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Study On The Novel Enzyme Biosensors And Their Applications For The Analysis Of The Environmental Toxic Substances

Posted on:2006-01-24Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y H YangFull Text:PDF
GTID:1118360182970253Subject:Analytical Chemistry
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There has been considerable interest in studying biosensors because they can be applied to a wide range of analytical tasks, such as clinical diagnosis, bioassay, environmental monitoring and industrial analysis, etc. Growing concern to environmental protection brings forward higher requirement for environmental monitoring. Enzyme biosensor might be favorable for the environmental monitoring in situ, which have been applied as useful devices in monitoring environment and the detection of chemical weapon in a faster and cheaper way. In the design and fabrication of electrochemical biosensors, the development of a simple and effective strategy for the construction of sensitive membrane on the electrode is a crucial step. Aiming at the problems existed in immobilization technique of biosensors, this research developed a series of new immobilization matrixes and strategies in order to improve the activity of immobilized biomolecules, and developed H2O2, glucose and urea biosensors based on the new immobilization strategies. In addition, for the purpose of solving the interference problem in real sample analysis and the regeneration of the biosensor surface, some enzyme electrodes were constructed to determine environmental toxic agents based on the size-exclusion effect of polyelectrolyte and the adsorption-desorption effect of gold nanoparticles. Furthermore, a specific sequence DNA (K-ras gene which is high association with colorectal cancer) sensor based on HRP-labeled probe was constructed. The details are summarized as follows: 1. Four kinds of new immobilization matrixes and strategies have been developed and successfully applied to immobilize enzymes and fabricate electrochemical biosensors. (1) In chapter 2, we presented for the first time a novel enzyme biosensor based on nanoporous ZnO/chitosan inorganic–organic composite film as immobilization matrix with good stability. This material combined the advantage of inorganic species, ZnO, and organic polymer, chitosan. The immobilization of enzyme is based on the adsorption of nanoporous ZnO; therefore, the usage of glutaraldehyde can be avoided. Well-studied horseradish peroxidase (HRP) was chosen as a model enzyme. The results obtained from scanning electrode microscopy indicated that ZnO/chitosan film is porous and highly homogeneous. HRP can be effectively entrapped in the film with well retained bioactivity comparing to that of cross-linking HRP by glutaraldehyde. The linear range was 5.0×10-6 to 2.0× 10-3 mol L-1 with a sensitivity of 43.8 μA L mmol -1The detection limit was 2.5×10-6 mol L-1 at 3σ. (2) In our previous study, we found the dispersion of inorganic species needed to be improved. In chapter 3, a surface treated nanoporous ZrO2/Chitosan composite matrix was developed to fabricate the glucose biosensor. The nanoporous ZrO2 was treated with anionic surfactant (sodium dodecylbenzene sulfonate ) to improve the dispersion of ZrO2 in chitosan solution. The immobilization of enzyme is based on the adsorption on nanoporous ZrO2. GOx can be effectively entrapped in the film with a higher bioactivity compared to that of GOx cross-linked by glutaraldhyde. The linear range was 1.25×10-5 to 9.5× 10-3 mol L-1 with a detection limit of 1.0 ×10-5 mol L-1 and a sensitivity of 0.028 μA L mmol -1. (3) In chapter 4, a urease sensor based on PVC-NH2 matrix pH-sensitive membrane has been proposed. A new method of urease immobilization based on the strong lection-glycoprotein was developed, Con A and urease were assembled layer by layer. The effects of experimental conditions and the response characteristics of sensor have been investigated in detail. The sensor showed a linear response to urea from 6.9×10-5 to 1.0×10-3 mol L-1 with a detection limit of 4.5×10-5mol L-1. The sensor was applied to the determination of urea in milk samples with satisfactory results. (4) It is more difficult to achieve the direct electron transfer between enzyme and electrode comparing to common redox reaction involving protein (such as cytochrome). The electrochemical researches have paid their efforts toward this goal continuatively. Based on the excellent features of gold nanoparticles such as large surface area, high catalysis effect, strong adsorption ability, nice biocompatibility and conductive ability,a mediator-free horseradish peroxidase (HRP) biosensor has been developed in chapter 5. First, HRP was adsorbed by gold nanoparticles, then, it was immobilized on the bare gold electrode surface by activated concanavalin A (Con A). The experimental conditions were optimized. The electrode provided a linear response to H2O2 over a concentration range of 5.0×10-6~1.2×10-2 mol L-1 with a detection limit of 2.9×10-6 mol L-1. The system was applied to the determination of samples, the results were satisfactory. 