Magnetic Fe 3 O 4 nanoparticles (Fe 3 O 4 NPs) were synthesized by co-precipitation (of Fe 2+ and Fe 3+ ) and surface-carboxylated by poly(styrene-co-acrylic acid) (PSA). Then they were thoroughly characterized by Scanning Electron Microscope and Transmission Electron Microscope analyses. Using glutaraldehyde cross-linking immobilization strategy, cholesterol oxidase (ChOx) was immobilized onto Fe 3 O 4 /PSA microspheres. The Fe 3 O 4 /PSA-ChOx has been successfully synthesized on the planar platinum polyaniline (PANi) modified electrode. PANi/Fe 3 O 4 /PSA-ChOx sensing properties were studied using cyclic voltammetric and chronoamperometric techniques. Under the optimized experimental conditionsthe biosensor shows excellent features, such as wide linear range, 0.2 to 1.8 mM (R 2 ¼ 0.9901), low detection limit (0.02 mM), short response time (5-10 s), and high sensitivity (8796 mA mM À1 cm À2 ). The interference test with selected organic compounds, such as glucose, ascorbic acid, uric acid, and acetaminophen indicated high selectivity of the above sensor. It can be proven that the proposed PANi/ Fe 3 O 4 /PSA-ChOx based sensing concept could be successfully used for other biodetections.

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