| The contamination of food by harmful microorganism is still a non-negligible threat in food safety. Diarrhea-related diseases lead to thousands of deaths in the world every year. Moreover, it is estimated that more than seventy percent of these diseases are caused by microorganism-contaminated food and drinking water. The microorganisms commonly found in raw material and finished food products are catalase positive bacteria including S. aureus, escherichia coli (E, coli), vibrio parahaemolyticus, listeria and salmonella. Therefore, the analysis for the presence of both pathogenic and spoilage bacteria is a very necessary practice for ensuring food safety and quality especially in food industry. Conventional bacteria testing methods are complex, labor-intensive and not suitable for on-sites use regarding to the procedures of culture preparation, plates inoculation, colony counting and biochemical characterization. In this study, four types of biosensing system were established for the detection of catalase-positive bacteria in food sample and the evaluation of the analytic performance was also conducted. The fabrication of the sensor and the detecting results were summarized as follows:1. Establishment of optical sensor for detection of catalase-positive bacteria:Horseradish peroxidase and hydrogen donor (o-tolidine dihydrochloride hydrate) was immobilized on a test strip. After the incubation of the samples containing catalase-positive bacteria with hydrogen peroxide, the resulting mixture was added on the test strip and the color-developing reaction for the detection of the remaining H2O2 was performed. Since the catalase in the bacteria can catalyse the H2O2, a negative correlation was present between the count of the catalase-positive bacteria and the amount of the remaining H2O2, which exhibited positive correlation with the density of the chromatic reagent. The optical sensor was established conveniently by inserting the test strip into a portable reflectometer. The signal of the optical sensor, which used a light-emitting diode as the monochromatic light resource, was based on the detection of the reflecting red light from the reacting area on the test strip. It was observed that a linear correlation was indicated between the logarithmic value of colony-forming unit and the inhibitory rate of the light density over the range from 103 to109 CFU/mL with the detecting time of 2 min under the optimized experimental conditions ( pH =6, 5mmol/L H2O2, and 60 s for color-developing). The regression equation could be represented by (x-6.777)/0.379y=- 0.9040.9521+e + (r2=0.988, for n=5). The test strip demonstrated favorable stability at 4℃for 6 months. In addition, the data from this technique was in accordance with that from the methods based on the spectrometer determination. The advantage of the optical sensor included satisfactory sensitivity, simplicity and short detecting time. 2. Fabrication of biosensor system based on chemiluminescence for fast detection of catalase-positive bacteria. A traditional chemiluminescence reaction was set up using luminol-H2O2-HRP-NaTPB system. Due to the consumption of H2O2 by the catalase from the catalase-positive bacteria, the more the count of the catalase-positive bacteria in the food sample, the less the intensity of chemiluminescence was produced. Thus a fast technique for detection of catalase-positive bacteria was established based on the above reaction system using the electronic signals converted from the chemiluminescence.The results showed that a linear correlation was indicated between the logarithmic value of colony-forming unit and the inhibitory rate of the chemiluminescence over the range from 103 to109 CFU/mL with the detecting time of 10 min under the optimized experimental conditions (0.5×10-4g/L HRP, 5 min for incubation of bacterial sample with luminol reaction system). The regression equation could be represented by y = ? 1 + e0 ( x.?97.8223 )/ 0.631 +1.055 (r~2= 0.99, for n = 5). The results from this sensing system were in accordance with that from the technique based on the RFL-1 Chemiluminescence spectrometer. This technique showed satisfactory sensitivity and multi-sample determination could be performed in a synchronous way, providing a practical tool for the screening of the catalase-positive bacteria in food sample.3. Development of amperometric sensor for rapid detection of catalase-positive bacteria. Self-made screen printed electrode (SPE) modified with chitosan-peroxidase was connected to a portable self-made amperometric sensor. The determination of catalase-positive bacteria was carried out by adding the reaction product, which was obtained from the hydrogen peroxide dismutation catalyzed by the bacterial catalase, to the reacting area of the SPEs. A working potential of 0.55 V was applied in the sensing system and the current value displayed on the amperometric sensor was used as the detection signal.The results indicated that a linear correlation was manifested between the logarithmic value of colony-forming unit and the amperometric response over the range from 104 to108 CFU/mL with the detecting time of 10 min under the optimized experimental conditions (pH = 6.5, 5min for the incubation of the bacteria with hydrogen peroxide). The regression equation could be represented by y = -3.29x + 31.79,r2=0.997. The test strip demonstrated favorable stability at 4℃for 6 months. In addition, the data from this technique was in accordance with that from the determination based on the CHI630C Electrochemistry Working Station. The advantage of the optical sensor included satisfactory sensitivity, simplicity and low cost. Due to the application of disposable SPE, the sensing system could be used as a practical tool on the sites.4. Establishment of biosensor system for specific detection of E. Coli and Salmonella in food based on ATP-luciferase reaction system. Polyclonal antibody against E. Coli and Salmonella were employed to capture the target bacteria in food samples. The ATP in the bacteria was released using the extraction agents and the chemiluminescence based on ATP-luciferase reaction system was used as the sensor signal.It was indicated that there was a linear correlation between the logarithmic value of colony-forming unit and the intensity of the chemiluminescence over the range from 103 to108 CFU/mL with the detecting limit of 102CFU/mL under the optimized experimental conditions (pH 7.8, 0.025% DDAB). The regression equations for detection of E. Coli and Salmonella could be represented by ( 6.22)/0.61121.1 129.9y = ? 1 + e x?+ (r = 0.986, for n = 5) and y = ? 1 + 1e6 ( x8?.62.445) /0.7+178.58 (r = 0.993, for n = 5), respectively. This technique demonstrated satisfactory sensitivity and specificity and provided basic approaches for the development of novel sensor based on ATP-luciferase reaction system. KEYWORDS: Catalase-Positive Bacteria, biosensor,...
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