| Salmonella pullorum (S. pullorum) generally caused pullorum and fowl typhoid, which is a kind of epidemic septicemias in chicken and cause heavily economic losses to the poultry industry.Detection of microbial pathogens in food is the key to the prevention and identification of foodborne diseases. Electrochemical immunosensor for its high sensitivity, simplicity and rapidity has been rapidly developed, but it also existed a deficiency—its reproducibility was not as good as we want, that affected the accuracy of the test results, although many researchers had done a lot of research work and been reported, it was still difficult to be really applied in practice.In order to further enhance the sensor’s accuracy and stability, a crucial assessment regarding implementation of reproducibility and stability analysis of a range of immunosensor is provided. First, analyze and find out the possible reasons of the bad repeatability and accuracy. Second, design a set of specific immunosensors and analyae several performance through testing. Third, study on four new types of sensors which based on different materials and modified methods, conduct several experiments includes sensitivity, specificity, accuracy, economy and convenient to get the result of what kind of immune electrode combination has a high reproducibility and accuracy. The last one, on the basis of the above research and analysis, design a better immune sensor. Aimed to provide a model for pathogenic bacteria detection, as well as promote the electrochemical immunosensor really applied to the actual detection for pathogenic bacteria.This paper mainly revolves around the following three aspects:1. Enzyme-functionalized electrochemical immunosensor based on electrochemically reduced graphene oxide/polyvinyl alcohol/Polydimethylsiloxane for detection of Salmonella pullorumA novel and ultrasensitive electrochemical enzyme immunosensor based on the electrochemically reduced graphene oxide (ERGO) and the polyvinyl alcohol (PVA)-multlayer polydimethylsiloxane (PDMS) film modified Screen-printed Carbon Electrode (SPCE) for rapid detection of Salmonella pullorum (S. pullorum) was proposed in this study. The electrochemical characteristics of the stepwise modified electrodes and the determinations of S. pullorum were investigated by Cyclic Voltammetry (CV). As shown in the results of this study, the conductivity of the electrode, electron transfer and communication of electrons were all promoted by the electrodeposition of graphene oxide, thus the sensitivity of the immunosensor was enhanced and strengthened. Moreover, the binding capacity and biocompatibility of ERGO modified electrode was elevated greatly by the PVA-PDMS film, which contributed to the integration of the following modifications and amplification of the current response signal. Further insight into the composite by Fourier Transformed Infrared Spectroscopy (FTIR).Under the optimized working conditions, the sensor showed a good performance with a linear response range from102cfu mL-1to10cfu mL-1, the detection limit was1.61×102cfu mL-1(S/N=3). The proposed enzyme immunosensor with high sensitivity, good specificity, acceptable accuracy and reproducibility, and low detection limit characteristics could be a promising analytical tool in detection of S. pullorum in practical samples and a model for the development of immunosensor of other bacterium of interests.2. Comparative research on the accuracy of four kinds of immunosensor for rapid detection of Salmonella pullorumIn order to improve the uniformity of modified electrode and difficulty of control for electrochemical immunosensor, which lead to poor repeatability and accuracy, this study compared four kinds of methods for modification of immunosensor. Screen printing electrode (SPCE) was choosen as supporting electrode, and modified with different materials include Graphene Oxide (GO); Nanogold Particles (AuNPs) and Ionic Liquids (ILs) by electrodeposition method, then made a detection of S. pullorum immune electrode after HRP-Anti-S. pullorum incubation. The determinations of the target were carried out by cyclic voltammetry (CV). Electrode homogeneity, immobility, sensitivity, specificity and stability of all the four kinds of modified electrode were compared in the experiment. The results show that AuNPs, ILs has an obvious promoting effect on various properties of the electrode, also have an significant difference with other groups, it’s suitable for rapid detection of S. pullorum and the actual application prospect is good.3. Immunosensor based on electrodeposition of gold-nanoparticles and ionic liquid composite for detection of Salmonella pullorumIn order to increase the reproducibility and stability of electrochemical immunosensor, which is a key issue for its application and popularization, an accuracy and stable immunosensor for rapid detection of Salmonella pullorum (S. pullorum) was proposed in this study. The immunosensor was fabricated by modifying Screen-printed Carbon Electrode (SPCE) with electrodeposite gold nanoparticles (AuNPs), ionic liquid (ILs) and HRP-labeled anti-S. pullorum (AuNPs/ILs/HRP). AuNPs/ILs/HRP immunosensor electrode was constructed by electrochemically depositing Au nanoparticles on working electrode surface and then modifying with ILs and HRP-labeled anti-S pullorum. The electrochemical characteristics of the stepwise modified electrodes and the determinations of S. pullorum were investigated by Cyclic Voltammetry (CV). As shown in the results of the experiments, AuNPs with unique electrochemical properties as well as biocompatibility characteristics have been proven to be able to strengthen the antibodies combination effectively and to increase the electrochemical response signal. In addition, a crucial assessment regarding implementation of reproducibility and stability analysis of a range of immunosensor is provided. We found that application of AuNPs/ILs in the immune modified electrodes showed obvious improvement when compared with other groups. Given high level of reproducibility, stability, target specificity and sensitivity, AuNPs and ILs were considered to be excellent elements for electrode modification. |
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