Study On Electrochemical Biosensor Based On Nanomaterial Composite

Nanomaterials play an important role in the design and fabrication of a new generation of biosensors, due to their unique physical and chemical properties, which can provide a platform for the transformation of optical and electrical signal. As typical biomacromolecules and special catalysts, protein and enzyme have been widely applied to fabricate a biosensitive membrane to develop more sensitive and selective detection methods. Among them, direct electrochemistry and biosensing of redox proteins have been investigated more extensively and become a hot topic in the bioelectrochemistry and life science fields. In recent years, nanomaterials have been gradually introduced into electrochemical biosensors. The large surface area and excellent electrocatalytic activity of nanomaterials enable them to promote the direct electron transfer (DET) between electrode materials and redox active biomolecules and the electron transfer between protein and substrate. Additionally, the special structure and morphology of nanomaterials can prevent biomolecules from aggregating and make the orientation of deposited protein more suitable for retaining its physiological activity and stability. Since2000, ionic liquids (IL) have been applied in the fields of electrochemistry and electroanalysis. Ionic conductivity, wide electrochemical window, very low volatility, excellent thermal stability and biocompatibility are among the properties of ionic liquids making them attractive for electrode modification. In this thesis, inorganic nanomaterials, IL and chitosan were used to fabricate different kinds of modified electrodes. The direct electrochemistry of heme proteins and the application of the modified electrodes to biosensors were studied in details. The thesis can be summarized as follows:1. A carbon ionic liquid electrode (CILE) was fabricated by mixing carbon powder, ionic liquid1-butyl-3-methyl-imidazolium tetra fluoroborate ([BMIMIBF4) and paraffin together. By combing the flower-like SnS2nanoparticles synthesized through hydrothermal process with [BMIM]BF4, a composite was fabricated and further used to immobilize hemoglobin (Hb) on the CILE. The structure and morphology of SnS2nanoflower was characterized by scanning electron microscopy (SEM), Energy Dispersive X-Ray (EDX) and X-ray powder diffraction (XRD). Ultraviolet-visible (UV-vis) and Fourier transform infrared (FTIR) spectroscopic results indicated that Hb molecules retained the native structure in the SnS2-[BMIM]BF4composite. A couple of well-defined redox peaks of Hb appeared on cyclic voltammogram of Nafion/Hb-SnS2-IL/CILE, which indicates that the direct electron transfer between Hb and the underlying electrode was realized. The electrochemical parameter of electron transfer coefficient (a) was calculated to be0.387, and heterogeneous electron transfer rate constant (ks) was0.725s-1. The fabricated electrode showed good electrocatalytic ability for the reduction of trichloroacetic acid (TCA) with a wider linear range from0.8to21.0mmol L-1and a lower detection limit as0.27mmol L-1(3σ), due to the the synergistic effects of SnS2nanoflower and IL.2. Carbon coated Fe3O4nanospindles (C@Fe3O4) were synthesized by partial reduction of monodispersed hematite nanospindles with carbon coatings. A novel modified electrode was prepared by applying carbon coated Fe3O4nanospindles, myoglobin (Mb), ionic liquid (IL) l-ethyl-3-methylimidazolium ethylsulfate ([EMIM]EtOSO3) and chitosan (CTS) step by step on the surface of CILE with another ionic liquid1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4) as a binder. UV-vis absorption and FTIR spectra results indicated that Mb remained its native structure in the composite film. Electrochemical behavior of the CTS/IL/Mb/C@Fe3O4/CILE was investigated with the presence of a pair of well-defined redox peak, which indicated the direct electron transfer of Mb was realized with the underlying electrode. The fabricated CTS/IL/Mb/C@Fe3O4/CILE showed good electrocatalytic activity for the reduction of TCA with a linear range from1.0to20.0mmol L-1, and a detection limit as0.3337mmol L-1(3a). The apparent Michaelis-Menten constant was estimated to be1.390mmol L-1. Furthermore, the biosensor possesses satisfactory stability and good reproducibility.3. Hierarchically ordered macro-mesoporous anatase TiO2with lamellar morphology was successfully prepared by using natural rose petals and triblock copolymer EO20P03oE02o (P123) as dual templates. In the synthetic process, P123was employed as structure-directing agent, and inexpensive and environment-friendly rose petals were used as a biotemplate for the fabrication of laminar macroporous structures. The structure and morphology of TiO2was characterized by SEM, transmission electron microscopy (TEM) and XRD. It indicates that the macro-mesoporous structure maintained perfectly after calcination at450℃. A novel modified electrode was prepared by droping IL, CTS and as made TiO2on the surface of glassy carbon electrode (GCE) layer by layer and then electro-deposition of Pt nanoparticles finally. The electrocatalytic behavior of the NO2-on the CTS-IL/TiO2/Pt/GCE was investigated by cyclic voltammetry (CV) and differential pulse voltvammetry (DPV). The proposed biosensor exhibited a good linear response in the range from1to14500μmol L-1, with a detection limit of0.87μmol L-1(3σ). The sensor was successfully applied for nitrite detection in milk and ham sausage samples with satisfactory results.4. Hierarchically ordered macro-mesoporous anatase TiO2with lamellar morphology was prepared by using natural rose petals and P123as dual templates, and Au nanoparticle (nano Au) sol-gel was synthesized successfully with sodium borohydride as a reducing agent and sodium citrate as a protective agent. By combing TiO2, nano Au, CTS and dibutylo-phthalate (DBP), a composite was fabricated to develop a Au/TiO2/DBP/CTS modified electrode. Finally, bovine serum albumin (BSA) was used to block the non-specific adsorption sites to obtain a competitive electrochemical immunosensor based on antigen-antibody specific interaction. The immunosensor achieved shows a low detection limit as0.063ng mL-1(3a) and a wide linear range from0.1to100000ng mL-1. Furthermore, the biosensor possessed satisfactory stability and good reproducibility.