Chitosan (CS, 95% deacetylation) was bought from Shanghai Biochemical (Shanghai, China). d-glucose and 30% H2O2 were obtained from Guangzhou Chemical (Guangzhou, China). Disinfectant was purchased from South Land Pharmaceutical. Other reagents were of analytical reagent grade. All selleck solutions were prepared with doubly distilled water. HQ was purchased from Sinopharm Chemical Co. Ltd., China. The exact concentration of H2O2 was determined by titration against a standard potassium permanganate solution.2.3. Synthesis of Fe3O4 Nanoparticles [37]Fe3O4 nanoparticles were synthesized by co-precipitation of Fe2+ and Fe3+ ions in the presence of alkaline solution under hydrothermal treatment. 5.2 g of FeCl3?6H2O and 2.0 g of FeCl2?4H2O were dissolved in 25 mL of 0.4 M HCl.

The solution of the mixed iron-salts was added dropwise into a solution of 250 mL 1.5 M NaOH with vigorous stirring under an atmosphere of nitrogen gas. Then the obtained black precipitate was heated at 75 ��C for 30 min. The precipitate was collected through centrifugation at 4,000 rpm, washed sequentially with distilled water and ethanol. Inhibitors,Modulators,Libraries A black colored powder (Fe3O4 nanoparticles) was obtained upon drying under vacuum at 60 ��C Inhibitors,Modulators,Libraries for 6 h.2.4. Preparation of Magnetic Microspheres (Fe3O4/CS) [31]The suspension cross-linking technique was used for the preparation of magnetic microspheres. A 20 mL 2.5% chitosan solution containing 0.4 g Fe3O4 nanoparticles was prepared using a 3% acetic acid solution. Then it was poured, dropwise, into the dispersion medium composing of 80 mL paraffine oil and 5 mL Span-80 (a type of nonionic surfactant).

At the same time, the dispersion medium was stirred with a magnetic Inhibitors,Modulators,Libraries stirrer at 1,500 rpm at room temperature. After thirty minutes, 1.0 M Inhibitors,Modulators,Libraries NaOH was added slowly to the dispersion medium to adjust the mixture to pH 10.0 at the stirring rate of 1200 rpm. Fifteen minutes later, 0.4 mL of epoxychloropropane was added to the medium and reacted for 40 min at 50 ��C. Similarly, an additional 0.4 mL of epoxychloropropane was added to the dispersion and the stirring rate was lowered to 1,000 rpm. After 1 h, the mixture was continuously reacted for a further 2 h with a lower stirring rate of 800 rpm at the temperature of 60�C70 ��C. At the end of this period, the magnetic microspheres were collected using a magnet and washed consecutively with ether, acetone, 10% ethanol and doubly distilled water, then vacuum dried at 50 ��C prior to storage for further analysis and use.

2.5. Configuration of Fe3O4/CS-Hb-Fe3O4/CS-GCEA glassy carbon electrode (GCE, 3 mm in diameter) was polished with 1.0, 0.3, and 0.05 ��m Al2O3 slurry, and rinsed thoroughly GSK-3 with doubly distilled water in order to obtain a mirror-like surface. Then it was sonicated in 1:1 nitric acid, absolute ethanol and doubly distilled water for 5 min, respectively, and allowed to dry at room temperature. 2.0 mg Fe3O4/CS was dispersed into truly 1.