Study On Several Electrochemical Sensors For Reactive Oxygen Species And DNA

Reactive oxygen species (ROS), are highly unstable molecules, generated in vivo during metabolic processes. At high ROS concentration or an overproduction state, ROS can cause oxidative stress which can induce damage of lipids, proteins or DNA, impeding normal cell functioning and leading to numerous human diseases, as well as to the aging process.Deoxyribonucleic acid (DNA) plays a crucial role in the storage of genetic information and protein biosynthesis. It is well known that the abnormal changes of bases in organisms imply the deficiency and mutation of the immunity system, and can be used as the indicator of various diseases.Therefore, tracking and probing the concentration of ROS and DNA is challenging and essential. Because of their simplicity, fast response, relatively cheap cost, and low power requirement, electrochemical methods have aroused great interests. Therefore, in this dissertation, a few electrochemical sensors for ROS, DNA and ROS induced DNA damage have been developed and the main points are summarized as follows:(1) A superoxide anion (O2·-) biosensor has been developed based on layer-by-layer assembled poly-(diallyldimethylammonium chloride)(PDDA) and superoxide dismutase (SOD) on the L-cysteine (Cys) modified gold (Au) electrode. In25mM phosphate buffer solution (pH7.2), the (SOD/PDDA)n/Cys/Au electrode shows a pair of well-defined and nearly reversible peak at ca.84mV vs SCE, confirming the direct electrochemistry of SOD. Based on the bifunctional enzymatic catalytic activities of SOD to the oxidation and reduction of02·-, the developed (SOD/PDDA)5/Cys/Au electrode exhibits good analytical characteristics in the detection of02·-, such as low detection limit, good stability, reproducibility, selectivity and particularly a wide linear range (0.5-546μM).(2) A novel non-enzymatic hydrogen peroxide (H2O2) sensor based on the nitrogen doped porous carbon nanopolyhedrons (N-PCNPs) modified glassy carbon electrode (N-PCNPs/GC electrode) was developed. The N-PCNPs were prepared from direct carbonization of ZIF-8nanopolyhedrons. The results show that the N-PCNPs/GC electrode exhibits excellent electrocatalytic activity toward H2O2. Optimization of measurement parameters such as the applied potential and pH value were studied in detail. Under the optimum conditions, the calibration curve for H2O2determination is linear in the range from0.001to11mM with a detection limit of0.2μM (S/N=3).(3) By using silver staining enhancement of Au nanoparticles, we have successfully constructed a DNA-based biosensor for sensitive electrochemical detection of hydroxyl radical (·OH). The DNA biosensor was fabricated by directly assembling thiolated DNA1(SH-DNA1) on the planar gold electrode through the "Au-S" bond.·OH generated from Fenton reaction could induce serious oxidative damage of the DNA layer adsorbed on the electrode surface. In order to enhance the sensitivity of the biosensor, DNA2-functionalized Au nanoparticles (DNA2-AuNPs) and silver staining enhancement were used to amplify the response signal. Based on the distinct signal amplification by AuNPs-catalyzed silver staining, the resulting biosensor exhibits a good analytical performance with a wide detection linear range from0.2to200μM, and a low detection limit of50nM, and exhibits satisfactory selectivity and stability. Moreover, this electrochemical biosensor has potential applications in the evaluation of antioxidant capacity.(4) A sensitive method for electrochemical detection of hydroxyl radical (·OH) was successfully developed based on magnetic beads (MBs)-DNA-Ag nanoparticles (AgNPs)(MBs-DNA-AgNPs) nanocomposite. MBs were utilized for bioseparation and AgNPs as signal indicator. When·OH broke the DNA strands, AgNPs were detached from the surface of the MBs-DNA-AgNPs nanocomposite. Magnetic particles could be easily removed from the solution and the detached AgNPs were collected. Therefore, the anodic stripping voltammetry (ASV) signal of dissolved silver on glassy carbon (GC) electrode depended on the amounts of·OH. The results show that the ASV signal linearly increases with the increase of the concentration of·OH in the range of0.05～4μM, and the detection limit is as low as10nM. In addition, the developed method has potential application in the screening of antioxidants.(5) Based on electro-immobilization of guanine on graphene nanoribbons (GNRs) modified glassy carbon(GC) electrode, a new electrochemical DNA biosensor was developed for the evaluation of total antioxidant capacities (TAC) in fruit juices. The biosensor relies on the guanine damage that is induced by hydroxyl radical (·OH) generated by Fenton-type reaction. Ascorbic acid (AA), which has the ability to scavenge the·OH and to protect the guanine immobilized on the electrode surface, was used as the standard antioxidant to evaluate the TAC in fruit juice. Under optimized conditions, the proposed biosensor has excellent analytical performance for antioxidant capacity assessment:wide linear range(0.1to4mg L-1), high sensitivity (4.16mA mg-1L) and low detection limit (0.05mg L-1). Additionally, the biosensor was successfully applied to the determination of the TAC in fruit juices.(6) Based on the wide potential window and good electrochemical properties of the modified electrode, the direct electrochemical oxidation of four free DNA bases on the graphene nanoribbons (GNRs) modified glassy carbon (GC) electrode was achieved. Compared with bare GC and carbon nanotubes (CNTs) modified electrodes, the GNRs/GC electrode exhibited obviously enhanced electrocatalytic activities towards the oxidation of guanine (G), adenine (A), thymine (T) and cytosine (C). Under the optimum conditions, the GNRs/GC electrode has low detection limit, high sensitivity and wide linear range for the detection of G, A, T and C. On the other hand, the electrochemical oxidation of quaternary mixture of G, A, T, and C on the GNRs/GC electrode was investigated. The values of the peak potential difference between G and A, A and T, T and C are290mV,200mV,140mV, respectively. The peak separations are large enough for their recognition in mixture and simultaneous detection of G, A, T and C. The GNRs/GC electrode also displays good stability, reproducibility and excellent anti-interferent ability, and good capability for the analysis of Single-Nucleotide Polymorphisms (SNPs) without hybridization or labeling.