| Homogeneous immunoassays do not require separation of bound from free labeled antigens (antibodies)or free labeled antibodies(antigens)after immunological reaction.This technique has some merits including simple and timesaving because of the analysis in homogeneous solutions. Capillary electrophoresis(CE)combined with immunoassay(IA)is characterized by high resolution and less sample,and will become a powerful tool in clinical diagnosis,environmental and food analyses.Some problems such as selectivity and sensitivity in homogeneous immunoassays have been improved by CE-IA coupled with chemiluminescence(CL)detection.In addition,CE-IA method based on CL is low cost,high sensitivity,good reproducibility and easily to achieve automatic analysis.Therefore,this joint technique has more potential in homogeneous immunoassays.CE is developed as a kind of separation technology since 1980s.CE is used for drug analysis extensively due to its higher separating capability,shorter migration time than other separating machines and the development of joint techniques with different detection.CE has already exerted a significant influence on drug metabolism in vivo,pharmacokinetic study,pharmaceuticals quality control and the study of pharmaceuticals target,especially in treating nanolitre samples and single cell analysis.It is very significant to investigate the pharmaceuticals absorbability,distribution, metabolism,transformation and decreasing the side effect,imprving the molecular structure as well as promoting the positive effect by CE.CL detection is very suitable to employment in high performance liqiud chromatography(HPLC) and CE,while the main problem is dilution effect behind chromatographic column.In general,the signal peak could become broad as a result of the large dead volume in flow cell and have an effect on the resolution.This is the one of main reasons that commercial instruments have not been still come out in CE analysis.In this study,a simple and micro reactor was designed so extraordinary that it can obtain the most luminescent intensity as soon as the CL reaction was carried out.Moreover, there wasn't any dead volume and dilution effect.The new assemble technique based on CE coupled with CL detection has been successfully applied in determination of luteinizing hormone, follicle-stimulating hormone,hepatitis B surface antigen and antibody in serum samples at nanolitre grade,and expanded the fields of homogeneous immunoassays.Furthermore,this thesis has developed the new technique in CE-CL detection and established new methods in determination of levodapa and its metabolite in human blood,fluoroquinolones in urine,clenbuterol,salbutamol and diethylstibestrol in porcine urine,promethazine and chlorpromazine in human serum.1.CE immunoassay based on CL detectionIn the first part of the dissertation,the research work was made up of three sections of immunoassay based on different immunoassay modes and different chemilminescence enhancers. These different methods have been successfully applied to quantitative analysis of virus and hormones in human serum.(1)A sensitive immunoassay for determination of hepatitis B surface antigen and antibody in human serum using capillary electrophoresis with chemiluminescence detectionA sensitive and homogeneous immunoassay(IA)based on capillary electrophoresis(CE)with enhanced chemiluminescence(CL)detection has been developed for the determination of hepatitis B surface antigen(HBsAg)and antibody(HBsAb)in human serum.The conditions for the CL reaction and electrophoresis were investigated in detail using horseradish peroxidase(HRP)labeled HBsAg(HBsAg*)as a marker because of its catalytic effects on the luminol-hydrogen peroxide reaction.The CL reaction was enhanced by para-iodophenol and the CL detector was designed uniquely without any dead volume or diluents effect.The present method has been used for assaying HBsAg and HBsAb in human serum using a competitive format and a noncompetitive format,respectively.Under the optimal conditions,the linear ranges were from 1 to 400 pmol/L (R=0.9988)for HBsAg and 2 to 200 mIU/mL(R=0.9981)for HBsAb.The detection limits were 0.4 pmol/L and 1 mIU/mL for HBsAg and HBsAb,respectively.The relative standard deviations of peak area were 4.2%and the errors of it were from - 0.03%to + 0.05%for 80 pmol/L HBsAg* (n=7).In this study,the free HBsAg* and the bound HBsAg*(HBsAg*- HBsAb)were separated in the separation capillary within 6 min using a borate run buffer.To verify the experimental reliability, the result was comparable with that of enzyme linked immunosorbent assay(ELISA)and demonstrated the feasibility of the CE-CL immunoassay method for clinical diagnosis.(2)Noncompetitive immunoassay for luteinizing hormone in human serum using capillary electrophoresis with chemiluminescence detectionA noncompetitive immunoassay based on capillary electrophoresis(CE)with chemiluminescence(CL)detection has been developed for the determination of luteinizing hormone (LH)in human serum.The work involved the development of separation and CL conditions allowing for routine analysis of serum samples.In this study,horseradish peroxidase(HRP)labeled monoclonal anti-LH can catalyze the luminol-hydrogen peroxide reaction.The determined LH can react with excessive amount of HRP labeled anti-LH.Within 14 minutes,free enzyme conjugate and immune complex could be separated in alkaline borate buffer by high voltage of 15 kV.To improve the sensitivity,a series of measures were adopted including the choice of para-iodophenol as a CL enhancer,unique design in detect window.Under the optimal conditions,calibration curve for LH was established in the concentration range of 1-200 mIU/mL and the detection limit was 0.08 mIU/mL.Compared with ELISA,this method decreased the detection limit for about 12 times and it has been successfully employed on determination of the LH in human serum.