Two Kinds Of Urease-mediated Immunoassays For The Sensitive Detection Of S.choleraesuis And Cronobacter

Traditional enzyme-linked immunosorbent assays(ELISA)method is generally based on horseradish peroxidase(HRP)or alkaline phosphatase(ALP)catalyzed substrates to produce colorimetric signals for quantitative detection of the target analytes.However,the low molar extinction coefficient and single color of colorimetric substances lead to a relatively low sensitivity,which obstructs its widely applications for the detection of the trace amount of target analytes or for the visual detection of target analytes.To address these issues,some signal transducers,such as fluorescence,chemiluminescenc,electrochemistry,and plasmon resonance,has been used as novel signal outputs for improving the sensitivity of conventional ELISA.Dynamic Light Scattering(DLS)has been widely used for detecting the size distribution of particles in suspension liquid.As we know,the scattering intensity of the particles is in proportion to the sixth power of their size.Thus,in theory,the DLS signal of particles can be considered as an ultra-sensitive signal converter to amplify the detection signal of traditional immunoassay.In recent years,foodborne diseases caused by the foodborne pathogens are extensive and serious food safety problem in China.In China,there are about 200,000 to 400,000 food poisoning cases each year,and most of them are caused by foodborne pathogens.In the present study,two kinds of novel immunoassays,namely plasmonic enzyme-linked immunosorbent assays(pELISA)and dynamic light scattering immunoassay,were developed for highly sensitive detection of the Salmonella choleraesuis and Cronobacter,in food samples,respectively.Firstly,pELISA was established based on the urease-induced metallization of gold nanorods(AuNRs),in which the urease catalyzes urea to produce ammonia,and the ammonia further reacts with silver nitrate to form a hydroxated silver diaminohydride complex.Then,the complex is reduced by glucose to deposite the silver shell on the surface of AuNRs,and to form a metallized Au@Ag core-shell structure.Owing the localized surface plasmon resonance(LSPR)of AuNR and silver shell,the AuNR@Ag can show different colors based on the difference of the metallic silver shell thickness on the surface of AuNRs.The various parameters that influence the detection performance of pELISA were systematically optimized.Under the optimal conditions,The visual limit of detection(vLOD)for S.choleraesuis detection by naked eye was 1.21×102 CFU/mL,while the LOD by microplate reader was 1.21×101 CFU/mL,which is about 2-3 orders of magnitude higher than that of conventional ELISA.In addition,the proposed pELISA exhibited a negligible cross-reaction with other nine foodborne pathogens.The average recovery and variable coefficient of the proposed pELISA was further evaluated by the analysis of S.choleraesuis spiked milk samples.The results showed that the proposed method has an accepted accuracy and precision for S.choleraesuis in real milk samples.Secondly,a novel DLS immunosensor was developed for the sensitive detection of Cronobacter.In the proposed method,the urease induced the metallization of silver nitrate on the surface of gold nanoflowers(AuNFs),which further cause the change of light scattering intensity of AuNFs.To the best our knowledge,the DLS-based immunosensor for Cronobacter detection has not yet been reported.Furthermore,the proposed method combined with the immunomagnetic separation technology has been successfully applied for the sensitive detection of C.muytjensii ATCC 51329 in real milk samples.Under the optimal experimental conditions,the proposed method exhibited a wide quantitative linear range for C.muytjensii ATCC 51329 detection from 3.4×102 CFU/mL to 3.4×108 CFU/mL with a LOD value of 3.9×101 CFU/mL,which is about three orders of magnitude higher than that of conventional ELISA.The accuracy and precision were evaluated by analyzing the C.muytjensii ATCC 51329 spiked milk samples.The results showed the average recoveries ranged from 87.9%to 107.91%with the coefficient of variation ranging from 2.72%to 9.32%,indicating the proposed method can be used for C.muytjensii detection in real milk samples.