The Preparation Of SOD Molecular Assembled Systems With Imidazole And Their Actions Toward Superoxide Anion

Self-assembled monolayers (SAMs) have been considered as the most advanced form among the various methods to modify electrode surface. Biosensors can be fabricated through fixing the biologically active species (such as enzyme, antibody, cell etc.) to the surface of electrode modified with self-assembly monolayers (SAMs), and their potential applications in the fields of chemistry, biology, clinic diagnestics as well as environmental monitoring have been proved. Superoxide dismutase (SOD) is a common metalloenzyme in organism. However, the direct ultilization of natural SOD have many problems such as stability, affinity with biological film, bioavailability and antigenic property, etc. The model compounds of superoxide dismutase (MSOD), designed and synthesized according to the structure of the active site of natural superoxide dismutase, have low molecular weight, high stability, relatively long half-time, good solubility in liposome and always show good superoxide dismutase like activities . Electrodes midified with MSOD are expected to be used as biosensors to detect superoxide, which will have potential applications in the fields of chemistry, biology, clinic diagnestics as well as environmental monitoring.In our work, we synthesized imidazole derivatives. Constructed MSOD system containing imidazole derivatives with self-assembly ability on gold electrodes. The strcture and the property of these modified elctrodes were studied by electrochemical methods. SOD-like activity of these modified elctrodes towardes superoxide was measured. The main contents are listed as the following:1. 6-(lH-imidazol-2-yl)hexane-1-thiol (IHT) was synthesized. The molecular structures of the intermediates and the target compound were confirmed by ~1HNMR, MS and IR etc.2. Various self assembled mnolayers have been constructed on gold electrodes including 6-(1H-imidazol-2-yl)hexane-1-thiol (IHT)/Au, IHT/1-hexanethiol (HT)/ Au mixed, N-(2-(1H-imidazol-5-yl) ethyl)-5-(1,2-dithiolan-3-yl) pentanamide (IEDTA, Synthesized by our group)/Au, and mixed SAMs of IEDTA/ HT/ Au, 2-(1H-imidazol-5 -yl) ethanamine (IEA)/5-(1,2-dithiolan-3-yl) pentanoic acid (TA)/ Au and IEA/ TA/ HT/ Au, by the self-assembly monolayer technology. The electrochemical cyclic voltammetry and electrochemical impendence spectra were employed to characterize the structures and the properties of the above SAMs on gold. The results show that closely packed and well ordered SAMs have been fabricated and their surface coverages are very high. The surface coverages of the mixed SAMs on gold are higher than that of the corresponding pure SAMs and increased with the increases of the molar ratio of HT in the SAMs.3. Copper ion was coordinated to the terminal imidazole group confined at the interface of the above mentioned SAMs systems. The results show that, the concentration of copper ion coordinated to the pure SAMs are lower than of the corresponding mixed SAMs. As for the IHT/HT/Au modified electrodes, the amount of coordinated Cu2+ reaches to the highest concentration, 2.3 X 10'nmol/cm2 with the molar ratio 1:4 for the mixed IHT/HT monolayer. For the mixed BEDTA/HT and ffiA/TA/HT monolayers, the maxmum coodinated copper ion concentrations reach 2.33X10"1 mol/cm2 and 2.00xl0"10 mol /cm2, respectively at the molar ratio 1:4. Because the existence of the dulte medium molecule HT greatly decrease the surface concentration and the close packing state of the terminal imidazole at the interface of SAMs, and higher coodinate copper ion can be expected.4. Xanthine-XOD was employed as the generation system of superoxide, then cyclic voltammetry was employed to investigate the SOD-like activity of Cu2+/IEDTA(HT)/Au and Cu2+/EEA/TA(HT)/Au modified electrodes. Under the existence of oxygen, Xanthine is oxidized to uric acid catalysed by XOD, and superoxide is produced at the same time. Superoxide disproportionates influenced by MSOD and the subsequent redox current is measured in electrochemical system. Thus the electrochemical cyclic voltammetrical plots of MSOD system before and after adding Xanthine-XOD in buffer are compared, and an apparent redox current increase is observed after adding Xanthine-XOD to MSOD system. The result demonstrates the MSOD system on gold electrode promotes the disproportionation of superoxide and its SOD-like activity towards superoxide. This work establishes the possibility by use of MSOD immobilized on the surface of electrode for the biosensor fabrication.