*4.3. Functionalisation of the Sensor Surface*

The immunoreagents were immobilised onto the biosensor transducer, as depicted in Figure 6, using PAH as a polycation electrostatically deposited on the biosensor surface. A thin (10–20 nm) layer of SiO<sup>2</sup> was left of the surface of the Si3N<sup>4</sup> core to provide a negative surface charge of OH− for binding PAH polycations; this was achieved using ellipsometry thickness measurements to calibrate the etching time. The coating layer was deposited by incubating the sensor for 20 min with a 1 mg/mL aqueous solution of PAH. After removal of the solution, the sensor was rinsed three times with de-ionised water, and it was coated by incubating the sensor with a 0.01 mg/mL solution of ProtA in 35 mM Tris-HCl buffer (pH = 7.5) for 15 min. After being rinsed three times with Tris-HCl buffer, incubation with polyclonal antibodies to ZON at a concentration of 1 µg/mL in Tris-HCl buffer was carried out for 15 min. Finally, after final rinsing the cell three times with Tris-HCl buffer, biosensing tests were performed by injecting ZON aliquots at increasing concentrations into the cell and recording the PW sensor responses.

**Figure 6.** Steps in functionalisation of the sensor surface, and detection of zearalenone (ZON) with the functionalised sensor. Bare planar waveguide sensor (**a**); electrostatic immobilisation of a polyallylamine hydrochloride (PAH) layer (**b**); binding protein A (Prot A) to PAH (**c**); oriented anchoring of ZON-specific antibodies by Prot A (**d**), detection of ZON by its specific binding to the anchored antibodies (**e**).
