On-line Preconcentration Of Biomarker By Capillary Electrophoresis And Its Application

Due to environmental pollution or the body’s natural aging, the body may contain excessive reactive oxygen species. These ROS can damage DNA, proteins and other biological macromolecules, and also damage to normal physiological activities. Guanine, in the body, is the most susceptible to attack by hydroxyl radicals and singlet oxygen, which oxidize hydroxy of C-8in Guanine and generate prodution of8-hydroxy-2’-deoxyguanosine (8-OHdG).8-OHdG is the main metabolites by the damage to oxidised DNA. In recent years, the most common method, using in detection8-OHdG, is the high performance liquid chromatography-electrochemical detection (HPLC/EC), gas chromatography-mass spectrometry method (GC/MS), liquid chromatography-mass spectrometry detection (HPLC/MS) and so on. Becuse of, howere, many of detection steps and some expensive equipment, these methods are difficult to carry out in the laboratory. The on online concentration in capillary electrophoresis technology is not to extend the analysis time of sample analysis and the same time makes the detection sensitivity greatly enhanced. We selected the typical biomarker8-OHdG and typical amino acids and developed a on-line preconcentration in capillary electrophoresis for the determination of8-OHdG and amino acids.This thesis includes five parts:Chapter1:The first part of preface introduction, the basic theory, capillary electrophoresis and on online concentration techniques. Second, introduction,8-hydroxy-2’-deoxyguanosine (8-OHdG) origin、The significance of the research and testing methods. Finally, expounded on the research purposes and contents for this paper.Chapter two:Optimize the condition of sweeping under SDS-borate buffer solution and SDC-borate buffer solution for the8-OHdG. By comparing the enrichment conditions under the SDS-borate buffer and SDC-borate buffer solution, showed that8-OHdG can be concentrated better under SDS-borate buffer. Selection SDS-buffer system for optimal conditions:Separation voltage of10KV, buffer solution30mmol/L borate and80mmol/L SDS,8-OHdG and dG in the best injection time for the90s. Under SDS-borate buffer,8-OHdG was concentrated and the sensitivity compared with zone electrophoresis increased by34times and the detection limit is490nmol/L. However, this method can not be used for the determination of8-OHdG in urine for its low concentration, and8-OHdG also can not be completely separated from other sample matrix. Therefore, further study is needed to improve the sensitivity of the method for the determination of8-OHdG in urine.Chapter three:Determination of8-OHdG in urine by capillary electrophoresis using gold nanoparticles instead of SDS. Studies showed analyte can be enriched by which being interation with gold nanoparticles and the sensitivity compared with zone electrophoresis was increased by136times, the detection limit was120nmol/L Combination with solid phase extraction, under the best conditions, the method can be applied to the analysis of actual urine samples of cancer patients. However, due to the complicated sample pretreatment, the further research was focused on the avoiding of pretreatment step, developing a higher sensitivity and more efficient method.Chapter four:A method of using gold nanoparticles and surfactant was established for enrichment of8-OHdG. In the synthesis process, a series of different size gold nanoparticles solution were synthesized through changing the volume of solution with different HAuCl4. After analysis, nano particles with surfactant M had a good enrichment on8-OHdG. the method can be used for the determination of8-OHdG in urine from healthy persons and cancer patients without pretreatment step. the sensitivity compared with zone electrophoresis was increased by350times, the detection limit is39nmol/L.Chapter five:A method of moving reaction boundary enrichment with capillary electrophoresis was established for enrichment phenylalanine and tryptophan. The optimal conditions of enrichment was that:50mmol/L, pH2.3formic acid for the background buffer,50mmol/L, pH9.8ammonium formate-ammonia for the sample buffer, pressure injection50mbar, the injection time140s, voltage15KV. the detection limit of Tryptophan and phenylalanine was16nmol/L and64nmol/L, respectively. The sensitivity compared with zone electrophoresis was increased by144times and83times, respectively. This method can be used for urine of cancer patients adding standard and can eliminate other substances in urine. Metabolites with disease may contain some typical amino acids or other zwitterion, but it would be difficult for the qualitative of samples. So the next research would be the use of CE-MS with mobile Reaction boundary method to detect some of the characteristic amino acids and other components in human metabolites.