| Brucellosis and Schistosomasis are two wide-disseminated and serious diseases threatening animals and human beings. The existing methods used for clinical analysis suffer from the common problems of insufficient sensitivity, requiring qualified personal, being time consuming and complicated. The development of sensitive, fast, robust, selective assay methods for the diagnosis of Schistosomasis and Brucellosis is necessary for health protection and for further medical treatments. In this thesis, the fluorometric enzyme immunosensing approaches and the electrochemical immunosensors based on different immobilization methods and different transducers have been developed for Brucellosis and Schistosomasis diagnosis. Additionally, electrochemical DNA sensors based on different immobilization method for DNA probes and hybridization indication for DNA detection are also reported in this thesis. The detail materials include the following:Part I: (1) In chapter 2, a disposable biocomposite support body based on PVC-nano-Au particle has been developed for the immobilization of Brueclla antibody. Using 8-hydroxyquinoline/Mg2+ ternary complex as a substrate for HRP and 4-aminoantipyrine as an enzymatic reaction disturbant, Brueclla melitensis antigens were determined by a "sandwich immunoassay" procedure. Owing to the introduction of 4-aminoantipyrine in the HRP-catalyzed reaction, an obvious extra-amplification was obtained with the proposed fluorometric enzyme immunosensing system, resulting in a linear detection range of 0.35-120ng/mL, with a detection limit of 0.35 ng/mL. (2) In chapter 3 and 4, renewable biocomposite support bodies derived from sol-gel and paraffin/cellulose matrixes have been used for the immobilization of Schistosomia japonicum and Brucella, respectively. The surface of matrix-biocomposite could be renewed by simply polishing. In the aid of HRP-labeled antibody and TMB, the regenerated surface of the matrix-biocomposite serves as a platform for the competitive immuno-reaction and the enzymatic reaction for the determination of SjAb and BrAb, resulting in the detection limit of 4.5ng/ml for SjAb and 2.7ng/mL for BrAb. Such materials for immobilization matrix retain the activity of theimmunoreagents well. (3) In chapter 5, a natural substrate ferulic acid which possesses an outstanding feature of relatively high stability toward H2O2 in the absence of HRP was employed in the production of fluorescence signal in the same immunosensing devices as the aforementioned in chapter 3. The concentration of Schistosomia japonicum antibody (SjAb) was evaluated by calculating the fluorescence decrease of ferulic acid solution induced by HRP-SjAb, resulting in a linear detection range of 45-150ng/ml, with a detection limit of 45ng/ml.Part II: In chapter 6, a renewable amperometric immunosensor has been developed for the determination of Brucella melitensis antibody (BrAb) in serum samples. Attributing to the sol-gel technique for the encapsulation of Brucella melitensis antigen, the activity of immobilized antigen obtained a great retention. The surface of sensors can be renewed by simply polishing. Using BrAb labeled with HRP and o-aminophenol as a substrate, amperometric detection at -150 mV (vs.SCE) results in a linear detection range of 3.5-200 ng/ml, with a detection limit of 3.5 g/ml. In chapter 7,a sol-gel matrix based amperometric immunosensor has been constructed by encapsulating newcastle virus. In the use of NcAb-HRP conjugate and TMB, the newcastle antibody (NcAb) in serum samples could be determined using amperometrc finish at 100 mV, resulting in a linear detection range of 11.1-190 g/ml, with a detection limit of 11.1 g/ml. In such an approach, the activity of the entrapped immunocomposite could well be retained and the matrix-immunocomposite layer could be regenerated by simply polishing.Part III: In chapter 8, a specific sequence DNA sensor based on the covalent immobilization of DNA probes via chitosan incorporated in carbon paste electrode and an indicator, neutral red was dev...
|
PDF Full Text Request