*2.2.1 Phage-QCM-based sensors*

Quartz crystal microbalance (QCM) sensors are mass-based sensors that are highly sensitive with the ability of detecting nanogram variations in mass. QCM

#### **Figure 3.**

*Schematic representation of* S*.* typhimurium *detection on tomato and spinach leaves on magnetoelastic-E2 phage-based biosensor system, adapted from [34].*

**181**

*Applications of Phage-Based Biosensors in the Diagnosis of Infectious Diseases, Food Safety…*

biosensors are functionalized by a very thin piezoelectric film having both sides coated with two conductive electrodes. Mechanical resonance is stimulated by

and sensitive platform for *Salmonella typhimurium* detection. This immobilized

[35]. Other reports of phage-based QCM sensor applications in detection of infectious bacteria in food safety and environmental monitoring are briefed in **Table 1**.

Magnetoelastic sensors are prepared from materials having magnetoelastic property, i.e., magnetism and elasticity, and they contract/extend on excitation by alternative-current-magnetic field. The resonance frequency depends on the viscosity/mass adjacent to the surface of the resonating material. Magnetoelastic devices are used for detection of biological and chemical analytes by integration of bio-probes like phages on the biosensor surface and might be functional in gaseous, static, liquid, or flowing condition [21]. Likewise, E2 bacteriophage was genetically modified for specific detection of *S. typhimurium* in samples of food [36], on spinach leaves [37], and in apple juice, tomato, or milk [38], and all these magnetoelastic biosensors displayed outstanding selectivity and specificity. In addition, E2 bacteriophage-based magnetoelastic biosensors expressed tremendous stability when exposed to severe environmental conditions [39]. ME-lytic phage-based biosensor was reported to detect MRSA bacteria. In the evaluation based on varied immobilization times (10, 30, 90, 270, 810, and 2430 min) and bacteriophage con-

lished for optimal conditions. The optimal immobilization time and concentration in PFU/mL for effective binding of phage to ME sensor surface was calculated as 30 min and 1011 PFU/mL, respectively. This ME-based biosensor approach was used

A schematic representation of electrochemical biosensor of nanoflowers— AuNPs and Thi-phage composite—for *E. coli* detection is illustrated in **Figure 4**.

Among the electrochemical detection methods, amperometry has been most commonly used for detection of pathogenic bacteria and offered an improved sensitivity platform related to other electrochemical approaches. Electrochemical amperometric biosensor involves a working electrode (having bio-probe) and a reference electrode. For current production in the analyte sample, a bias potential is passed on these electrodes. The produced current is directly dependent on the degree of electron transfer that fluctuates with changes in analyte's ionic concentration. Simply, amperometric sensors detect ionic changes in the solution by determining the variations in electric current. Several approaches have been established for detection of foodborne pathogenic bacteria based on phage-amperometric

successfully for detection of MRSA bacteria with LOD of 103

**2.3 Phage-based electrochemical biosensors**

*2.3.1 Phage-amperometric biosensors*

Consequently, QCM-based biosensors could be established to quantify the mass of many target analytes by immobilization of individual bio-probes on the surface of sensor. Phages as bio-probes can be conjugated with QCM biosensors for selective screening of bacterial cells. For instance, physically adsorbed bacteriophages around

on the surface of piezoelectric transducer provided a very rapid

CFU/mL and a quick reaction and detection time of less than 3 min

–1012 PFU/mL), lytic phage binding to ME sensor surface was estab-

CFU/mL having a broad linear

CFU/mL [40].

*DOI: http://dx.doi.org/10.5772/intechopen.88644*

3 × 1010 PFU/cm−<sup>2</sup>

–107

*2.2.2 Phage magnetoelastic sensors*

range of 100

centrations (108

electrical field application through the quartz crystal.

bacteriophage on QCM biosensor had a LOD of 102

#### *Applications of Phage-Based Biosensors in the Diagnosis of Infectious Diseases, Food Safety… DOI: http://dx.doi.org/10.5772/intechopen.88644*

biosensors are functionalized by a very thin piezoelectric film having both sides coated with two conductive electrodes. Mechanical resonance is stimulated by electrical field application through the quartz crystal.

Consequently, QCM-based biosensors could be established to quantify the mass of many target analytes by immobilization of individual bio-probes on the surface of sensor. Phages as bio-probes can be conjugated with QCM biosensors for selective screening of bacterial cells. For instance, physically adsorbed bacteriophages around 3 × 1010 PFU/cm−<sup>2</sup> on the surface of piezoelectric transducer provided a very rapid and sensitive platform for *Salmonella typhimurium* detection. This immobilized bacteriophage on QCM biosensor had a LOD of 102 CFU/mL having a broad linear range of 100 –107 CFU/mL and a quick reaction and detection time of less than 3 min [35]. Other reports of phage-based QCM sensor applications in detection of infectious bacteria in food safety and environmental monitoring are briefed in **Table 1**.

