Surface modification of polyacrylonitrile film by anchoring conductive polyaniline and determination of uricase adsorption capacity and activity
Introduction
The immobilization of enzymes on electrically conductive polymer films have been extensively studied [1], [2], [3], [4]. For example, conductive polymer polyaniline has been used for grafting of various polymers due to the high conductivities achievable by doping and their potential as highly tailorable semiconductive materials for use in electrochromics and biosensors [5], [6], [7]. Polyacrylonitrile does not have functional groups for immobilization of biological molecules therefore, its surface can be modified by anchoring with various functional group carrying monomers or ligands to alter the final surface properties [8], [9], [10]. Enzyme immobilized polyaniline-based polymeric films have been frequently used as a part of biosensors [11], [12], [13].
Since the first enzyme biosensor was described in 1962, numerous publications on biosensor technology have prospered in different fields, from medicine, environment, food and process industries, to security and defense [11], [12], [13], [14], [15]. A large number of immobilized enzymes have been used for construction of biosensors. Immobilized uricase is an important part of the enzyme-based sensor for uric acid determination in biological and other fluids. Uric acid is basically an organic compound of carbon, nitrogen, oxygen and hydrogen with the formula C5H4N4O3 and, it is a heterocyclic purine derivative originating from nucleic acids catabolism. The normal level of uric acid in serum is 0.21 and 0.83 mM. This range is considered normal by the American Medical Association. Uric acid concentrations in blood plasma above and below the normal range are known, respectively, as hyperuricemia and hypouricemia. Excess serum accumulation of uric acid can lead to a type of arthritis known as gout [16], [17] This painful condition is the result of needle-like crystals of uric acid precipitating in joints and capillaries. Elevated serum uric acid (hyperuricemia) can result from high intake of purine-rich foods, and/or impaired excretion by the kidneys. Saturation levels of uric acid in blood may result in one form of kidney stones when the urate crystallizes in the kidney. Thus, high concentration of uric acid in the blood can cause damage to body organs. Therefore, its analysis is of considerable importance. Accurate and rapid measurement of uric acid is very important in clinical and forensic analysis in order to analyze human body fluids, e.g. blood and urine, among others [18]. Uricase (EC 1.7.3.3) is an enzyme participating in the final step of purine degradation and can be used enzymatic determination of uric acid in biological fluids.
In order to make enzyme possess enough effective interact and high activity yield, the polyacrylonitrile film was grafted with conductive polyaniline with varying surface density. The grafting technique can create a functional film surface for interaction biological macromolecules (Fig. 1). First, polyacrylonitrile membrane was prepared by polymer casting and then polyaniline was grafted onto the surface of PAN film. Thus, we developed an economical, high loading capacity polyaniline grafted polyacrylonitrile films which were used for reversible immobilization of uricase. The grafting efficiency, the surface electrical resistances and contact angles of composite films were investigated. Accurate determination of uric acid in biological fluids using immobilized-uricase was performed and obtained results indicate that the uricase-immobilized system can be easily adapted to a biosensor for clinical application.
Section snippets
Materials
Uricase (Urate oxidase; EC 1.7.3.3; Arthrobacter globiformis (about 2 U/mg), catalyzes the oxidation of uric acid to 5-hydroxyisourate), peroxidase (POD, EC 1.11.1.7, Type II from horseradish), bovine serum albumin (BSA), polyacrylonitrile (PAN, Mn 150,000 g/mol), o-dianisidine dihydrochloride, aniline and dimethyl sulfoxide, uric acid were purchased from Sigma–Aldrich Chemical Co. (St Louis, MO, USA) and used as received. Coomassie Brilliant Blue G-250 for the Bradford protein assay, was
Characterization of conductive composite film
In this work, conductive polyaniline (PAN) film was prepared using the conventional casting method in DMSO solution containing CaCl2. The PAN film was grafted with polyaniline (PANI) in the presence of potassium dichromate as an oxidant for graft polymerization (Fig. 1). A maximum grafting yield of 2.4% was observed with an aniline concentration of 5.0 mol/L (Table 1). As seen in this table, an increase in the contents of grafted polyaniline (from 1.2 to 2.4%) on PAN film led to a decrease in
Conclusion
In this study, a novel colorimetric method for determination of serum uric acid was developed using immobilized uricase, which is based on measurement of H2O2 by coupling reaction with peroxidase/o-dianisidine as chromogenic system. This study offers a novel and economical protocol for uricase immobilization on PAN/PANI-1–3 films via adsorption. The (PAN/PANI)-3 composition was used for immobilization of uricase. Temperature, pH, thermal and storage stabilities were enhanced for immobilized
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