The Study Of Enzyme Immunosensor For Detection Of Salmonella Pullorum And Salmonella Gallinarum

Pathogenic microorganism infections of livestock and poultry have common, widespread and severe impact to their health and food safety. In the poultry industry, pullorum and fowl typhoid diseases are two kinds of epidemic sepsis that has the most threats to the health of chickens, and they are generally caused by Salmonella pullorum and Salmonella gallinarum. They cause great economic losses to the poultry industry with heavy transmissibility and serious pollution, and at the same time seriously threaten safety of the poultry products which are also as food of the people, thus causing widespread concerns at home and abroad. It is greatly necessary to strengthen the detection of these two pathogens. Accurate, simple, economic and rapid detection methods are urgently needed for prevention and control of diseases and guarantee of food security. Currently, standard method and rapid detection method are mainly conducted for detection of these two pathogens, they both have their advantages and drawbacks, which promotes the constant exploration and pursuit of novel and rapid detection techniques to meet the expectation of desirable detection effect.Electrochemical enzyme immunosensor combining the double amplification of enzyme and electrochemistry, with the specificity of the immune reaction, which is endowed with the advantages of simple equipment, easy to use, flexible method, easily integrated system and miniaturization, is superior to the general immunosensor. Hitherto the development of the enzyme immunoassay sensing technology, the key links of electrode selection, maintenance of the activity and stability of the fixed biological components, and signal amplification of reaction of the immune conjugates and so on which had been considerably improved but not yet reached the ideal level need further research and melioration. Therefore, research and improvement in these aspects of the enzyme immunosensor are carried out in this work, for the purpose of exploring and researching novel and better enzyme immunosensor for rapid detection of Salmonella pullorum and Salmonella gallinarum.The research tasks conducted in this paper were described as follows: 1The fabrication of enzyme immunosensor for detection of Salmonella pullorum and Salmonella gallinarum based on the multi-walled carbon nanotubes/gelatin-trehalose complex modified four channel screen-printed carbon electrodeA complex solution involved gelatin polymer and trehalose was prepared firstly, which was used for dispersing the multi-walled carbon nanotubes that have excellent characteristics such as high conductivity and quick electron transfer but without good dispersion. And then the horseradish peroxidase-labeled anti-Salmonella pullorum&Salmonella gallinarum was entrapped in the multi-walled carbon nanotubes/gelatin-trehalose complex modified four-channel screen-printed carbon electrode to construct novel enzyme immune electrode for detection of Salmonella pullorum and Salmonella gallinarum, so as to improve its sensitivity and stability. The surface morphology of the modified electrodes was characterized by atomic force microscopy. The electrochemical characterizations of the enzyme immune electrodes were determined by cyclic voltammetry, as well as the detection of Salmonella pullorum and Salmonella gallinarum and the performance determination of the immune electrode. The results showed that under the optimal assay conditions, a good linear response occurred in the concentration range of103to109cfu/mL of Salmonella pullorum and Salmonella gallinarum, with a low detection limit of6.42×102 cfu/mL (S/N=3). The stability test of the fabricated immunosensor showed that it remained89.6%of its original response signal after storage of28days under4℃. The performance evaluation of the proposed immunosensor showed that it had high stability, good specificity, acceptable reproducibility and accuracy. The preparation method is simple, sensitive, stable, economic and operable, which made it possess a certain potential application value.2The fabrication of enzyme immunosensor for detection of Salmonella pullorum and Salmonella gallinarum based on the ionic liquid of [BMIM]PF6/multi-walled carbon nanotubes/Sodium alginate-carboxymethyl chitosan complex modified four channel screen-printed carbon electrodeIn this study, the combination of excellent properties of ionic liquid [BMIM]PF6with the good electrical characteristics of multi-walled carbon nanotubes which were dispersed in sodium alginate (SA) and carboxymethyl chitosan (CMC) complex was conducted to immobilize the horseradish peroxidase-labeled anti-Salmonella pullorum&Salmonella gallinarum onto the surface of the four channel screen-printed carbon electrode to propose an enzyme immunosensor with double amplified response signal for detection of Salmonella pullorum and Salmonella gallinarum. The surface morphology of the modified electrodes was characterized by atomic force microscopy, while the electrochemical properties were determined by cyclic voltammetry. The results showed that under the optimal assay conditions, a good linear response occurred in the concentration range of10to109cfu/mL of Salmonella pullorum and Salmonella gallinarum, with a low detection limit of3.93×102cfu/mL (S/N=3). The stability test of the fabricated immunosensor showed that it remained90.15%of its original response signal after storage of28days under4℃. The performance evaluation of the proposed immunosensor showed that it had high stability, good specificity, acceptable reproducibility and accuracy. The results demonstrated that the proposed enzyme immunosensor with combination of multi-walled carbon nanotubes and ionic liquid achieved the effect of double amplification of detection signal which enabled it highly sensitive in detection. Ionic liquid was also helpful for maintaining bioactivity of enzyme labeled antibody, enhancing its stability, thereby prolonging effective time of the immune electrode. The method has certain reference value and potential application value to the construction of enzyme immunosensor for detection of other analytes of interests.3The fabrication of enzyme immunosensor for detection of Salmonella pullorum and Salmonella gallinarum based on the streptavidin-biotin system/electrodeposited gold nanoparticles modified four channel screen-printed carbon electrodeAn electrically active layer was produced on the four-channel screen printed electrode surface by the electrodeposition of gold nanoparticles, then the streptavidin was adsorbed onto it, a multi-stage amplification system of streptavidin-biotin fonned immediately after the binding of biotinylated anti-Salmonella pullorum&Salmonella gallinarum with high affinity. The antigens of Salmonella pullorum and Salmonella gallinarum and horseradish peroxidase-labeled anti-Salmonella pullorum&Salmonella gallinarum were immobilized onto the electrode through immunoreaction of antigen and antibody to build up a novel enzyme immunosensor for detection of Salmonella pullorum and Salmonella gallinarum. The characterizations of the stepwise modified electrodes were conducted by cyclic voltammetry and electrochemical impedance spectroscopy, whereas the determination of the targets of Salmonella pullorum and Salmonella gallinarum was carried out by cyclic voltammetry. The results showed that the electrodeposited gold nanoparticles promoted the electron transfer, thus enhanced the conductivity of the electrode, simplified modification procedure of gold nanoparticles and improved stability of immobilization. The immobilized amount of the immune molecules was increased due to the high affinity of streptavidin-biotin system, which probably had a certain directional effect to the immobilized immune molecules. In the dual roles of the above-mentioned modified materials, the current response signal of the enzyme immunosensor was greatly enhanced. As a result, it was observed that a good linear response occurred in the concentration range of102to109cfu/mL of the analytes, with a low detection limit of1.95×102cfu/mL (S/N=3). The stability test of the fabricated immunosensor showed that it remained88.37%of its original response signal after storage of28days under4℃. The proposed enzyme immunosensor of rapid, high sensitivity, good specificity and stability, and acceptable accuracy, reproducibility and low detection limit characteristics could be a promising analytical tool in detection of Salmonella pullorum and Salmonella gallinarum and a model for the development of enzyme immunosensor of other analytes of interests.