| Due to the high catalytic efficiency and substrate specificity of natural enzymes, they have received tremendous attention in different fields. However, the natural enzymes, a kind of biological catalysts, are easy to be inactive by the external environment. Accordingly, the investigation of enzyme mimics has been a research focus. In recent years, porphyrins, host reagents, molecular imprinting polymers, and membrane systems and the complexes have been used as enzyme mimics. Owing to their surface effect, quantum size effect, volume effect and macroscopic quantum tunneling effect, nanomaterials have attracted considerable attention in the fields of chemistry, physics, biomedicine, life science and environmental protection. However, the inorganic nanoparticles are generally considered to be biologically and chemically inert and their catalytic activity has been neglected for a long time. Since first report on the intrinsic peroxidase-like activity of ferromagnetic nanoparticles by Yan's research group in 2007, various nanomaterials, such as oxide nanoparticles, carbon-based nanomaterials, single metal nanoparticles and double metal composite nanomaterials, have been found to possess catalytic activity of enzyme mimics. Previous reports demonstrated that the nanoparticles are a kind of sensitive materials, one can be obtained nanoparticles in different compositions and different morphologies with different synthetic method to meet specific catalytic purposes. In view of this, three nanoparticles, namely, ferrite magnetic nanoparticles, carbon nanodots and cerium dioxide nanoparticles were investigated as mimetic peroxidases and used to construct optical analytical methods for detecting hydrogen peroxide in present thesis. This thesis includes seven chapters: The first chapter:In this chapter, we summarized the research progress of enzyme mimics.The second chapter:In this chapter, we dealed with the peroxidase mimic properties of six ferrite magnetic nanoparticles, namely, CoFe2O4, CuFe2O4,γ-Fe2O4 MnFe2O4, precursor of NiFe2O4 and precursor of magnesium ferrite using uminol chemiluminesent reaction as a model system. The results were compared with that of horseradish peroxidase (HRP). Our results are:(ⅰ) like HPR, the six ferrite magnetic nanoparticles are able to catalyze the oxidation of luminol by hydrogen peroxide to produce enhanced chemiluminescence, and the catalytic activity of the studied six ferrite magnetic nanoparticles depends on the pH value, temperature and substrate (H2O2) concentration. Also, they could catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide to produce a typical colour reaction. These results show that the ferrite magnetic nanoparticles possess intrinsic peroxidase properties; (ⅱ) the apparent Michaelis constants (Km) using hydrogen peroxide as substrates were higher than that of HRP, in the order CoFe2O4< y-Fe2O3< precursor of NiFe2O4< CuFe2O4< MnFe2O4< precursor of magnesium ferrite; (ⅲ) the ferrite magnetic nanoparticles show better pH and temperature tolerance than HRP.The third chapter:In this chapter, we choose CoFe2O4 nanoparticles as a typical example to establish a method for the determination of hydrogen peroxide. Under the optimal conditions, the present chemiluminescence (CL) system showed a linear range for the detection of hydrogen peroxide from 0.1-10μM with a detection limit (3σ) of 10 nM. The proposed method was successfully applied to determine hydrogen peroxide in water samples and glucose in serum samples with satisfactory results. It is believed that the nanostructured CoFe2O4 may be useful artificial peroxidase for a wide range of potential applications in environmental analysis, bioanalysis, and chemiluminescence.The fourth chapter:In this chapter, a new CL flow hydrogen peroxide biosensor based on sol-gel-derived CoFe2O4 nanoparticles-based peroxidase mimic biocatalyst has been developed. In this biosensor, the sol-gel containing CoFe2O4 nanoparticles (CoFe2O4 NPs) were packed into a glass column (i.d.40 mm x 3 mm) as a CL flow cell. The CoFe2O4 NPs exhibited peroxidase-like catalytic activity and catalyzed the oxidation of luminol by hydrogen peroxide. When luminol and hydrogen peroxide passed through the CL flow cell, the hydrogen peroxide was sensed and with CL emission. The resulting hydrogen peroxide-sensitive biosensor was characterized in a flow injection system to evaluate their main parameters, such as