Research Of Detection E. Coli O157:H7Based On Different Size Au Nanoparticle Enhanced SPR

Nowadays, food safety has attracted much attention. According to statistics, the most alimentary toxicosis is caused by bacteria. Escherichia coli0157:H7(E. coli0157:H7) is the most common pathogens of bacterial in alimentary toxicosis incident. Therefore, a rapid and simple method of analysis is needed for detect microbiological contamination in food, such as E. coli O157:H7. Surface plasmon resonance (SPR) technology has become an important analytical technique in food analysis.The sandwich method based on gold nanoparticles (AuNPs) labeled antigen or antibody has been reported using SPR biosensor for detecting E. coli0157:H7. However, The investigation of the enhancement SPR signal for different size AuNPs has not been reported based on sandwich assay of different size AuNPs labeling E. coli O157:H7polyclonal antibody (PAb) as the second antibody. In this paper, the direct method and sandwich method based on different size AuNPs labeling E. coli O157:H7PAb were used to detect E. coli O157:H7using the dual-channel SPR sensor in order to select the optimal size of AuNPs. The minimum detection concentrations for direct assay and sandwich assay of optimal size AuNPs are103cfu/mL and10cfu/mL respectively, in addition, the specificity of this biosensor also investigated. The main contents were as follows:1. Synthesis for AuNPs of different sizeThe AuNPs of three sizes were synthesized using the reduction of citrate sodium, and were characterized by the transmission electron microscope(TEM) and UV-Vis spectrophotometer. The results showed that the most AuNPs are spherical, and are evenly dispersed in the solution. The sizes of AuNPs were17.79nm,28.22nm,34.05nm, respectively.2. The biological function and characterization of AuNPsThe optimal amounts of AuNPs labeling PAb were determined by visual method. Then, the compound of the AuNPs-PAb were produced by adjusting the pH value, slowly dropping PAb to the AuNPs solution in the state of the magnetic stirring, and twice centrifugation. The bare site of AuNPs surface are blocked by adding1%bovine serum albumin (BSA). The AuNPs-PAb compounds were charactered by UV-Vis spectrophotometer.3. The direct method and AuNPs enhanced sandwich assay to detect E. coli0157:H7In this paper, the direct assay and sandwich assay based on different size AuNPs labeled E. coli O157:H7PAb as the second antibody, the PAb immobilized on the sensor surface as the first antibody were used to detect E. coli0157:H7, respectively. The results showed the SPR signal of the sandwich assay is larger than that of direct method. The most optimal AuNPs size was selected by comparing SPR signal enhancement of the three sizes of AuNPs. The minimum detection concentrations of direct method and optimal particle size(17.79nm) of AuNPs sandwich assay were103cfu/mL and10cfu/mL, and the optimum particle size of AuNPs sensitivity was100times higher than that of direct detection. These are mainly due to the surface area of AuNPs increasing the amount of labeled PAb and enhancing refractive index of the biosensor surface.4. Specific and recovery assayE. coli O157:H7and Gram-negative bacteria of the Enteropathogenic Escherichia coli (EPEC) were detected by AuNPs enhanced sandwich assay. Results showed that the detection method had a high specificity. The recovery assay results showed that the recovery rate was84.5, RSD was1.372%. This method had high accuracy, good reproducibility.