Study On The Electrochemical Detection Of Escherichia Coli Gene Sequence With High Performance Using DNA Sensor

As one of the typical bacterial pathogens,Escherichia coli(E.coli)mainly exists in the intestinal tract of human and other warm-blooded animals and can pollute various drinks and foodstuffs through multiple channels.Most E.colis can not cause diseases,however,some of them produce enterotoxin and are the major cause of infectious outbreaks with serious consequences.The high sensitive DNA detection maybe an effective indirect way to achieve the detection of its pathogen.Therefore,developing a novel,fast and specific detection technique of E.coli gene is an important issue which is of great theoretical and practical significance.Based on the previous work,two similar but slight different sensor architectures using target induced signaling probe shifting(TISPS)signaling mechanism were designed and fabricated in this paper.The existing forms of two capture probes(CPs)relative to Au electrode surface are different for two sensors with the CP unattached for S1 and attached to the electrode surface through Au-S bond for S2.Two proposed sensor systems were finally used for the analysis of E.coli lac Z gene.The corresponding works are listed as follows:1.S1 exhibits a simple structure with only composed of three probes.A 12-base CP1/AP duplex can be formed through the hybridization of the CP1 and AP,and the AP is attached to the Au electrode surface using Au-S bond.The methylene blue(MB)modified signaling probe(SP)is free in the detection solution.In the absence of target,the proposed sensor shows a low background current signal because of the MB labels far away from Au electrode surface in the solution with low efficient electron transfer;in the presence of target,the longer and more stable 24-base CPI/target duplex formed between CP1 and the target gene sequence undoes the former duplex to release the AP,which induces the formation of another 8-base AP/SP duplex.With the happening of DNA hybridization displacement-based reaction,the hybridization resistance decreasing(HRD)way is adopted because CP1 can be taken into the solution by the CPI/target duplex.Such structure change can drive the MB labels close to the electrode surface with much more efficient electron transfer,thereby producing a large detection current.Some main parameters which affect the fabrication of sensor were optimized through designing proper experiments.These parameters are the ratio between CP1 and AP,the probe coverage on the electrode surface,SP concentration and the sensor interrogation temperature.Two electrochemical detecting technologies,alternating current voltammetry(ACV)and cyclic voltammetry(CV)were used to characterize the sensor system.When used for the analysis of perfect match target DNA,a good linear relationship is obtained between detection signal and the target concentration(log c/M)within a broad range from 0.01 pM to 1 nM with the equation of y=31.203x+441.34,the correlation coefficient(R2)of 0.9787,and a detection limit of-7 fM for the proposed sensor.This linear range can be used for the quantitative detection of target for this sensor system.When used for the analysis of 1 and 2 point mutation in pure working medium,the obtained discrimination factors are 1.29±0.2 and 2.95±0.4 respectively.In addition,this sensor system also exhibits an excellent working performance and anti-fouling ability when used in other 4 different real samples,50%tap water,50%pure water,50%milk,50%(100%)calf serum.2.Based on S1,we redesign and fabricate another sensor(S2)with a slight difference,in which a 24-base thiolated CP2 and a 12-base thiolated AP are co-immobilized on the Au electrode surface via the Au-S chemistry.Such change in structure results in the hybridization resistance increasing(HRI)way opposite to S1 for this sensor,further causing the changes of sensor behaviors and comprehensive performances.Under the optimized fabrication parameters and detection conditions,the 24-base perfect match target DNA can be quantified over the range from 0.01 pM to 0.5 nM with the equation of y=45.563x+55.5,the linear correlation coefficient of R2=0.9753 and the detection limit of?10 fM for the proposed sensor.When used for the analysis of 1 and 2 point mutation in pure working medium,this sensor system shows a pecular specificity tunable ability and better discrimination factors of 1.52±0.2(Fsi)and 3.90±0.5(FS2)than those obtained from S1.In addition,S2 also exhibits an excellent working performance when used in different real samples.