| In the chapter one of the thesis, first of all, the principle of immunoassay was introduced simply. Then capillary electrophoretic immunoassay (CEIA) was presented. It included that the formats of CEIA such as non-competitive, competitive and on-line immunoassay. The detection methods of CEIA including UV, LIF, particle counting detection and electrochemical detection and applications of CEIA for detection of proteins, drugs, hormones, viruses and other complex biological samples were reviewed. In the chapter two, a method of capillary zone electrophoresis with catalysis-electrochemical detection was developed and applied to determine horseradish peroxidase (HRP). This method combined an on-line enzyme catalysis reactor with capillary electrophoresis. The on-line enzyme catalysis reactor with a separation capillary and a reaction capillary was designed. Isoenzymes of HRP was separated by capillary zone electrophoresis, and then catalyzed the enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB(Red)) and H2O2 in the reaction capillary. The reaction product, TMB(Ox), could be determined at the outlet of the reaction of the reaction capillary. The optimum conditions of the method are 1.4×10-2 mol/L H3BO3-3.8×10-4 mol/L Na2B4O7 (pH 7.4) for the run buffer, 1.0×10-2 mol/L Na2HPO4-5.0×10-3 mol/L C6H6O7 (pH 5.0) for the substrate solution, 2.0×10-4 mol/L for the concentration of TMB(Red), 2.0×10-3 mol/L for the concentration of H2O2, 40 cm for the liquid pressure height, 20 kV for the separation voltage, 0.10 V (vs. SCE) for the detection potential. Three isoenzymes of HRP were separated and detected. Their linear ranges were 2.4×10-112.4×10-8, 4.8×10-102.4×10-8 and 4.8×10-112.4×10-8 mol/L respectively. Their limits of detection (LODs) were 4.8×10-12, 7.3×10-11 and 1.6×10-11 mol/L (or 0.048, 0.72 and 0.16 amol), respectively. The response for a series of ten injections of 1.2×10-9 mol/L HRP resulted in a relative standard deviations of 2.12.4% for the detected electric charges, 2.93.2% for the migration times. Using this method, a commercial HRP was measured. The result agreed with the value given by the specification. In the chapter three, capillary electrophortic enzyme immunoassay with electrochemical detection (CE-EIA-ED) was developed and applied to detect thyroxine 4 (T4). To our knowledge, this is the first report for CE-EIA-ED. In the method, T4 competed with HRP labeled-T4 (T4*) for a fixed number of antibody (Ab) binding sites. Free T4* and bound complex (Ab-T4*) in the solution were separated by capillary zone electrophoresis (CZE). They could catalyze the reaction of enzyme substrates (TMB(Red) and H2O2) in the reaction capillary. The reaction product TMB(Ox) was determined using amperometric detection at the outlet of the reaction capillary. Since the concetration of TMB(Ox) was much higher than that of T4* due to the enzyme application, the LOD of the method should be very low. The detected electric charge of T4* was used for quantitation in the assay. The linear range was 0.550 μg/L and the LOD of T4 is 3.8×10-9 mol/L (or 23 amol). The concentration of T4 in two controls were determined. The results agreed with the stated values. In the chapter four, tumor marker CA15-3 (Ag) was detected by CE-EIA-ED. In the experiment, the immunoassay protocol was a non-competitive format. An excess amount of HRP labeled monoclonal antibody (Ab*) was added to the system to ensure the completion of the immuno-reaction. Ab* and Ag-Ab* were separated by CZE in the separation capillary, both catalyzed the reaction of TMB(Red) and H2O2 in the reaction capillary. Their product TMB(Ox) was detected by using amperometric defection. The electrophoretic peak corresponding to Ag-Ab* was used for quantitation of CA15-3. The CE-EIA-ED offered several advantages such as simply operation, high selectivity, low reagent consumption and short incubation time over other conventional immunoassays. The linear range of the method for T4 was from 0.20 U/mL to 41.7 U/mL. The concentration LOD (3σ) of CA15-3 is as low as 0.024 U/mL (or 1.3×10-7 U) dueto the enzyme amplification, which was about one order of maguitude lower than radioimmunoassay. The response for a series of eight injections of 20.8 U/mL CA15-3 resulted in a relative standard deviation of 4.4% for the migration time, tm and 6.3% for the electric charge detected, q respectively. The detected results of two breast cancer serum samples agreed with the values determined by ELISA. The recovery of the method is between 95 and 104%. In the chapter five, tumor marker CA125 (Ag) was detected by capillary electrophoretic enzyme immunoassay with electrochemical detection. The immunoassay protocol was a non-competitive format and the signal of Ag-Ab* was used for quantitation in the assay. Like chapter four, CE-EIA-ED overcomes the shortcomings of conventional immunoassay such as complicated operation, time waste and high sample consumption. The linear range was between 1.67 and 83.3 U/mL and the concentration LOD (3σ) of CA125 was 0.29 U/mL (or 1.6×10-6 U). The response for a series of six injections of 6.67 U/mL CA125 resulted in a relative standard deviation of 4.8% for tm and 3.9% for q. Two serum samples were detected and the results were consistent with the values detected by ELISA. The recovery of method for CA125 was between from 93 and 106%. In the chapter six, we developed an analysis technique of individual cells for surface antigent. Using this technique, the continuous injection of intact cells, which had the antigent-antibody complex with labeled enzyme on the surface was performed through an introduction capillary. In the capillary, the labeled enzyme catalyzed the reaction of the enzyme substrate. The reaction product was then detected at the outlet of the capillary with amperometric detection. The technique has been used for the determined of tumor marker CA15-3 on the surface of individual breast cancer cells.
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