2. Several kinds of enzyme electrodes have been used to detect environmental toxic agents based on the inhibition of toxic agents to enzymes. (1) An amperometric nicotine inhibition biosensor has been substantially simplified and used for determination of nicotine in real sample in chapter 6.Choline oxidase was immobilized on the carbon paste electrode through cross-linking with BSA by glutaraldehyde. Besides the use of single enzyme choline oxidase to replace bienzyme, the use of 1, 4-benzoquinone as an electron mediator makes it possible to avoid the use of oxygen or hydrogen peroxide sensor as the internal transducer. Nicotine inhibits the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode provided a linear response to nicotine over a concentration range of 2.0 ×10-5 to 9.2 ×10-4 mol L-1 with a detection limit of 1.0 ×10-5 mol L-1. The system was applied to the determination of samples, the results were satisfactory. (2) An amperometric horseradish peroxidase (HRP) inhibition biosensor based on a thiolate self-assembled monolayer (SAM) has been developed for the determination of sulfides in chapter 7. Cysteamine SAM was applied to the gold electrode surface and HRP was then immobilized on the electrode by cross-linking with glutaraldehyde. Cysteamine monlayer only can bind the relatively low mass loading of the enzyme, which makes it possible to achieve lower detection limit for the inhibitor. The determination of sulfides can be achieved in the range of 0.5 to 12.7 μmol L-1 with a detection limit of 0.3 μmol L-1. The kinetic parameters such as apparent Michaelis-/Menten constant ( K ampp) and maximum current density (Imax) with and without inhibitor have also been calculated and possible mechanism has been deduced according to the variation of K ampp The recovery of sulfides in a real samples was satisfactory. The low detection limit, low-cost, simple and fast fabrication of the sensor makes it superior to other techniques. (3) In chapter 8, an amperometric pesticides inhibition biosensor has been developed and used for the determination of pesticide in vegetable samples. To eliminate the interference of ascorbic acid, multilayer films of polyelectrolyte (chitosan/ polystyrensulfonate) were coated on the glass carbon electrode. As a modified substrate, acetylthiocholine was hydrolyzed by acetylcholinesterase and produced thiocholine which can be oxidized at + 700 mV versus SCE. Pesticide inhibits the activity of enzyme with an effect of decreasing of oxidation current. The electrode provided a linear response over a concentration range of 6.6×10-6 to 4.4×10-4 mol L-1 for phoxim with a detection limit of 1.3×10-6 mol L-1, over a range 1.0× 10-8 to 5.9× 10-7 mol L-1 for malathion; over a range of 8.6× 10-6 to 5.2× 10-4 mol L-1 for dimethoate. This biosensor has been used to determine pesticides in a real vegetable sample. (4) It was shown that the inhibition of urease by heavy-metal ions wasirreversible. The activity of urease is hard to recover entirely with regeneration solution after contacting inhibitors for many times. For this reason, a renewable urease biosensor is highly desired. In chapter 9,a renewable potentiometric urease inhibition biosensor based on self-assembled gold nanoparticles has been developed for the determination of mercury ions. Gold nanoparticles were chemically adsorbed on the PVC-NH2 matrix membrane pH electrode surface containing N, N-Didecylaminomethylbenzene (DAMAB) as a neutral carrier and urease was then immobilized on the gold nanoparticles. The linear range of determination of Hg2+ was 0.09 to 1.99 μmo L-1with a detection limit of 0.05 μmol L-1. The advantages of self-assembled immobilization are low detection limit, fast response and easy regeneration. 3.The incidence of colorectal cancer is very high in North America, west Europe, Australia and New Zealand, ranked the second in visceral cancer. The common used methods to detect colorectal cancer are not sensitive. Therefore, it is hard to diagnose colorectal cancer in its early stage, which is unfavorable for the treatment. In chapter 10, a high-sensitive DNA sensor based on HRP-labeled probe to detect specific sequence of K-ras gene which is associated with colorectal cancer was reported. Capture probe modified with –SH was firstly chemically adsorbed on the gold electrode through self-assembly. Then, the hybridization of a complementary nucleic acid (target DNA : K-ras gene) and HRP labeled oligonucleotide detection probe occurred in a sandwich way. Finally, H2O2 electroreduction current catalyzed by HRP was measured amperometrically in the presence of hydroquinon as mediator. The linear range is 1.17×10 -11~1.17×10 -7 mol L-1.The electrode with capture probe can be reused after regeneration in boiling water.
Keywords/Search Tags:Electrochemical biosensor, Nanoparticles, Inorganic-organic composite film, Biomaterials immobilization, enzyme inhibition, environmental toxic substances, colorectal cancer, DNA biosensors
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