Amperometric DNA Sensors Based On Two Complex Nanoparticles

More and more attentions had been payed to the electrochemical biosensor which is an inexpensive way that can sensitively detect biological macromolecules. Electrochemical DNA sensor has advantages such as simple fabrication, long life of used, reproducibility well, high sensitivity, low cost and easy implement miniaturization and so on, and being one of the hotspots of international and domestic research in recent years. The immobilization of DNA on the electrode surface is one of the top prior technology of the preparation of electrochemical DNA sensor, the different way of immobilization will lead to different specific reaction of the hybridization of single-stranded DNA (ssDNA) and its complementary strand DNA (cDNA), and will directly affect the sensitivity of electro chemical DNA sensors. In recent years, nanomaterials such as carbon nano-materials, magnetic nano-materials has been used in the preparation of biosensors, and has became a hot topic of biosensor research field. In this paper, gold nanoparticle composites has been fixed on glassy carbon electrode (GCE) or gold disk electrode (Au) surface, used the fuction of cooperation of the AU-NH2bond to fix the DNA on the surface of GCE or Au, and fabricated two current electrochemical DNA biosensor by taking advantage of the excellent electron transport properties of prussian blue (PB) or carbon nanotubes (CNTs). The major contents in this thesis include three sections as follows:(1) Amperometric DNA sensor based on gold coated magnetic prussian blueThionine (Th) was electropolymerized on the GCE, then PTh/GCE electrode was gained, then use the cooperation of the AU-NH2bond to fix golden coated magnetic prussian blue (Au@MPB) to PTh/GCE electrode surface, then use the cooperation of the Au-NH2bond to fix ssDNA to the GCE electrode surface, and the amperometric DNA electrochemical sensor was gained. The process of ssDNA immobilization and hybridization reaction are characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV), the influence of fixed ssDNA time, pH value, potential, hybridization reaction time and hybridization reaction temperature act on the performance of sensor were studied. Under optimization experiment condition, the slope of the DNA working curve of determined by pulse voltammetry was-10.52A·mL/μg, the linear range was from5×10-7～1×10-5μg/mL, the lower detection limit of3.07×10-8μg/mL, the detection limit of1.04×10-8μg/mL, and correlation coefficient r=0.9936.The relative standard deviation of10identical sensors were less than5%,the working curve slope decreased by12.6%after the11days’ storage, and the curve slope decreased by9.85%after three times reused.(2) Amperometric DNA electrochemical sensor based on the carbon nanotubes arraysMixed self-assembled monolayer (MSAM) was prepared firstly on Au electrode, then fixed the CNTs to the MSAMs surface by the diazotization reaction, and CNTs arrays were got on the surface of gold electrode. Then successively fixed the4-ATP、Au@MPB and ssDNA to modified electrode surface of the CNTs array, and a electrochemical DNA sensor (Au/MSAMs/CNTs/4-ATP/Au@MPB/ssDNA) based on CNTs array was gained. Immo-bilization and hybridization of DNA were characterized with EIS, CV and DPV by using Fe(CN)63-/Fe(CN)64-as an electrochemical hybridization indicator, the results showed that the slope of the working curve determined by DPV was-7.34A·mL/μg with the linear range from5.0×10-8～1.0×10-5μg/mL, the lower detection limit of1.31×10-8μg/mL and the correlation coefficient r=0.9924. The sensor has good reproducibility, and the work curve slope of the sensor decreased20%after13days.(3) Electrochemical DNA biosensor based on carbon nanotube arrays coated gold nanoparticlesFirst, CNTs array was fixed on MSAMs surface by diazotization. Then the electrode modified nano-Au/CNTs/MSAMs/Au was prepared by electrodeposited gold nanoparticles (nano-Au) on modified electrode surface, and fixed single-stranded DNA to GCE surface by the Au-NH2bond. Immobilization and hybridization of DNA were characterized with EIS, CV and DPV by using methylene blue (MB) as an electrochemical hybridization indicator, and the electrochemical DNA sensor was determined by DPV. The effects of the pH value, hybridize time and hybridize temperature were investigated. Under optimization experiment condition, the slope of the working curve of determined by pulse voltammetry was-9.66A mL/μg, the linear range was from1×10-13～1×10-6μg/mL with the lower detection limit of4.34×10-14μg/mL and the detection limit of5.42×10-14μg/mL, the correlation coefficient r=0.9918. The relative standard deviation of8identical sensors were less than5%, the sensor has good stability and reproducibility, and the work curve slope of the sensor decreased27.8%after15days, the curve slope decreased by12%after five times reused.