Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (21): 4186-4195.doi: 10.3864/j.issn.0578-1752.2017.21.013

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

An Electrochemical Aptasensor for Detection of Salmonella Based on Composite Nanomaterial and Enzymatic Recycling for Amplification

XU LianYing, WANG BiNi, ZHANG FuXin   

  1. College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710119
  • Received:2017-05-17 Online:2017-11-01 Published:2017-11-01

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Abstract: 【Objective】 Salmonella is an important detection target of pathogeny bacteria in food. In order to overcome the shortcomings of traditional Salmonella detection methods, a novel assay of electrochemical aptasensor for quantitative detection of Salmonella with better practicability was established.【Method】Reduced Graphene Oxide (rGO) solution and toluidine blue (Tb) solution were mixed together to obtain the Tb-rGO nanocomposite, and then the Tb-rGO nanocomposite was dispersed in gold nanoparticles (AuNPs) colloidal solution to obtain the AuNPs-Tb-rGO nanocomposite. Then, the as-prepared AuNPs-Tb-rGO nanocomposite was incubated with amino-DNA to obtain the DNA-nanocomposite (S1-AuNPs-Tb-rGO). The complementary strands of the aptamers of Salmonella (S2) were attached to the surface of gold electrode by Au-S-bond, and then the electrode surface was blocked with HT. Subsequently, the aptamers of Salmonella (Apt) were dripped onto the modified electrode to make Aptbind with S2. The modified electrode was immersed into the mixture containing Salmonella and exonuclease I (Exo I). In terms of the characteristics of Exo I that could amplify electrical signals and the aptamers that could exclusively bind with Salmonella, the aptamers were taken away from S2 circularly. Then, the S1-AuNPs-Tb-rGO composite was attached to the surface of electrode by the hybridization of S1 and S2. Finally, the conditions of the incubation time in bacteria liquid, the Exo I concentration and the S1-AuNPs-Tb-rGo composite concentration were optimized and the electrical signals of the electrode surface was monitored to construct the aptasensor. This electrochemical aptasensor was used to test Escherichia coli, Staphylococcus aureus, Shigella, Listeria monocytogenes and Enterobacter sakazakii to ensure the electrochemical aptasensor’s specificity. The electrochemical aptasensor was used to detect 6×102-6×106 cfu/mL Salmonella to ensure the electrochemical aptasensor’s sensitivity. Then this electrochemical aptasensor was used to detect the goat milk to evaluate the practical use of electrochemical aptasensor.【Result】The optimization of the electrochemical aptasensor incubation time in bacterial liquid, the Exo I concentration and the S1-AuNPs-Tb-rGo composite concentration were studied in detail, and the optimal conditions were 1 h, 0.6 U?µL-1 and 200 nmol?L-1. When Salmonellas were tested existent, they had specific binding with Apt and the S1-AuNPs-Tb-rGo composite was attached to the electrode surface. So the linear sweep voltammetry curve of the electrochemical aptasensor showed a rise of oxidation peak. The developed aptasensor was specific to Salmonella and did not react with non-target bacteria. The electrochemical aptasensor detected the Salmonella target at a titer higher than 200 cfu/mL. A good recovery of Salmonella in the range of 91.6%-106.3% was obtained in goat milk by electrochemical aptasensor assays developed.【Conclusion】This electrochemical aptasensor can detect Salmonella with a easy operation, a wide linear range, a high sensitivity and a low cost, which provide good application prospects in the field of rapid quantitative detection of salmonella.

Key words: Salmonella, electrochemical aptasensor, reduced Graphene Oxide, toluidine blue, gold nanoparticles, Exonuclease I

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