| Since the development of the first glucose biosensor in 1967 by Updik and Hicks, biosensors have received considerable attention and much more progress due to its convenicene and high sensitivity. Recently, enzyme biosensors have used to test multi-analyte. H2O2 is product of industry or middle production of many industrial processes, and also a side product of oxidases, so the detection of H2O2 is of great significance in industrial setting environmental and biological chemistry. Hb has commonly been employed to construct H2O2 biosensors because of its commercial availability and peroxidase activity. However, as their redox centers are usually deeply buried within the protein molecules, it is difficult for electron to transfer between proteins and electrode. Therefore, it is a challenge to develop matrix to immobilize the redox protein, in which the protein can keep stability and its direct electron-transfer rate can be enhanced In this paper, we fabricated some biosensors with hollow zirconium dioxide microspheres (HZMS)-sodium alginate (SA), graphene-zinc oxide, acerate ZnO whiskers (AZW)-sodium alginate (SA) and ZnO hollow spheres (ZHS). The resulting biosensors exhibit direct ekectrochemical reaction and can be used to determine hydrogen peroxide in low potential with high sensitivity. The details are summarized as follows:(1) Direct electrochemistry and electrocatalysis of hydrogen peroxide using hemoglobin immobilized in hollow zirconium dioxide spheres and sodium alginate filmsA biocompatible composite made from hollow zirconium dioxide microspheres (HZMS) and sodium alginate (SA) is presented and used for the construction of a biosensor for hydrogen peroxide. The composition, morphology and size were studied by transmission electron microscopy. FT-IR and UV-vis spectroscopy revealed that hemoglobin (Hb) entrapped in the ZHMS retains its native structure. A pair of stableandwell-defined quasi-reversible redox peaks of Hb is obtained, with a formal potential of -0.15 V at pH 7.0. The apparent heterogeneous electron transfer rate constant is 1.15 s-1, indicating facile electron transfer that may result from the unique nanostructures and larger surface area of the HZMS. The amperometric response of the sensor varied linearly with the concentration of hydrogen peroxide in the range from 1.75×10-6 mol/L to 4.9×10-3 mol/L, with a detection limit of 6×10-7 mol/L (at S/N=3). The biosensor possesses high sensitivity, good reproducibility, and long-term stability.(2) Direct electrochemistry and enhanced electrocatalytic activity of hemoglobin entrapped in graphene and ZnO nanosphere composite filmA biocomposite film for sensing hydrogen peroxide (HP) is described that is based on nanospheres made from hemoglobin (Hb), graphene, and zinc oxide. The composition, morphology and size of the film were studied by transmission electron microscopy. UV-vis spectroscopy revealed that the Hb entrapped in the graphene and ZnO nanosphere retains its native structure. A pair of stable and well-defined quasi-reversible redox peaks of Hb was obtained, with a formal potential of -30 mV at pH 6.5. Hb exhibits excellent a long-term bioelectrocatalytic activity towards HP. The apparent heterogeneous electron transfer rate constant is 1.0 s-1, indicating that the presence of graphene in the composite film facilitates the electron transfer between matrix and the electroactive center of Hb. The sensor responds linearly to HP in the range from 1.8μmol/L to 2.3 mmol/L, with a detection limit of 0.6μmol/L (at S/N=3). The apparent Michaelis-Menten constant is 1.46 mmol/L. The biosensor displays high sensitivity, good reproducibility, and long-term stability.(3) Acerate ZnO whiskers and sodium alginate films:Preparation and application in bioelectrochemistry of hemoglobinA novel biocompatible acerate ZnO whisker (AZW) has been prepared.And we explored AZW and sodium alginate (SA) for the construction of electrochemical biosensors. The composition, morphology and size were studied by scanning electron microscopy (SEM). UV-vis spectra revealed that hemoglobin (Hb) adsorbed in the acerate ZnO whiskers and sodium alginate retained its native structure. The amperometric response was measured as a function of H2O2 concentration, at a fixed potential of -0.25 V in phosphate-buffered saline (pH 7.0). The electrochemical parameters of Hb in acerate ZnO whiskers and sodium alginate were calculated with the results of the electron transfer coefficient (a) and the apparent heterogeneous electron transfer rate constant (ks) as 0.5 and 2.5 s-1 respectively, indicating good facilitation of the electron transfer between Hb and the modified electrode, which may result from the unique nanostructures and larger surface area of acerate ZnO whiskers. The hydrogen peroxide biosensor showed a fast response of less than 5 s of linear range 2.1μmol/L-4.8 mmol/L, with a detection of 0.7μmol/L (S/N=3). The apparent Michaelis-Menten constant Kmapp is 0.8 mmol/L. The biosensor possesses high sensitivity, good reproducibility, and long-term stability.(4) A Biosensor for Hydrogen Peroxide Based on hemoglobin immobilized in zinc oxide hollow spheresA new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping Hemoglobin (Hb) in the ZnO hollow spheres (ZHS) nanoparticles. The sensitivity of the biosensor was enhanced by ZnO hollow spheres. Transmission electron microscopy (TEM) used to characterize the composition, morphology and size. Several techniques, including UV-vis absorption spectroscopy, cyclic voltammetry, chronoamperometry were employed to performance of the biosensor. The ZnO hollow spheres nanoparticles had high stability and good retention of bioactivity of the immobilized enzyme. The electrochemical parameters of Hb in the ZHS was calculated with the results of the electron transfer coefficient (a) and the apparent heterogeneous electron transfer rate constant (ks) as 0.5 and 3.1 s-1 respectively, indicating good facilitation of the electron transfer between Hb and the modified electrode, which may result from the unique nanostructures and larger surface area of the ZHS. The resulting biosensors showed a linear range from 2.1μmol/L to 5.18 mmol/L, with a detection limit of 0.6μmol/L (S/N=3) under optimized experimental conditions. The results demonstrate that the ZHS matrix may improve the protein loading with the retention of bioactivity and greatly promote the direct electron transfer, which can be attributed to its high specific surface area, and biocompatibility. The biosensor obtained from this study possesses high sensitivity, good reproducibility, and long-term stability.
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