**2.3 Synthesis of magnetite and laccase immobilization**

Magnetite nanoparticles were synthesized by coprecipitation of 20 mL of 1 M FeCl2 and 20 mL of 2 M FeCl3 under agitation at 1,500 rpm and 90 °C. 40 mL of 8 M NaOH and 40 mL of 2% (v/v) TMAH were then added to the mixture during 3.5 h at a flow rate of 12 mL/h. Nanoparticles (Magnetite) were magnetically separated aided by a strong permanent magnet, then washed thoroughly with 2% (v/v) TMAH, and finally sonicated for 100 min in a VibraCell ultrasonic bath (Sonics, USA).

Magnetite nanoparticles were buffered by adding a NaOH solution until pH approached 11, then sonicated for 10 min. 50 μL of 2% (v/v) TMAH was added and dispersed and then the mixture was sonicated for 10 min. Silanization of the nanoparticles was carried out by adding 50 μL of 2% (v/v) APTES followed by sonication for 20 more min. 50 μL of 2% (v/v) glutaraldehyde was added to the mixture as the crosslinker, and left to react for 30 min. Finally, 50 μL of 960 U/L laccase was added and left overnight to immobilize the enzyme on the nanoparticles. The resulting bionanocompounds (i.e., Lac-Magnetite) were separated by magnetism and washed thoroughly with MilliQ water.
