| The biosensor is a novel analytical measurement, which has been a most burgeoning and one of the most flourish research realms in the electrochemistry and electroanalytic chemistry, since it was developed in the middle of 70's. The advantages of the biosensor including their inexpensive instrumentation, simple-design, high-sensitivity, low-cost and fair selectivity attract substantial research efforts directed to the developments of some new electrochemical biosensors and have been used in clinical, environmental control, and food quality control, etc. In this thesis, the second generation amperometric hydrogen peroxide biosensor is prepared by conducting polymers and electron mediator. Meanwhile, exploring new materials and methods for preparation of new direct electron transfer biosensor with high response, broader linear range and without the aid of an electron mediator. UV-Vis spectra, Atomic force microscopy and electrochemical techniques are used as tools for studying the properties of interface. The main works are included as follows:The first part are hydrogen peroxide biosensors:1. A novel hydrogen peroxide biosensor has been prepared. HRP/ nano- Au/ thionine / nano -Au were constructed by the self-assembly technique on the electrode modified by poly 2-aminopyridine, which was electropolymerized onto a platinum (Pt) electrode to form a positively charged surface. The negatively charged nano-Au absorbed on poly 2-aminopyridine was employed as matrix to immobilize thionine as an electron mediator and horseradish peroxidase. The H2O2 biosensor prepared displayed a quick and sensitive response to hydrogen peroxide. The linear response of detection of the biosensor to hydrogen peroxide is in the concentration range of 6.0×10-7～1.3×10-3 mol·L-1 with a detection limit of 2.1×10-7mol·L-1. Moreover, the studied biosensor exhibited good repeatability and high selectivity.2. A mediator-free amperometric hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) enzyme on colloidal Au modified platinum (Pt) wire electrode, which was modified by poly 2,6-pyridinediamine(pPA). The modified process was characterized by electrochemical impedance spectroscopy (EIS) and the electrochemical characteristics of the biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The biosensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator, the linear range was 4.2×10-7～1.5×10-3 mol·L-1 (r=0.9977), with a detection limit of 1.4×10-7 mol·L-1. Moreover, the performance and factors influencing the resulted biosensor were studied in detail. The studied biosensor exhibited permselectivity, good stability and good fabrication reproducibility.3. Alternate adsorption of positively charged colloid-Au nanoparticles (nano-Au⊕) and negatively charged hemoglobin (Hb) from its pH 8.0 buffers on L-cysteine (L-cys) modified gold electrode resulted in the assembly of {Hb /nano-Au⊕}n layer-by-layer films /L-cys modified gold electrode. The nano-Au⊕was characterized by transmission electron micrograph (TEM) and microelectrophoresis. The modified electrode interface morphology was characterized by electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), cylic voltammograms (CV) and chronoamperometry. Direct electron transfer between hemoglobin and gold electrodes was studied and the apparent Michaelis-Menten constant (kmapp) of the modified electrode was evaluated to be 0.10 mmol·L-1. This approach for assembly of {Hb /nano-Au⊕}n layer-by-layer films showed higher surface concentration of electroactive proteins than the simple assembly monolayer protein method, and the activity of proteins in the nano-Au⊕films could be retained higher compared with the electropolymerization membrane. Furthermore, the proteins in nano-Au⊕films retained their near-native structure, and electrochemically catalyzed reduction of hydrogen peroxide, the linear range was from 2.1×10-8 to 1.2×10-3 mol·L-1 (r = 0.994) with a detection limit of 1.1×10-8 mol·L-1. The second part is ascorbic acid sensor:A novel electrochemical sensor for the detection of ascorbic acid (AA) has been constructed. Initially, a layer of poly-2,6-pyridinediamine (pPA) was fastened on the glassy carbon (GC) electrode surface through electro-polymerization technique, then negatively charged nano-Au and positively charged 2,6-pyridinediamine (PA) were alternately adsorbed on the pPA-modified GC electrode by layer-by-layer technique to prepare PA / naono-Au / pPA / GC modified electrode. The preparation and electrochemical characteristics of the modified electrode were described through cyclic voltammetry and chronoamperometry. The modified electrode exhibits enhanced electrocatalytic behavior to the oxidation of AA for 2,6-pridinediamine bilayer films provide abundant amino groups and positive charge, which promote the electron transfer between electrode and AA, and enhance the sensitivity of electrode. Moreover, the oxidation peak potential of ascorbic acid shifts from 0.5 V on the bare glassy carbon electrode to 0.2 V on the PA / nano-Au / pPA / GC modified electrode, i.e. the oxidation potential of ascorbic acid shifts by 0.3 V in the negative direction, exhibiting hard evidence for the electrocatalytic oxidation of ascorbic acid. Under optimal conditions, the modified electrode was highly sensitive to oxidation of AA with a detection of 3.3×10-8 mol·L-1 and the linear range was from 1.0×10-7to 5.5×10-3mol·L-1 (r = 0.9983) by cyclic voltammetry. |
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