**3. Phage-based biosensors in food safety and environmental monitoring**

Bacteriophage-based biosensors have been established for broad range of applications in food and environmental contaminant detection, for example, pathogens, toxins, and other environmental pollutants. Pathogens causing food contaminations are the supreme common objects of bacteriophage-based biosensors. One more field wherever bacteriophages are utilized as bio-recognition probes is clinical diagnostics of infectious diseases as explained in Section 2. **Table 1** sums up various whole phage/phage component-based biosensor applications in food safety, environmental monitoring, and infectious disease diagnosis. As this chapter does not cover all the reported methodical explanations and applications, therefore interested bibliophiles are referred to the latest literature. For potential future on-site applications, few of the most recent phage-based biosensors for pathogen detection in food and water are briefed as follow.

### **3.1 Food safety**

*Biosensors for Environmental Monitoring*

current generation [43].

**Figure 4.**

*2.3.2 Phage impedimetric sensors*

*electrochemical screening, adapted from [41].*

biosensors. Amperometric method integrated with bacteriophage typing was reported to specifically detect bacteria like *E. coli* K12, *Bacillus cereus*, *and* 

*Illustration of the establishment of composites (nanoflowers—AuNPs and Thi-phage) and* E. coli

*Mycobacterium smegmatis* [42]. The working principle of this biosensor was phage infection that resulted in bacterial cell lysis, subsequently releasing bacterial cell contents, like enzymes and other cell debris, into the test sample. This enzymatic release can be sensed and measured involving particular substrate. The reaction product is oxidized or reduced at working and reference electrodes, resulting in

Electrochemical impedance spectroscopy (EIS)-based sensors determine the fluctuations in impedance as a result of interactions between bio-probe and the analyte. EIS-based sensors have been utilized for bacterial detection by observing the variations on interface of solution-electrode because of the microbial capture on the biosensor surface. The target analyte binding on the sensor surface typically raises the impedance because of the insulating behavior. Phages have been utilized as a sandwiched cross-linker between bacterial cell and the electrode surface. An effective phage-EIS-based platform was reported for recognition of *E. coli* bacterial cells by T4 bacteriophage immobilization on the surface of activated carbon screen-printed electrode with LOD of ~104 CFU/mL [44]. By increasing bacterial concentration, a decrease in impedance was observed, which was differing from ordinary binding of intact bacterial cells on EIS biosensor. The motive behind this type of observations was because of the lytic activity of bacteriophages that directed cell lysis and the release of ionic cellular contents and alternatively a rise in conductivity. The detection was specific, and they confirmed the specificity by using *Salmonella* as a negative control. Other reports of impedimetric phage-based detections are summarized

**182**

in **Table 1**.

Magnetoelastic (ME) phage-based biosensor was compared with TaqMan-based qPCR for *Salmonella typhimurium* detection on cantaloupe surface. LOD of both approaches was calculated by successive inoculation of cantaloupe surfaces with *S. typhimurium* suspensions. LOD of *S. typhimurium* was 2.47 ± 0.50 log CFU/2 mm2 and 1.35 ± 0.07 log CFU/2 mm2 area of cantaloupe surface and 6.28 and 2.41% by ME phage-based biosensor and qPCR, respectively. This comparison revealed that phage-based ME biosensor is more encouraging and an on-site applicable method to detect *S. typhimurium* on fresh fruit and vegetable surfaces [4]. In another report that was based on fluorescence imaging, *Salmonella* detection was reported involving bacteriophage-derived peptides that bind to *Salmonella enterica* (serotype Typhimurium) cells. In this report, ME biosensor coated with C4–22 phage was used to evaluate and detect *Salmonella* in/on chicken meat. In the case of on-surface detection approach, phage C4–22-based biosensor confirmed *Salmonella* binding capacity 12 times higher than control with no-phage-based sensor, while *Salmonella* cells at concentration of 7.86 × 105 CFU spiked per mm2 area. In the case of inchicken meat approach, phage C4–22 biosensors were inserted at varied depths below the surface of chicken meat (0.1, 0.5, 1.0 cm) after inoculation of *Salmonella* on the surface. The latter approach presented 23.27–33% of *Salmonella* cell absorption up to 0.1-cm deep under the surface [45].

*P. aeruginosa* was detected by lytic phage PaP1 displaying high specificity. For label-free *P. aeruginosa* detection, ECL biosensor involving PaP1 was developed. Biosensor was fabricated on glass carbon electrode surface through deposition of PaP1-conjugated carboxyl-graphene. Adsorption of PaP1 tail fibers and baseplate to bacterial cell wall resulted in a decrease of ECL signal, since the accumulation of non-conductive bio-complex on electrode surface disrupted the electron transfer. ECL signal dropped linearly with 1.4 × 102 –1.4 × 106 CFU/mL concentration of *P. aeruginosa*, with biosensing time of 30 min and very low LOD of 56 colony-forming units per mL. With the help of this biosensor, *P. aeruginosa* was quantified in milk with varying values of recovery from 78.6 to 114.3% [3]. Similarly, phage P100 and magnetic particle composite were established to separate *L. monocytogenes* from food samples. Varied sized magnetic particles (150, 500, and 1000 nm) were used for phage P100 immobilization either physically or chemically. The coupling ratio of composites was investigated, and the capturing efficiency of *L. monocytogenes* was evaluated for each composite. The authors reported that composites developed by physical immobilization of P100 attained a greater efficiency of capture and

selectivity toward *L. monocytogenes*. These composites of phage and magnetic particles were further used to selectively isolate *L. monocytogenes* from real sample of food like whole milk and ground beef [46].
