**5.2 Phage display technologies**

*Biosensors for Environmental Monitoring*

optical density was 19 phages/ mm<sup>2</sup>

integrity during immobilization.

the following context.

**bonding**

of high number of phage particles creates a steric interruption between phage particles [39]. Thus number of phage particles immobilized on solid surface should not surpass a specific threshold per surface area [48]. For T4 phage, estimated

resulting in reduced signal [49]. Phage particles can be simply oriented on the surface of electrode as they bear positive and negative potential on their tail fibers and head respectively. Phage immobilization strategies are briefly highlighted in

**5.1 Immobilization by physisorption, electrostatic bindings and covalent** 

*Graphical representation of bacteriophage random immobilization and electrostatic, charge-directed orientated immobilization of T2 phage onto CNT electrode surface functionalized with polyethyleneimine* 

Most common approach used for immobilization of phages is physisorption [50, 51]. This approach is very artless, but then again the adsorbed phage may possibly detach as of substrate surface because of shear, changes in pH, or temperature, or ionic concentrations caused in the medium that reduces principally their biosensing applications. Subsequently most phage particles having net negative charge at pH 7 [52], a number of investigators successfully used electrostatic binding for phages immobilization **Figure 4,** [52]. Also this methodology suffers due to variability and bacteriophage detachment in turn to the physico-chemical fluctuations in the analyte medium. Covalent bonding of phages offered a more stronger attachment and is not at risk to easy detachment of phages [53, 54]. Proper chemical studies can make easy selection of suitable substrate and then potential application. Covalent attachment resulted in a sophisticated bacteriophage surface mass that is principally necessary for phage application in biosensor development [55, 56]. To design bioactive surfaces with phages, phage infectivity is important or at least phage should be able to interact with host bacteria or analyte; therefore, optimization is needed to reduce the effect on bacteriophage

area beyond that clogging was happened,

**164**

**Figure 4.**

*(PEI) [57].*

Bacteriophage-display tools can enable scientist to display peptides of choice present on the phage exterior, that is, phage envelope. Phages expressed peptide can consequently be adsorbed on material surfaces that are coated with peptides specific ligands **Figure 5**. Phage-display-libraries are produced by introducing DNA segments into specific phages to facilitate each phage to display a specific peptide expressed by the DNA segment inserted [58]. Technology of phage display developed as a combined influence of two central thoughts, fusion phage and combinatorial peptide libraries [59]. The first theory allows display of external peptides on bacteriophage surface [59]; while the second idea hires libraries of numerous peptides achieved in corresponding production as contrasting to production of single specific peptides [60]. Merging these two theories stemmed progress in phage-display-tools, multibillion clone alignments of self-assembled and self-amplified bio-components [54]. It is significant to keep in mind that genetic alteration may alter the characteristics properties of bacteriophages. For example, biotin-carboxylcarrier-protein (BCCP) gene or the cellulose-binding-module (CBM) gene to the small-outer-capsid-protein (SOCP) gene of T4 bacteriophage was attached, affecting bacteriophage infectivity, and result was decline in burst size, as well as extended latent period [61].

#### **Figure 5.**

*Current applications of phage display technologies as imaging agents. Icosahedral phages are mostly used as, aiming on moieties for bacterial detection, and substrates for signal amplification. While filamentous phages are mostly used as multifunctional probes, and a variety of sensors [67].*

## **5.3 Phage entrapment in porous matrix**

Bacteriophages immobilization in micro-porous matrices permits them functionally and also structurally stable, keeping them active for long time period. Phages immobilization by entrapment in a porous hydrogel, (bio)polymeric agar and alginate matrices, is a tool for selection of applications where protection of phage particles essential against severe environmental conditions [62]. Additionally, entrapment might aid to maintain moisture, which is important for many phages infectivity, or keep phage particles in lyophilized condition [63]. A fruitful marketable case in point of entrapped bacteriophage in matrix is PhagoBioDerm [64] that is 0.2 mm thick, porous-polymericwound-dressing saturated with a mixture of biocides and lytic phages [65]. The matrices used for bacteriophage entrapment, that might possibly delay interaction of entrapped bacteriophage particles with host bacterial cells or analytes that are present in the vicinity of medium [66], marking inefficiency of phage bioactive surface.

### **5.4 Phage layer by layer organization**

Many investigators discovered to possibly immobilize phages by alternative layering with polyelectrolytes having oppositely charges, and claimed observation of enhanced phage particle surface coverage [68, 69]. For instance, a layer by layer methodology for M13 bacteriophage was reported, and phage was sandwiched between oppositely charged layers of weak poly-electrolytes, that was capable to diffuse freely form a nearby packed phage monolayer [69].

### **5.5 Efficiency of immobilized phages in biosensing platforms**

The effectiveness of bio-sensing approaches is mostly measured in terms of minimum limit of detection (LOD) of bacterial or other analyte. Thus researchers attempted and focused to improve the bacteriophage surface coverage for pushing detection limits. Significantly keep in mind that the LOD has not been improved biosensors where phages are immobilized by covalent binding, in comparison to the approaches where phage is immobilized by physisorption [22]. Thus, bacteriophage surface coverage is not only the factor to necessarily increase and improve the sensitivity and LOD of bacteriophage-based biosensor. Limit of detection of biosensors, based on various transduction approaches can be different depending on the working principle of selected transduction platform.
