| It is important and valuable to develop highly sensitive assay in the immuno-analytical field. Amperometric immunosensor, combined electroanalytical chemistry with immunologic methods, is an important analytical method which possessed good selectivity, high sensitivity, fast, simple operation, the versatility of construction method, and so on. In the fabrication process of immunosensor, the sensing platform construction for the immobilization of biomolecules firmly with activity kept well and the design of bioconjugate nano-probe for signal amplification are two of the most important point. With the rapid development of nano-technique, all kinds of nano-matericals have been used in the field of biosensor due to their unique electricity property, large surface-to-volume area, highly surface free energy, good biocompatible, and so on. This dissertation focuses on the construction of nano-active sensing interface for the immobilization of antibody and the design bioconjuate nano-probes using nano-material as carrier of enzyme and antibody for the analyte recognization and signal amplification, and were further used to prepare amperometric immunosensor. The research details are described as follows:1 Multilayer structured amperometric immunosensor based on gold nanoparticles and Prussian blue nanoparticles/nanocomposite functionalized interfaceIn this paper, a novel strategy for the fabrication of sensitive reagentless amperometric immunosensor was proposed. Firstly, Prussian blue nanoparticles (PBNPs) as redox probe were immobilized on three dimensional structured membrane of the gold colloidal nanoparticles (AuNPs) doped chitosan-multiwall carbon nanotubes (CS-MWNTs) homogeneous composite (CS-MWNTs-AuNPs) by electrostatic interactions between the negatively charged PBNPs and the positively charged amino groups of CS and strong binding interaction between GNPs and nitrile group (-CN) of PBNPs. Subsequently, the gold nanoparticles (GNPs) was electrodeposited on the surface of the composite by electrochemical reduction of gold chloride tetrahydrate (HAuCl4) to immobilize antibody biomolecules (anti-CEA) and avoid the leakage of PBNPs. The stepwise assembly process was characterized by means of cyclic voltammetry (CV) and electrochemical impendance spectroscopy (EIS). Furthermore, the morphology of the prepared nanomaterials was researched by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Under the optimized conditions, the decrease of CVs current of determination CEA was proportional to concentration ranges from 0.3 to 120 ng·mL-1 with a detection limit of 0.1 ng·mL-1 at a signal-to-noise of 3. Moreover, the proposed immunosensor exhibited good accuracy, high sensitivity and stability.2 Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor markerA novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Aushell@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab2) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Aushell@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Aushell@GOD were performed by one-pot assembly of Fc@Ab2 and GOD on the surface of Aushell@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng·mL-1 with the detection limit down to 6.7 pg·mL-1. The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease.3 Horseradish peroxidase-functionalized Pt hollow nanospheres and multiple redox probes as trace labels for sensitive simultaneous multianalyte electrochemical immunoassayIn this work, a novel strategy for the fabrication of sensitive simultaneous multianalyte amperometric immunosensor was proposed for the first time based on the use of different redox probes as tracers to label the different signal antibodies. Each biorecognition event yielded a distinct voltammetric peak, which position and size reflected the identify and level, respectively, of the corresponding antigen. In the meanwhile, the multianalyte simultaneous detection capability was coupled to the amplification feature of Pt hollow nanospheres and HRP in the tracers, which both could amplify the detectable signal for the sandwich-type immunoassay by their synergistic action for the reduction of H2O2. This concept was demonstrated for simultaneous immunoassay of AFP and CEA protein in connection with thionine and ferrocene-monocarboxylic, respectively. The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values, which could provide a promising sensitive simultaneous multianalyte immunoassay approach for protein diagnostics and biosecurity. Although the present assay system was focused on the determination of target antigen molecules (AFP and CEA), it could be easily extented to the measurement of other antigens or biocompounds. Moreover, the proposed method didn't require sophisticated fabrication and provided a promising potential in clinical application.
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