Research On Electrochemical Aptamer Sensor Of Enterobacter Sakazakii Based On Signal Amplification

Enterobacter sakazakii is an important genus of Cronobacter,which can cause various infectious diseases,especially for newborns,which can cause serious fatal diseases.The study found that infant formula is the main source of infection of E.sakazakii in newborns.Therefore,rapid and specific detection of E.sakazakii is an important way to prevent food safety incidents.The common detection methods of E.sakazakii are:plate counting method,enzyme-linked immunosorbent assay(ELSA),polymerase chain reaction(PCR),etc.,which are time-consuming,costly and sensitive in detecting target substances.Defects such as detection of the target bacteria cannot be directly performed.Based on the specific recognition function of aptamers,this study combined with graphene oxide,enzyme digestion and hybridization chain reaction amplification signal technology to design three electrochemical aptsensors with higher performance for quantitative,more sensitive and more conveniently and directly to detect E.sakazakii.This not only monitors and prevents the contamination of E.sakazakii,but also provides new detection strategies for other pathogens.The main research contents are as follows:1.To construct a signal-attenuated E.sakazakii electrochemical aptamer sensor based on exonuclease I was constructed to achieve rapid detection of E.sakazakii in goat milk powder.The gold electrode modified with the thiolated DNA strand S1 was incubated in a solution containing a methylene blue(MB)-labeled aptamer chain(Apt)at 24? to bind S1 to Apt to form a DNA double-stranded structure.In the presence of E.sakazakii,the electrode modified with DNA double strands is incubated in a mixed solution containing E.sakazakii and exonuclease 1(Exo ?)for a period of time,at which time E.sakazakii The aptamer chain specifically binds and carries it away from the electrode surface,and the electrochemical response signal is attenuated.Under the action of Exo ?,the aptamer chain was cleaved,and the released E.sakazakii again carried away the aptamer chain from the electrode surface,thereby amplifying the electrochemical response signal.At the same time,the detection conditions such as incubation temperature,the incubation time of aptamer chain,the incubation time of target bacteria and Exo ? concentration were optimized.Under the optimal detection conditions,the constructed sensor detects the linear range of E.sakazakii from 1×102 cfu/mL?1×106 cfu/mL,and the minimum detection limit is 33 cfu/mL.The sensor can complete the detection of E.sakazakii within 1h,and can detect the specificity of E.sakazakii,and can be successfully used in the detection of E.sakazakii in goat milk powder.The recoveries of standard addition are 94.7%?107.2%.2.An electrochemical aptamer sensor of based on graphene oxide signal amplification was constructed.First,the thiolated E.sakazaki aptamer chain(Apt)was modified on the surface of the gold electrode,and the electrode was incubated in a certain concentration of E.sakazakii solution for a period of time,and then immersed in a certain concentration of GO and MB,respectively.Incubate in solution.Due to the specific binding of E.sakazakii to its aptamer chain,the adsorption of GO and aptamer chains is hindered,which reduces the adhesion of MB on the electrode surface,resulting in a weakening of the electrochemical signal.The optimum incubation time for optimized graphene oxide was 4 h,and the optimal incubation time for methylene blue was 1 h.Under the optimal conditions,the constructed sensor detects the linear range of E.sakazakii from 2×101 cfu/mL?2×106 cfu/mL,and the minimum detection limit is 7 cfu/mL.It also has strong specificity and good repeatability for detection of E.sakazakii.In the detection of E.sakazakii in fresh goat milk,the spiked recovery of the sensor was 96.2%-106.2%,indicating that it can be successfully used in the detection of Enterobacter sakazakii in actual samples.3.An electrochemical aptamer sensor based on the hybridization chain reaction and exonuclease I signal amplification was developed to achieve double amplification of the sensor response signal.The thiolated aptamer complementary strand S1 is modified on the surface of the gold electrode,after which the electrode is incubated in the aptamer chain(Apt)solution for a period of time to allow S1 to fully bind to the aptamer strand to form a DNA double-stranded structure.Then,the modified electrode is incubated for a certain period of time in a mixed solution of E.sakazakii and Exo ?,and the aptamer chain is carried away from the electrode surface by Exo ?,and the aptamer chain is excised to release E.sakazakii.Achieve a preliminary amplification of the signal.Next,the electrode is incubated in a solution of DNA strand S2 for a period of time such that S1 and S2 taken away from the aptamer strand hybridize.Finally,the modified electrode is incubated in a mixture containing the ferrocene(Fc)modified DNA strand S3 and the DNA strand S4 for a period of time to achieve secondary amplification of the signal.The constructed sensor showed a good linear relationship between 2×101 cfu/mL?2×107 cfu/mL for the detection of The minimum detection limit was 7 cfu/mL,and the specificity was strong.In the spiked recovery test of Enterobacter sakazakii in fresh goat milk,the spiked recovery rate was 93.8%?110.0%,indicating that the sensor can be used for the detection of actual samples.