Study On Analytical Methods With High Selectivity In Biological And Environmental Samples

The composition in real words samples, such as biological and environmental samples, is relatively complicated. The interference of matrix to target compound is serious when directly analyzed, so analytical methods with strong selectivity are recommended in real word sample analysis. Using methods with high specificity to integrate sample separation and preconcentration is the key step to solve this problem. Solid phase extraction (SPE) and solid phase microextraction (SPME) are sample pre-treatment technologies developing rapidly these years. They have many advantages over traditional methods. Development of different high specific adsorption materials for different target compounds is the hot spot nowadays. In this research, four important compounds in biological and environmental samples, including antibiotic sulfamethoxazole (SMX), glucoside, bovine serum albumin (BSA) and bovine serum fibrinogen (BFg), are chosen as the target compounds. In consideration of special characteristics of each compound, specific SPE stationary or SPME fiber coating is developed. Combining with such new analytical methods as molecular imprinting, immunoassay and interaction between enzyme and its specific inhibitor, a series of new analytical methods with high selectivity is established. There are five chapters in this dissertation and the content of each is as follows:Chapter 1:The basic concepts of SPE and SPME are briefly described. Develo-pment of research in extraction and adsorption materials is reviewed. Then, some new analytical methods with high selectivity, including molecularly imprinting, immunoassay and specific interaction between enzyme and its inhibitor, are introduced. At last, how to bring forward the research idea and main contents of the research are introduced.Chapter 2:A new indirect inhibitive immunoassay using surface plasmon resonance (SPR) coupled with molecularly imprinting technology for SMX.A SMX-MIP coating capillary was produced. The preparation conditions, such as ratio of monomer to imprinting molecule, ratio of monomer to cross-linker, polymerization time and so on, were optimized. The scanning electron microscope (SEM) image illustrated that the thickness of the coating was 198nm. The capillary was connected to a biosensor BIAcore3000 and used as an in-tube SPE and on-line preconcentration device before immunoassay. A SMX sensor chip was produced and used to detect the concentration of uninhibited anti-SMX monoclonal antibodies (MAbs). The anti-SMX MAbs was inhibited by SMX extracted by the MIP coating and the detected concentration by the sensor chip decreased accordingly. The reduced MAbs concentration was linear with extracted SMX amount. The calibration curve was generated by linear fit over the range of 0.04-10 ng/mL. The limit of detection was 0.01 ng/mL. This method was applied to the detection of low concentration samples successfully. It is easy to be operated, and has high sensitivity and automatization.Chapter 3:A new protein imprinting method of imprinting BSA on the surface of CdS quantum dots.Protein imprinting is a new method for specific recognition of target proteins. For BSA, surface imprinting is most favorable. But the adsorption capacity is not good enough. Semiconductor nanocrystals, also called quantum dots (QDs), are new nanoscale materials. They have large specific surface area and many surface deficiencies, which is in favor of adsorption. In this study, a BSA surface imprinting method was developed by the incorporation of water soluble L-cysteine-QDs into molecularly imprinted polymers, which can offer shape selectivity. Preparation and adsorption conditions were optimized. Size of the QDs and QDs-MIP particles was nanoscale illustrated by SEM images. Photoluminescence emission of CdS was quenched when rebinding of the template. The quenching of photoluminescence emissions is presumably due to the fluorescence resonance energy transfer between quantum dots and template molecule BSA. The adsorption is compiled with Langmuir isotherm and chemical adsorption is the rate-controlling step. The maximum adsorption capacity was 142.4 mg/g larger than that of undoped BSA MIP method. This study demonstrates the validity of QDs coupled with MIP technology to analyze BSA.Chapter 4:Preparation, characterization and evaluation of new stationary phase for SPE ofβ-D-glucosides.Glucosylamidine is highly potent and selective inhibitor of glucosidase and glucosides are substrate of glucosidase catalytic reaction. So, it is presumed that there might be specific interaction between glucosylamidine and glucosides. In this study, a positively charged P-D-glucosylamidine was immobilized through a one-pot synthesis procedure involving the addition ofβ-glucosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give a SPE stationary phase for P-D-glucosides. During synthesis, infrared spectroscopy, nuclear magnetic resonance spectroscopy and ultraviolet-visible spectroscopy was used to characterize products. The synthesizedβ-D-glucosylamidine stationary phase is stable and can interact withβ-D-glucosides selectively and reversibly. The retainedβ-D-glucopyranoside could only be eluted byβ-D-glucose solution. It indicates that the binding of theβ-D-glucoside was of specific nature that corresponds to the glycon substrate specificity of theβ-D-glucoside.Chapter 5:A new SPME method of extracting BFg from bovine plasma using carbon nanotubes (CNTs) coating fiber.CNTs are a kind of novel and interesting carbon material with high hydrophobicity and large specific surface area. In this investigation, commercial polydimethylsiloxane (PDMS) fiber was coated with single-wall nanotubes (SWCNTs) and multi-wall nanotubes (MWCNTs) by organic bonding agent, to study their adsorption and extraction ability of proteins, and bovine fibrinogen (BFg) and BSA were selected as the target proteins. The adsorption conditions were optimized. The adsorbed proteins could be desorbed when changing the pH and ionic strength of the adsorption buffer. The desorption conditions were also optimized. Either SWCNTs or MWCNTs adsorbed bigger protein BFg more strongly than. BSA. They can selectively adsorb BFg in certain conditions. The fibers coated with CNTs had advantages over PDMS fiber in selectivity. It could be used to separate and purify BFg in bovine blood plasma and quantify its concentration. This method is easy to be operated and hopeful to be directly applied to in-vivo detection.