(3)Analysis of follicle-stimulating hormone by CE with chemiluminescence detection based on competitive immunoassayA competitive immunoassay for determination of FSH in human serum was established by capillary electrophoresis with enhanced chemiluminescence detection.The method is based on the competitive immunochemical reaction between the FSH and HRP- labeled FSH with the anti-FSH, CE separation of the antibody bound and free HRP- labeled FSH,followed by the chemiluminescence detection.The detection limit is governed by the stability of the immune complex,which related to analytical time and the design of detector,and the choice of the chemiluminescence enhancer,which dominated the chemiluminescence intensity.Therefore,a unique chemiluminescence detector without any dead volume or diluents effect was employed and sodium tetraphenylboron was selected as the optimal enhancer,thus the sensitivity was effectively improved.Within 15 minutes,free HRP-labeted FSH and the immune complex could be separated in alkaline borate buffer by high voltage of 15 kV.Under the optimal conditions,calibration curve for FSH was established in the concentration range of 0-100 mIU/mL and the detection limit was 0.06 mIU/mL.Compared with ELISA,this system enhanced the concentration sensitivity more than 30 times.2.The study of CE pharmaceuticals analysisIn the second part of the dissertation,the research work was divided into four sections of pharmaceuticals analysis based on laser-induced fluorescence(LIF)detection and CL detection methods.In CE-CL system,different CL reactions were employed in determination of fluoroquinolones,feed additive and thiophenylamine in biological fluids.The major contents in second part are described as follows:(1)Simultaneous determination of levodopa and its metabolite in human blood by capillary electrophoresis with laser-induced fluorescence detectionA rapid,sensitive and reproducible method is described for the analysis of levodopa and its metabolite dopamine(DA)in human blood.The influence of carbidopa as the inhibitor againist the decarboxylase activity on the metabolism has been also studied.After derivatization in a dark pulsator for 12 h at room temperature,the fluorescein isothiocyanate(FITC)derivative of levodopa and other components were separated by capillary zone electrophoresis(CZE)within 13 min and detected with laser-induced fluorescence(LIF).Under the optimum analysis conditions,the linear range is 3.0×10-8- 4.0×10-6mol/L and 1.0×10-8-2.0×10-6mol/L for levodopa and DA,respectively. The detection limits of levodopa and DA were 7.8×10-9mol/L(39.0 amol)and 3.1×10-9mol/L(15.5 amol),respectively.The method was successfully applied to monitoring the levodopa and DA in human blood after one took tablets orally.(2)Simultaneous determination of salbutamol,clenbuterol and diethylstilbestrol by capillary electrophoresis with chemiluminescence detectionThis paper describes the separation and quantification of salbutamol(SBA),clenbuterol(CLB) and diethylstilbestrol(DES)in porcine urine by capillary electrophoresis with chemiluminescence detection.The method was based on its enhanced sensitivity effect on a luminol-potassium hexacyanoferrate(Ⅲ)(K3[Fe(CN)6])CL reaction.Three pharmaceuticals were well separated by capillary zone electrophoresis(CZE)utilizing 50mmol/L Na2HPO4/NaH2PO4 phosphate buffer(pH 6.5)as running electrolyte within 350 s.The results obtained include a detection limit from 0.8x10-9mol/L to 2.0×10-9mol/L and good repeatability(R.S.D.= 2.8%for n = 7 for a 5.0×10-8 mol/LSBA solution).The proposed method has been successfully applied to the determination of SBA,CLB and DES in porcine urine,the reliability of the assay method was established by parallel determination and by standard-addition method(recoveries = 96%-103%).(3)Effective separation and simultaneous determination of three fluoroquinolones by capillary electrophoresis with chemiluminescence detectorA rapid capillary electrophoresis-chemiluminescence method is proposed for the simultaneous determination of fluoroquinolones such as ofloxacin(OF),norfloxacin(NF)and ciprofloxacin(CPF) in human urine samples.The method is based on the sensitizing effect of the compounds on the chemiluminescence oxidation of cerium(Ⅳ)and sodium sulfite in acidic medium.In the optimum conditions,the results obtained include a linear dynamic range of OF,NF and CPF were 2.0×10-8-6.0 x10-6g/mL,2.0×10-8- 4.0×10-6g/mL and 5.0×10-8- 5.0×10-6g/mL.The detection limits for them were 3.3×10-9g/mL,4.5×10-9g/mL and 5.8×10-9g/mL,respectivelly.The R.S.D.for eleven parallel measurements of 2.0×10-7g/mL OF was 3.1%.The proposed method has been successfully applied to the determination of OF,NF and CPF in man-made and natrual urine samples.(4)Simultaneous determination of chlorpromazine and promethazine using capillary electrophoresis with cerium(Ⅳ)-rhodamine 6G chemiluminescence detectionA flow-injection analytical method by capillary electrophoresis with chemiluminescence detection for the simultaneous determination of chlorpromazine and promethazine is presented.The method is based on the chemiluminescence reaction of chlorpromazine and promethazine with cerium(Ⅳ)in nitric acid,sensitized by the fluorescent dye rhodamine 6G.The mutual internal standard method was employed in this work due to the similar molecular weight and structure.The proposed procedure allows quantitation of chlorpromazine and promethazine in the concentrationrange of 4.0×10-8-2.0×10-6g/mL and 2.0×10-8-2.0×10-6g/mL,with a detection limit of 5.6 ng/mL and 3.4 ng/mL,respectivelly.An RSD of 2.8%(n = 9)at 2.0×10-6g/mL for promethazine. The method was successfully applied to the simultaneous determination of Chlorpromazine and promethazine in human serum. |
PDF Full Text Request