| Biosensor is a modern analytical apparatus. It uses bioactive unit (such as: enzyme, antibody, nucleic acid, cell) as sensitive motif, to highly selectively detect analyte. In 1962, Clark first proposed: "add enzymes to sensitive film of chemical modified electrode, in order to implement selectively determining and analyzing objective". In recently 30 years, biosensor has gained rapid development, because of the advantages of the biosensor including high-sensitivity, fair selectivity, their inexpensive instrumentation, simple-design and easily carrying out real-time living-body detecting, and again because of it potentially extensively applying in clinical medicine, environmental control and food industry, etc. At the same time, it becomes a very active research topic. However, the immobilization of biorecognition molecule is most important and crucial factor in study and design of biosensors. Its immobilization effects directly affect performance of biosensors (such as: sensitivity, selectivity, and life-time). This paper is just based on the immobilization of biorecognition molecule and combination with inorganic nanomaterials of today's material science, to construct many biosensors. The main works are included as follows:Part I investigated the peroxide hydrogen biosensors based on nano-Au and Pt hollow nanospheres modified electrodes1. Sol-Gel is a favorable material for the immobilization of biomolecules. Gold nanoparticles is provided with large surface area and strong adsorption possibility. It can strongly adsorb big biomolecules and retain their bioactivity. Combined above both merits, we developed a peroxide biosensor. Concretely, a stable Prussian blue (PB) film was electrodeposited on platinum electrode by applying constant potential. After that, horseradish peroxidase (HRP) was entrapped to colloidal gold combined with the gelatin, which were immobilized onto surface of Prussian blue modified platinum electrode. Then the HRP-nano-Au-gelatin film was treated with glutaraldehyde (GA). Cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS) were used to characterize the electrochemical properties of the biosensor. The factors influencing the performance of the biosensor were investigated in 0.05 mmol·L-1 phosphate buffer (pH 6.0), such as applied potential, pH, temperature, interfering substances. The biosensor exhibited fine selectivity, good repeatability, high sensitivity and good stability. The linear response of the biosensor to hydrogen peroxide is in the concentration range of 1.2×10-6～1.3×10-3 mol·L-1 with a detection limit of 6.0×10-7 mol·L-1.2. Hollow material exhibits remarkably catalytic activities different from their solid counterparts with the advantages of low density, saving of material, and reduction of costs. Recently, hollow material has received wide attention. Thus, a facile, efficient, and economical route was used for synthesizing homogeneous size Pt hollow nanospheres whose microstructure has been characterized by transmission electron microscopy (TEM), and it was immobilized on the surface of bare glass carbon electrode. In phosphate buffer solution (PBS), electrochemical catalysis of modified electrode to H2O2 was studied. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry were used to characterize the electrochemical properties of modified electrode. The factors influencing the performance of the biosensor were investigated in 0.1 mol·L-1 PBS (pH 7.5), such as applied potential, pH, temperature. In addition, stability, repeatability and possibility of anti-interfering was studied. The linear response of the modified electrode to H2O2 is in the concentration range of 3.33×10-6～1.03×10-3 mol·L-1 with a detection limit of 1.09×10-6 mol·L(-1). The modified electrode was used to detect recoveries of experimental samples, the result is good.Part II investigated immunosensor based on nano-Au, nano-TiO2 and nickel hexacyanoferrates nanoparticles modified electrodes.1. Nano-Au can be deposited on the surface of TiO2 colloids, which can enhance the stability the gold nanocatalysis and improve the properties of both. Meantime, both exhibit good biocompatibility. Moreover, nano-Au can strongly adsorb biomolecules and retain their bioactivities. So, a novel reagentless amperometric immunosensor for the determination of alpha-fetoprotein (AFP) was prepared by immobilizing TiO2 colloids on Prussian blue (PB) modified platinum electrode, which yielded a positively charged interface with strong adsorption to deposit gold nanoparticles for immobilization of alpha-fetoprotein antibody (anti-AFP). The factors influencing the performance of the proposed immunosensors were studied in detail. Under optimal conditions, cyclic voltammograms (CV) determination of AFP showed a specific response in two concentration ranges from 3.0 to 30.0 ng·mL-1 and from 30.0 to 300.0 ng·mL-1 with a detection limit of 1.0 ng·mL-1 at a signal-to-noise ratio of 3. The proposed immunosensor exhibited high selectivity, good reproducibility, long-term stability (>2 months) and good repeatability.2. When sensors were constructed, addition of electroactive substance can enhance performance of the biosensor. So we developed a novel immunosensor based gold nanoparticles (nano-Au) with advantages of good biocompatibility and strong adsorption possibility, and favorable electroactive substance nickel hexacyanoferrates nanoparticles (NiHCFNPs) for determination of carcinoembryonic antigen (CEA) in clinical immunoassay. The fabrication steps of the immunosensor as follow: firstly, nano-Au was immobilized on the surface of bare glass carbon electrode (GCE) by a simple electrochemical reduction of HAuCl4 solution; secondly, NiHCFNPs as an electroactive substance, were immobilized on the layer of gold nanoparticles. Microstructure and surface morphology of NiHCFNPs have been characterized by Transmission electron microscopy (TEM) and scanning electron microscope (SEM); thirdly, nano-Au was again immobilized on the surface of NiHCFNPs, which can offer a favorable microenvironment and biocompatibility to immobilize anti-CEA. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were applied to characterize the electrochemical properties of modified process. Effect of deposition time of nano-Au, pH of working buffer, incubation temperature and time were studied in detail for optimization of analytical performance. Under optimal conditions, the peak current of CV of the immobilized anti-CEA decreased linearly with increasing CEA concentration in two ranges from 0.5 to 10.0 ng·mL-1 and from 10.0 to 160.0 ng·mL-1, with a detection limit 0.1 ng·mL-1 at three times background noise. The proposed immunosensor show good repeatability and reproducibility, acceptable accuracy, high sensitivity and would be valuable for diagnosis and monitoring of carcinoma. |
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