Application And Construction Of DNA Electrochemical Sensor Based On Fe₃O₄ Magnetic Nanoparticles

Gold is a rare and stable metal on earth and has a high commercial value.With the increasing price of gold in the market,humans exploited large amounts of gold mining areas,which leads to serious ecological damage.Therefore,it is necessary to find a method for energy-efficient exploration of minerals.In recent years,there was a green environmental exploration method involving microorganisms.Research found that bacterial concentration of B.cereus in the soil of the mining area was related to the content of gold,so it is possible to use the content of B.cereus in the soil to track the auriferous regions.Traditional analytical methods have been designed for the detection of B.cereus including plate counting,multi-tube fermentation,and membrane filtration.However,these methods are time-consuming,complicated and cannot be used for rapid detection.Therefore,there is an urgent need to establish an environmentally-friendly,rapid,highly selective and low-cost method for the detection of B.cereus.The method of electrochemical DNA sensors combines the advantages of three technologies:nanotechnology,biological science and electrochemical analysis technology.So it is a sensitive,inexpensive,and short-time method.This is a new type of detection method using electrochemical DNA sensors for the detection of bacteria.Thus,we synthesized the nanocomposites Fe₃O₄-NH₂@Au and Fe₃O₄/MWCNTs-COOH according to the excellent biocompatibility and magnetic properties of Fe₃O₄.Then we used them to modify the electrodes,and loaded with the specific DNA of B.cereus,constructed a new and efficient B.cereus sensor.The dessertation consists of five parts:In the first part,we mainly introduced the biological characteristics of B.cereus and the principle for microbiological prospecting.Meanwhile,traditional detection methods of bacteria,modern molecular biological methods and biosensor methods were reviewed.Additionaly,we also analyzed the advantages and disadvantages of various methods.Furthermore,the principle development and application of DNA sensors were introduced.This part also explained the significance and innovation of this work.In the second part,3 nm Fe₃O₄ nanoparticles was synthesized by green,inexpensive chemical coprecipitation method from the solution of ferrous/ferric solution.The synthesized Fe₃O₄ NPs was characterized by FTIR,?-potential,TEM and XRD methods.It was successfully compounded with chitosan?CS?and used for modification of glassy carbon electrode?GCE?to obtain Fe₃O₄/CS/p-DNA electrode.A label-free method based on electrochemical DPV method for quantitative analysis of B.cereus DNA.The sensitivity of the sensor was improved because the lager surface area and biocompatibility of Fe₃O₄ greatly increased the electron transfer rate,with a detection limit of 1.2×10^-13 M?S/N=3?.In the third part,we obtained about 13 nm Au NPs by classic reducing the HAuCl₄·3H₂O solution with the sodium citrate.It was characterized by FTIR,UV and TEM.The synthesized nanocomposites were used as electrode modification to obtain Fe₃O₄-NH₂@Au/CS/p-DNA electrode.Characterized the electrochemical behavior of the electrode by CV and EIS,and the current response was compared with the Fe₃O₄/CS/p-DNA electrode.DPV method was used to analysis the concentration of B.cereus specific DNA with a lower detection limit of 1.7×10^-14 M?S/N=3?.This was because that the synergy effect between Fe₃O₄-NH₂ and Au increased the electron transfer rate.However,the comparing of sensors based on the Fe₃O₄ nanoparticles indicated that the signal increase was not significant.So we need to modify the material.Carbon nanotubes are a good matrix material for composite materials because of their excellent electron transport and unique tubular structure.Therefore,in the fourth part,we successfully loaded Fe₃O₄ and MWCNTs-COOH for sensor construction,and characterized the Fe₃O₄/MWCNTs-COOH nanocomposites by FTIR,XRD,TEM and EDS.To characterize electrochemical behavior of the electrodes,CV and EIS were carried out.B.cereus DNA concentration was detected with DPV.At the same time,adsorption mechanism of DNA on electrode material was proposed that is caused by electrostatic attractive forces.As we know,CS has a positive charge under acidic conditions which comes from protonation,so it can be adsorbed positively charged DNA on its surface via electrostatic interaction.The sensor has a higher current response compared to previously constructed sensors,because Fe₃O₄/MWCNTs-COOH has excellent stability,electron transport properties and biocompatibility,with a detection limit of 2.0×10^-15 M?S/N=3?.Using this sensor to detect B.cereus in the real sample,the recovery rate reached 94.5%¹05.0%,and the RSD was very small,indicating the sensor can be used for the detection and analysis of real sample.In the last part,we summarized the full text and looked forward to the future work.