| Chemiluminescence（CL）and fluorescence（FL）are belonging to molecular luminescence analysis.Chemiluminescent method does not need excitation source,thus the detecting instrument is much simpler and generating low background signal.At the same time,CL has exerted high sensitivity and wide linear range.Since the excitation source and photodetector are not collinear,FL has high signal to noise ratio and low detecting limit.CL and FL combined with molecule recognition agents functionalized magnetic particles can simplify the pretreatments,eliminate matrix effects,enhance detect specificity and further improve the sensitivity and accuracy,thus is suitable for detection of Staphylococcus aureus（S.aureus）.Herein,competitive CL and sandwich FL for specific detection of S.aureus were established by combining magnetic separation and two specific molecule recognition agents-Immunoglobulin G（IgG）and phage-displayed peptide（PDP）.Part 1 Ultra-sensitive chemiluminescent detection of S.aureus based on competitive binding of Staphylococcus protein A-modified magnetic beads to IgG A competitive CL was established based on Staphylococcus protein A（SPA）and IgG binding for directly detection of S.aureus.SPA can bind to the Fc region of IgG.As SPA almost expresses naturally in the cell walls of all S.aureus and rarely present in other kinds of bacteria,the binding between S.aureus and IgG becomes more specificity.SPA-modified magnetic beads and S.aureus were competitively binding to rabbit IgG which previously labeled with horseradish peroxidase（HRP）.The beads were then magnetically separated,and CL was generated by adding the reagents luminol and H2O2.Under optimal conditions,the intensity of CL decreases with increasing concentration of S.aureus over a very wide linear range(1.0×101 to 1.0×109 cfu·m L-1),with a limit of detection of 6.0cfu·m L-1（S/N=3）.This method exerts application potential in clinical diagnosis,food and drug safety control,as well as environmental monitoring,as it owns merits like rapid detection,high specificity and sensitivity,low cost and simple manipulation.Part 2 Sandwich fluorimetric method for specific detection of S.aureus based on dual peptides recognition strategyHerein,a sandwich fluorimetric strategy was established based on two peptides for detecting S.aureus specifically.In this protocol,a phage-displayed peptide and fluorescein isothiocyanate（FITC）tagged magainin I were utilized as specifically molecule recognition agent to targeted bacterium and high sensitive fluorescent probe,respectively.Also,phage-displayed peptide tagged magnetic particles was used to separate,capture and enrich the target bacterium specifically from assay sample,as to eliminate matrix effects and enhance detect specificity.Compared with bacteriophage and antibody,phage-displayed peptide and magainin I exert merits like stable,inexpensive,easy to synthesis and modification.The intensity of FL increased with increasing concentration of S.aureus from 1.0×101 to 1.0×105 cfu·m L-1,with a limit of detection of 9.0 cfu·m L-1（S/N=3）.This proof-of-concept work could also be extended to detect other pathogens by screening phage-displayed peptides specific to other bacteria. |
