Study On New Methods For Separation And Determination Of Biological Active Substances

The separation and determination of biological active substances have become more and more important in modern analytical chemistry,which has widely been used in food safety,environmental monitoring,clinical diagnosis,pharmaceutical analysis,medicine sanitation,quality control and other fields.One or several components in a complex system should be separated and analyzed in these fields,such as the quantity determination of some effect components in Chinese medicine or detection of biomarks for some diseases in clinical diagnosis or the determination of melamine in milk in food analysis.Thus,a simple,fast,sensitive and low cost method for separation and determination shows great importance.Based on separation and determination of active substances in biological samples, this work focuses on development of new methods showed as follows.1.Simultaneous determination of tryptophan and kynurenine in plasma samples by high-performance liquid chromatography with programmed wavelength ultraviolet detection A simple,fast,sensitive and specific high-performance liquid chromatography(HPLC) method is developed for simultaneous determination of kynurenine(Kyn) and tryptophan(Trp) with ultraviolet (UV) detection setting programmed wavelength.The separation was carried out on an Agilent Hypersil ODS column(125 mm×4.0 mm,5μm) in less than 6 min and the eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 to 4 min for Kyn,and at 278 nm from 4 min to 6 min for Trp in a single run with UV detector.The linearities of the method were from 0.269 to 21.20μmol/L for Kyn and 3.35 to 678.0μmol/L for Trp,and the detection limits were 0.028μmol/L for Kyn and 0.053μmol/L for Trp,respectively.Satisfactory precisions and recoveries were obtained by this method.The assay was employed to analyze plasma samples of children patients with Kawasaki disease(KD), allergic purpura(AP),nephrotic syndrome(NS) and nephritis.Compared with the control group,KD group showed great difference while other groups showed little difference.2.Functional paramagnetic beads applied to the detection of tumor markerAn electrochemical immunoassay method using a novel magnetic field fixation of biological substance by paramagnetic beads combined with sreen-printed carbon electrode was proposed for the separation and detection of tumor marker using CA199 as a model analyte.The immunosensor was prepared with streptavidin immobilized epoxysilane modified paramagnetic beads,and the biotinlated antibody was connected to the surface of the paramagnetic beads though the reaction of streptavidin and biotin,and then after the off-line incubation the sample and HRP-anti-CA199 were added.Thus,the HRP-anti-CA199 was connected to the surface of the paramagnetic beads successfully,which was then added to the surface of the work electrode of the screen-printed carbon electrode.The electrochemical detection was performed after adding the mixture of o-phenylenediamine(OPD) and H₂O₂ as substrate under the magnetic field.The electrochemical response signal was proportional to the concentration of CA199 in the range of 5.0～120 U/mL.No reagent for immobilization was needed in this method,and it also has the characteristic of simple,sensitive and low cost.3.Microfluidics applied to immunoassay by electrochemical detectionIn this research,a microfluidic immunoassay method was developed by performing an immunoreaction in a microchannel and integrating the screen-printed electrodes in a chip for electrochemical determination of tumor markers.The convenient carry-home microchip congregates the functions of sampling,separation and detection for immunoassay in detection of tumor markers.