Immobilization of horseradish peroxidase (HRP) enzyme on Silica nanoparticles

Immobilized enzymes have several advantages over soluble enzymes such as retention and repeating of their catalytic activities. The stabilization of enzyme activity was the most important character for its immobilizing. The immobilized enzymes are useful in diagnostics, bio-affinity chromatography, and biosensor applications. In the present study, horseradish peroxidase (HRP) was immobilized on silica nanoparticles. For achieving this purpose, firstly, silica nanoparticles were synthesized. Then, the nanoparticles were conjugated with HRP (1 µL, 1000 µg mL-1) using 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.5 mg mL-1 ) and N-hydroxysuccinimide (NHS, 0.5 mg mL-1) at 25 ◦C for 12h. The silica-HRP complex was rinsed three times with H2O. Afterwards, the silica-HRP complex structure was screened by SEM and DLS. The activity of silica-HRP complex was measured by adding tetramethylbenzidine (TME) to mixture reaction. The reaction was stopped with adding 50 µL HCl (10 mM) and the result was evaluated by UV-Vis spectrophotometry. The result revealed that the structure and size of silica nanoparticles were spherical and 121-172 nm, respectively. Also, the stability and activity of enzyme were increased up to 1.7 and 1.2 times, respectively. Overall, silicon nanoparticles could be used as immobilizing agents in order to enhance the stability and activity of the enzyme.