Application Of Novel Room Temperature Ionic Liquids And Nanomaterials In The Third-Generation Biosensors Based On Direct Electron Transfer

In this dissertation, the third-generation electrochemical biosensors based on direct electron transfer between the enzyme and the underlying electrode were fabricated and developed, which may be widely used in chemical, biological, clinical, food, and environmental fields. Room temperature ionic liquids (RTILs) and nanomaterials have grabbed researchers' interest in recent years because of their unique properties and potential applications in many areas. However, the applications of these two kinds of novel materials in biosensors are still in the beginning. There are many unknown fields waiting for scientists to explore. We explored the application of RTILs and nanomaterials in the fabrication of the third-generation biosensors, and the performance of these novel materials and the resulting biosensors was evaluated. These researches may open up new opportunities for designing novel biosensors and enhancing the performance of existing bioanalytical devices. The main points are summarized as follows:1. A novel composite material based on RTIL (1-butyl-3-methylimidazolium tetrafluoroborate) and chitosan (biopolymer) was obtained for the first time. Hemoglobin (Hb) and horseradish peroxidase (HRP) were chosen as model metalloenzymes to investigate the composite material. Results revealed that the composite material had good biocompatibility for entrapped enzymes, and enhanced thermal stability of entrapped enzymes was observed. Furthermore, dramatically facilitated electron transfer between the metalloenzymes and the underlying electrode was achieved, which may be due to the good ionic conductivity of RTIL and the interaction between the composite and the metalloenzyme (especially the formation of numerous hydrogen bond between the RTIL and the metalloenzyme). The efficient direct electron transfer between the metalloenzymes and the electrode offers excellent prospect for designing third-generation biosensor with good performance. The biosensors based the composite material displayed good performance for the detection of environmental pollutant (trichloroacetic acid) and biological intermediate (hydrogen peroxide). This is the first successful example to use RTIL-based biopolymer for the fabrication of third-generation biosensor. The entrapment of RTILs in chitosan may open up new opportunities for the application of RTILs in many fields (such as biosensors, biocatalysis and solid-state electronics). The RTIL-based composite material is a promising matrix for the immobilization of metalloenzymes, which provides a new platform for the study of redox-active proteins, and may find wide potential applications in the fabrication of the third-generation biosensors.2. Considering the desirable properties of nanomaterials, we synthesized and selected several kinds of nanomaterials with novel nanostructure and properties. These nanomaterials were used for the first time to fabricate nanobiosensors and the biosensing performance of these nanomaterials was investigated. We studied the effect of biocompatible nanostructured film on the bioactivity of biomolecules and their role in the immobilization of biomolecules, explored the effect of composition, size, structure, functional group, biocompatibility and signal transducing ability of nanomaterials on the direct electron transfer and bioactivity of metalloenzymes, which may establish a foundation for the construction of the third-generation biosensor based on the direct electron transfer. Three kinds of representative nanomatetials were explored.(1) Hydroxyl-containing antimony oxide bromide nanorodsA hydroxyl-containing antimony oxide bromide (AOB) nanorods was synthesized and explored for the application in biosensors for the first time. The AOB nanorods contained abundant functional hydroxyl groups, which was advantageous for the immobilization of biomolecules because the aqueous-like environment provided by the hydrophilic surfaces could stabilize the immobilized proteins. The AOB nanorods were combined with chitosan to form an organic-inorganic hybrid material, which was used as an immobilization matrix to entrap horseradish peroxidase. With advantages of organic-inorganic hybrid materials, dramatically facilitated direct electron transfer and excellent bioactivity could be readily achieved by entrapping metalloenzymes into the Chitosan-AOB composite. The prepared reagentless mediator-free third-generation biosensor displayed good performance along with good long-term stability. The AOB nanorods based composite material could be used efficiently for the entrapment of other proteins, and may find wide potential applications in biosensors, biocatalysis, bioelectronics and biomedical devices.(2) Layered spongy Co₃O₄ nanoflakesLayered Co₃O₄ nanoflakes with spongy nanostructure were synthesized for the first time. The Co₃O₄ nanoflakes were integrated with conductive polymer to be used as an enzyme immobilization matrix, the large surface area of which could greatly enhance the active surface area available for protein binding. Besides, the layered porous structure of the nanomaterial could provide a protective microenvironment for the immobilized enzymes to retain their bioactivity; meanwhile it provided convenience for the substrate to access the immobilized enzyme. Fast direct electron transfer between hemoglobin and the underlying electrode (k_s=2.9 s^-1) was achieved on the Co₃O₄-based biosensing interface. The small apparent Michaelis-Menten constant (0.136 mM) and the high sensitivity (396 mA cm^-2 M^-1) of the enzyme electrode indicated that Hb in the composite possessed high enzyme-like peroxidase activity. The biosensor based on Co₃O₄ nanostructure may provide a new vision for the development of third-generation nanobiosensor.(3) Carbon nanofibersCarbon nanofibers (CNFs), with typical diameters of ～80 run, were successfully combined with Nafion to construction a reagentless mediator-free Hb-based H₂O₂ biosensor. The results revealed that Hb retained its native secondary structure in the CNF-based composite film, and exhibited dramatically facilitated direct electron transfer and excellent bioactivity. The prepared H₂O₂ biosensor displayed high sensitivity, wide linear range, low detection limit, fast response, good reproducibility, selectivity and long-term stability. The CNF-based composites were proved to be an ideal biosensing platform for the construction of mediator-free biosensors, and may find wide potential applications in the near future.