Conductive Nano-materials Applied For Preparation Of The Hydrogen Peroxide Sensor

Since the development of the first glucose biosensor in 1967 by Updik and Hicks,biosensor have received considerable attention and made much more progress due to its convenience,high sensitivity and selectivity.Recently,enzyme-based biosensors have used to test multi-analyte.H₂O₂ is a raw material or middle production of many industrial processes,and an outgrowth of catalase's reaction,so the detection of H₂O₂ is of great significance in the food,industrial setting,clinical,medicine and environmental.As we all known,horseradish peroxidase and hemoglobin-based electrodes are one of the most commonly investigated biosensors for bioelectrocatalytic reduction of H₂O₂,because these enzymes are highly selective to H₂O₂.However,the enzyme moleculars have the following disadvantages:(1) effective immobilization of enzyme to solid electrode surface is very difficult;(2) the electron transfer rate between the electrode and the redox activity center of enzyme is very slow because the redox activity center of enzyme(HRP and Hb) is deeply buried inside insulated protein shells;(3) the immobilized enzyme(HRP and Hb) on the surface of the electrode is facilitated to denature by many kinds of factors in the ambient environment,which leads to such H₂O₂ biosensor suffer from a poor enzyme activity,a low reproducibility and stability.Therefore,how to find suitable materials and methods to maintain the enzyme activity, enhance the volume of immobilized enzyme,as well as to accelerate the electron transfer rate or the development of a new type of non-enzyme-based sensors are very important for obtaining highly sensitive and selective H₂O₂ sensor.Based on this,the main research works in this paper are included as follows:PartⅠ:Amperometric hydrogen peroxide biosensor based on covalently immobilizing thionine as a mediatorThe introduction of the mediator and metal nano-materials can significantly accelerate the electron transfer rate and increase the immobilization of biomolecules,as well as improve the sensitivity of the sensor.So,in this part a simple and novel amperometric hydrogen peroxide biosensor has been constructed. Firstly,the 2,6-pyridinedicarboxylic acid,acted as a support of the redox mediator to provide enough carboxyl,was initially electro-polymerized on to glassy carbon electrode surface,and then we concentrated on covalent modification that involves the coupling of the amino groups of the thionin to the carboxyl groups of the 2,6-pyridinedicarboxylic acid,using carbodiimide reaction that selectively forms amide linkages only with carboxyl groups.Subsequently,nano-Au was electro-deposited on to thionin-modified electrode surface.Finally,hemoglobin as a model simulation enzyme was adsorbed on to the nano-Au layer to obtain biosensor for the detection of H₂O₂.The proposed biosensor exhibited fast amperometric response to H₂O₂,and linear range was from 9.1μmol/L to 5.0 mmol/Lwith the detection limit of 2.6μmol/L (S/N=3).The apparent Michaelis-Menten constant was evaluated to be 3.15 mmol/L,indicating that Hb possessed a high biological affinity to H₂O₂.PartⅡ:Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on DNA-silver nanohybrids and PDDA-protected gold nanoparticlesUp to now,many materials have been used to immobilize enzyme on the surface of electrodes,such as quantum dots,polymers,mesoporous materials and nanomaterials.Among these materials,nanomaterials, especially functionalized nanocomposites have attracted great research interest in biosensor because of their versatility of the physical and chemical properties and other properties.However,the conductivity of some functionalized nanocomposites is not very good,which resulted in the low sensitivity and the low accuracy in some degree,thereby they are limited in the biosensing application.To our best knowledge, although many applications of conducting metal nanoparticles modified by other materials have been reported,very few studies have been carried out for construction of hydrogen peroxide biosensor by taking advantage of the merits of composite materials which combine together the different properties of components and lead the way to a new,tunable behavior.So,in this part,we fabricated a hydrogen peroxide biosensor formed with DNA-Ag and PDDA-Au to entrap HRP.First,we electrochemically reduced the DNA-Ag⁺ complex to obtain negatively charged immobilization matrix to immobilize PDDA-Au.Then,the second layer of DNA-Ag was assembled onto the modified electrode based on the electrostatic force and excellent film-forming ability of DNA.Finally,the positively charged HRP was adsorbed tightly onto the DNA-Ag layer to obtain hydrogen peroxide biosensor.Because these composite combined the advantages of inorganic metal species(Au,Ag) and organic polymer(PDDA,DNA),the resulting biosensor showed a linear response to H₂O₂ over a concentration range from 7.0μmol/L to 7.8 mmol/L with a detection limit of 2.0μmol/L(S/N=3) trader optimized conditions.The apparent Michaelis-Menten constant was evaluated to be 1.3 mmol/L.The biosensor exhibited high sensitivity and an acceptable stability.PartⅢ:Electrochemical sensor based on Prussian blue nanorods and gold nanochainsBecause the enzyme based-sensor existing some shortcomings,such as poor stability and reproducibility,as well as low sensitivity.To minimize or eliminate these limitations,chemically modified electrodes which needn't any enzyme are gaining an increasing attention and will become a trend to detect H₂O₂.So,in this part,we used the MWCNTs as a template to synthesize Prussian blue nanorods (PB@MWCNTs).Then we used the PB@MWCNTs as an "artificial peroxidase" to construct a new non-enzyme based sensor by using chitosan(Cs) to immobilize them onto the surface of the gold electrode. But Cs has its own inherent obstacle such as the poor electrical conductivity.Thus,to further overcome this problem,gold nanochains have been successfully introduced for the construction of the sensor.We all know that gold nanoparticles not only possess larger specific surface area,good biocompatibility,but also possess good conductivity.So when they are synthesized to a sort of gold nanochains,they can make possible conducting Channels to reduce the effective electron transfer distance and to facilitate charge transfer.In combination with the advantages of PB@MWCNTs and gold nanochains in the sensor would result in the improvement of analytical performance and lower detection limit for H₂O₂ determination.In addition,the absence of any step of enzyme immobilization onto the surface of electrode not only leads to a more simple assay system,but also possesses long-term stability,good repeatability and low cost.