New Nano-materials And Polymer As Applied To Sensing-interface Fabrication For Amperometric Biosensors

Electrochemical biosensors based on the specific recognition of biomaterials with the magnification function of electrochemical determination present some outstanding advantages including high sensitivity, nice selectivity, low cost and easy miniaturization. The key aspect of the development of the biosensors is the methodology of immobilization of biomolecules on the electrode surface. This dissertation focuses on developing new immobilization strategies of biomaterials for the purpose of improving the performance and long-term stability of biosensors. Phenolic compounds together with some bio-chemical species were chosen as the representative analytes in our sensor design research. Phenolic compounds are important contaminants in environment, which are mainly from wastewaters of industries and petroleum refining. Because of the detrimental effect of phenolic compounds on human health, the identification and quantification of such compounds are very important. The conventional methods of assay of these compounds suffer from low sensitivity, complicated sample pretreatment and unsuitable on-site monitoring. Accordingly, the development of chemical sensors that are simple, sensitive and suitable for field applications for these analytes is of considerable interest. The detailed materials are summarized as follows: 1. The new immobilization methods based on core-shell magnetic nanoparticles have been developed and successfully applied to immobilize enzymes or immunoreagents for fabricating amperometric biosensors. In chapter 2, a phenol biosensor was developed based on the immobilization of tyrosinase on the surface of modified magnetic MgFe2O4 nanoparticles. First, the solid-state reaction was used to synthesize magnetic MgFe2O4 nanoparticles, then core-shell magnetic nanoparticles (MgFe2O4-SiO2) was formed via SiO2 coating Thus tyrosinase was covalently attached onto the surface of silanized core-shell (MgFe2O4-SiO2) magnetic nanoparticles in the presence of glutaraldehyde, forming magnetic bio-nanoparticles. Finally the magnetic bio-nanoparticles were immobilized on the surface of carbon paste electrode in the presence of magnetic field. The resulting phenol biosensor produced fast and sensitive response towards phenol. The linear range for phenol determination was from 1 × 10-6 to 2.5 × 10-4 mol L-1 with a detection limit of 6.0 × 10-7 mol L-1, and a high sensitivity of 54.2 μA.?mmol-1 L was obtained. The biosensor was found to be stable over a month. Because the modified core-shell magnetic nanoparticles can attach antibody, in chapter 8, an amperometric immunosensor was developed based on antibody immobilizing on the surface of core-shell magnetic nanoparticles (CdFe2O4-SiO2). The immunoassay included following steps: the human IgG antibody was first covalently immobilized onto the surface of the modified core-shell magnetic nanoparticles, forming magnetic bio-nanoparticles, Then the resulting bio-magnetic nanoparticles were attached to the surface of carbon paste electrode with the help of a permanent magnet. A sandwich immunoassay was performed to determine the analyte IgG. That is to say, analyte IgG and HRP-labeled anti-IgG were successively immobilized onto the surface of magnetic bio-nanoparticle modified carbon paste electrode. Finally the electrochemical detection was conducted. The linear range for IgG assay was 0.51 ~ 30.17 μg mL-1 with a detection limit of 0.18 μg mL-1. The immunosensor showed no significant reduction in activity over 2 weeks. The determination procedure with core-shell (CdFe2O4-SiO2) magnetic nanoparticles showed some advantages, such as simple manipulation, easy modification with bio-molecule, low cost and the possibility of repeated regeneration. 2. Two kinds of novel strategies for forming active nano-Au interface on the gold electrode have been developed and successfully applied to immobilize tyrosinase for fabricating amperometric biosensors. In chapter 3, a tyrosinase biosensor was proposed based on enzyme-labeled nano-Au immobilized on cystamine/chitosan modified gold electrode. The biosensor exhibited a fast response (~ 20 s), and the order of the sensitivity of three phenolic compounds was as follows: p-cresol > phenol > catechol. The response of the biosensor decreased to 75 % of the initial response after two weeks when used every other day. In chapter 5, a phenol biosensor was developed based on 1,6-hexanedithiol and nano-Au self-assembled layers. The biosensor showed good and fast response (~ 10 s), and the sensitivities of the enzyme electrode for catechol, phenol and p-cresol were 26.7, 25.8 and 14.5 μA.??mmol-1 L, respectively. The enzyme electrode maintained 72 % of its original activity after intermittent use for one month when storing in PBS (pH 7.0) at 4 oC. 3. Nanomaterials have good biocompatibility. They can keep activity of biomolecules due to the desirable microenviroment, and enhance the direct electron transfer between the enzyme's active sites and the electrode. In chapter 4, a novel phenol biosensor was proposed by immobilizing tyrosinase to nano-ZnO particles. The low isoelectric point tyrosinase was first adsorbed on the surface of high isoelectric point nano-ZnO particles in the presence of electrostatic interactions,then immobilized on the glassy carbon electrode via the film-forming of chitosan. The resulting biosensor showed fast response and high sensitivity towards three phenolic compounds. For catechol, the sensitivity was 114 μA mmol-1 L, and the linear range was 8.0 × 10-8 ~ 7.5 × 10-5 mol L-1 with a dection limit of 0.02 μmol L-1. 4. In recent years, much interest has attracted to the use of sol-gel materials for biosensor construction. However, two notable drawbacks limited the practical application of pure Si sol-gel matrix in electrochemical biosensors, one was the brittleness and the other was the decrease of enzyme activity because of the presence of acid catalyst during sol-gel preparation. Developing some non-silica sol-gel materials is an effective way to solve the existing problems. In chapter 6, an amperometric biosensor was first reported based on new ZnO sol-gel film as an immobilization matrix of tyrosinase for the deterimination of phenolic compounds. The ZnO sol-gel film could overcome the brittleness of the pure sol-gel-derived silicate matrix and increased the long-term stability of the biosensor. Our results proved that ZnO sol-gel matrix provided an advantageous microenvironment in terms of its favorable isoelectric point for tyrosinase loading and the immobilized tyrosinase retained its activity to a large extent. The resulting biosensor showed good response towards phenol and the sensitivity was as high as 168 μA.?mmol-1 L. The linear range for phenol determination was from 1.5 × 10-7 to 4.0 × 10-5 mol L-1 with a detection limit of 8.0 × 10-8 mol L-1. 5. Poly(amidoamine) (PAMAM) dendrimers are dendrimers with amine terminated groups. The large area of dendrimers allows an increase in the number of immobilized functional units, producing "amplification effect", and thus increases the sensitivity of the biosensor. In chapter 7, a sensitive HRP biosensor combining fourth generation (G4) PAMAM dendrimers with nano-Au was proposed. PAMAM dendrimer contained lots of amine groups, which had affinity for nano-Au, so the nano-Au monolayers could be formed on the surface of PAMAM dendrimer/ cystamine modified gold electrode. By immobilizing HRP on the surface of nano-Au monolayers, a H2O2 biosensor could be developed. The resulting biosensor showed good and fast response towards H2O2, and the linear range was from 1 × 10-5 to 2.5 × 10-3 mol L-1 with a sensitivity of 0.53 A L mol-1 cm-2.