Study On The Rapid Detection Of Vibrio Parahaemolyticus By Immunomagnetic Bead Separation And Quantum Dot Labeling

In recent years,thepopulation exposure scale and food poisoning which was caused by Vibrio parahaemolyticus showed a significant upward trend,and has exceeded the food poisoning caused by Salmonella,becoming one of the primary food borne pathogens in China.At present,the traditional laboratory detection method can not meet the need of the detection of Vibrio parahaemolyticus for its long test period,low specificity,low sensitivity and complicated operation.Therefore,it is necessary to find a method of detection which is rapid,accurate,specify and sensitive for Vibrio parahaemolyticus in food to strengthen the prevention and control of food borne diseases.In this paper,we combined the technology of immuno-magnetic nanobeads separation with the labeling technology of quantum dots.Based on the principle of antigen-antibody specific reaction,the IMB can separated the target bacteria from the sample,which own the target antibody and can bind the target bacteria specifically.Then the quantum dot biosensor was used for labeling the sediment to obtain the complex sandwich structure “IMB-VP-QDs”.After that,we detected the fluorescence intensity by using a fluorescence spectroscopy to achieve a Vibrio parahaemolyticus detection method which is rapid and quantitative.As a result to establish a method of detecting Vibrio parahaemolyticus by combining immunomagnetic separation technique and quantum dot fluorescent labeling technique.1.Preparation and characterization of functional Fe₃O₄ nanoparticlesIn this experiment,Fe₃O₄ magnetic nanoparticles were synthesized by the method of hydrothermal synthesis firstly.The Tween-20 modified aggregates were prepared by the method of micro-emulsion and the silicon shells were coated on thesurface though St?ber method.After got the Fe₃O₄@SiO2 nanoparticles,the carboxyl groups were provided by embedding the Acrylic acid in the surface though the polystyrene high polymerization reaction and finally got the carboxyl-functionalized nanobeads.The structure and properties of the synthesized magnetic nanoparticles were characterized by transmission electron microscopy,infrared spectroscopy,Zeta potential and magnetometer.The results of TEM show that the Fe₃O₄@SiO2@st nanoparticles are spherical and own an average particle size of 217 nm on average with a good monodispersity.The results of infrared spectroscopy show a stretching vibration peak at 1714.753 cm-1in the detection of functionalized Fe₃O₄ nanoparticles,indicating the successfully modification of carboxyl group on the surface of Fe₃O₄@SiO2 nanoparticles.The potential value of the nanoparticles reduced from-17 mv（Fe₃O₄）to-36 mv（Fe₃O₄@SiO2）and finally to-46.8 mv（Fe₃O₄@SiO2@st）at pH 7,which laterally indicated that SiO2 and carboxyl are successfully modified on the surface of Fe₃O₄.With the coating of shells,the saturation magnetic of Fe₃O₄@SiO2@st decreases from 60.586 emu/g（Fe₃O₄）to21.264 emu/gand still own a good magnetic response.2.Preparation of immunomagnetic beads and its enrichment efficiencyThe carboxyl-functional Fe₃O₄ nanoparticles,1μm IMB and 180 nm IMBwere respectively conjugated with the polyclonal-antibody IgG of Vibrio parahaemolyticusto obtain the IMBs which could enrich and isolate Vibrio parahaemolyticus.In order to evaluate the Vibrio parahaemolyticus capture efficiency of IMBs which were synthesizedand determined which kind of IMB could be used in next step of experiment,the capture efficiency of the magnetic beads was calculatedthough the plate counting method before and after the enrichment.The enrichment test results showed thatthe synthetic MNBs（28.57%）<180 nm MNBs（41.43%）<1 μm MNBs（57.14%）.What we can find is that,the synthetic nanobeads were less likely to capture Vibrio parahaemolyticus than another two kinds of nanobeads,which probably due to their surface carboxylation insufficient and the fluffy porous structure of polystyrene.So 1μm MNBs and 180 nm MNBs wereselected for the next step in the detection work.3.Establishment and evaluation of VP detection methodsWe combined the 1 μm MNBs and 180 nm MNBs with polyclonal antibody IgG respectively to obtain the IMB,and combined the quantum dots with IgY for preparing a kind of QDs-probe which could identify VP bacteria specifically.At the time of detection,we added the IMB into the sample to enrich and separate the target bacteria according to the principle of antigen-antibody specific reaction.After the QDs-probe was injected,the sediments were labeled and a kind of sandwich structure,"IMB-VP-QDs",was obtained.Fluorescence intensity of the sandwich structure was detected by using a fluorescence spectroscopy,and the quantitative detection of Vibrio parahaemolyticus was achieved though the linear relationship between fluorescence intensity and bacterial concentration.The amount of immunomagnetic beads,the time of enrichment,the amount of quantum dot coupled IgY,the amount of quantum dot fluorescent probe and the time of labeling were optimized.Vibrio rapid detection method,and the sensitivity,specificity and reproducibility of the detection method were evaluated,and the artificial simulated samples were detected by the established detection method.The results showed that the optimal polyclonal antibody IgG conjugation at 1 μm and180 nm MNBs were 100 μg and 30 μg respectively.The optimal using amount of1μm and 180 nm IMB were 50 μL and 40 μL respectively.The best enriching time of 1 μm and 180 nm IMB were 60 min and 90 min respectively.The optimal polyclonal antibody IgY conjugating amount with QDs was 120 μg,and the best labeling time of QDs-probe was 90 min while the best using amount of QDs-probe was 300 μL and 400 μL for 1 μm and 180 nm respectively.The sensitivity of this method could reach a detection limit level at 102cfu/mL with a total reaction time of150 min.The result own a good linear relationship.The average recoveries were90.61% and 95.26% respectively showed that the method indicated a well accuracy,and the specific experiment（included Vibrio parahaemolyticus,Shigella bogdii.,E.coli O157: H7 and Brucella）indicated that no cross-reaction exist.The 1 μm and180 nm IMB’s coefficient of variation（CV）in one day and during three days were less than 10%,4.03%-7.41% 、 2.03%-7.83% 、 3.30%-6.09% and 2.75%-5.53%respectively,indicated that the method own a well repeatability.The two kinds of IMB on the test of simulation showed that the minimum detection limit（LOD）were102cfu/mL,and could be applied well in the detection of simulated samples.In summary,this paper establishes a rapid detection method for Vibrio parahaemolyticus based on immunomagnetic bead separation technology and quantum dot fluorescent labeling technique.The detection method,which own a low detection limit,a good specificity and a good stability,lays the foundation for the rapid detection of Vibrio parahaemolyticus.