Abstract

Bacteriophages (or phages) are parasites that infect specific bacteria. This host-selectivity can be useful to identify bacterial contaminants in food, water, environment etc. In the present study, phage was isolated from stagnant water and cultivated by overlay method against host bacteria that is E. coli. Phage titer was calculated to be 10⁷ pfu/ml using 10-fold dilutions. Plaque reaction activity was observed within 4 to 6 h against host bacteria by spot test.  Morphological identification of phage by transmission electron microscopy (TEM) using uranyl acetate staining revealed about 78 nanometers (nm) in wide phage capsid and tail length (527 nm). The isolated phage was classified into order Caudovirales since it possessed a long non-contractile tail and icosahedral capsid head, thus is a member of the family Siphoviridae.  Also, the phage identified followed a lytic life cycle since plaque reaction activity was observed within 4-6 h against host bacteria. Gold immuno-functionalization using self-assembled monolayers (SAM) has been widely used for the detection of small targets, but there are limited reports available describing the detection systems for bacteria by using phages. Thus, in order to develop a suitable detection system for identification of specific bacteria, it is suitable to exploit the close association and selectivity between bacteria and bacteriophage. Bacteriophages were immobilized onto gold surface by SAM using a stable acyl amino ester intermediate generated by 1-ethyl-3(3-dimethylaminoproply) carbodiimide (EDC) and N-hydrosuccinimide (NHS) to condense the bacteriophage. Fourier transformation infrared (FTIR) microscopy presence of different functional groups present in each layer formation. Bacteriophage immobilization over the gold surface was verified through by scanning electron microscope (SEM). Electrochemical analysis was performed for a rapid and specific detection of E. coli cell. The present bio-sensing system comprises of quick and specific detection of host bacteria and possesses a very low detection limit (10⁴ cfu/ml). We propose phages utilization as a bio-component in biosensor development for bacteria capture.

Key words: Bacteriophage, biosensors, electrochemical, microscopy, immune-functionalisation and Self-assembled molecular monolayer.