Investigation Of Electrochemical Biosensor Based On Boron-Doped Diamond And Newly Synthesized Nano-Material

1. Boron-doped diamond (BDD) electrodes have recently attracted considerable interest, especially for electrochemical analysis due to its outstanding characteristics: (1) very low background current density; (2) a wide potential window in aqueous solution; (3) good activity toward some redox analytes without any pretreatment; (4) long term response stability. At present, the investigation of BDD electrode is at a starting phase. References have reported with respect to some chemicals electrochemical activity on bare BDD electrode. But the biosensors based on BDD electrode have rarely developed and need the further investigation. In chapter 2-6, we fully utilized the characteristics of the deficient electron structure (carbon sp3 and boron) and easy oxidation introducing -OH on BDD electrode surface to preparation of biosensors, which were used to investigate the electrochemistry of biological molecules and be rapid, sensitive. The detail materials are shown as follows: (1) The preparation and electrochemical characteristics of oxidized boron-doped diamond electrode modified by ruthenium tris (2, 2') bipyridyl (Ru(bpy)33+) are described (In chapter 2). The BDD electrode was subjected to electrochemical oxidation in a 1 mol/L H2SO4 aqueous solution to introduce hydroxyl groups to its surface. The hydroxyl groups thus generated were electrochemically modified with Ru(bpy)33+ in Ru(bpy)33+ solution. The Ru(bpy)33+ modified boron-doped diamond electrode has strong electrocatalytic effects on the redox of vitamin B6 (VB6). In the presence of a large amount of vitamin B1 (VB1) and vitamin B2 (VB2), the redox peak potentials of VB6 shift slightly while VB1 and VB2 are not reduced or oxidized on the modified electrode. A sensitive square wave voltammetric response for VB6 is obtained covering a linear range from 2.8×10-7 to 3.7×10-4 mol/L with a detection limit of 6.3×10-8 mol/L in the presence of a large amount of VB1 and VB2. The determination of VB6 in tablets using this modified electrode showed promising results compared with the traditional methods. (2) Catechins are strong autoxidants in alkali solution. In chapter 3, we for the first time developed a electrochemical method based on ruthenium tris (2, 2') bipyridyl (Ru(bpy)33+) modified boron-doped diamond (BDD) electrode to investigate the electrochemical reduction of catechin autoxidation intermediate which exists steadily as verified by the electron spin resonance (ESR) evidence. Ru(bpy)33+ film on the BDD electrode can accelerate formation of the intermediate, due to Ru(bpy)33+ working as a one-electron oxidant. Carbon sp3 and boron possess the deficient electron structure, which is subject to combine with free radical intermediates to be accumulated on BDD electrode surface. So the reduction process of catechin autoxidation intermediate can be monitored on the Ru(bpy)33+ modified boron-doped diamond (BDD) electrode. The reduction peak potential was observed at –855.5 mV. Moreover, the effects of pH, ascorbic acid, Cu2+, Fe2+ and autoxidation time are investigated, demonstrating the reduction peak being really the reduction of catechin autoxidation intermediate. Sensitive amperometric response were obtained covering linear range 3.3×10-7 mol/L ～ 1.6×10-4 mol/L. The determination of catechin sample using this method shows satisfactory results compared with the traditional methods. (3) The direct detection of clenbuterol (CL) in pig liver without any extraction separation using pyrrole-DNA modified boron-doped diamond (BDD) electrode is reported (In chapter 4). The BDD electrode surface was electrochemically modified with pyrrole polymer. After being rinsed carefully with doubly distilled water, the electrodes were immersed in DNA solution to assemble DNA membrane on pyrrole polymer. The pyrrole-DNA modified BDD electrode has strong electrocatalytic effects on the redox reaction of CL. A sensitive cyclic voltammetric response for CL is obtained covering a linear range from 3.4×10-6 mol/L to 5×10-4 mol/L with a detection limit of 8.5×10-7 mol/L. The mean recovery the CL in pig liver sample solution is 102.7% and a good reproducibility is obtained. (4) An tyrosinase biosensor based on a boron-doped diamond (BDD) electrode as the base electrode has been developed (In chapter 5). BDD was treated with p-amino phenol then coated with a tyrosinase film cross-linked with glutaraldehyde. The enzyme biosensor catalyzes phenolic compounds to get quinines which can be reduced to produce reduction current. The enzyme electrode provided a linear response to catechol over a concentration range of 1.0×10-8 to 1.0×10-5 mol/L with a detection limit of 5.2×10-9 mol/L. The enzyme biosensor has good responses to phenol and p-cresol, the linear calibration ranges to them are 5.0×10-8～×2.0×10-5mol/L and 5.0×10-8～5.0×10-6mol/L, respectively. The biosensor was evaluated with satisfactory analytical performance in terms of the repeatability and stability. (5) The electrochemical assay of bromide and iodide ions at boron-doped diamond (BDD) electrode was investigated (In chapter 6). The BDD electrode exhibited well-resolved and irreversible reduction voltammograms, while the GC electrode provided only ill-defined response. Cyclic voltammetric signals at BDD electrode for Br-and I-were observed at 561 mV and 125 mV vs SCE; the values shifted negatively for 228.7 mV and 187.5 mV respectively, comparing