The Configuration And Application Of Salmonella Electrochemical Aptamer Sensor Based On Digestion Signal Amplification

As a common foodborne pathogen,Salmonella has a strong environmental adaptability,a wide range of contamination and a strong pathogenicity.The resulting intestinal toxins can cause abdominal pain,nausea,fever,vomitin,etc.Salmonella is an important detection target of pathogeny bactera in food.A novel assay of electrochemical aptasensor for quantitative detection of Salmonella based on aptamer recognition and enzymatic recycling with better practicability was constructed and investigated in order to overcome the shortcomings of traditional Salmonella detection methods,such as time-saving,sensitivity,simplicity,etc.The aptasensor we constructed can be used for the on-site detection of Salmonella,and it can also provide a new idea for the detection of other foodborne pathogens.The research is divided into four chapters,and the main contents of this research are as follows:1.The as-prepared AuNPs-Tb-rGO nanocomposite was incubated with amino-DNA(S1)to obtain the DNA-nanocoposite(S1-AuNPs-Tb-rGO).And then the aptamers of Salmonella(Apt)were dripped onto the complementary strands of the aptamer of Salmonella(S2)modified electrode to make Apt bind with S2.Subsequently,the modified electrode was immersed into the mixture containing Salmonella and exonuclease I(Exo I)at 37?.The aptamers were taken away from S2 by Salmonella,and the Exo I could amplify electrical signals.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 aptasenor.Under the optimal conditions,the peak current of the aptasensor was in good linear relationship with the base-10 logarithm of Salmonella concentration.The linear range was from 6×102?6×106 cfu/mL,and the detection limit was 200 cfu/mL.The detection could be finished within 1 h.The result also showed that the developed aptasensor was specific to Salmonella and did not react with non-target bacteria.A good recovery of Salmonella in the range of 91.6%-1063%was obtained in goat milk by electrochemical aptasensor assays developed.2.The reduced graphene oxide(rGO)and Au nanoparticles(AuNPs)were modified on the glassy carbon electrode(GCE)suface.And then the complementary strands of the aptamers of Salmonella(S)were attached to the surface of rGO/AuNPs GCE.Subsequently,the aptamers of Salmonella(Apt)were dripped onto the modified electrode to make Apt bind with S.After the above processing,the modified electrode was immersed into the mixture containing Salmonella and exonuclease I(Exo I)at 37?.The aptamers were taken away from S by Salmonella,and the Exo I could amplify electrical signals.Finally,the modified electrode was immersed into methylene blue(MB)solution for a while.The conditions of the incubation time in bacteria liquid and the Exo I concentration were optimized and the electrical signals of the electrode surface was monitored to construct the aptasensor.Under the optimal conditions,the peak current of the aptasensor was in good linear relationship with the base-10 logarithm of Slmonella concentration.The linear range was from 2×102?2×107 cfu/mL,and the detection limit was 67 cfu/mL.The detection could be finished within 40 min.The result also showed that the developed aptasensor was specific to Salmonella and did not react with non-target bacteria.A good recovery of Salmonella in the range of 97.3%-106.7%was obtained in pork by electrochemical aptasensor assays developed.3.The complementary strands of the aptamers of Salmonella(S)were attached to the surface of gold electrode,and then the modified electrode was immersed into the aptamer of Salmonella(Apt)solution to make Apt bind with S.Subsequently,the modified electrode was immersed into graphene oxide(GO)solution and methylene blue(MB)solution to fully react in order to mark MB on the electrode.Finally,the modified electrode was immersed into the mixture containing Salmonella and exonuclease I(Exo I)at 37?.The aptamers were taken away from S by Salmonella,and the Exo I could amplify electrical signals.Consequently,GO and MB were fallen off the electrode extensively.The conditions of the incubation time in bacteria liquid and the Exo I concentration were optimized and the electrical signals of the electrode surface was monitored to construct the aptasensor.Under the optimal conditions,the peak current of the aptasensor was in good linear relationship with the base-10 logarithm of Salmonella concentration.The linear range was from 1×102?1×107 cfu/mL,and the detection limit was 33 cfu/mL.The detection could be finished within 40 min.The result also showed that the developed aptasensor was specific to Salmonella and did not react with non-target bacteria.A good recovery of Salmonella in the range of 94.7%-106.1%was obtained in eggs by electrochemical aptasensor assays developed.