Studies On Novel Redox-mediated Modification Of Enzyme Biosensors Based On Nano-materials

Since the development of the first glucose biosensor in 1967 by Updik and Hicks,biosensor have gained widely attention and made much more progress.The enzyme biosensor is usually an analytical device that consists of a biological recognition element with enzyme immobilization,a signal transducer and target analyte.So it has the functions of molecule recognition and selective catalysis.As also,this electrode responds to detection substance fastly,prepares simply and needs little sample.At present,enzyme biosensors have used to test multi-analyte.Thereinto,hydrogen peroxide(H₂O₂)biosensor based on catalase or mimetic enzyme has been researched widely.H₂O₂ is a raw material or middle production of many industrial process,and an outgrowth of catalase's reaction,so the detection of H₂O₂ is of great interest to biochemists because of its importance in the foodstuff,medicine,environment,clinical and industrial setting.With a continued interest in electrochemical mediated-enzyme biosensors,there are great efforts made to focus on the search of new mediators and the modification of the electrode surface in order to develop new enzyme biosensors with high sensitivity,selectivity,and stability.It has been reported that the crucial aspect in the fabrication of a biosensor is the immobilization of biorecognition molecule.Thus the selection of the electronic mediator,preventing the leaking of the mediator,the choice of condign material for the biorecognition molecule immobilization,the retention of the enzyme bioactivity and the construction of a novel enzyme biosensor model had been developed in this thesis.The main works and conclusions are included as follows:PartⅠ:Hydrogen Peroxide Biosensor Based on Azure I and Nano-Au through Layer by Layer Electrostatic Adsorption.With the consideration of the immobilization quantity of nano-Au and enzyme,a novel hydrogen peroxide biosensor was constructed by means of electropolymerization and layer-by-layer self-assembled technique.Firstly,p-aminobenzene sulfonie acid was electropolymerized to glassy carbon electrode surface,which used to electrostatic interaction with the positively charged Azure I. then,layer-by-layer self-assembly technique is used to form Azure I and nano-Au multilayer films (nano-Au/AI),.Finally,the positively charged horseradish peroxidase is successfully immobilized on the gold nanoparticles surface.The electrocatalytie behavior of the modified electrode to H₂O₂ was investigated by cyclic voltammetry and chronoamperometry.The experiments showed that the modified electrode had excellent electrocatalytic activity for the reduction of H₂O₂.The linear range of this biosensor is 3.5×10^-6～3.6×10^-3mol/L with a detection limit of 1.2×10^-6mol/L(S/N=3).In this experiment,HRP can remain a higher bioactivity with the Miehaelis-Menten Constant of 1.5 mmol/L.PartⅡ:Immobilization of horseradish peroxidase on silica sol-gel / chitosan hybrid film for the preparation of hydrogen peroxide biosensor.Fe(CN)₆^3-/4-,the most widely used as inorganic electron mediators,has been used extensively in biosensor because of its excellent electron transferability and a high degree of reversibility.However, analytes are usually detected using Fe(CN)₆^3-/4-in the detection solution,in which the reference, counter electrodes and the sample solution may be polluted,so it would be preferable to immobilize Fe(CN)₆^3-/4-on the electrode surface,which can provide several advantages such as reducing the analytical time and the consumption of the reagent.In chapter 3 of this paper,a simple and effective strategy for fabrication of hydrogen peroxide(H₂O₂)biosensor has been developed by entrapping horseradish peroxidase(HRP)in chitosan/silica sol-gel hybrid membranes(CSHMs)doped with Fe(CN)₆^3-/4-and gold nanoparticles(GNPs)on platinum electrode surface.The experiment suggest that the adoption of the hybrid films and nano-Au improve the sensitivity of the biosensor.The studied biosensor exhibited good reproducibility,selectivity and high sensitivity.Linear calibration for hydrogen peroxide was obtained in the range of 3.5×10^-6to 1.4×10^-3mol/L under the optimized conditions with the detection limit(S/N = 3)of 8.0×10^-7mol/L.The apparent Michaelis-Menten constant of the enzyme electrode was 0.93 mmol/L.The system was applied for the determination of the samples,and the results obtained were satisfactory.PartⅢ:Reagentless electrochemical hydrogen peroxide biosensor based on toluidine blue-derived organic material and functionalized gold nanoparticles.At present,redox-mediated modification of enzyme biosensors have been extensively researched.However,the problem of mediator leaking has been puzzling.In this work,PTCDA, which is considered to be the archetype for organic materials and has become more and more important in the application of electronics and photoelectronics in resent years,was hydrolyzed to 3,4,9,10-perylenetetracarboxylic acid(PTA)with four carboxylic acid groups.The carboxyl of PTA could interact with the NH₂ of toluidine blue(TB)in the process of mixing the two solutions.This strategy can increase the molecular weight of mediators,and composite leakage was avoided.To our knowledge,thus far no attempt has been made to use 3,4,9,10-perylenetetmcarboxylic acid-toluidine blue(PTATB)molecules as a material for fabricating enzyme biosensor.Moreover,in the construction of biosensor,the retention of enzyme bioactivity is a important factor.Nano-Au and have gained much more attention in electroanalytical studies because of their unique properties such as easy preparation,good biocompatibility,and absorption.However,nanoparticles would tend to aggregate or precipitate during their synthesis and storage.To avoid them,we exploit O-carboxymethyl chitosan(CMCS)as a stabilizer for protecting nano-Au.In this work,we aimed to improve the stability and bioactivity of enzyme biosensors and combine the advantageous features of PTATB and CMCS-GNPs to develop a highly stable and sensitive H₂O₂ biosensor.