Capillary Electrophoresis With Electrochemical Detection Of Red Tide Toxins And Analysis Of DNA Mismatch

In this work, capillary electrophoresis (CE) electrochemical methods and enzyme-linked immunoassay were combined. It took HRP as the marker enzyme, and selected a hydrogen donor of high sensitivity as the substrate. Enzyme-catalyzed reaction products were separated by capillary electrophoresis with a micro-electrode amperometric detection. This work achieved the rapid on-line detection of three redtide toxins including the paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP). The detection time was reduced from the routine immunization of more than 1 d to a few minutes. Moreover, it was easy to operate on, had less reagent consumption, and could meet the on-sense testing requirements. Through the metal ions of Hg2+ and Ag+, we respectively could combine the two DNA strands in the C and T nucleotide bases. We used the ferrocene and Tris-HCl solution for the system to achieve a single base mismatch identificationin in DNA and investigate the electrophoresis separation effects of different mismatch sites. The paper is divided into six chapters.The first chapter is an introduction. It introduced briefly the situation and prospects of capillary electrophoresis technology, CE Electrochemical enzyme-catalyzed study and the progress of red tide toxins, and explained the significance of the topic and the main content.In ChapterⅡ, horseradish peroxidase (HRP) was detected by capillary electrophoresis electrochemical enzyme-linked immunoassay. Through HRP catalyzing, H2O2 was oxidated from o-aminophenol (OAP) to 3-amino phenoxazine (AP). Then AP occurred redox reactions in certain conditions. We could detect the content of HRP through the indirect detection of enzymatic reaction products AP by electrochemical detection. Optimal detection conditions of HRP were optimized to finalize the separation voltage was 15 kv, the injection pressure and injection time were 10 kv and 6 s, and the buffer solution is a concentration of 1.0×10-2 mol·L-1 with pH=5.0 BR solution. In this state, the detection limit of free HRP was 1.09×10-12 mol·L-1 (S/N=3), and the linear range is 2.3×10-12-3.5×10-10mol·L-1.In ChapterⅢ, the paralytic shellfish poisoning (PSP) and diarrheic shellfish poisoning (DSP) were separated and detected through a competitive mode. In experiment, series of different concentrations of PSP standard antigens or the actual samples (Ag) respectively and quantification of anti-OA antibodies, enzyme-labeled antigen Ag* were added in micro-enrichment tubes to incubate. The enzyme-labeled antigen Ag* and the standards or actual samples competed with the limited antibodies, then were injected into the separation capillary. The excess Ag* and the immune complex [Ab-Ag*] in order reached the enzymatic reaction pond and catalyzed H2O2 oxidating substrate reaction. It would be detected in the Pt electrode with two electrophoretic peaks detected.In the method, the linear range of PSP was 0.8～16.0 pg/mL, and the detection limit was 0.32 pg/mL,5 times lower than spectrophotometric enzyme-linked immunosorbent assay (ELISA); The linear range of DSP was 1.0～50.0 pg/mL, the detection limit was 0.40 pg/mL. We took the method to detect the samples infected shellfish toxins and compared with the ELISA method, and the correlation was good.In ChapterⅣ, we took non-competitive mode. HRP labeled amnesic shellfish poison(ASP) antibody (Ab) was in excess. The sample solution was incubated with both HRP-labeled the amnesic shellfish toxin antibodies-antigen conjugates [Ag-Ab*] and unreacted amnesic shellfish toxin antibodies (Ab*). The amnesic shellfish toxin-HRP enzyme complex of the mixture and the remaining amnesic shellfish toxin antibodies in accordance with the different migration rates were individed in separation capillary into different zones, with sequentially into the reaction capillary. They were separated by capillary electrophoresis, then respectively in the reaction capillary occurred catalytic oxidation of H2O2 substrate o-aminophenol to the 3-Amino-phenoxazine with the electrochemical activity detected by electrochemical detection cell. Two electrophoretic peaks could be detected. Owing to the differences of Amnesic shellfish toxin concentrations, the binding amount of amnesic shellfish toxin antigen and antibody was different, so catalytic H2O2 oxidation products of o-aminophenol to 3-amino-phenoxazine concentration is different, would product different eletrochemical signals. We could have quantitative analysis in amnesic shellfish poisoning enzyme-antibody complex and the amnesic shellfish poisoning in shellish samples.Amnesic shellfish poisoning (ASP) was separated by non-competitive mode. In the method of ASP determination, the linear range was 5.0～500.0 pg/mL, and the detection limit was 2.0 pg/mL.In Chapter V, we combined CE and electrochemical analysis, to achieve a single base mismatch DNA recognition, and investigated the influence of different mismatch sites electrophoresis separation. Through the metal ions Hg2+ and Ag+ respectively, double-stranded DNA could combine the two T bases and C bases. When the Hg2+ was present,it could form a T-Hg2+-T interaction; when Ag+ was present, it could forme a C-Ag+-C interaction, to achieve single-base T or C mismatch in DNA testing. We took ferrocene as a marker, and marked the ssDNA probe with target DNA hybridization, then produced the double-stranded DNA (dsDNA). Through the electrochemical property of ferrocene we could detected the mismatch DNA.In Chapter VI, we combined capillary electrophoresis and electrochemiluminescence, and discussed the Ru(bpy)32+ CE-ECL technique for the detection of DNA mismatch. We took Ru(bpy)2 (dcbpy)NHS as a marker, and marked the ssDNA probe with target DNA hybridization, then produced the double-stranded DNA (dsDNA). Through experiments, we using 0.02 mol·L-1 with pH=7.0 PBS (containing tripropylamine of 0.02 mol·L-1 concentration) as buffer,8 kv as the separation voltage,10 kv and 10 s as injection voltage and injection time,1.1 v as the detection potential, and 900 v as the photomultiplier tube high pressure. If it took a well dark conditions, DNA could be separated and detected better. In the detection of single base mismatch ssDNA, the linear range was 4.0×10-8-3.0×10-6 mol·L-1, and the detection limit was 1×10-8 mol·L-1.