| The rapid and accurate determination of hydrogen peroxide(H2O2)is of great importance because it is not only the product of the reactions catalyzed by many highly selective oxidases but also an essential compound in food,pharmaceutical and environmental analyses.Among these techniques employed for hydrogen peroxide analysis,such as titrimetry,photometry, chemiluminescence,high performance liquid chromatography and electrochemistry,amperometric enzyme-based biosensors have received considerable interest,because this class of technique is characterized by sensitivity,convenience and high selectivity.Electron transfer between an electrode and the redox activity center of an enzy me is the basis for developing various enzyme-based biosensors.It can be achieved through two different pathways.One involves electron-shuttling mediators to establish an electrical communication between redox proteins and the underlying electrodes.In this scheme,there are still several challenges to keep mediators from diffusing away from the electrode surface into the bulk solution.The other pathway involves direct electron transfer between redox proteins and the electrode.On the other hand,the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability,sensitivity and selectivity of biosensors.In this paper,nano-Au,with large surface area,high-surface free energy, was chosen to immobilize toluidine blue by layer-by-layer(LBL)assembly technique,and thionine was covalently bound onto the bovine serum albumin film with glutaraldehyde as cross-linker to achieve the immobilization of electron-shuttling mediators.For the leakage significantly decreases, the proposed mediator-based hydrogen peroxide biosensors exhibit good analytical performance.In addition,attention has been paid to the nano-Au/MWNTs composite and core-shell organosilica@chitosan/MWNTs composite.The application of these composites as an immobilization matrix for fabricating mediator-free hydrogen peroxide biosensors has been preliminarily investigated.The main points of this dissertation are summarized as follows:Part one:Study of the hydrogen peroxide biosensor based on the layer-by-layer assembly films of gold colloidal nanoparticles and toluidine blue The precursor film was first formed on the Au electrode surface based on the self-assembly of L-cysteine and the adsorption of gold colloidal nan oparticles(nano-Au).Layer-by-layer(LBL) assembly films of toluidine blue(TB)and nano-Au were fabricated by Au-N covalent bond and electrostatic adsorption between TB and nano-Au.Finally,horseradish peroxidase(HRP)was assembled onto {nano-Au/TB}nmultilayer films to fabricate a novel hydrogen peroxide biosensor. Cyclic voltammeter(CV)and quartz crystal microbalance(QCM)were adopted to monitor the regular growth of {nano-Au/TB} bilayer films.Morphologies of the films were characterized with atomic force microscopy(AFM).The effects of applied potential,temperature and pH on the current response were investigated.Repeatability,stability and anti-interference were also researched.The proposed biosensor responds rapidly to H2O2 in the linear range from 1.5×10-7mol/L to 8.6×10-3 mol/L with a detection limit of 7.0×10-8mol/L(S/N=3).Part two:Study of the hydrogen peroxide biosensor based on immobilizing HRP on thionine-bovine serum albumin conjugate and gold colloidal nanoparticlesA novel enzyme immobilization technique based on thionine-bovine serum albumin conjugate (Th-BSA)and gold colloidal nanoparticles(nano-Au)was developed.Thionine was covalently bound onto the BSA film with glutaraldehyde(GA)as cross-linker to achieve Th-BSA conjugate. The free amino groups of thionine were then used to attach nano-Au for the immobilization of horseradish peroxidase(HRP).Such nano-Au/Th-BSA matrix shows a favorable microenviment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme.Several techniques,including atomic force microscopy(AFM),X-ray photoelectron spectroscopy(XPS),Infrared spectra(IR)and electrochemical impedance spectroscopy(EIS)have been employed to characterize the assembly process.The proposed biosensor displays excellent catalytic activity and rapid response for H2O2. The linear range for the determination of HO2 is from 4.9×10-7to 1.6×10-3mol/L with a detection limit of 2.1×10-7mol/L at 30 and a Michaelies-Menten constant value of 0.023 mmol/L.Part three:Amperometric third-generation hydrogen peroxide biosensors