Studies Of Novel Techniques For Immobilization Of Biomolecules And Their Applications In Developing Biosensors

Electrochemical biosensors are widely used for the assay of biological analytes. The advantages of this approach including their simple-design, high-sensitivity, low-cost, sensitivity and inexpensive instrumentation attract substantial research efforts directed to the developments of some new electrochemical biosensors. However, the method of immobilization, the prevention or elimination of nonspecific interactions still remains to be the key steps. In this thesis, the electrochemical biosensors based on different immobilization methods and different transducers have been developed for protein determination. The main works and conclusions are included as follows:Part I investigated the hydrogen peroxide biosensor based on different mediator.1. A hydrogen peroxide biosensor was constructed by immobilizing of horseradish peroxidase (HRP) with gold nanoparticles (nano Au), which chemisorbed onto the thiol groups both inside the network and on the surface of three-dimensional sol-gel on a Prussian blue-modified platinium electrode. The introduction of silica sol-gel matrix could increase the assembled nanoparticles and proteins, but also avoid the leaching of mediator from the electrode. Under the optimized conditions, the linear calibration for H₂O₂ was obtained in the range 7.0×10^-6 to 6.6×10^-3mol.L^-1 with a detection of 3.0×10^-6 mol.L^-1 at a signal-to-noise ratio of 3. The studied biosensor exhibited high sensitivity, selectivity and stability.2. A novel approach by means of self-assembled technique and opposite-charged adsorption to immobilize horseradish peroxidase (HRP) on gold nanoparticles, which were immobilized on glassy carbon electrode modified with poly-2-aminobenzoic acid (PABA) and toluidine blue (TB). Aminobenzoic acid was electropolymerized to glassy carbon electrode surface for the first time, which used to electrostatic interaction with the positively charged TB. The effects of experimental conditions such as the polymerization time of poly-ABA, assembling-time for mediator, pH, temperature and applied potential on the electrode performance have been investigated. Under optimal conditions, the linear response of the sensor to H₂O₂ is in therange of 1.5><10'5 1.3><10'3molL'1 with a detection limit of 5.6x1 O^molL"1. The current response time is 10 s. The biosensor retained 83.6 % of its original activity after three weeks of use. Moreover, the studied biosensor exhibited good reproducibility, selectivity and high sensitivity.Part II studied the direct electrochemistry and electrocatalytic of the third-generation biosensor.The direct electrochemical behaviors of Cytochrome c (Cyt c), which layer-by- layer electrostatic adsorbed with gold nanoparticles on glassy carbon electrode modified with poly-2-aminobenzoic acid were investigated. The modified electrode showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for heme Fe (III)/Fe (II) redox couple in phosphate buffered saline solution (pH 6.5). The modified electrode showed a stable and significant catalytic activity for the electrochemical reduction of hydrogen peroxide. By varying the number of the layers, we can control the immobilized Cytochrome c amount thus enhance the linear range and sensitivity of the sensors. Under the optimized conditions, the linear response of detection to H2O2 is in the concentration range of 9.8x10"* to UxlO^molL*1 with a detection limit of 6.6x 10"8 mol-L*1.Part III investigated the novel immunosensor based on HRP instead of BSA as blocking agent.1. A new current amplified immunosensor for carcinoembryonic antigen (CEA) was demonstrated. The electrode was fabricated by positively charged toluidine blue coated on negatively charged poly-sulfanilic acid (PSAA) modified GCE through electrostatic interactions to assemble gold nanoparticles for immobilization of anti-CEA and horseradish peroxidase instead of bovine serum albumin (BSA) to block sites against nonspecific binding. The parameters, which influence the amperometric detection of immunoassay, included using HRP instead of BSA as blocking agent and amplify the current response;the choice of PSAA anionic precursor;the electrostatic adsorption time of TB mediator;the pH;the concentration of H2O2;the immunochemical incubation temperature and time were investigated. The CVs reduction current of the immunosensor changed linearly in two concentration ranges of CEA from 0.5 to 5.0 and 5.0 to 120.0 ng-mL'1 in presence of 0.3 mmol-L"1 H2O2 in analyte solution, and the detection limit was 0.2 ng-mL'1 at three times background noise. The proposed method is economical, efficient and potentially attractive for clinical immunoassays.2. A novel immunosensor for direct amperometric determination of ot-1-fetoprotein (AFP) was constructed by anti-AFP immobilized on multilayers of HRP/nano Au through electrostatic adsorption onto L-cysteine modified gold electrode surface. The design of the immunosensorsignificantly simplified the immunoassay procedure and amplified the amperometric reduction current through multilayers of HRP/nano Au, the assay format avoided the introducer of an electron transfer mediator, and nano Au efficiently retained the biological activity of protein. Under optimal conditions, the immunosensor was highly sensitive to AFP with a detection of 0.5 ng-mL"1 and the linear range was at two concentrations from 1 to 10 and from 10 to 200 ng-mL"1 with 0.7 mmol-L"1 H2O2 in analyte solution. More than 86.7 % of the results of the human serum samples obtained by this method were in agreement with those obtained by radioimmunoassay. So it would be a potential application on clinical determination of AFP level.