| Food safety issues have been already considered the fourth social problem following Population, Resource and Environment now in. And it was one of the main reasons that the foods were contaminated by pathogenic bacteria or conditional pathogens. These pathogens include mainly Enterohemorrhagic Escherichia coli O157: H7 (E. coli O157:H7), Salmonella, Vibrio parahaemolyticus and Proteus, etc. And food poisoning caused by E. coli O157:H7 was worth particularly concern. E. coli O157:H7 is the main serotype of Enterohemorrhagic Escherichia coli, which can cause diseases such as diarrhea, Hemorrhagic enteritis. Since 1982 an outbreak of food poisoning caused by E. coli O157:H7 in the United States, and then E. coli O157:H7 infection prevalence continuously appeared in many countries in the world. At present, there are many method of detecting E. coli O157:H7, but many of them have associated disadvantages; for example, many methods are complicated, time consuming, and have a low sensitivity. The epidemiological data showed that the infectious dose of E. coli O157:H7 was possibly fewer than 100 cells. Therefore, the sensitive methods for detecting this bacterium are urgently required.Piezoelectric sensor as a new bio-sensor, the detection theory is that the frequency change is proportional to the mass change, which in turn is linked to the test material. Piezoelectric sensors has been widely applied in the detection of pathogenic bacteria, but its sensitivity is commonly 104-106 CFU/mL. However, the detection limit of the piezoelectric sensor was not less than 102 CFU/mL with"quality amplification effect"of the magnetic particles and colloidal gold, it was unable to meet the needs of some high-pathogenic bacteria detection.To address the issue of E. coli O157:H7 infection dose low and the low sensitivity of detection piezoelectric sensor, the method of quartz crystal microbalance immunosenosr (QCM immunosensor) will be developed to extremely improve the detection limit of E. coli O157:H7 with multiple enhancement of frequency based on the mass enhancement, separation concentration with beacon immunomagnetic nanoparticles (BIMPs), functional gold nanoparticles, and the technique of gold growth. 1. Preparation of BIMPs: Magnetic nanoparticles were prepared by a chemical coprecipitation method with FeCl3, FeCl2, ammonia solution, and dextran (T-40), the nanoparticles had a uniform size, contained an electron-dense core of 5 nm by transmission electron microscope (TEM). The prepared nanoparticles were purified using gel filtration and high-intensity magnetic separation method, and the results of the two purification methods were compared. Finally we selected the high-intensity magnetic separation method to purify nanoparticles for its advantages, such as easy, fast, and cheap. The concentration of rabbit polyclonal anti-E. coli O157:H7 antibody (target antibody, T-Ab) and biotin-IgG (beacon antibody, B-Ab) was 0.5 mg/mL, and BIMPs were packed by magnetic nanoparticles loaded with T-Ab and B-Ab at an ratio of 1︰0,1︰5,1︰10,1︰20,1︰40,1︰60,1︰80, and 1︰100 (T-Ab : B-Ab). Comprehensive consideration the separation concentration and connecting colloidal gold of BIMPs function, the optimized ratio of T-Ab : B-Ab was 1 : 60 on BIMPs. The recovery of BIMPs (1 : 60) were further determined in a different concentration of E. coli O157: H7. BIMPs were only applied when the concentration of the E. coli O157:H7 solution was less than 106 CFU/ml, and its suitable volume was 20μL. It was found that BIMPs could be stored at least 12 months at 4℃with a hith recovery.2. Preparation of functional colloidal gold and growth solution: About 18 nm colloidal gold particles were prepared by sodium citrate reduction, and the peak wavelength with narrow space was 518 nm when the colloidal gold solution was scanned between 400 nm-800 nm with UV spectrophotometer. The minimum amount of streptavidin required was 16μg/mL by the wavelength scan method. The quality of streptavidin-gold was verified by a dot immunofiltration method, and it could be used in the following experiment of QCM immunosenosr. The growth solution was prepared using cetyltrimethylammonium bromide (CTAB), and the most suitable heating temperature and time was 100℃and 10 min. The ratio of growth solution and colloidal gold was 9︰1 with the volume. The shape change of colloidal gold particles were observed before and after addition of the growth solution by TEM, the particles had enlarged in size to approximately 40 nm after growth.3. Detection of E. coli O157:H7 by QCM immunosensor: In this study, the frequency of the crystal was measured in the gas phase, and the frequency was recorded when the variation of frequency measurement was less than 1 Hz. The QCM immunosensor was fabricated by Protein A from Staphylococus aureus (SPA) for the antibody immobilization. The most suitable immobilization concentration and time for SPA was 1.2 mg/mL and 40 min. The mouse monoclonal anti-E. coli O157:H7 antibody (McAb) was prepared using subtractive immunization, and its specificity was 98%. McAb was immobilized on the crystal by SPA, and the most suitable immobilization concentration and time was 1.0 mg/mL and 60 min. After E. coli O157:H7 was separated and concentrated by BMIPs, the free BIMPs were removed using the Ultrafree-MC microcentrifuge filters; On the basis of the biotin-avidin system, the streptavidin-gold was linked with BIMPs, the free streptavidin-gold was removed by a magnetic plate; The gold particles on BIMPs were enlarged by the growth solution prepared fresh, the unreacted growth solution was separated using a magnetic plate and discarded; Finally, the frequency was measured by QCM immnosensor when the complex containing E. coli O157:H7, BIMPs, and enlarged streptavidin-gold was added to the crystal fabricated with McAb, the limit of detection was 23 CFU/mL. Thirty-nine food samples were collected and detected by QCM immunosensor and National standard methods, E. coli O157:H7 was not detected using 2 methods. However, E. coli O157:H7 were detected in the food samples added E. coli O157:H7, and the limit of detection was 53 CFU/mL, and false negative results appeared in 3 food samples.The entirely new BIMPs were prepared based nanotechnques, antigen-antibody reactions, the biotin-avidin system, and gold growth in this study. BIMPs had 2 functions of connection and mass enhancement besides the traditional separation. The novel method for detecting E. coli O157:H7 by using QCM immunosensor and BIMPs, the frequency of QCM immunosensor was enhanced by 3 times. Finally, we obtained the method with high sensitivity for detecting E. coli O157:H7, and the total analysis time was approximately 4 h. In this study, we developed a rapid, accurate, and sensitive method for detecting E. coli O157:H7. The method is very important to improve the level of guarantee food safety and prevent food poisoning in China. |
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