Scanning Electrochemical Microscopy Determination Of Peroxidase And Nano-electrodes In Single-cell Applications

In chapter one of this thesis, first of all, the basic apparatus, the operation modes, and the theory of scanning electrochemical microscopy (SECM) were introduced briefly. Then, the tips in SECM including ultramicroelectrode (UME), chemical modified electrodes, biosensors, micropipettes and nanopipettes etc, and the technique for obtaining SECM imaging such as constant current mode and constant height mode were discussed. Finally, the applications of SECM in the investigation for enzyme, antigen/antibody complex, DNA and cells were reviewed.In chapter two, horseradish peroxidase (HRP) molecules were immobilized on a insulator substrate with the insolublc cross-linked polymer of β-CDP and glutaraldhyde. The catalytic oxidation of hydroquinone (HQ) to p-benzoquinone (BQ) on the surface of immobilized MRP was studied using the feedback mode. The one-order enzyme-mediator kinetics was established and the apparent heterogeneous rale constant was evaluated. Then I1Q produced by the diffusion controlled tip reaction was oxided on the substrate surface by HRP, when the tip close to the spots of HRP. The tip current on the approach curve was greater than the value for a purely insulating substrate. Because the reaction speed of BQ on the lip was very fast, the tip current is mainly controlled by the heterogeneous reaction of IIRP. By fitting the experiment approach curves with theoretical values, the one-order apparent heterogeneous rate constant was evaluated. Using this method, the heterogeneous rate constants in the solutions with different pH values were determined easily. Our result showed that the activity of IIRP was highest in the buffer of pH 7.0.In chapter three, a method for detecting myeloperoxidase (MPO) in a single lymphocyte by SECM was developed. The single layer of lymphocytes on a glass slide was formed by the slide method and they were then immobilized on the glass slide. After dry, the permeability of the cell membrane changed so that low mass molecules (substrates of enzymes, products of enzyme-catalyzed reactions) could pass through the cell membrane while enzyme and other maeronioleeules were kept within the cell boundy at uncharged concentrations. HQ and H₂O₂ as the substrates of MPO, passed through the cellmembrane and reacted under the catalysis of intracellular MPO, and the products, BQ and H2O, were formed. As the speed of this catalysis reaction was very fast, the concentration of BQ around cells would become higher and higher. Therefore, BQ would diffuse from the cell surface to the buffer. The tip potential was fixed at -0.2 V where BQ could be reduced. As the tip approached to the substrate closely and moved on the substrate under constant height mode, it could detect the changes of the concentration of BQ in the solution on the substrate. When the tip moved near to a lymphocyte, the current would increase greatly. Therefore, we could detect the activity of intracellular MPO by SECM. The peak current in four lymphocytes ranged from 4.15 to 7.04x10'10 A, and corresponding peak volumes were 2.98 to 11.6><10"6 urn2-A in physiological buffered saline (PBS) containing of 3.00x10-3 mol/LHQ and H2O2.In chapter four of this thesis, a method for detecting MPO in single living cell by SECM was developed. First of all, the optimum condition of detection was l.OOxlO*3 mol/L HQ, 1.00x 10'3 mol/L H2O2 for substrate and PBS for buffer. Digitionin (Dig) in PBS could dissolve cholesterol on the cell membrane and form microhole on it. Such low mass molecules could pass through the membrane while macromolecules such as enzyme were still within the cell. Consequently, HQ and H2O2 could enter the cell and react under the catalysis of MPO to produce BQ and H2O. When the tip moved above the surface of substrate, which immobilized cells with collagen, it could determine the changes of the concentration of BQ, find cells and detect the activity of intracellular MPO by SECM. The peak current in 8 lymphocytes ranged from 1.4 to 4.3 xlO"10 A, and corresponding peak volumes were 1.0 to 6.6x 10"6 um -A. The peak currents in 3 monocytes were 1.8 to 7.2x 10'u A, and the peak volumes were 6.4 to 11 x 10"7 um2A.In chapter five, we fabricated two kinds of gold nanoelectrodes (GNEs). One kind is plating shield adhesive insulated GNEs (PSAGNEs) and the other is phenol-allylphenol copolymer insulated GNEs (PACGNEs). The differences between the two kinds of GNEs were compared using microscope, cycle voltammetry and SECM tip approach curve. The plating shield adhesive layer on PSAGNEs was thicker and coarser than that of PACGNEs. PACGNEs had better voltammeric response and more stable than PSAGNEs. The radii of...