Construction Of Bienzyme Biosensor Based On One-Step Electrodeposition

There are three chapters in this thesis. A simple and controllable electrodeposition approach was established for fabricating bienzyme biosensor based on covalent-coupled sol-gel process (the first chapter) and biospecific affinity of Concanavalin A (Con A) (the second chapter) by in-situ formation of biocomposite film on electrode. A reagentless hydrogen peroxide (H₂O₂) biosensors was also fabricated based on immobilization of horseradish peroxidase (HRP) on layered calcium carbonate-gold nanoparticles (CaCO3-AuNPs) inorganic hybrid composite in the third chapter.1. HRP and glucose oxidase (GOD) bienzyme biosensor was constructed by in-situ formation of the organic–inorganic biocomposite film based on one-step electrodeposition and covalent-coupled sol–gel process. The electrodeposition was performed in the solution containing functional inorganic precursor possessing the epoxy groups,γ-glycidoxypropyltrimethoxysiloxane (GPTMS), a biopolymer chitosan (CS), HRP and GOD. The covalent-coupled sol-gel process was formed by self-hydrolysis and self-condensation of GPTMS, followed by in-situ covalent cross-linking of CS, HRP and GOD through covalent reaction between amino groups and epoxy groups. The developed bienzyme biosensor presented high stability in acidic solution owing to the covalent-coupled organic-inorganic hybridization. Compared with the non-hybrid HRP-GOD/CS/Au electrode, the bienzyme biosensor of HRP-GOD/GPTMS/CS/Au showed improved sensitivity and a wider linear range for the determination of glucose. The linear response of the developed HRP-GOD/GPTMS/CS/Au biosensor for the determination of glucose ranged from 1μM to 351μM with a detection limit of 0.3μM.2. One-step construction of HRP and GOD bienzyme biosensor was established based on a simple and controllable electrodeposition approach by in-situ formation of biocomposite film on electrode. With H₂O₂ instantly generated by GOD-catalyzed oxidation of glucose, quantitative measurement of glucose could be achieved. In the proposed electrodeposition strategy, Con A was applied as bifunctional cross-linker for in-situ incorporating of HRP-GOD in biocompatible CS matrix due to its biospecific affinity interaction with CS and glycoproteins. Scanning electron microscopy showed that the HRP-GOD/Con A/CS biocomposite film possessed highly porous surface. The developed bienzyme biosensor exhibited a fast amperometric response for the determination of glucose. The linear response of the developed biosensor for the determination of glucose ranged from 1.0×10?6 to 2.2×10?4 M with a detection limit of 6.7×10?7 M. The biosensor can be used to determine glucose in serum directly. The biosensor presented high stability owing to the design of the introduction of Con A and instant generation of H₂O₂ with bienzyme system. The one-step construction of biosensor based on non-manual technique in biocompatible environment was direct and facile.3. A mediator-free H₂O₂ biosensor was fabricated based on immobilization of HRP on layered CaCO3-AuNPs inorganic hybrid composite. The proposed biosensor showed a strong electrocatalytic activity toward the reduction of H₂O₂, which could be attributed to the favored orientation of HRP in the well-confined surface as well as the high electrical conductivity of the resulting CaCO3-AuNPs inorganic hybrid composite. The hybrid composite was obtained by the adsorption of AuNPs onto the surfaces of layered CaCO3 through electrostatic interaction. The key analytical parameters relative to the biosensor performance such as pH and applied potential were optimized. The developed biosensor also exhibited a fast amperometric response (3 s), a good linear response toward H₂O₂ over a wide range of concentration from 5.0×10?7 to 5.2×10?3 M, and a low detection limit of 1.0×10?7 M. The facile, inexpensive and reliable sensing platform based on layered CaCO3-AuNPs inorganic hybrid composite should hold a huge potential for the fabrication of more other biosensors.