The Assembly And Applications Of Bio-Composites Based On Redox Proteins And Nano-Materials

Direct electrochemistry of redox proteins has aroused great interest in biological and bioelectrochemistry fields. Study on direct electrochemistry of redox proteins is of great importance both for studying the intrinsic redox behaviors of proteins and fabricating new kinds of biosensors without addition of mediators.Nanotechnology is revolutionizing the development of electrochemical biosensors. The nanostructure materials can greatly enhance the active surface available for protein binding over geometrical area, and maintain the proteins' physiological activity without detectable denaturation. In this dissertation, electrodes are modified with several bioaffinitic nanomaterials (such as carbon nanotube, mesoporous silicate and quantum dots) to investigate the electrochemical and electrocatalytic properties of heme-proteins. These researches may open up new opportunities for designing novel third generation of biosensors. More details are summarized below:(1) A novel multi-walls carbon nanotubes (MWNTs) based hybrid material with sandwich structure (DNA-Hb-MWNTs), was fabricated by alternative assembly of hemoglobin (Hb) and DNA on MWNTs. The unique sandwich-like nanostructures not only provided a favorable microenvironment to keep the bioactivity of Hb but also prevented the bound protein leakage. Compared with Hb-MWNTs/GC electrode, the prepared DNA-Hb-MWNTs/GC electrode displayed better ability to keep the bioelectroactivity of Hb and exhibited high electrocatalytic performance to H₂O₂ with fast response, wide linear range, good sensitivity and excellent stability. Such avenue, which integrated of MWNTs, metalloproteins and DNA into an organized multi-components platform, maybe open up a new route for the fabrication of biosensors, biofuel cells and other bioelectrochemical devices.(2) An organized multi-components hybrid material, constructed by mesopores cellular foam silicate (MCFs) and quantum dots (QDs), was designed for the immobilization and biosensing of protein. The negative CdTe QDs were assembled on the surface of mesopores in amino group functionalized MCFs through electrostatic interaction to form QDs-MCFs hybrid material. Since QDs-MCFs possessed mesostructure, good biocompatibility and large surface area, Mb was consequently immobilized into the matrix to construct Mb-QDs-MCFs. Compared with the Mb-MCFs/GC electrode, the Mb-QDs-MCFs/GC electrode could not only realize enhanced direct electrochemistry but also display better sensitivity and wider linear range to the detection of hydrogen peroxide. The experiment results demonstrated that the hybrid matrix provided a biocompatible microenvironment for protein and supplied a necessary pathway for its direct electron transfer.(3) For the first time, we reported the covalent immobilization of Mb into amino-functionalized mesostructured cellular foams with large mesopores by the glutaraldehyde method. Compared with the physical adsorption method, such chemical binding method could efficiently avoid the leaching of the immobilized Mb. FTIR and UV-vis spectra demonstrated the native structure of Mb was well preserved after covalently immobilized in the mesoporous matrix. The Mb-MCFs/Nafion/GC electrode exhibited fast direct electron transfer and showed a good electrocatalytic performance to H₂O₂ with high sensitivity, wide linear range and low detection. The excellent electrochemical performance of the Mb-MCFs/Nafion/GC electrode was possibly contributed to biocompatibility of the mesoporous silicate together with the peculiar structure of MCFs, whose large pores with 30 nm avoid the mass-transfer limitations. Furthermore, due to compatible micro-environment and the protective effect provided by the mesoporous matrix, the Mb-MCFs/Nafion/GC electrode exhibited enhanced thermal stability that the immobilized Mb retained 82% initial activity after even heating at 80℃for 20 min.(4) A novel organoclay covalently modified with imidazolium salt, which was 1-propyl-3-methyl-imidazolium chloride-functionalized magnesium phyllosilicate (pmim⁺Cl^--clay), was firstly synthesized and used as an exfoliated precursor for the intercalation of hemoglobin (Hb). Intercalation was achieved by in-situ exfoliation of the organoclay in water without any additive and then re-assembly with negatively charged Hb under mild conditions. XRD profiles indicated that Hb was intercalated into the gallery of the hybrid matrix. TEM and SEM images showed that lamellar structure was well preserved during the synthetic and assembly procedures. The activity study indicated that the immobilized proteins preserved most of their catalytic activities. Furthermore, the immobilized Hb showed excellent high thermal stability under extremely thermal condition because of the synergetic affects of layered matrix and the imidazolium salt. The Hb-pmim⁺Cl^--clay retained about 72.5%, 50.5% of the apparent activity after thermal treatment at 70℃, 100℃for 20 min respectively.