Fabrication Of Enzyme Micro-pattern By Scanning Electrochemical Microscopy(SECM)

In chapter one of this paper,the basic apparatus and operation modes of scanning electrochemical microscopy(SECM)were first introduced briefly.Then,the applications of SECM the applications of SECM in biologic system,including the detection and imaging of enzyme,antigen/antibody complex,DNA and cells,micropatternning of multiplicate surfaces with different materials,especially biological material were reviewed in detail.In chapter two,we developed a new SECM method for characterization of horseradish peroxidase(HRP)by using a ultromicro-electrode that was modified with glucose oxidase (GOx).For this method,a gold disc micrometer electrode modified with GOx was fabricated for characterising HRP in the solution containing glucose and H₂Q.In this system,glucose and O₂ reacted under the catalysis of GOx at the electrode to produce H₂O₂.Then,H₂O₂ and H₂Q reacted under the catalysis of HRP that had been immobilized on the substrate to produce BQ and H₂O,and BQ was reduced on the tip.When the tip was moved close to the HRP,the current would increase.Therefore,we could charaterise the activity of the HRP immobilized on the substrate.The results obtained using this method were compared with those using the SECM generation-collection mode and feedback mode.Since H₂O₂,one of the two substrates of HRP,was not in the solution,but was generated on the electrode,so the lateral resolution using this method was higher than that using generation-collection mode,and this method was more sensitive than the feedback mode.In chapter three,micrometer dots of GOx were fabricated on the platinum substrate electrode using a concave platinum micrometer electrode.First,concave platinum micrometer electrodes were fabricated by means of phenol-allylphenol copolymer insulation and electrochemical etching.Then,the GOx at the platinum electode was electrochemically adsorbed on a platinum substrate.When the concentration of GOx was 10 mg/mL,the concentration of Triton X-100 was 0.8 mmol/L,the electroadsorption potential on the Pt substrate was 1.2 V(vs.Ag/AgCl),and the electroadsorption time was 60 min,GOx could be electroadsorbed on the Pt substrate well.The patternning of GOx was achieved by the electroadsorption.In this system,the concave Pt micrometer electrode was used as reference and counter electrode and the Pt electrode substrate was used as the work electrode.The diameter of the microarray fabricated by this method was about 20μm characterised with scanning eletron microscope(SEM).In chapter four,GOx micropatterns were fabricated by SECM "Dip-pen" technique.We utilized the elements of "Dip-Pen nanolithography"(DPN)of AFM in SECM.In this work, a dual micrometer platinum electrode(DMPE)was fabricated first.Then,GOx was electroadsorbed on one platinum electrode of the DMPE under the conditions which had been selected in chapter three.After the DMPE was placed upon the platinum electrode substrate,the microeleetrode without GOx in the DNPE was served as reference and counter electrode,the microelectrode with GOx in the DMPE was served as work electrode, and the platinum electrode substrate was served as another work electrode.Appropriate potentials were applied on the two work electrodes,then GOx was desorbed from the microelectrode with GOx and adsorbed on the platinum electrode substrate at the same time.So the activated patterns were fabricated in the process of electroadsorption - electrodesorption - electroadsorption,and the patterns were characterised with SECM.For fabricating smaller micropatterns of GOx,a thin solution between the DMPE and the platinum electrode substrate was used to minimize the diffusion of the GOx in solution.In chapter five,the micropatterns of HRP were fabricated by means of SECM "Dip-Pen" technique.First,the adsorption and desorption of Bio-HRP on platinum electrode were investigated by electrochemistry.Then,Bio-HRP was adsorbed on the platinum microelectrode under the conditions selected before.The electrode was approached to substrate modified with the streptavidin.Finally,Bio-HRP was desorbed from the platinum microelectrode and reacted with the streptavidin.The activity of the pattern was characterised with SECM.A thin solution between the microelectrode and the substrate was also used to minimize the diffusion of HRP in solution.