# A Photonic Label-Free Biosensor to Detect _Salmonella_ spp. in Fresh Vegetables and Meat
## 3 Results and Discussion
### Study of Bioreceptor In immobilization  
The antibodies employed as bioreceptors for detection can be tagged with fluorescent compounds [50; 51]. These probes are then deposited onto the biosensor surface at different concentrations. The suitability of different antibodies as bioreceptors can be evaluated using the resulting fluorescence data. Laser scanners are crucial in the analysis and quantification of the fluorescence signal generated by molecular excitation. These scanners provide an accurate and controlled way of measuring the fluorescence emitted from samples. This approach enables comparison of diverse immobilization methods and helps select the most effective antibody to be immobilized on biosensor surfaces. Following immobilization of the selected antibodies for _Salmonella_ spp. on silicon nitride surfaces, the results were consistent with expectations. In Figure 6a, the drops in Line 1 corresponded to the results of the immobilization of the monoclonal antibody. This antibody subsequently reacted by binding to the secondary anti-mouse antibody. In this second row (Line 2) a serial dilution of the positive control LPS of _Salmonella_ spp. confirmed a successful interaction with the monoclonal antibody. This outcome was consistent with the experimental design because the secondary antibody was the monoclonal GAM (goat anti-mouse Alexa Fluor 647). Consequently, this secondary antibody specifically reacts to the monoclonal antibodies and did not bind with the polyclonal antibody on the same surface. On surface 3 (Figure 6c), the four drops in the first row signified the immobilization of the polyclonal antibody, with subsequent addition of the secondary GAR (goat anti-rabbit Alexa Fluor 647) antibody. In the second row (Line 2), the four drops also corresponded to the binding with a dilution of the positive control LPS of _Salmonella_ spp. This reaction indicated successful interaction with the immobilized polyclonal primary antibody, and surfaces 2 (Figure 6b) and 4 (Figure 6d) remained non-activated. They exhibited lower fluorescence intensity than the activated surfaces (Figure 4a,c). This discrepancy highlighted the importance of surface activation in facilitating antibody immobilization and subsequent binding. The signal for both covalently immobilized antibodies (pAb and mAb) through EDC/NHS activation on surfaces 1 and 3 was significantly higher than the signal for the non-specific control surfaces 3 and 4 (no EDC/NHS treatment), as shown in Figure 7. The higher fluorescence intensity measurements for both antibodies on activated surfaces 1 and 3 can be attributed to the formation of covalent bonds between the antibodies and the chemically treated surface. As a result, this immobilization control experiment provided confirmation that both antibodies were securely covalently attached to the chemically treated surface of the PICs, which acted as immunosensor surface. The monoclonal antibody against _Salmonella_ spp. yielded superior results in terms of fluorescence intensity and covalent attachment. The differences in fluorescence intensity between activated and non-activated surfaces indicated that immobilization was specific and that the antibodies contributed to the observed fluorescence signals. Statistical analysis was conducted to evaluate differences in fluorescence intensity values between activated and non-activated surfaces, as well as between monoclonal and polyclonal antibodies on activated surfaces. For surfaces with the immobilized monoclonal antibody (surfaces 1 and 2), statistically significant differences were observed, with a \(p\)-value of 0.0016 (less than the alpha value of 0.05). The result for the F statistic exceeded the critical value from the tables. This finding suggested significant differences in fluorescence intensity between activated and non-activated surfaces for the monoclonal antibody. For surfaces 3 and 4, with the immobilized polyclonal antibody, statistically significant differences were observed.