kinetic parameters, detection limit, linear range, reproducibility, operational and storage stability. Under the optimal conditions, the CL response to hydrogen peroxide concentration was linear in the range of 0.01-10μM with a detection limit (3a) of 3.2 nM. The biosensor showed some advantages of high reproducibility, stability and robustness. The biosensor had been successfully applied to the determination of hydrogen peroxide in rainwater. When combined with a glucose oxidase column as injection loop, the proposed biosensor had been successfully applied to determine glucose in human serum by a stopped-flow mode with satisfactory results.The fifth chapter:In this chapter, the plant tissue enzyme and CoFe2O4 nanoparticles-based mimetic peroxidase were used to construct a novel flow injection chemiluminescence (CL) biosensor for the determination of oxalic acid in urine samples. The spinach (spinacia oleracea) leaves and CoFe2O4 nanoparticles (CoFe2O4 NPs) were exploited as molecular recognition element and mimetic peroxidase, respectively. In this biosensor, oxalic acid was oxidized by oxygen under the catalysis of oxalate oxidase in the tissue column (TC) to produce hydrogen peroxide, thereby reacts with Luminol in the presence of the mimetic peroxidase generating CL signal. In this case, two most common unnatural enzymes were combined for a useful application. The CL reaction conditions, tissue-based biosensor recognition conditions, stability and lifetime of the biosensor have been studied in detail. Results obtained in this biosensor revealed that the sensitivity was significantly improved in the presence of the mimetic peroxidase. In the optimum conditions, the CL intensity was linear with oxalic acid concentration in the range of 1.0×10-2-1.0 x 102μM with a detection limit (DL) of 3.2 nM. The relative standard deviation (RSD) was 2.9% for 1.0μM oxalic acid (n-11). The biosensor has been applied to determine oxalic acid in urine samples with satisfactory results.The sixth chapter:In this chapter, the highly stable and dispersible carbon nanodots have been successfully synthesized. The as-prepared caborn nanodots can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide, and exhibit peroxidase-like activity. Like the natural horseradish peroxidase (HRP), the catalytical activity of the carbon nanodots was dependent on the temperature, pH and hydrogen peroxide concentrations. The results showed that the maxium catalytical effect was obtained at 35℃, pH 3.5 and 300 mM hydrogen peroxide. The Michaelis-Menten kinetics study demonstrated that the Michaelis constant (Km) of corbon nanodots to TMB is 0.039 mM which is smaller than that of HRP indicated that the affinity between TMB and the corbon nanodots is stronger than that between HRP and TMB. Based on the hydrogen peroxide concentration dependent-catalytic activity of the carbon nanodots, a simple, sensitive, selective and cheap colorimetric assay to detect glucose in serum samples was constructed. Also, a catalytic mechanism was discussed.The seventh chapter:In this chapter, we have successfully synthesized the stable and good water-souble polyacrylic acid functioned CeO2 nanoparticels. The data of dynamic light scattering (DLS) showed that the hydrodynamic diameter of the carbon nanoparticles was about 5 nm. The results of Fourier transform infrared spectroscopy (FTIR) confirmed the polyacrylic acid coating was an integral part of CeO2 nanoparticels. The X-ray photoelectron spectroscopy (XPS) spectrum revealed that the synthesized CeO2 nanoparticels have a mixed valence state of Ce3+ and Ce4+. The prepared CeO2 nanoparticels can catalyze the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB) oxidized by hydrogen peroxide and display peroxidase-like activity. In the present work, we proposed that TMB and hydrogen peroxide were first adsorbed on the surface of CeO2 nanoparticels, then TMB donated lone-pair electrons in the amino groups to CeO2 nanoparticles, and the electrons are captured by the surface peroxide species to produce OH and O2·-/HO2·to oxidize TMB. On the basis of the dependency of CeO2 nanoparticels-based peroxidase-like activity on hydrogen peroxide concentration and the conversion of glucose to hydrogen peroxide by glucose oxidase, a simple, sensitive and selective colorimetric method for the determination of glucose in serum has been constructed.
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