Development Of A High Sensitive And Selective Method For Wide-bore Electrophoresis And Its Applications In Bioanalysis

Tumor and normal cells can be separated by electrophoresis based on the different charge to mass ratio,but the commonly used diameter of capillary were 50 ?m or 75 ?m,so the difference of size between tumor and normal cells was difficult to show in capillary channels.At the same time,because of the size and its own gravity of cells,it was easy to adhere to tube walls and even cause blockage.In addition,the loading volume of capillary electrophoresis(CE)was too low to cell analysis and the Joule heat would affect the activity of cell or protein.So we applied wide-bore electrophoresis(WBE)to cell separation and cell content analysis.On the basis of the WBE technology researched by predecessors,we evaluated the refrigeration performance of the vertical device and then used the rat erythrocyte and human erythrocyte as the model sample to evaluate the analytical ability of WBE.The effects of additive concentrations,electrophoresis voltages and the buffer concentration were investigated and the optimum experimental conditions were determined by calculating the reproducibility and theoretical plate number.Finally,three kinds of acids were separated by vertical WBE successfully.Secondly,a capillary was inserted into wide-bore tube as the transport channel,so the integration device of the WBE-CE for sample separating and transporting was constructed.The polystyrene microspheres(PS,2-3 ?m)and human erythrocytes were used as the model samples.In gravity only and both gravity and electric modes,the transfer performance of the device was investigated by changing the placement angle of the device,the concentration of buffer additive and electrophoresis voltage.The WBE-CE device separated and transferred of three kinds of alkaline proteins successfully.It lays the groundwork for cell content online analysis.Finally,the feasibility of on-line cell lysis,purine nucleotide phosphatase(PNP)enzyme reaction and detection of human erythrocyte was investigated by CE,the capillary diameter we used was 200 ?m.The influence of pH and concentration of the buffer and voltage were investigated.The higher electrophoresis current was easily loss the buffer capacity and cause bubbles to form,in addition,we need more time to change buffer and clean capillary.So the optimum conditions of PNP online CE analysis were applied to WBE-CE system by setting two injection sites for RBC and substrate respectively.The substrate was injected and then reacted with PNP released by erythrocyte,the reaction complex were separated by electrophoresis and detected by UV.WBE-CE system integrated the cell sampling,lysing,online enzyme reaction,separation and detection will provide help for to cell content analysis.