Antibacterial Peptide-based Colorimetric Bioassays For Rapid Detection Of E.Coli O157:H7

Escherichia coli 0157:H7 is one of major foodbome pathogens that not only seriously threaten human health but also cause serious economic losses.As conventional culture-based methods,PCR-based methods,and immunological methods are used for detection of E.coli 0157:H7,biosensor method has become a promising alternative due to its rapid analysis and simple operation.In this research,antimicrobial peptides(AMP),one of the recognition elements applied in bacteria detection was investigated for their function as a bioreceptor as well as a signal amplifier by having AMP free in solution and flexibly bond onto the surface of E.coli O157:H7 with a high density.Based on this principle,three colorimetric bioassays were developed in this study for rapid and sensitive detection of E.coli 0157:H7 as summarized in the follows.1.The immunomagnetic separation of E.coli 0157:H7The magnetic nanoparticles conjugated with specific antibody were used to capture,separate and concentrate E.coli 0157:H7.The capture efficiencies were up to 90%in the bacterial concentration range from 103 to 105 cfu/mL.Combined with the developed magnetic separation,the immunoassay method was able to detect as low as 989 cfu/mL.Moreover,in order to reduce the non-specific adsorption and the steric hindrance of magnetic beads,the magnetic capture-release procedure was introduced and a recovery of 85%was achieved.These two magnetic separation methods provided a foundation for the development of new colorimetric bioassays.2.Detection of E.coli 0157:H7 based on competitive reaction between biotin-antimicrobial peptide and biotin-horseradish peroxidaseThe detection of E.coli 0157:H7 was based on competitive reaction between biotin-AMP and biotin-horseradish peroxidase(bio-HRP)where biotin acted as the label,and AMP could bind to the bacteria surface with high density to amplify signal.In the test,bio-AMP attached to the surface of bacteria and precipitated along with bacteria after centrifugation.The concentration of bio-AMP had a negative correlation with the concentration of E.coli 0157:H7,and could be quantified by the competitive reaction with bio-HRP on the streptavidin coated magnetic beads.In this way,E.coli O157:H7 could be detected as low as 721 cfu/mL in a range of 103 to 106 cfu/mL.The result showed that there were about(1-1.4)× 107 AMP molecules on the surface of one bacterial cell,indicating a 107 fold amplification achieved by AMP.3.Rapid detection of E.coli 0157:H7 based on antimicrobial peptide and horseradish peroxidase-catalyzed signal amplificationAn AMP-HRP based colorimetric bioassay was developed for rapid and sensitive detection of E.coli O157:H7 considering AMP could bind to bacterial cells rapidly with high density and HRP as the direct label could be highly efficient in signal amplification.AMP-HRP could anchor on the surface of target bacteria rapidly through electrostatic and hydrophobic interactions.After unbound probes were removed by filtration,HRP catalyzed 3,3,5,5-tetramethylbenzidine to colored product,which was measured to quantify E.coli 0157:H7.The immunomagnetic capture-release procedure was introduced to improve the specificity.Due to the abundant AMP-binding sites on the bacterial surface,the proposed bioassay could detect E.coli 0157:H7 as low as 13 cfu/mL with a linear range of 102-105 cfu/mL in 45 min without pre-enrichment.The results showed the limits of detection for E.coli 0157:H7 in spiked apple juice and ground beef samples were 119 and 451 cfu/mL,respectively.4.Rapid and sensitive detection of E.coli 0157:H7 based on antimicrobial peptide functionalized magnetic nanoparticles and urease-catalyzed signal amplificationTo simplify the procedure,a dual-functionalized AMP modified magnetic nanoparticles(MNP-AMP)were fabricated.Then,a simple method for rapid and sensitive detection of E.coli 0157:H7 was developed based on MNP-AMP and urease-catalyzed signal amplification.In the presence of bacteria,the bacteria were captured by MNP-AMP,and the binding sites for urease on the surface of MNP-AMP were blocked.Therefore,a large amount of urease retained in the supernatant after magnetic separation to induce the color change by efficient catalytic hydrolysis of urea into ammonium.Due to the high capture affinity,efficient amplification and simple manipulation,the proposed bioassay could detect E.coli 0157:H7 as low as 12 cfu/mL within 30 min.This novel method was successfully applied to detect E.coli 0157:H7 in spiked apple juice and ground beef samples with a limit of detection of 84 and 233 cfu/mL,respectively,demonstrating its potential in testing food samples.