Preparation And Application Of Electrochemical Biosensor Based On Nanomaterial-Protein Composite

Protein is an important biological macromolecule and the direct electrochemical behavior of redox proteins or enzymes on the electrode can provide a model for the electron transfer processes study of life in vivo, which is important to understand the mechanism of electron transfer, explore the physiological mechanisms of life and develope new third-generation biosensors and bio-fuel cells. Ionic liquids (ILs) have been utilized in the fields of electrochemistry and electroanalysis due to their unique physicochemical properties including higher ionic conductivity, wider electrochemical windows, non-volatile and non-toxic. The special characteristics of nanomaterials such as high surface area and high activity also make them the most promising matrials used in biosensor. In this thesis IL and nano-material are used as modifiers to fabricate four kinds of heme proteins modified electrodes and the direct electrochemistry of heme proteins are studied in details. The thesis can be summarized as follows:1. An urchinlike MnO2 nanoparticle was synthesized by hydrothermal method and applied to the protein electrochemistry. By using a carbon ionic liquid electrode (CILE) as the basal electrode, hemoglobin (Hb) was immobilized on the surface of CILE with chitosan (CTS) and MnO2 nanoparticle composite material. The characteristics of Hb in the modified films were investigated by UV-vis spectrum, scan electron microscopy (SEM), FT-IR spectrum and electrochemical methods. Spectroscopic results indicated that Hb molecules retained its native structure in the composite film. The electrochemical behaviors of Hb in the modified electrode were carefully investigated and the electrochemical parameters were calculated. The modified electrode showed excellent electrocatalytic behaviors to the reduction of trichloroacetic acid (TCA) and the apparent Michaelis-Menten constants (KMapp) was calculated.2. A new electrochemical biosensor was fabricated by using chitosan (CTS), multi-walled carbon nanotubes (MWCNTs), ionic liquid (IL) and hemoglobin (Hb) nanocomposite materials modified carbon ionic liquid electrode (CILE) as the working electrode. CILE was prepared by using 1-ethyl-3-methylimidazolium ethylsulphate ([EMIM]EtOSO3) as the binder with graphite powder. Hb was immobilized on the surface of CILE with the mixture of CTS, MWCNTs, IL to fabricate the modified electrode of CTS-Hb-CNT-IL/CILE. The characteristics of Hb in the modified films were investigated by UV-vis spectrum and FT-IR spectrum, which indicated that Hb in the film can retain its native structure. The EIS experiment showed the high conductivity of the modified film, which indicated the synergistic effect of CNTs and IL present in the composite film further accelerated the electron transfer rate. A pair of well-defined and quasi-reversible cyclic voltammetric peaks appeared and the electrochemical parameters were calculated. The modified electrodes showed excellent electrocatalytic behaviors to the reduction of trichloroacetic acid (TCA) and sodium nitrite (NaNO2) with the apparent Michaelis-Menten constants (KMapp) calculated, respectively.3. Two CILEs were constructed based on the substitute of paraffin with 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM]EtOSO3) and N-butylpyridinium hexafluorophosphate (BPPF6) to mix with graphite powder, respectively. Horseradish peroxidase (HRP) and myoglobin (Mb) were immobilized on the surface of CILE with different film-forming substrates such as dextran (De), sodium alginate (SA) and different nanomaterials such as V2O5 and Fe2O3 to fabricate different modified electrodes denoted as De-HRP-V2O5-IL/CILE and SA-Mb-Fe2O3-IL/CILE. The characteristics of these modified electrodes were investigated by UV-Vis spectrum, scan electron microscopy (SEM), FT-IR spectrum and electrochemical methods. The modified electrodes showed excellent electrocatalytic behaviors to the reduction of trichloroacetic acid (TCA). Under the optimum conditions, a linear relationship between the catalytic reduction peak currents and the TCA concentration was obtained in the range from 0.4 to 16.0 mmol/L and 0.6-12.0 mmol/L with the detection limit (3σ) as 0.3 and 0.4 mmol/L, respectively.