## *2.2.2 Phage magnetoelastic sensors*

*Biosensors for Environmental Monitoring*

*2.1.5 Phage-colorimetric sensors*

**2.2 Phage-based micromechanical sensors**

*2.2.1 Phage-QCM-based sensors*

*Bacillus globigii*, MS2 bacteriophage, and also SEB [27]. The typically reported sensitivity until now is about 20 CFU/mL by epi-fluorescent microscopic platform

Sensing based on changes in color allows the use of simple diagnostic systems like spectrophotometers, or even involving smartphones, and both of them are comparatively common and feasible. Designed colorimetric phage-based biosensors are mostly based and integrated on the utilization of reporter bacteriophages that carry genes coding for reporter enzymes. The foremost colorimetric sensor based on phage was to detect *Salmonella* ice nucleation sensor using reporter gene *inaW* [29]*.* Expression of ice nucleation protein was induced upon infection, interrupting the cell, and was consequently observed by the addition of an indicator dye (orange colored) [30]. Other serviceable reporter genes that have been successfully used with various colorimetric substrates are *celB* and *lacZ* segments encoding β-galactosidase and β-glycosidase [31]. More recently enhanced phage-based colorimetric technique has been reported to be integrating and coupling with novel technologies like surface plasmon [32], macroscope and smartphone [33], and lateral flow assay [11]. Other colorimetric phage-based biosensors established in recent years are briefed in **Table 1**.

Representative micromechanical biosensor (magnetoelastic) is expressed in **Figure 3**,

Quartz crystal microbalance (QCM) sensors are mass-based sensors that are highly sensitive with the ability of detecting nanogram variations in mass. QCM

involving E2 phage for detection of *S*. *typhimurium* on tomato and spinach leaves. Further micromechanical-phage-based biosensors are briefed in the following context.

[25] and is 1 CFU/mL by flow cytometric recognition approach [28].

**180**

**Figure 3.**

*phage-based biosensor system, adapted from [34].*

*Schematic representation of* S*.* typhimurium *detection on tomato and spinach leaves on magnetoelastic-E2* 

Magnetoelastic sensors are prepared from materials having magnetoelastic property, i.e., magnetism and elasticity, and they contract/extend on excitation by alternative-current-magnetic field. The resonance frequency depends on the viscosity/mass adjacent to the surface of the resonating material. Magnetoelastic devices are used for detection of biological and chemical analytes by integration of bio-probes like phages on the biosensor surface and might be functional in gaseous, static, liquid, or flowing condition [21]. Likewise, E2 bacteriophage was genetically modified for specific detection of *S. typhimurium* in samples of food [36], on spinach leaves [37], and in apple juice, tomato, or milk [38], and all these magnetoelastic biosensors displayed outstanding selectivity and specificity. In addition, E2 bacteriophage-based magnetoelastic biosensors expressed tremendous stability when exposed to severe environmental conditions [39]. ME-lytic phage-based biosensor was reported to detect MRSA bacteria. In the evaluation based on varied immobilization times (10, 30, 90, 270, 810, and 2430 min) and bacteriophage concentrations (108 –1012 PFU/mL), lytic phage binding to ME sensor surface was established for optimal conditions. The optimal immobilization time and concentration in PFU/mL for effective binding of phage to ME sensor surface was calculated as 30 min and 1011 PFU/mL, respectively. This ME-based biosensor approach was used successfully for detection of MRSA bacteria with LOD of 103 CFU/mL [40].

### **2.3 Phage-based electrochemical biosensors**

A schematic representation of electrochemical biosensor of nanoflowers— AuNPs and Thi-phage composite—for *E. coli* detection is illustrated in **Figure 4**.

#### *2.3.1 Phage-amperometric biosensors*

Among the electrochemical detection methods, amperometry has been most commonly used for detection of pathogenic bacteria and offered an improved sensitivity platform related to other electrochemical approaches. Electrochemical amperometric biosensor involves a working electrode (having bio-probe) and a reference electrode. For current production in the analyte sample, a bias potential is passed on these electrodes. The produced current is directly dependent on the degree of electron transfer that fluctuates with changes in analyte's ionic concentration. Simply, amperometric sensors detect ionic changes in the solution by determining the variations in electric current. Several approaches have been established for detection of foodborne pathogenic bacteria based on phage-amperometric

#### **Figure 4.**

*Illustration of the establishment of composites (nanoflowers—AuNPs and Thi-phage) and* E. coli *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 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 current generation [43].

## *2.3.2 Phage impedimetric sensors*

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 in **Table 1**.

**183**

*Applications of Phage-Based Biosensors in the Diagnosis of Infectious Diseases, Food Safety…*

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

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

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*

CFU spiked per mm<sup>2</sup>

chicken 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 absorp-

*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.

*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

–1.4 × 106

area of cantaloupe surface and 6.28 and 2.41% by

area. In the case of in-

CFU/mL concentration of *P.* 

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

*DOI: http://dx.doi.org/10.5772/intechopen.88644*

detection in food and water are briefed as follow.

**3.1 Food safety**

and 1.35 ± 0.07 log CFU/2 mm2

cells at concentration of 7.86 × 105

tion up to 0.1-cm deep under the surface [45].

ECL signal dropped linearly with 1.4 × 102

*Applications of Phage-Based Biosensors in the Diagnosis of Infectious Diseases, Food Safety… DOI: http://dx.doi.org/10.5772/intechopen.88644*
