Paul, David W.

Fifth Committee Member

Tung, Chao-Hung Steve

Keywords

Applied sciences; Pure sciences; Cytokines; Heparin; Magnetic beads; Microfluidics; Polystyrene beads; Pre-concentration

Abstract

A proof-of-concept microfluidic device combined with heparin-immobilized magnetic beads was created to concentrate cytokine proteins collected from microdialysis samples. Cytokines are known to be related to several diseases such as cancer, and Parkinson's diseases, so to be able to develop more effective diseases treatments their interactions have to be well understood. Amine-functionalized polystyrene and carboxyl-functionalized magnetic microspheres of ~6.0 ìm in diameter were used to immobilize heparin. The amount of heparin immobilized on polystyrene beads was 5.82 x 10-8 ± 0.36 x 10-8 M per 1.0 x 106 beads and for magnetic beads was 0.64 x 10-8 ± 0.01 x 10-8M per 1.0 x 106 beads. The minimum initial heparin concentration needed to bind ~ 100% cytokines was 36.8 ìM based on estimations for a fixed initial concentration (1.0 nM) of cytokines. For polystyrene beads, it was found that 0.1 and 1.0 nM ratCCL2 (MCP-1) bound to immobilized heparin at levels of 94.50 and 83.67%, respectively. For heparin immobilized magnetic beads, experimental percentages of cytokine bound to heparin were 70.38 ± 1.71 % (ratCCL2, 0.57 nM) and 11.07 % (ratTNF-á, 0.09 nM). The differences between experimental and estimated percentages of cytokine bound to heparin were 28.31 and 31.56% for ratCCL2 and ratTNF-á. A microfluidic system was designed and made of polydimethylsiloxane (PDMS) with soft lithography. The dimensions were as follows: a) Inlet channel width of 0.1 mm, b) circular trapping area of 3.6 mm in diameter, and c) outlet channel width of 0.2 mm. The equivalent circuit theory was used to estimate the pressure drop for each channel at a flow rate of 1.0 ìL/min. Estimated Reynolds numbers for each channel were low (0.17, 0.01, and 0.11) in agreement with the theory. Estimated pressure drops were 112.2, 0.20, and 30.28 Pa. Using different flow rates, the infusion of magnetic microspheres into the device and their "spreading" behavior within circular channel was observed and quantified. "Spreading" behavior of magnetic microspheres on a circular channel could be controlled by changing their flow rate. Controlling the behavior of magnetic microspheres is very crucial for pre-concentration of cytokine proteins on bead-based microfluidic devices. This microfluidic device is now ready for testing of the trapping and preconcentration of cytokines in real microdialysis samples.

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