The Application Of New Biomolecular Immobilization Methods And Materials In The Fabrication Of Biosensor

Electrochemical biosensors are widely used in food, clinical, industrial and environmental analyses because of their simple-design, high-sensitivity, low-cost, sensitivity and inexpensive instrumentation. However, the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability, sensitivity and selectivity of biosensor. In this thesis, the electrochemical biosensors based on different immobilization methods and materials have been developed in this paper. The main works and conclusions are included as follows:1. A highly efficient H₂O₂ biosensor was fabricated based on the mixture of Horseradish Peroxidase (HRP), ZrO₂ sol-gel and nano-Au immobilized on gold electrode. ZrO₂ sol-gel had high biocompatibility feature and provide a suitable microenvironment to keep HPR bioactivity. The presence of nano-Au in matrix can effectively enhance electron transfer between enzyme and electrode since the formation of electrostatic complex of nano-Au-HRP. The biosensor demonstrated excellently electrocatalytical ability for H₂O₂. The linear range of biosensor for the detection of H₂O₂ was 7.0μmol/L to 3.9 mmol/L with a detective limit of 4.0μmol/L. The performance of biosensor and effects of applied potential, solution pH, temperature and interferents on the biosensor were examined in this paper.2. A novel hydrogen peroxide biosensor had been developed based on layer-by-layer (LBL) assembly of gold nanoparticles(nano-Au) and poly-diallyldimethylammonium chloride (PDDA) on polymerized o-phenylenediamine (o-PDA) film modified platinum electrode surface. The o-PDA film has been utilized to associate nano-Au with its amino groups and polycation PDDA bond to nano-Au for their electrostatic attraction. At last, hemoglobin (Hb) was immobilized on Au colloid by electrostatic adsorption. The immobilized Hb displayed excellent electrocatalytical response to the reduction of hydrogen peroxide. The performance and factors influencing the resulted biosensor were studied in detail. The resulted biosensor exhibited fast amperometric response (within 5 s) to H₂O₂. Linear calibration for H₂O₂ was obtained in the range of 1.3μmol/L to 1.4 m mol/L with a detection limit of 0.8μmol/L under the optimized conditions. Furthermore, the resulted biosensor exhibited high sensitivity, good reproducibility and long-term stability.3. A novel H₂O₂ biosensor is described which is based on immobilization of horseradish peroxidase (HRP) on DNA/ZrO₂/Au electrode. The DNA is attached via its 5' end to ZrO₂ and this provides a microenvironment for the immobilization of various biomolecules and promotes electron transfer between HRP and the electrode surface. Under optimized conditions, the biosensor reduced H₂O₂ linearly between 3.5μmol/L-10 mmol/L with a detection limit of 0.8μmol/L at a signal-to-noise ratio of 3. In addition, the developed biosensor shows an acceptable stability and repeatability. Importantly, the analytical methodology could be further developed for the immobilization of other proteins and biocompounds.4. A very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO₂ thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP).The HRP in HRP-ZrO₂ thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H₂O₂. Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO₂/Au electrode) showed a linear response to H₂O₂ over a concentration range from 0.02 mmol/L to 9.45 mmol/L with a detection limit of 2μmol/L based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K_M^app) was evaluated to be 8.01 mmol/L. What's more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.5. The preparation and performance of a good amperometric biosensor for hydrogen peroxide (H₂O₂) was fabricated based on immobilization of hemoglobin (Hb) on DNA/Poly-2,6- pyridinediamine (PPD) modified Au electrode. PPD thin films were firstly electro-deposited on Au electrode surface, which provide a well platform to attach negative charged DNA molecules by electrostatic attraction. The adsorbed DNA network provides good microenvironment for the immobilization ofbiomolecules and promotes electron transfer between the immobilized Hb and the electrode surface. Experimental conditions influencing the biosensor performance such as pH, potential and temperature were optimized and assessed. The proposed biosensor displayed a good electrocatalytic response to the reduction of H₂O₂ and the linear range is 1.7μmol/Lto 3 m mol/Lwith a detection limit of 1.0μmol/L based on the signal-to-noise ratio of 3 (S/N=3) under the optimized conditions. The Michaelis-Menten constant K_M^app of Hb immobilized on the electrode surface was found to be 0.8 mmol/L, the developed biosensor shows an acceptable sensitivity and stability.