Flow Injection Chemiluminescence Analysis

This thesis includes a review and a research section.The review describes the kinds of enhanced chemiluminescence (ECL) system, enhancer and mechanism of enhanced chemiluminescence, flow-injection technology, and magnetic particle technology and automation technology in enhanced chemiluminescence immunoassay (ECLIA). The application of ECLIA in recent years is summarized. Chemiluminescence (CL) analysis is becoming increasingly important in various fields owing to its simple instrumentation, high sensitivity, wide dynamic rage, reproducibility, simplicity and rapidity. ECLIA is becoming increasingly promising method for Immunoassay, DNA probe hybridization assay, Western blotting and Southern blotting owing to the enhancer which increases the intensity of the light emission by several orders of magnitude and reduces the background light emission. This leads to a dramatic increase in the signal-to-blank ratio. The light emission is long-lived glow (>30 min).The research section involves three subsections. In the first subsection, a novel flow-injection chemiluminescence method for the determination of resveratrol glucoside has been developed based on the inhibition of the chemiluminescence of Luminol-H2O2-KIO4 in alkaline aqueous solution. The linear range of resveratrol glucoside is from 4.6×10-9 g/mL-2.0×10-6 g/mL with the detection limit of 2.0×10-9 g/mL. The relative standard derivation is 1.9% for 1.0×10-7 g/mL resveratrol glucoside (n=11). This method developed has been applied to determining resveratrol glucoside in Polygonwn Cuspidatum of Chinese traditional drugs and red wines with satisfactory results.In the second subsection, novel flow-injection chemiluminescence (CL) system for the determination of hydrogen peroxide is described, which is based on a catalytic effect of hydrogen peroxidase for a luminol-hydrogen peroxide CL reaction. A designed capillary column immobilized horseradish peroxidase has been utilized as both reactor and flow CL cell. The CL emission detected is generated from the reaction between the sample of hydrogen peroxide and luminol passed through the column under the catalysis of HRP. The calibration graph for hydrogen peroxide is linear in the rangefrom 1.0×10-8 mol/L to 1.0×10-4 mol/L, and the detection limit is 9.0×10-9 mol/L hydrogen peroxide. Interfering ions in rainwater are effectively separated on-line by an upstream ion exchanger. A complete detection process could be performed in 1 min with a relative standard deviation of 1.4% (c=1.0×10-6 mol/L, n=9). The system developed has been applied successfully to the determination of hydrogen peroxide in rainwater.In the last subsection, a capillary-column-based reactor has been designed for detection of IgG antibody. IgG antigen is covalently immobilized onto an inner wall of a column. When the sample (IgG antibody) and the HRP-labeled antibody pass through the column, a competitive binding for IgG antigen immobilized on the wall occurs, resulting the ECL signal deceases with a increase of sample concentration using luminol-hydrogen peroxide-p-iodophenol system. The effect of the blocking agent, flow rate of samples on the ECL signal is investigated. The linear range from the dilution of concentration 1:1×1011 to the dilution of concentration 1:1×1010 with a relative standard deviation of 6.5% is obtained using the system designed. The total assay time is less than twenty minutes.