Rapid Colorimetric Detection Of Escherichia Coli Based On T4 Phage-displayed β-galactosidase

The contamination of food and water by pathogenic microorganisms is an eternal challenge to the global medical system and food hygiene.We need a quick and specific method to detect pathogen urgently.As a new generation of detection method,biosensor is widely used for its advantages of good specificity,high sensitivity and fast analysis speed.Phages can bind to host bacteria specifically and be used as recognition elements of biosensors.But wild-type phages themselves do not usually produce optical signal that can be easily detected.Therefore,we displayed the enzyme protein on the phage surface in vitro by assembly,and the enzyme-catalyzed chromogenic substrate provided the optical detection signal.In this thesis,soc-β-galactoside(soc-β-gal)protein was displayed on the capsid surface of T4 phage by affinity immobilization to obtainβ-gal T4.Then magnetic beads coupling with polyclonal antibody for early capture separation,followed by specific capture of target bacteria byβ-gal T4 which catalyze the chromogenic substrate to achieve the purpose of rapid and quantitative detection of bacteria.The detailed research content includes following two parts.(1)The specificity of soc-β-gal was specifically combined to the soc end of the capsid surface of T4 phage by affinity binding.The characterization of SDS-PAGE and substrate colorimetry indicated the enzyme was immobilized on the surface of T4 phage successfully.Transmission electron microscopy and bacterial infection assay showed that the nature properties of theβ-gal T4 did not change.In addition,the stability under different conditions and enzyme kinetic constants of the two forms ofβ-gal were compared.The results showed thatβ-gal T4 significantly improved the stability and affinity of the substrate compared with freeβ-gal,which demonstrated the performance advantage ofβ-gal T4 in enzyme labeling and its potential as a bacterial detection tool.(2)E.coli K12 was detected by sandwich method based onβ-gal T4 and magnetic separation technology.The specific binding of T4 to E.coli K12 captured by magnetic beads could be separated under magnetic field.And then chlorophenol redβ-D-galactopyranoside(CPRG)was added and incubated.The concentration of E.coli K12 was quantitatively determined according to the colorimetric results.Results showed that the system of different concentrations of E.coli K12 have positive linear response and the concentration of 10~3cfu/m L could be detected within 2 h.The detection results of other common non-target bacteria showed that the method had good selectivity,and it had been successfully applied to the actual sample detection,indicated its application potential in practical scene.