Preparation Of Chitosan Magnetic Nanoparticle And Prelimilary Study On Immobilization Of NAD(H)

Magnetic nanoparticle is easily to be separated due to its superparamagnetic.Chitosan is biomaterial with goodbiocompatible property. It is valuable topreparation of chitosan coated magnetic nanoparticles which can combine theexcellent characteristics of two materials,. In this research, the different chitosancoated magnetic nanoparticles were prepared by the adsorption/cross-linking method,furthermore all these prepared nanoparticles were used to adsorp BSA andimmobilize nicotinicacyl coenzyme Ⅰ (NAD (H)) for estimation its potentialapplication in immobilization of enzyme and coenzyme.Firstly, a chitosan-coated magnetic nanoparticles were prepared. At first,surface carboxyl functional magnetic nanoparticles (MNP-COOH) were prepared byco-precipitation; then the chitosan were adsorbed on the surface of particles byelectrostatic interaction; afterwards hydroxyl group on the chitosan werecross-linked by glutaraldehyde or epichlorohydrin under the protection of primaryamine group with formaldehyde. The chitosan magnetic nanoparticles with differentsurface function were prepared by using functional reagents. The magneticnanopartieles were characterized by scanning electron microscope (SEM), X-raydiffraction (XRD), Fourier transform infrared (FTIR), thermogravimetric analysis(TGA), vibrating sample magnetometry (VSM) and Zeta potential analyzer．Theresults show that the magnetic nanoparticles were coated by chitosan successfullyand can be completely dispersed in the aqueous solution of various pH1~14to formuniform sol. The diameter of these nanoparticles with the saturation magnetizationof64.5emu/g distributes between10and20nm. The remnant magnetization andcoercivity of the nanoparticles were nearly zero, which demonstrated it has typicalsuperparamagnetic characterization.Secondly, the property of BSA adsorption on chitosan nanoparticle was studied.The different chitosan coated magnetic nanoparticles were investigated to obtain theproperties of BSA adsorption on them. The kinetics and isotherm adsorption curveswere studied respectively. The results show that the adsorption equilibrium could be reached in a short time of20minutes. The interaction of BSA and particles is amonolayer adsorption model at low concentration of BSA and will transit tomultilayer adsorption with the excess of BSA. The maximum adsorption capacityreached330mg BSA/g for MNP-CS-O-EPO, which was produced by EPOmodification of chitosan crosslinked with glutaraldehyde.Finally, the immobilization of NAD+and NADH on chitosan nanoparticles wasstudied. The crosslinking agents of carbodiimide(EDC) and epichlorohydrin wereused to immobilize NAD(H) by different chitosan coated magnetic nanoparticles.The influence of time and NAD(H) concentration on immobilization were studiedrespectively. The results shows that the epoxy groups on surface of nanoparticlecould immobilized the reduced coenzyme(NADH) effectively but not the oxidizedcoenzyme(NAD+) when use epichlorohydrin as the crosslinking agent. The amountof NADH immobilized on MNP-CS-N-EPO and MNP-CS-O-EPO reached33mg/gand149mg/g, respectively. Wherenever NADH and NAD+could be fixed on theparticles when EDC was used as the crosslinking reagent, but the efficiency ofNADH fixing is much greater than NAD+fixing. The NADH and NAD+fixied onMNP-CS-N-AA particles reached120mg/g and43mg/g, respectively. All of the fourdifferents immobilized NAD (H) can assist the ADH catalytics the redox reactioneffectively.