based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticlesA convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes(MWNTs)and gold colloidal nanoparticles(nano-Au)by using proteins as linker is proposed.In such a strategy,Hemoglobin(Hb)was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNTs and nano-Au.Acid-pretreated,negatively charged MWNTs was first modified on the surface of glassy carbon(GC)electrode,then,positively charged Hb was adsorbed onto MWNTs films by electrostatic interaction.Finally,nano-Au and Hb were successively assembled onto the modified electrode to obtain the Hb/nano-Au/Hb/MWNTs/GC electrode.The assembly of Hb and nano-Au was characterized with cyclic voltammetry(CV), electrochemical impedance spectroscopy(EIS)and transmission electron microscopy(TEM).The direct electron transfer of Hb is observed on the Hb/nano-Au/Hb/MWNTs/GC electrode,which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third generation mediator-free H2O2 biosensor.As compared to those H2O2 biosensors only based on carbon nanotubes,the proposed biosensor modified with MWNTs and nano-Au displays a broader linear range and a lower detection limit for H2O2 determination.The linear range is from 2.1×10-7to 3.0×10-3mol/L with a detection limit of 8.0×10-8mol/L at 3σ.The Michaelies-Menten constant KMappvalue is estimated to be 0.26 mmol/L.Moreover,this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.Part four:A hydrogen peroxide biosensor based on multi-wall carbon nanotubes/chitosan composite and multilayer films of hemoglobin and colloidal gold nanoparticlesAn amperometric biosensor for H2O2 was developed based on multilayer assembly of hemoglobin(Hb)and colloidal gold nanoparticles(nano-Au)on multi-wall carbon nanotubes/chitosan composite.Chitosan(CS)was chosen for dispersing multi-wall carbon nanotubes(MWNTs)to form a stable CS-MWNTs composite.This composite was first coated on the surface of glassy carbon electrode to provide a containing amino groups interface for assembling nano-Au,followed by the adsorption of Hb to form a bilayer of {Hb/nano-Au}.Repeating the assembly step of nano-Au and Hb resulted in {Hb/nano-Au}n multilayers.The resulting system brought a new platform for electrochemical devices by using the synergistic action of the electrocatalytic activity of nano-Au and MWNTs.The assembly of nano-Au onto CS-MWNTs was confirmed by transmission electron microscopy.The consecutive growth of {Hb/nano-Au}n multilayers was confirmed by cyclic voltammetry and UV-vis absorption spectroscopy.The resulting electrode displays excellent electrocatalytic activity and rapid response for H2O2.The linear range for the determination of H2O2 is from 5.0×10-7to 2.0×10-3mol/L with a detection limit of 2.1×10-7mol/L at 3σand a Michaelies-Menten constant KMappvalue of 0.19 mmol/L.At the same time,the effects of applied potential and pH on the sensor were examined.Part five:A hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on core-shell organosilica@chitosan nanospheres and multiwall carbon nanotubes compositesThe application of a composite of multiwall carbon nanotubes(MWNTs)and core-shell organosilica@chitosan crosslinked nanospheres as an immobilization matrix for a construction of a novel mediator-free amperometric hydrogen peroxide(H2O2)biosensor was described.MWNTs were dispersed in a suspension of positively charged organosilica@chitosan nanospheres in acetic acid solution(0.6 wt%)to achieve an organosilica@chitosan/MWNTs composite,which was cast onto a glass carbon electrode(GCE)surface directly.And then,horseradish peroxidase(HRP),as a model enzyme,was immobilized onto it through electrostatic interaction between oppositely charged organosilica@chitosan nanospheres and HRP.The direct electron transfer of HRP is achieved at the HRP/organosilica@chitosan/MWNTs/GCE,which exhibits an excellent electrocatalytic activity for the reduction of H2O2.The catalysis currents increased linearly to H2O2 concentration in a wide range of 7.0×10-7tO 2.8×10-3mol/L with a detection limit,of 2.5×10-7mol/L at 3σ.A Michaelies-Menten constant KMappvalue is estimated to be 0.32 mmol/L,indicating a high-catalytic activity of HRP.Moreover,the proposed biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.When used to detect H22 concentration in disinfector sample,it shows satisfactory results.
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