Two Novel Enzyme-linked Immunosorbent Assay For The Ultrasensitive Detection Of Listeria Monocytogenes And Escherichia Coli O157:H7

Enzyme-linked immunosorbent assay（ELISA）is a non-radioactive immunoassay technique based on immunoenzyme reaction.However,the conventional ELISA based on colour-reaction induced by horseradish peroxidase（HRP）catalyzed substrates commonly suffers from low sensitivity.Currently,developing novel signal output to improve the sensitivity of ELISA has attracted much attention in the field of analytical chemistry.Foodborne pathogens can cause food poisoning in contaminated meat,eggs,aquatic products,fruits,vegetables and dairy products.The contamination of foodborne pathogen has become a major problem in food safety and aroused 30%⁹0% food poisoning events.Therefore,it is necessary to establish a simple,rapid and sensitive method for the ultrasensitive detection of foodborne pathogens.In this study,two kinds of ELISA methods based on catalase（CAT）-mediated novel signal transduction patterns were proposed for ultrasensitive detection of Listeria monocytogenes（L.monocytogenes）and Escherichia coli O157:H7（E.coli O157:H7）,respectively.Firstly,a plasmonic ELISA（pELISA）based on CAT-mediated gold nanoparticle growth was developed for ultrasensitivity detection of L.monocytogenes,where the silica nanoparticles（SiO₂）coated with poly（acrylic acid）brushes（PAA）synthesized by surface-initiated reversible addition-fragmentation chain transfer polymerization was designed as a “CAT container” to increase enzyme loading,and the streptavidin（SA）-biotin system was used as a bridge to introduce the SiO₂@PAA@CAT into the immunoassay.When there was L.monocytogenes in the solution,the hydrogen peroxide（H₂O₂）was consumed by the CAT,and thus the chloroauric acid was reduced to generate irregular gold nanoparticles（AuNPs）,resulting in a blue solution;while when the L.monocytogenes was absent in the solution,excessive amounts of H₂O₂ catalyzed the chloroauric acid to generate regular spherical AuNPs,and the resulting solution shown red colour.According to this principle,the calibration curve of the developed p ELISA was constructed for sensitive detection of L.monocytogenes.The linear range for quantitative detection of L.monocytogenes ranged from 8×100 to 8×106 CFU/mL.The regression equation was described as y =-0.015Log（x）-0.2057 with a reliable correlation coefficient of 0.985.The limit of detection（LOD）of this proposed method for detection of L.monocytogenes in PBS buffer was 8×101 CFU/mL,which was 5 orders of magnitude lower than that of HRP-based conventional ELISA.The LOD for detection of L.monocytogenes-spiked lettuce samples was 8×101CFU/g.The recovery rates of L.monocytogenes spiked samples at concentrations of 8×100CFU/g,8×101CFU/g,8×102CFU/g,and 8×103CFU/g were 50±12.5%,101%±22.6%,68.3%±6.13%,and 76.8%±21.8%,respectively.The concentrated solution（1mL）prepared fro m samples spiked with 8×100CFU/g of L.monocytogenes was divided into 10 parts and then detected by the proposed pELISA method.The results of p ELISA and plate counts both showed that some of these parts were positive,indicating the capability of proposed p ELISA for detecting a few of cells in the lettuce sample.The specificity of the p ELISA was evaluated and showed a negligible cross-reaction with other non-target bacterium.Secondly,we deveoped a fluorescence immunoassay based on CAT-mediated fluorescence quenching of mercaptopropionic acid modified CdTe QDs（MPA-QDs）for the high sensitive detection of E.coli O157:H7 in milk.The CAT was introduced into ELISA through SA-biotin system.When the E.coli O157:H7 was present in the solution,the CAT bound to the surface of the E.coli O157:H7 could consume H₂O₂,resulting a reduced fluorescence quenching rate of MPA-QDs.The parameters included the concentrations of anti-E.coli O157:H7 polyclonal antibody,biotinylated anti-E.coli O157:H7 monoclonal antibody and SA@CAT,the hydrolysis temperature and time of CAT to H₂O₂,the incubation time between H₂O₂ and MPA-QDs,were systematically investigated.Under the optimal conditions,a three-parameter logistic regression calibration curve(y=-56.2206+5.1263×Ln（x+6.95×104）was established（R2=0.9925）.The linear range of the quantitative detection of E.coli O157:H7 ranged from 8×100 to 8×106 CFU/mL.The LOD was 5×102 CFU/mL,which is 140 times lower than that of the conventional HRP-based ELISA.The average recoveries for intra-assay,inter-assays and actual samples ranged from 84.6% to 111.2%,71.6% to 108.4% and 65.88% to 105.6%,with a coefficient of variation（CV）ranging from 6.7% to 24.9%,6.2% to20.1 % and 4.5% to 25.7%,respectively.The specificity of the fluorescence ELISA was evaluated and showed a negligiblelo cross-reaction with other non-target bacterium.