Molecularly imprinted polymer layer using Navicula sp. Frustules as core material for lysozyme recognition
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Date
2016-06-01
Authors
Lim Guat Wei
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Abstract
Molecularly imprinted polymer (MIP) is a robust and cost-effective recognition material with the potential to replace antibody and enzyme. Conventionally, MIP prepared via bulk imprinting method usually showed poor binding site accessibility for targeted protein molecules. To overcome this drawback, significant amount of research works have been carried out using nanoparticles as core material for protein imprinting. This method has improved the template accessibility as well as rebinding kinetics, but the rebinding capacity achieved is still relatively low owing to the polymer layer generated is extremely thin which was not able to encapsulate the entire protein and the recognition cavities generated therein is limited. Hence, diatom frustules with higher surface were used as replacement of core material for MIP in order to improve rebinding capacity. In this regard, Navicula sp. frustules were used for the first time as core material due to their unique properties of good mechanical strength, nanostructure pores, biocompatible, reproducible with exactness in massive number, and easy to functionalize in order to address the limitation of nanomaterials. In this study, the MIP layer was synthesized on Navicula sp. frustules using acrylamide (AAM), methacrylic acid (MAA), and 2-dimethylamino ethylmethacrylate (DMAEMA) as functional monomers. Lysozyme (LYZ) was used as template to be imprinted as it acts as important index in the diagnosis of various diseases. The polymerization conditions were examined and the optimal conditions were attained at percentage of crosslinker of 50 wt % and monomers molar ratio AAM/MAA/DMAEMA of 1/0.5/0.5. Based on rebinding result, this optimal MIP exhibited enhancement in both rebinding capacity (124 ± 1.09 mg g−1) and imprinting factor (2.99) with a rapid rebinding kinetic (15 minutes) when compared to other researchers that use nanoparticle core material. The result also demonstrated that affinity of MIP towards LYZ is 2.32 times higher than Cyt C and 8.87 times higher than BSA. This confirmed that conformation and configuration of recognition cavities created in MIP matches with template LYZ. The MIP could also be regenerated and reused at least 5 repeated cycles via mild washing with 0.5 M NaCl which suggested that LYZ interact non-covalently with MIP. This protein-MIP interaction was further investigated by extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) analysis which indicated that van der Waals, electrostatic and steric interactions played dominant role in dictating the adsorption of protein on MIP. The predicted attractive interaction of less than -1.693 kT suggested that LYZ adsorption onto MIP is generally weak and could be disrupted easily. Apart from these, the practicability of MIP to rebind LYZ in protein mixture solution was also performed. The test demonstrated that MIP is able to discriminate LYZ from competing proteins when exposed to various pH solution or solution with high concentration of competing protein. Lastly, the MIP was incorporated with fluorophore (FITC-MIP) to enhance the utility of MIP as potential recognition element for biosensor. The developed FITC-MIP was able to quantify LYZ within concentration range of 0 to 0.025 mg mL-1 with a lower limit of detection of 0.0015 mg mL-1 and a response time within 30 minutes. The outstanding recognition and rebinding performance suggested that Navicula sp. frustule is a good core material for protein imprinted polymer layer which and has promising application in biomedical and diagnostics.