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Horseradish Peroxidase (HRP) is ranked as one of the most important industrial enzymes that is extensively used in industry. Cholesterol is routinely detected indirectly by cholesterol oxidase in the presence of O 2 , liberating H 2 O 2  as a by-product. The H 2 O 2  content is determined through the HRP activity in the presence of a redox dye, producing a red colored quinoneimine which can be measured quantitatively. Herein, we have designed a magnetic nanoparticle for reusing and easily separating HRP as the most expensive compartment for the low-cost cholesterol assay. The gum Arabic coated magnetic nanoparticles were functionalized with L-lysine linker for maintaining protein flexibility on nanoparticle. Enzyme-loaded nanoparticles were characterized by TEM, FTIR, DLS, VSM and XRD analysis. The immobilization efficiency was ∼65% and the immobilized HRP retained 60% of its activity after 8 times reuse. The optimum pH and thermal stability shifted from 7.0 to 8.0 and 60 to 70 °C after immobilization, respectively. Storage stability of HRP was improved by 10%, at 4 °C for 60 days. Immobilized HRP showed more catalytic activity in presence of Fe 2+ , Ca 2+  and Na + . The designed system has cholesterol detection linearity range from 0.2 to 5.0 mM and detection limit of 0.08 mM and acceptable correlation coefficient of 0.9973 and 0.9982 on sample serum using both chromogens. The HRP-loaded magnetic nanoparticles are capable of being used as a cost-effective system for cholesterol determination in laboratory due to its reusability and stability benefits.

Immobilization of horseradish peroxidase on lysine-functionalized gum Arabic-coated Fe 3 O 4 nanoparticles for cholesterol determination