Study And Application Of Capillary-based Enzyme Assay And Immunoassay

Due to its unique advantages,such as short diffusion distances,low reagent consumption,easy to package,capillary tubes have been widely applied in a variety of research fields of analytical chemistry.Focusing on the fused silica capillary tubes in enzyme on-line continues monitoring and fabrication of immunosensor,the present paper carried out the following research work:1.We developed an enzyme analysis system for on-line enzyme reaction process based on capillary electrophoresis(CE).We fabricated a simple and easy-to-operate sequential CE analysis system,which was constructed by coaxially aligning two capillaries through a sample vial with a distance of 15?m between the capillary ends.Continuous sample introduction and CE analysis were easily achieved by an extra positive voltages that were applied in the sample vial.With combination of rapid on-line derivatization and CE technique,the activity of urease was studied by continuously measuring the amount of the product(ammonia)from the beginning to the end of the enzymatic reaction.In the process of the enzyme reaction,only ammonia can be derivatized and detected at 340 nm.The slope from linear fitting of the electrophorograms is used to calculate the initial reaction rate of urease.The resulting value of Km and Vmax is 2.79 ± 0.15 mM and 4.32 ± 0.20 mM/min(n = 3),respectively.The results are in good agreement with those obtained using off-line enzyme assay,demonstrates the feasibility and reliability of the present continuous on-line enzyme analysis.The inhibition constant and inhibition mechanism of Cu2+ were investigated using the present method.According to the change of Km and Vmax,indicating that Cu2+ is a noncompetitive inhibitor on urease.The Ki value of the inhibitor is determined to be 0.34 ± 0.02 ?M(n = 3).This study could be of potential application in controlling urease activity in therapies of diseases induced by ureolytic bacteria and in agriculture for proper nitrogen management in soils.2.We present sequential CE analysis of the whole process of enzyme reaction by combing optically gated(OG)injection and commercial-available UV-Vis detection.OG injection has been proved to be a fast,serial,and reproducible sample injection method.Laser with corresponding wavelength was applied for photobleaching the sample.The UV absorption of the photobleached plug decreases in comparison to the background,resulting in a negative peak in the electropherogram.Several experimental conditions including laser power,injection time,voltage,derivate reagent were investigated.With the application of the OG-UV/vis CE analysis,L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction.The Km and Vmax values were 0.25 mM and 0.24 mM/min,respectively,which are in good agreement with the results of traditional off-line enzyme assays,indicating the feasibility and reliability of integrating the OG injection with UV/vis detection.Our study indicates that the OG-UV/vis method could be of potential value for online monitoring various chemical reaction and bioprocesses.3.We have developed a multichannel microstructure capillary-based immunosensor and studied its performance.Capture antibody was immobilized to the inner surface of capillary by covalent bonds.Fluorescence was measured by a LIF after the sandwich type immunoreactions.Compared to convention capillary,the multichannel microstructure capillary has large area for molecule recognition and immobilization,resulting a 5-fold improvement in sensitivity.In addition,we optimized the concentration of the blocking reagent and capture antibody.Under the optimized conditions,the intra and inter-assay coefficients of variation of the immunosensor obtained from three replicates were 4.43% and 7.14%,respectively.These results reveal the acceptable reproducibility of the proposed immunoassay.4.Multichannel microstructure capillary-based immunosensor for detection of tumor marker for hepatocellular carcinoma-alpha fetoprotein(AFP).The calibration curve of AFP,the liner range and limit of detection were compared with the results of the conventional capillary-based immunosensor and enzyme-linked immunosorbent assay(ELISA).The strong anti-interference ability of the immunosensor demonstrates the suitability in the analysis of complex samples.The developed method was used to detect AFP in spiked serum samples and real serum samples collected from Beijing Friendship Hospital.The recovery ranged from 84.3% to 119.0% in the spiked samples,which shows that the PCF-based immunosensor has acceptable accuracy in detecting AFP in spiked samples.The results were compared with those measured in the clinical laboratory,the relative errors of are less than 20.73%,which meet the accuracy requirements for real sample analysis.We further show a scatter diagram of the correlation of the two methods,the best-fit regression equation is y=0.888x+0.056.The immunosensor is expected to applied in the detection of the other tumor marker,have potential application in clinical analysis.