# A Photonic Label-Free Biosensor to Detect _Salmonella_ spp. in Fresh Vegetables and Meat
## 2 Materials and Methods
### Biodetection Method for Salmonella spp. Multiple experimental tests were conducted to determine the optimal detection limit and the highest sensitivity of the alternative developing technique. This performance and reliability of this detection technique were evaluated by flowing different samples of Salmonella_ spp. over previously functionalized photonic biosensors (PICS). This crucial step enabled comprehensive assessment of the technique's effectiveness in detecting and responding to various _Salmonella_ strains. Some of these strains (i.e., those used to spike samples) were acquired from The Spanish Type Culture Collection (CECT). Others were wild strains that came from naturally contaminated meat and vegetable samples. This feature contributes to the validation and reliability of the technique. To compare immunosensor responses, two Salmonella strains (_Salmonella enterica Typhimurium_ CECT4266 and _Salmonella enterica Enteritidis_ CECT7236) from The Spanish Type Culture Collection (CECT) were used to spike red Batavia and fresh chicken. Different samples of fresh salad and fresh chicken from the same batch were naturally contaminated by _Salmonella_ spp. within a concentration range of 10 to 10\({}^{10}\) CFU/mL, as detailed earlier. The samples were then systematically flowed and subjected to bacterial sensing using a Lumensia Sensors setup detector optimized for PICs (Figure 4). After the photonic chips were successfully functionalized, the next step was to attach a microfluidic adhesive layer to enable the controlled flow of samples. The immunosensors were securely housed within the previously functionalized PICS, as detailed earlier. A peristaltic pump operating at a rate of 15 uL/min controlled the flow rate of the sample dilutions. This precise flow method meant that the prepared samples reached the immunosensors, where the crucial reaction between the _Salmonella_ spp. antigen and the functionalized antibodies specific to that antigen took place. This setup enabled comprehensive evaluation of immunosensor performance in detecting and responding to _Salmonella_ spp. Its efficacy and reliability in real-world food samples was thus verified. The current trend in biosensors is to streamline the identification process in order to enhance accessibility and robustness. The system for capturing the transduction signal emitted by the sensor during the analysis consisted of three essential components (Figure 4): a PIC (designed by Lumensia Sensors), a two-channel microfluidic system integrated with the PIC, and a peristaltic pump. Both microfluidic channels were connected to the microflu  
Figure 4: **Laboratory set-up reader: detection system to detect samples in the laboratory. ** Description of photonic components: laser source, polarization drivers, control PCB. Indication of microfluidic components: peristaltic pump, sample reservoir, fluidic channels, waste reservoir, sensing area (PIC + fluidics). The food target sample was pushed from its reservoir through external microfluidic channels connected to an internal microfluidic channel where the sensor structure (PIC) was housed. The laser source impacted the sensor area (PIC) to create resonance by passing the sample over the ring resonators that housed the PIC. The data are collected and directed by the PCB. idic system, which was attached to the PIC. This setup enabled the samples to flow over the sensor for analysis [44]. The coupled microfluidic system substantially enhanced detection capabilities by enabling the simultaneous measurement of two samples. Two samples could be measured simultaneously thanks to the arrangement of eight ring resonators across two channels, each consisting of four ring resonators (Figure 3). Consequently, this configuration enabled detection of sensitivity on the ng/mL scale, with response times of under 30 min [44]. The setup for reading resonance data from signal transduction is an integral part of a biosensor system.