Label-free Electrochemical DNA Biosensors And Electrochemical Determination Of Medicinal Molecules

Currently, bioanalytical chemistry on the nanometer scale is the forefront and hotspot in the field of international bioanalyticl research. DNA is a carrier of geneticinformation and the material basis of gene expression. DNA electrochemical biosensor isa analysis device. It immobilizes DNA on the electrode surface firstly as a molecularrecognition substance, and then changes the biological signal after DNA hybridization tothe electrochemical detection signal. DNA electrochemical sensors has been of greatconcern for researchers with several features, such as high sensiticity, good selectivity,rapid analysis and inexpensive detection equipment. DNA electrochemical sensorsdivides into lable and free-lable types with DNA mark ornot. Lable sensor has highsensitivity, but it is very time-consuming and lots of steps in the labing process whichinludes the synthesis, label, separation and purification. It also may affect DNA activityand conformational. Therefore, an urgent need to establish a lable-free DNAelectrochemical sensors with simple, fast, high selectivity and high sensitivity.When small medicines molecules into the body, it will affect DNA replication,transcription and protein synthesis with the role of a target DNA, to inhibit thedeterioration of the cell. Electrochemical analysis has a very important position in thefild of medicines analysis and medicines interaction with DNA research, because itincludes simple, sensitve, rapid, easy to implement in situ, on-line, real-time, live andon-site determination. The emergence of nanotechnology opens a new direction in thefield of analytical chemisty for the development and application of nanomaterrials.Combine of nanomaterials，high specificity of DNA and high sensitive electrochemicaltechniques effectively, which can improve the detection sensitivity and expand the range of applications of nanomatererials. Graphene, as a novel nanomaterial, has become a hotspot in the field of electrochemisty.The purpose of this work is to develop a new method to determinateelectrochemical DNA biosensors and drugs with gold and grapheme nanomaterials. Italso to open up the research and application range of the biosensor analysis device,which provides potential applications for disease diagnosis and clinical treatmentbetween DNA and medicines molecules. This thesis is divided into five chapters. Thefist chapter is an introduction. Chapters two and three are a lable-free DNAelectrochemical sensor, and the last two chapters are a electrochemical reaserch ofmedicines. The main contents were as follows:1. A novel electrochemical DNA biosensor incorporating a signal enhancement forthe determination of p53tumor suppressor gene was designed. The signal enhancementwas achieved by using gold nanoparticles (GNPs), which was electrodeposited onto agold electrode (Au), as a platform for the immobilization of the thiolated DNA. A thiollinker group at the termini of the DNA strand was self-assembled onto the surface ofgold nanoparticles. By using methylene blue (MB) as the electrochemical indicator, thehybridization reactions were monitored with the reduction peak current. Under theoptimal conditions, the reduction peak current of MB is linearly related to theconcentration of p53tumor suppressor gene from1.0to1000.0nmol/L with a detectionlimit of0.8nmol/L(S/N=3). The relative standard derivation was3.8%for50.0nmol/Ltarget DNA in11repeated determinations.It was found that the sensitivity of the DNAsensor with gold nanoparticles modification was higher than that of the DNA sensorwithout gold nanoparticles modification. This work demonstrates that gold nanoparticlesmodified gold electrode provides a promising platform for immobilizing DNA andenhancing the sensitivity. The analytic results were sensitive and specific. It holdspromise for the diagnosis and management of other DNA cancer.2. A label-free electrochemical biosensor with AuNPs/graphene composite for thesensitive detection of p53tumor suppressor gene was developed. The DNA probe (HS-DNA) was covalently linked to the surface of AuNPs/Gr via an Au-S bond, andelectrochemical impedance spectra were used to assemble the electrode surface.Differential pulse voltammetry was utilized to monitor the DNA hybridization event bymeasuring the changes in peak current of the intercalated MB. The detection of the p53tumor suppressor gene sequence can be achieved in the concentration range from0.1to1000.0nmol/L with the detection limit of0.012nmo/L (3σ) under the optimal conditions.The relative standard derivation was4.1%for10.0nmol/L target DNA in11repeateddeterminations.Compared with gold nanoparticles modified gold electrode, grapheneand gold nanonparticles modified sensor to improve the determination sensitivity of thep53tumo suppressor gene.3. The gold nanoparticles modified carbon paste electrode (GNPs/CPE) wasprepared, the electrochemical characteristics of methotrexate at GNPs/CPE were studiedusing cyclic voltammetric method, a novel electrochemical method for the determinationof methotrexate at GNPs/CPE was established and its interaction with herring spermDNA was developed at gold nanoparticles modified carbon paste electrode (GNPs/CPE)using linear sweep voltammetry (LSV) and square wave voltammetry (SWV). There wasa good linear relationship between anodic peak current and methotrexate concentrationin the rage from0.5to10.0mol/L, and the detection limit(S/N=3) was0.2mol/L.The relative standard derivation was4.7%for3.0mol/L methotrexate in11repeateddeterminations. The method was applied in the determination of methotrexate in theinjection samples with satisfactory results. In the presence of methotrexate, after addingvarious concentrations of herring sperm DNA, the peak potential shifted positively andthe redox peak current of methotrexate was decreased, which indicated that methotrexatecould react with herring sperm DNA and form an electrochemical inactive compound.The binding equilibrium constant and binding ratios were calculated as4.6×10~5L mol-1and2:1respectively.4. The electrochemical behaviors of clenbuterol hydrochloride (CLB) at graphenemodified glass carbon electrode (GP/GCE) were established and its interaction with herring sperm DNA (hs DNA) was developed using different pulse voltammetry (DPV).The drug was irreversibly oxidized at around1.0V, giving rise to a product whichexhibited a quasi-reversible couple at around0.4V. We speculated that the reactionmechanism of CLB at GP/GCE may be on the benzene ring amino group which wasoxidized to the imino and imino reacted in water to produce benzoquinone, thenreleasing the ammonium ion. The oxidation peak of CLB at around1.0V was decided tostudy for analysis, there was a good linear relationship between anodic peak current andCLB concentration in the rage from10to150mol/L, and the detection limit (S/N=3)was5mol/L. In the presence of CLB, after adding various concentrations of hs DNA,the redox peak current of CLB at1.0V was decreased and peak potential shiftedpositively, which indicated that CLB could react with hs DNA and form anelectrochemical inactive compound. The binding equilibrium constant and binding ratioswere calculated as1.4×10~5L/mol and2:1respectively.