The Construction And Research On Novel Electrochemical Biosensors

Electrochemical biosensors that combine the specific recognition of biological protein molecules(eg: enyzme, antibody) toward targets with electrochemical transduction methods have the advantages of high sensitivity, low cost, and simple instrumentation. The immobilization of biological protein molecules is one of the key steps in developing high-performance biosensors, since it will affect the loading as well as the bioactivity. Usually, nanomaterials has been used to effective immobilize biological protein molecules because nanomaterials has good biological compatibility to keep the enzyme activity. However, a fatal weakness of these nanomaterials is that they can form close-packed structures after they are assembled on an electrode surface, which will reduce their specific surface area, restrain their electrochemical performance and is unfavorable to maintain the activity of protein. Biomass-derived porous carbon materials have drawn much attention due to their extensive sources, low cost, environmentally friendly behaviors, large specific surface area, short diffusion pathway, good electrical conductivity and high porosity. Therefore, how to keep activity of biological molecules in electrochemical biosensors and how to increase the amount of biomolecules on the electrode has remain particularly attractive to research. Based on electrochemistry, this thesis mainly investigated the eleetroehemical behavior of varieties of biomolecules toward immobilization on the modified biomass-derived porous carbon materials and realized glucose detection and hydrogen peroxide detection. The main research content and results are shown as below:1. A simple, sensitive and effective method to detect glucose in ultra-low ionic strength solution containing citrate-capped silver nanoparticles(CCAgNPs) was developed by monitoring the change of solution conductance. Glucose was catalyzed into gluconic acid firstly by glucose oxidase in an O2-saturated solution accompanied by the reduction of O2 into hydrogen peroxide(H2O2). Then, CCAgNPs was oxidized by H2O2 into Ag+ and the capping regent of citrate was released at the same time. All these resulted Ag+, gluconic acid and the released citrate would contribute to the increase of solution ionic strength together, leading to a detectable increase of solution conductance. And a novel conductance glucose biosensor was developed with a routine linear range of 0.06–4.0 mM and a suitable detection limit of 18.0 ?M. The novel glucose biosensor was further applied in energy drink sample and proven to be suitable for practical system with low ionic strength. The proposed conductance biosensor achieved a significant breakthrough of glucose detection in ultra-low ionic strength media.2. A novel glucose biosensor was developed by immobilizing glucose oxidase(GOD) on a three-dimensional(3D) porous kenaf stem-derived carbon(3D-KSC) which was firstly proposed as novel supporting materials to load biomolecules for electrochemical biosensing. Here, an integrated 3D-KSC electrode was prepared by using a whole piece of 3D-KSC to load the GOD molecules for glucose biosensing. The morphologies of integrated 3D-KSC and 3D-KSC/GOD electrode were characterized by scanning electron microscopy(SEM) and transmission electron microscopy(TEM). The SEM results revealed 3D honeycomb macroporous structure of integrated 3D-KSC electrode. The TEM results showed some microporosities and defects in the 3D-KSC electrode. The electrochemical behaviors and electrocatalytic performance of integrated 3D-KSC/GOD electrode were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. The effects of pH and scan rate on the electrochemical response of biosensor have been studied in detail. The glucose biosensor showed a wide linear range from 0.1 mM to 14.0 mM with a high sensitivity of 1.73 ?A m M-1 and a low detection limit of 50.75 ?M. Furthermore, the glucose biosensor exhibited high selectivity, good repeatability and reproducibility, and nice stability.3. A novel ultrasensitive immunosensor to detect the tumor biomarker, carcinoembryonic antigen(CEA), was developed by using AuNPs-Ab2-glucose oxidase-Concanavalin A as tracing tag and albumin bovine V-Ab1/chitosan-AuNPs/three-dimensional macroporous carbon integrated electrode as signal collector based on a sandwich-type assay mode. The fabrication process of tracing tag and signal collector were characterized by atomic force microscopy, UV-vis absorption spectroscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. After a sandwich immunoreaction, the quantitative capture of tracing tag on the signal collector could greatly hinder the electron transfer of Fe(CN)63-/4- and result in the rapid increase of the electrical resistance owing to both the increase of the modified layer thickness and the electrostatic repulsion between Fe(CN)63-/4- and the negatively charged tracing tag. The process was verified by electrochemical impedance spectroscopy. Compared with previously designed immunosensors, here the integrated electrode as signal collector provided a large specific surface area to load a large number of Ab1 and the pH-dependent behavior of tracing tag also enhanced the sensitivity greatly. As a consequence, the proposed immunosensor for CEA showed a wide linear range of 5 pg m L-1 to 50 ng m L-1, a low detection limit of 1.3 pg mL-1 and a high sensitivity of 57.573 ?A ng m L-1 cm-2. In addition, the immunosensor also showed satisfactory reproducibility, stability and acceptable reliability.4. It was firstly combined Tb@mesoMOF and three-dimensional macroporous carbon integrated electrode(3D-KSCs) to provide a new platform to immobilize Microperoxidase-11(MP-11). MP-11 has been demonstrated that MP-11 molecules interact with framework of Tb@mesoMOF through ?…? interations between the heme of MP-11 and the conjugated triazine and benzene rings in the organic ligand of Tb@mesoMOF. The strong interactions facilitate the retention MP-11 molecules within the metal-organic framework(MOF) pores, which also could retain MP-11 catalytic activity toward hydrogen peroxide(H2O2). The three-dimensional(3D) macroporous carbon(3D-KSCs) derived from kenaf stem to prepare the 3D-KSCs electrode showed a 3D honeycomb porous structure. Such structure provides a large specific surface area, effectively supports a large number of Tb@mesoMOF, and greatly enhances the mass and electron transfer. The electrochemical behaviors and electrocatalytic performances of the MP-11/Tb@mesoMOF/CHIT-Au NPs/3D-KSC electrode were evaluated by cyclic voltammetry and the amperometric method. The resulted electrode MP-11/Tb@mesoMOF/CHIT-AuNPs/3D-KSC show good electrocatalytic performances toward the reduction of H2O2 with a wide range of 5 ?M ~ 0.5 mM and low detection limit of 1.27 ?M(R= 0.996; n=11). Therefore, the new method of mesoporous MOF combination with the 3D-KSCs has provided insightful information that will help in the design of new functional mesoMOF combination with other nanomaterials for enzyme immobilization with enhanced biocatalysis performances.5. This study firstly demonstrates the exploitation of carbon nanotubes(CNTs) with mesoporous metal-organic framework(MOF) to form a new composite material as the platform for constructing integrated dehydrogenase-based electrochemical biosensors for glucose detection. In this study, The CNTs-Tb@mesoMOF composite material are able to serve as a matrix for immobilizing electrocatalysts(methylene green, MG) and dehydrogenases glucose dehydrogenase, GDH) onto the electrode surface and an integrated electrochemical biosensor is readily formed. The CNTs-Tb@mesoMOF composite material shows excellent adsorption capacities toward both MG and GDH and is thus employed as the matrix for our glucose biosensor. To construct the biosensor, we firstly droped coat a CNTs-Tb@mesoMOF-MG composite onto a glassy carbon electrode and then coat GDH onto the CNTs-Tb@mesoMOF-MG composite. Therefore, The novel biosensor is very sensitive to glucose with a very wide linear range of 25 ?M~17 mM and a low detection limit of 8 ?M(R=0.998,n=10).