Study On The Fabrication Of Electrochemical DNA Sensor And Its Application In Discriminating Gene Point Mutation

Gene point mutation is one of the reasons which result in the variation of various forms of pathogenic bacteria.Developing high performance technology of point mutation discrimination has an important significance for the in-depth understanding the genetic characteristics of pathogenic bacteria,the exploitation of high-efficient and low-toxic targeted drugs and the protection of human's physical and mental health.So far,among various discrimination technologies developed,the traditional methods generally have some disadvantages of complicate and time-consuming operating procedure,expensive instrument price and weak discrimination ability,so they are of little value for the practical application;by contrast,among newly developing analytical methods,electrochemical DNA biosensor exhibits many advantages of low cost of fabrication,simple and fast operation,high sensitivity,good selectivity and strong anti-fouling ability.They are easily not only miniaturized but also used for on-line monitoring.Comparing with other technologies,this method has of greater practical application prospect and exploitation value.In this paper,taking the development of analytical method with strong point mutation discrimination ability and high sensitivity as the aim,using the technology of electroactive redox-modified probe and molecular beacon,two new-type E-DNA sensor were fabricated through assembling the capture probe and the signalling probe with different forms.The optimal proposed sensor was finally used for the analysis of point mutation of Escherichia coli lac Z gene.The corresponding works are listed as follows:1.One "signal on" E-DNA sensor was fabricated based on the design idea of competitive displacement-based hybridization-induced conformation change.This sensor is composed of two probes,one linear capture probe and one stem-loop signaling probe,attached to the surface of Au electrode.Before adding target DNA,a 6?12 base-pair duplex formed between two probes forces the electroactive redox methylene blue far away from Au electrode surface,thereby producing low background current signal;after adding target DNA,more stable duplex formed between capture probe and target DNA undo the former duplex to release the signaling probe.The released signaling probe recovers the original stem-loop structure which drives MB close to the electrode surface,thereby producing high detecting current signal.The change of current signal value is proportional to the concentration of target DNA.7 main parameters which play important roles in the fabrication of sensor were optimized through designing proper experiments.These 7 parameters are the ratio of two probes,the concentration of mixed probes,the hybridization temperature,the ion strength of working medium,pH value,the supporting base-pair duplex length and the passivation and supporting solvent chain length.Two electrochemical detecting technologies,alternating current voltammetry(ACV)and cyclic voltammetry(CV)were used to characterize the sensor system.The comprehensive performance was investigated and assessed from many aspects.This proposed sensor exhibits high sensitivity for target DNA with the detection limit of 1 pM and a linear relationship of 10-12?10-9 M;When used for the analysis of 1 and 2 point mutation,the discrimination factors,2.0±0.2,5.3±0.4 and 1.6±0.1,2.9±0.3,were obtained separately in pure working medium and 50%serum.2.Based on the intrinsic characteristics and researching results of first sensor,the structure of new second sensor's system was optimized again for further improving the detection sensitivity and point mutation discrimination ability.Using the working mode of capture probe-target-signaling probe(CP-T-SP),this sensor's system exhibits very simple structure with only 1 capture probe attached to the Au electrode surface and another signaling probe free in the working medium.Before adding target DNA,comparing with the first sensor,this sensor shows much lower background current signal resulted from the electroactive MB of signaling probe far away from electrode surface;after adding target DNA,two duplexes were formed with two portions of target DNA hybridizing with two portions of capture probe and signaling probe separately.This sandwich bridge type duplex structure can bring MB labels near to the electrode surface,thereby producing the detection current signal.The change of current signal is proportional to the concentration of target DNA.5 main parameters which play important roles in the fabrication of sensor were optimized through designing proper experiments.These 5 parameters are the concentration of capture probe,the concentration of signaling probe,the hybridization temperature,the ion strength of working medium and pH value.Two electrochemical detecting technologies,alternating current voltammetry(ACV)and cyclic voltammetry(CV)were used to characterize the sensor system.The comprehensive performance was investigated and assessed from many aspects.This proposed sensor exhibits high sensitivity for target,Escherichia coli lac Z gene sequence,with the detection limit of?30 fM and a linear relationship of 10-13?10-10 M;When used for the analysis of 1 and multi point mutation in 7 different location,the discrimination factors,7.6±0.8?95.0±14,7.9±0.6?106.0±16,8.0±0.6?107.6±16,7.9±0.5?116.6±18,8.1±0.8?108.0±18 and 8.0±0.6?124.1±20,were obtained separately in pure working medium and other 5 different real samples,tap water,pure water,milk,beer and peanut milk beverage.In addition to these,this sensor's system also exhibits an excellent working performance.