to those at GC electrode. Sensitive amperometric responses for Br-and I-were obtained covering linear range 6.7×10-7 ～ 1.0×10-3 mol/L and 1.3×10-8 ～ 1.0×10-3 mol/L with 5.3×10-7 mol/L and 1.7×10-9 mol/L, respectively, under the optimum pH and applied potential. The amperometric response was very reproducible and stable with satisfactory recovery results.2. In recent years, synthesis of nanocomposites has attracted great interest of researchers. Nanostructured materials often possess a combination of physical and mechanical properties not present in conventional composites. But few further researches on microstructure of the nanocomposite were published in the past years. The late three chapters in this paper investigate the synthesis of three kinds of nanocomposite and discuss their electrochemical characteristics. Further more, they were used for preparation of biosensors. The detail materials are shown as follows: (6) Polyaniline-intercalated graphite oxide nanocomposite(PAI/GO) was synthesized by a procedure similar to an exfoliation/adsorption process (in chapter 7). The micro-structure and conductivity of PAI/GO were investigated using Fourier transform infrared spectrometer (FT-IR), X-ray diffractometer (XRD) , transimission electron microscope (TEM) and atomic force microscope(AFM). The results show that polyaniline intercalated between interlayers of GO, changed structure of GO greatly forming nanograins in chain helix-like form. The resulting nanocomposite enveloped in carbon paste electrode showed electrochemical activity and two sharp peak potentials of square wave voltammometry (SWV) at 668.0mV and 207.0 mV. Such characteristics of PAI/GO have been employed to investigate the interaction between DNA and PAI/GO. The results indicate that single-strand DNA (ssDNA) and double-strand DNA(dsDNA) can change redox of PAI/GO on PAI/GO modified the carbon paste electrode (CPE), and the modified electrode has strong electro-catalytic effect on the redox of ssDNA and dsDNA. The SWV redox potentials of ssDNA and dsDNA are 90.99 mV and 18 mV, respectively, at PAI/GO modified CPE. A sensitive SWV response between ssDNA and the modified electrode without immobilized ssDNA is obtained covering linear range from 34 μg/mL to 241 μg/mL. And the modified electrode immobilized by ssDNA can be utilized to monitor the hybridization and detect complementary ssDNA, covering linear range from 275 μg/mL to 551 μg/mL. (7) C/Fe nanocomposite (CFN) was synthesized by a procedure similar to an exfoliation/adsorption process to intercalate Fe3+ into graphite oxide layers and be reduced in a H2 atmosphere (in chapter 8). The results of X-ray diffractometry (XRD) and transimission electron microscopy (TEM) show that the form of CFN is carbon nanotube-Fe nanoparticle compose with α-Fe distributed on the nanotube wall. Paste electrode has been constructed using CFN mixed with paraffin. The electrochemical characteristics of such carbon-Fe nanocomposite paste electrode (CFNPE) has been compared with that of carbon paste electrode (CPE) and evaluated with respect to the electrochemistry of potassium ferricyanide, ascorbic acid and cysteine by cyclic voltammetry. CFNPE can accelerate the electron-transfer to improve the electrochemical reaction reversibility. To fabricate the third-generation glucosebiosensor, glucose oxidase (GOD) was immobilized on CFNPE surface with Nafion covered after a pretreatment. The bionsor have great effect on glucose redox processes. After being added glucose, the reduction current increase greatly. It is found that the linear range covers from 6.7×10-6 to 1.0×10-2 mol/L with a detection limit of 3.2×10-6 mol/L. Oxygen, ascorbic acid and uric acid have no interference with the glucose detection. The determination of glucose in serum using this biosensor showed promising results compared with the traditional methods. (8)L-cysteine-nanogold nanocomposite (CNN) was synthesized by a procedure similar to self-assembled process to L-cysteine on nanogold. The results of scan electron microscopy (SEM) show that the form of CNN is abnormal. The diameter of CNN is around 30 nm. New electrode interface has been constructed using CNN to immobilize on Nafion membrane with the nanogridding structure on glassy carbon (GC) electrode, which is denoted as GC/Nafion-CNN (GC/NCNN). SEM results indicate that Nafion membrane is in favor of immobilization of CNN on electrode surface. The union phenomenon disappears and the distribution of CNN on Nafion membrane is well-proportioned. The electrochemical characteristics of such new electrode interface have been compared with that of GC electrode using PBS (pH 7) and potassium ferricyanide solution by cyclic voltammetry. The new electrode interface has low background current, and it has the good electron-transfer ability. Through compared with the cyclic voltammograms of different electrode interfaces (GC/Nafion, GC/Nafion-nanogold, GC/CNN, GC/Nafion-CNN) in Fe(CN6)3-solution, it is found that on GC/NCNN, the electrochemical redox reaction of Fe(CN6)3-is the best reversible one, that is to say, GC/NCNN has the highest ability of electron-transfer. And it further suggests that nanogridding structure of Nafion membrane play an important role for immobilizing CNN. Based on the good electrochemical properties, GC/NCNN was utilized to fabricate the third-generation horseradish peroxidase (HRP) biosensor. The HRP biosensor exhibits good response to H2O2, and displays the remarkable sensitivity and stability.