A Novel Impedimetric Biosensor Fabricated With Affinity Peptides For Selective Detection Of Pathogenic Bacteria

Rapid detection of pathogenic microorganisms is an effective means of controlling the outbreak of infectious diseases.Currently available methods for qualitative microbial detection generally require a tedious processing procedure of the bacteria in a sample,involving operations such as selective culturing,enzyme-linked immunosorbent assays(ELISA),as well as polymerase chain reaction(PCR).People have therefore been very interested in seeking alternative real-time fast detections in recent years.In particular,a variety of immunosensors have been developed based on the antigenicity of the target bacterial cells,using technologies including immunofluorescence,surface plasmon resonance(SPR),electrochemical impedance spectroscopy(EIS).Nevertheless,these methods are excessively time consuming,using biological agents that are subject to low availability and high cost.In recent years,phage display has been widely used to screen affinity peptides against biomolecule,whole cells,nano scale materials,and polymer.The use of affinity peptides for pathogenic detection applications can be advantageous in terms of high binding affinity,feasibility for chemical modification,and low-cost of production.In this work,we examined the use of a Ph.D.12 library for discovery of peptides that can specifically bind to E.coli O157:H7,and further studied biosensors fabricated with such peptides for selective bacteria detection.The main results obtained are as follows:1.To determine the binding capacity and affinity strength,4 identified phage clones showed up more than once were chosen from the last two rounds of biopanning for ELISA assay.The phage displaying peptides AP2(VVSPDMNLLLTN)and AP3(GLHTSATNLYLH)exhibited a 3-times higher binding capacity against E.coli O157:H7 than that achieved with peptide library.And the phage AP3 showed good selectivity toward E.coli O157:H7 as compared to other bacteria strains.The above results demonstrate that the screening process was effective and AP3 was accordingly chosen for subsequent studies as the ligand to E.coli O157:H7 for construction of APs-modified gold electrode biosensor.2.The sequence GLHTSATNLYLHGGGC used in this work with three-glycine(G)residues act as a spacer and an external cysteine(C)that can act as an anchor onto gold electrode.CV and EIS were further conducted to demonstrate the attachment of the APs and bacteria onto the gold electrode surface.The biosensor can detect the E.coli O157:H7 from 2×1023 to 2×106 CFU/mL and the low limit of detection(LOD)of the method appeared to 20 CFU/mL.To further investigate the selectivity of the AP-fabricated biosensor,the biosensor expressed preferential sensitivity for different bacteria in an order of:E.coli O157:H7>E.coli DH5?>B.subtilis>S.aureus.The biosensor can quickly detect and specifically identify E.coli O157:H7.The affinity peptide fabricated impedance sensor shows a simple chemical modification and can effectively save detection time and cost,and it is a scientific and convenient detection method.All of these observations make it attractive for in situ biosensing applications serving the rapidly growing demands in public health,environment,and food safety.