Study On Electrochemical Immunosesor Based On Functional Nanomaterials For Sensitive Detection Of Staphylococcus Aureus

In this paper, a new method for the rapid detection of foodborne pathogens in food products has been explored, which is based on the latest research results combined with nanotechnology, materials science, sensor technology and instrumental analysis technology. Staphylococcus aureus (S. aureus) was selected as the research object by using novel nanomaterials to solve the existing problems of the detection technology and provide the theory basis for seeking more efficient, higher performance detection method in S. aureus. Additionally, this study also used self-assembly technique to immobilize the antibodies on the electrode surface to improve the stability and efficiency of the immunosensors. The main contents, results, and conclusions are as follows:1. Self-assembled electrochemical immunosensor based on gold nanorods modified glassy carbon electrode for label-free detection of staphylococcus aureusA novel electrochemical immunosensor based on self-assembled gold nanorods modified glassy carbon electrode for sensitive detection of staphylococcus aureus in milk was designed. Firstly, PDDA, PSS and GNR ere layer by layer dropped on the surface of the electrode by using the interaction between the positive and negative charge. Next, the electrode surface was covered with anti-S. aureus antibodies, and then the antibody-modified electrode was treated with 1.0% BSA-PBS to block the non-specific sites. After binding with S. aureus cells, the immunoreactive complexes on the electrode hindered the electron transfer of the electrode surface, which made the electron transfer resistance increased. The electron transfer resistance was proportional to the concentration of S. aureus. In order to achieve excellent analytical performance of the immunoassay method, the effects of the factors were also investigated. Results showed that under the conditions of the antibody concentration of 100μg mL-1, incubation temperature of 37℃, incubation time of 50 min, the biosensor displayed the best detection performance. The concentration of target bacteria detected by this method was linear in the range from 1.8×103-1.8×107 CFU mL-1 with a correlation coefficient of 0.9932 (R2). The detection limit of the developed impedimetric immunosensor was estimated to be 100 CFU mL-1. The immunosensor showed no obvious detection signal to non-target bacterium such as E. coli, S. typhimurium, B. pumilus, and K. pneumoniae, indicating good specificity. In real milk samples, the average values obtained from the immunosensor were approximate to the standard results obtained from the plate count method, and the relative standard deviation was 2.96%-6.78%, suggesting the good accuracy of the developed method.2. Metal ion functional polystyrene-acrylic nanoparticles for highly sensitive electrochemical detection of Staphylococcusaureus in food sampleIn the experiment, a highly sensitive immunosensor based on PSA/PAH/(PSS-Cu)n to detect S. aureus was developed. Firstly, we synthesized the uniform morphology, stable polystyrene-acrylic nanospheres (PSA), followed by the use of polyallylamine hydrochloride (PAH), polystyrene sulfonate (PSS) to functionalize PSA. Then, a large amount of Cu2+ was assembled onto the surface of PSA by electrostatic adsorption to produce PSA/PAH/(PSS/Cu2+)n. Then anti-S. aureus antibody (Ab) was cross-linked to the nanosphere surface to successfully build PSA/PAH/(PSS/Cu2+)n/Ab by using glutaraldehyde. Then MUA, EDC, MIS, Ab, and BSA were separately dropped on the electrode surface by layer by layer self-assembled method, followed by a gradient of concentration of S. aureus, and then captured by the functional nanoprobes PSA/PAH/(PSS-Cu)n. With the increase of the concentration of S. aureus, the amount of Cu2 +released from the PSA/PAH/(PSS-Cu)n. was greater, so the value of the current signal was higher. The current response was proportional to the concentration of S. aureus. In order to achieve excellent analytical performance of the immunoassay method, the effects of some factors were also investigated. Results showed that under the conditions of 3 monolayers of (PSS/Cu2+)n, incubation time of 40 min, the biosensor displayed the best detection performance. The concentration of S. aureus detected by this method was linear in the range from 3.2×102-3.2×107 CFU mL-1 with a correlation coefficient of 0.9948 (R2). The detection limit of the developed immunosensor was estimated to be 70 CFU mL-1. The immunosensor showed no obvious detection signal to non-target bacterium such as E. coli, S. typhimurium, B. pumilus, and K. pneumoniae, indicating good specificity. In real milk samples, the relative standard deviation ranged from 1.41% to 6.67%, and the recoveries of standard additions were in the range of 96.1%-100.7%, indicating acceptable accuracy of the electrochemical immunosensor. Therefore, the results demonstrate that the proposed strategy can be applied to real samples and has great potential in practical applications. 3. Highly sensitive detection of Staphylococcus aureus based on the fast magnetic separation electrochemical immunoassayA fast and sensitive immunosensor based on fast magnetic separation to detect S. aureus was fabricated. Functionalized quantum dots (QDs-Ab) and functionalized magnetic beads (Fe3O4-Ab) could specifically combine S. aureus to form a sandwich composite structure to be detected. The complex concentration was enriched at the bottom by plusing a magnetic field at the bottom of the tube. The target could be completely separated from the impurities through removing the supernatant and appropriate washing, and then adding HNO3 under ultrasonication. With the increase of the concentration of S. aureus, the amount of Cd2+ in the pool was greater, so the value of the current signal was higher. The current response value was proportional to the concentration of S. aureus. In order to achieve excellent analytical performance of the immunoassay method, the effects of the some factors were also investigated, results showed that under the conditions of the incubation time of 40min, and incubation temperature of 37℃, the biosensor displayed the best detection performance. The concentration of S. aureus detected by this biosensor was linear in the range from 2.21×102-2.21×107 CFU mL-1 with the correlation coefficient of 0.9994 (R2). The detection limit of the developed impedimetric immunosensor was estimated to be 83 CFU mL-1.To non-target bacteria V. parahaemolyticus, E. coli and S.typhimurium no obvious signal detection, showed good specificity. In real milk samples, the recoveries of standard additions were in the range of 92.8%-104.4%, suggesting the good accuracy of the developed method and can be applied to real samples.