Immobilization of myoglobin from horse skeletal muscle (Mb) and hemoglobin (Hb1) from Lucina pectinata in hydrophilic polymer networks for H2S biosensor application
Castro Forero, Angelines A.
AdvisorTorres Lugo, Madeline
CollegeCollege of Engineering
DepartmentDepartment of Chemical Engineering
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Hemeproteins are known for their spectroscopic properties which change during the binding of specific ligands. This project envisioned the immobilization of myoglobin from horse skeletal muscle (Mb) and hemoglobin I from Lucina pectinata (HbI) in hydrophilic polymer networks to use them as recognition elements in biosensor applications. Two immobilization techniques were considered, adsorption and entrapment. Hydrophilic polymer networks of various morphologies were tailored to examine the maximum encapsulation efficiency of each. Anionic hydrogels composed of methacrylic acid (MAA), cationic hydrogels composed of dimethylamino ethyl methacrylate (DMAEM) and the neutral hydrogels composed of poly(ethylene glycol) monomethylether monomethacrylate (PEGMA) (n=200, 400, 1000), all crosslinked with poly(ethylene glycol) dimethacrylate (PEGDMA) (n=200, 600, 1000), were synthesized by free radical solution polymerization. Using adsorption immobilization method, MAA based hydrogels incorporated the highest amount of Mb when compared to PEGMA or DMAEM polymers. Evaluation of the correlation length of the networks revealed that MAA hydrogels possessed the highest correlation length (15.611-26.988nm) when compared to PEGMA containing matrices (0.254-0.342nm) or DMAEM hydrogels (1.461-1.645nm). The Mb hydrodynamic radius was reported to be approximately 2.04nm indicating that neutral and cationic hydrogels may have not adsorbed significant amounts of proteins due size exclusion effect. Release experiments performed in sodium phosphate buffer (PBS) at pH 5.8 and 7.0 showed that solute transport mechanism in anionic hydrogels was a combination of Fickian diffusion and chain relaxation process. However, Fickian diffusion predominated at pH 7.0, while chain relaxation ruled at pH 5.8. Myoglobin diffusion coefficients for MAA based hydrogels at pH 7.0 were in the magnitude order of 10-9 cm 2 /s, and they increased as crosslinker lengths diminished. The amount of protein released decreased significantly as a function of pH. The diffusion coefficients for myoglobin loaded MAA hydrogels at pH 5.8 were in the order of magnitude of 10-9 cm 2 /s and 10-11 cm 2 /s for hydrogels crosslinked with PEGDMA600 and PEGDMA1000 respectively. Myoglobin loaded MAA hydrogels showed to retain its biological activity after the immobilization process. The amount of HbI incorporated by adsorption inside anionic hydrogels was considerably lower than myoglobin loaded in the same polymer configurations. HbI loaded MAA-PEGDMA hydrogels was able to bind hydrogen sulfide evidencing that was biologically active after immobilization process. Immobilization by entrapment was not possible to achieve in neutral and cationic hydrogels, because the contact between myoglobin and these polymerization solutions produced protein precipitation due to the presence of ethanol and PEG. Changes in myoglobin spectroscopic properties were observed immediately after contact with MAAPEGDMA polymerization solution and after polymerization was performed, indicating a possible detrimental effect over myoglobin structure. Release of myoglobin incorporated by entrapment in MAA-PEGDMA hydrogels was highly influenced by chain relaxation process. The diffusion coefficients of myoglobin incorporated by entrapment in anionic hydrogels were two magnitude orders smaller than for myoglobin incorporated by adsorption, both evaluated at pH 7.0.