Study Of Biosensor Based On Colloidal Gold Amplifying For Detection Of The Staphylococcus Enterotoxin B

Staphylococcus aureus Enterotoxin is one of important etiological agents of causing food poisoning and hospital onset of infection.Staphylococcal Enterotoxin B(SEB) is a kind of superantigen active exotoxins,possessing powerful biologic activity all the same. The SEB fast detecting method is possessing practical significance for preventing food poison and safeguarding food security,but traditional methods are complicated and time consuming,inability of achieving rapid detection requirement.So it is very necessary to establish a sensitive and selective method.In this thesis,using specific binding reaction of antigens and antibodies,the main purpose is to develop a colloidal electrochemical biological sensor method for rapid detection of SEB in foods.The key point of high sensitivity detecting method is prepared highly specific antibody.Using genetic engineering means,this thesis is expressed a new type SEB protein of possessing histidine-tag,then SEB antibody is prepared on this foundation. Concrete operations were as follow:the encoding SEB gene of possessing histidine-tag was constructed into the prokaryotic expressed vector pET 32a(+) successfully,then SEB protein was expressed in E.coli BL21(DE3) plysS at a high level,then recombinant protein was purified by Ni-NTA His Bind Resin purification column and SDS-PAGE showed its molecular mass was about 31KDa,the immunogenicity and specificity were tested by Western blotting assays.Then albino rabbits were immunized by the purified SEB protein immunogen and the SEB polyclonal antibody,about 120KDa by SDS-PAGE,was purified by affinity chromatography.ELISA standard curves showed that the cross-reactivity of SEB antiserums was 1%with SEA and SEC₁,and was bellow 0.01%with botulinus toxin A,the sensitivity for detection of SEB was 0.001μg/L and IC₅₀ was 0.5μg/L.So this SEB antibody had highly specificity and affinity.A novel type biological sensor method for detecting SEB was established initially.In this experiment,colloidal gold was prepared by optimized conditions with two kinds of diameters(5nm and 16nm colloid gold particles) according to the UV-scanning curve and scanning electron microscope.Indicated results were as follow:the colloid gold particles with 5nm in diameter for labeling antibody show in color of reddish yellow and the particle size is uniform,then the minimal stable concentration of Immunogold-SEB (colloid gold labeled antibody complex) was 15μg/ml,and the suitable stable concentration was 18μg/ml,and the suitable pH for labeling was 7.8.The red spot in Diafiltration experiment of colloidal gold proved the SEB antibody had immunological activity and highly specificity.On this foundation,using Fe(CN)₆^3-/Fe(CN)₆^4- and colloidal gold magnification techniques as the indicator,three electrode detection system and the SEB antibody was immobilized on the gold electrode surface with agarose embedding techniques was applied in the biological sensor.The electrochemical characteristics of the modified electrode at different stages of modification was studied by cyclic voltammetry electrochemical methods with optimized the immobilized condition of antibody at different modification stages and agarose surface appearance with different immobilized antibody was indicated by environment scanning electron microscope.In a word,this colloidal electrochemical biological sensor for detecting SEB is prepared successfully,the lowest detectable limit of the SEB was 0.1μg/L and the corresponding response signal was satisfacting in 1μg/L antigen solution.Then this method has good quality of simple operation,results in short reaction time and remarkable selectivity being suitable for detection of the SEB in foods in the further.