Development Of Fluorescence Immunoassay For Detection Of Egg’s Spoilage Organisms Based On Quantum Dots

Eggs have rich nutrients and are essential for people’s lives,so,the safety of eggs has attracted much attention of researchers.Salmonella,Proteus,and Escherichia coli are the common pathogens that lead to egg’s safety incidents and there have been many researches about the detection methods for these pathogens,but relatively few studies have been conducted on spoilage organisms that caused egg’s spoilage（such as Deftia tsumhatensis,Proteus penneri,etc.）.Eggs become spoilage because of these bacteria during the storage process,resulting in relatively the large economic losses,therefore,it is necessary to establish a rapid and simple detection method for these spoilage bacteria.Fluorescence immunoassay（FLISA）is a detection method with high sensitivity,simple operation and high accuracy,while,quantum dots,because of its high fluorescence and good stability,has widely used in fluorescent immune method.In this study,Deftia tsumhatensis CM13 and Proteus penneri isolated from eggs were chose as research objects to establish a FLISA method,and compared with ELISA which had similar principle and operating process,providing a new method for bacterial detection.The research contents of this study include:The preparation of water-soluble CdTe:Zn/ZnSQDs:water-soluble CdTe:Zn/ZnSQDs was successfully prepared by water synthesis method and the synthesis conditions were optimized.The optimization results were as follows:[Cd²⁺]/[Te^2-]=5:1,[Cd²⁺]/[Zn²⁺]/[MPA]=1:1.5:3.6,the pH of reaction system was 7.5,reflow temperature of CdTe:ZnQDs was 100℃,reflux time was 2.5 h,the best way to add zinc precursor and sulfur precursor was zinc precursor firstly,subsequently sulfur precursor;The reflow temperature of ZnS shell was 85℃,reflux time was 3.5 h.The fluorescence quantum yield of CdTe:Zn/ZnSQDs was 40.78%.From TEMs,CdTe:Zn/ZnSQDs was more distributed than CdTe:ZnQDs and CdTe:Zn/ZnSQDs was arranged by crystal in a straight line and its particle size was about 3 nm.Moreover,the stability of CdTe:Zn/ZnSQDs at room temperature under dark was better than CdTe:ZnQDs.After storing for 50d,the fluorescence intensity of CdTe:Zn/ZnSQDs was 84.26%of initial fluorescence intensity,while the fluorescence intensity of CdTe:ZnQDs was 55.33%of initial fluorescence intensity.The preparation of polyclonal antibody:heat-inactivated Deftia tsumhatensis CM13 and Proteus penneri were prepared to immunize the New Zealand white rabbits by subcutaneous 5-point injection for three months to obtained serum,respectively.The titer of polyclonal antibodies were both 1:51200 through indirect ELISA method,the titers of Deftia tsumhatensis CM13 polyclonal antibodies and Proteus penneri polyclonal antibodies were 1:12800 and 1:25600 after purification,respectively.The protein concentrations of Deftia tsumhatensis CM13 polyclonal antibody and Proteus penneri polyclonal antibody were 2.90 mg/mL and 3.40 mg/mL,respectively.Molecular weights of two kinds of polyclonal antibody were about 148 kD.In addition,no reactions were observed between these polyclonal antibodies and Escherichia Coli O157:H7,Shigella soonei,Pseudomonas Aeruginosa,Bacillus subtilis,Staphylococcus aureus,and Listeria monocytogenes.No reaction was happened between Deftia tsumhatensis CM13 polyclonal antibodies and Proteus penneri,and no reaction was found between Proteus penneri polyclonal antibodies and Deftia tsumhatensis CM13,therefore,the preparation of Deftia tsumhatensis CM13 polyclonal antibodies and Proteus penneri polyclonal antibodies had good specificity.CdTe:Zn/ZnSQDs coupling with antibody:according to fluorescence emission spectra,the fluorescence intensity of CdTe:Zn/ZnSQDs-antibody was increased about 30 a.u.than CdTe:Zn/ZnSQDs.From the ultraviolet absorption spectrum,the characteristic absorption peak of antibody at 280 nm was disappeared after coupling with CdTe:Zn/ZnSQDs and the absorbed intensity was increased than pure CdTe:Zn/ZnSQDs,indicating that the CdTe:Zn/ZnSQDs and antibody were succseefully coupled with each other,preliminary.Further identifications were operated by agarose gel fluorescent image under UV light and TEMs.Through dot blot experiment,the biological activity of antibody was maintained after coupling with CdTe:Zn/ZnSQDs and with the increase of antigen,the brightness of the spot was increased under UV light.The optimal coupling conditions of CdTe:Zn/ZnSQDs and antibodies were as following:the optimal dosage of active agent EDC·HCI（0.1mol/L）and coupling agent NHs（0.1mol/L）were 80μL and 60μL respectively.The optimal pH of coupling was 9.0.The optimal dosage of antibody（1 mg/mL）was 120μL,and the optimal reaction time was 2 h,coupling rate were 53%.The stability experiment shows that CdTe:Zn/ZnSQDs-antibody could be stored for 2 months at 4℃,avoiding light.Establishing of fluorescence immunoassay（FLISA）for detecting of spoilage bacteria in eggs:FLISA was performed to detect Deftia tsumhatensis CM13 and Proteus penneri,respectively,and compared with ELISA.First,the detection conditions of ELISA and FLISA were optimized,the best conditions for ELISA method were:blocking agent was 10%BSA,blocking time was 2.0 h,rabbit serum dilution degrees was 1:3200,the reaction time was 1.0 h,dilution degrees of goat anti-rabbit IgG-HRP was 1:5000,reaction time was 1.0 h,chromogenic time for OPD was 10 min.The best conditions for FLISA method were:blocking agent was 10%BSA,blocking time was 1.5 h,rabbit serum concentration was 1.5 mg/mL,the reaction time was 1.5 h,the addition of CdTe:Zn/ZnSQDs labeled goat anti-rabbit IgG was 300μL,reaction time was 1.5 h,the centrifugal separation speed of excessive CdTe:Zn/ZnSQDs labeled goat anti-rabbit IgG and CdTe:Zn/ZnSQDs labeled goat anti IgG-rabbit serum-antigen was 1000 r/min,1min.ELISA and FLISA were used to detect Deftia tsumhatensis CM13 and Proteus penneri,respectively.The detection limits of Deftia tsumhatensis CM13 detected by ELISA and FLISA were 5 log CFU/mL and 3.097 log CFU/mL,respectively.The detection limits of roteus penneri detected by ELISA and FLISA were 5.688 log CFU/mL and 3.581 log CFU/mL,respectively,indicating that the sensitivety of FLISA was better than ELISA in detecting of egg’s spoilage bacteria,and the detaction limit of FLISA was decreased by 2 logarithms than ELISA.