# A Photonic Label-Free Biosensor to Detect _Salmonella_ spp. in Fresh Vegetables and Meat
## 2 Materials and Methods
### Antibody Immobilization Control on Silicon Nitride Surfaces  
An antibody immobilization control experiment was conducted. It specifically targeted _Salmonella_ spp. on four planar silicon nitride surfaces. Initially, these four pieces of silicon nitride followed an oxidation process by immersing them in a 0.1 M hydrochloric acid (HCI) solution (Scharlab, Barcelona, Spain) comprising 5 mL of solution. This immersion lasted 30 min on an orbital shaker set at 30 rpm (revolutions per minute). Later, the surfaces were washed with water and then dried using a stream of air. These surfaces were subjected to a drying process in an oven incubator at 110 \({}^{\circ}\)C for 30 min. Next, they were tempered in a desiccator for 10 min and were finally incubated at 4 \({}^{\circ}\)C overnight. Surfaces 1 and 2 were subjected to activation. They were immersed in 500 \(\upmu\)L of a solution with a 2:1 molar ratio of NHS (N-Hydroxysuccinimide and EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) in 0.1 M MES (2-(N-morpholino) ethanesulfonic acid) obtained from ThermoFisher (Waltham, MA, USA). This immersion took place at room temperature for 30 min. Surfaces 3 and 4 were not activated and were instead immersed in 500 \(\upmu\)L of water for 30 min at the same temperature. Following these treatments, all surfaces were thoroughly washed with 0.1 M MES and water and then air-dried. Each surface received 0.8 \(\upmu\)L of solution, which was carefully applied in drops. Four drops of the selected monoclonal or polyclonal antibody (Abcam, Cambridge, UK) were added to the first row of four surfaces (1, 2, 3, and 4) against _Salmonella_ spp. Four additional drops of Lipopolysaccharides (LPS) from _Salmonella_ spp. were applied to the second row. Moreover, four more drops of 1\(\times\) PBS were deposited in the third row as a negative control. Additionally, 0.8 \(\upmu\)L of polyclonal antibody was added to the fourth row of surface 1 (S1), whereas 0.8 \(\upmu\)L of monoclonal antibody was introduced to the fourth row of surface 3 (S3). At the same time, surfaces 2 and 4 contained the same content in their respective fourth rows. All antibody solutions used were prepared to reach a final concentration of 200 \(\upmu\)g/mL in 1\(\times\) PBS. Later, each surface was kept for 2 h in controlled humidity conditions and was subsequently washed with 1\(\times\) PBS. Finally, all surfaces were blocked by immersing them in a solution of 1% bovine serum albumin (BSA) in 1\(\times\) PBS. They were left to incubate overnight at a temperature of 4 \({}^{\circ}\)C inside a refrigerator. This comprehensive procedure ensured the systematic immobilization of antibodies on the silicon nitride surfaces, facilitating subsequent experiments and evaluations. A standardized solution of LPS (rough strains) from _Salmonella enterica_ serotype _Typhimurium_ SL1181 (L9516), sourced from Sigma Aldrich, was used as a positive control in the experiments. This control was introduced in the second row of all surfaces except surfaces 1 and 2 and surfaces 3 and 4. With surfaces 1 and 2, a prior functionalization step was conducted using the monoclonal anti-_Salmonella_ spp. primary antibody. With surfaces 3 and 4, the polyclonal anti-_Salmonella_ spp. primary antibody was applied. The primary aim was to compare the immobilization efficiency of these antibodies on the surface material and observe their binding efficiency with the target antigen. After adding the drops containing the positive LPS control for _Salmonella_ spp., the surfaces were incubated at room temperature for 30 min. This step was designed to evaluate the antibodies' performance in terms of surface immobilization and antigen binding efficiency. After a 30-min incubation period, all surfaces were thoroughly washed using 1\(\times\) PBS and then water. Subsequently, a conjugated secondary antibody was applied uniformly across all rows and was incubated at room temperature for 30 min. Following this incubation, the surfaces were again meticulously washed with 1\(\times\) PBS and water.