Study On The Adsorption, Catalysis And Self-assembly Calcification Of Biochemical Substances On Nanoporous Materials

The application of nanomaterial and related technology provides a new platform for the development of current biomedicine, functional material, energy science, and so on. Compared with the bulk materials, nanostructured materials composed of nanostructure building blocks possess unique properties in many aspects, such as magnetism, photoelectricity, chemical activity, catalysis, and so on. At present, nanobiodetection, synthesis of biomineralized material, nanoparticles functionalized by biological macromolecules (i.e., enzyme and DNA) and restricted reaction of biomolecules in nanometer space are the hotspots of current research. In this thesis, piezoelectric quartz crystal sensing combined with nanomaterial, membrane preparation technology and other surface analysis techniques has been employed to study the adsorption of bilirubin and bovine serum albumin, catalysis of urease and glucose oxidase, and biomimetic calcification of apatite on nanoporous materials, and we aim to cognize the special laws of biochemical action on nanoporous materials. The present study is essential for the development of new biochemical measurement technology, realization of high efficiency enzyme immobilization and catalysis, synthesis of biomineralized material, and so on. The main work could be summarized as follows:1. Deposition and accumulation of extra free bilirubin(BR) in body tissues will initiate disorders in the metabolism of BR and cause various diseases. The deposition process and affecting factors of BR on mimic cell membranes (phospholipid bilayer) were first investigated using QCM to know its mechanism of causing illness, and then mainly studied the adsorpton and photochemical decomposition of BR at the nanometer TiO₂, the purpose of the work would explore new way to treat these dicreases caused by BR. The adsorption of BR at nanometer TiO₂ was verified with UV/vis and IR spectra. QCM measurements indicated that the amount of adsorbed BR increased with increasing BR concentration and decreased with increasing temperature and ionic strength. The effect of pH was complicated, the amount of BR adsorbed increased slightly in the pH range of 2-4, and then increased rapidly in the pH range of 4-8, finally decreased at pH > 8. The photodegradation of adsorbed BR at nanometer TiO₂ during UV illumination showed that the TiO₂ films could be regenerated and used repeatedly. At present, the removal of BR by naometer TiO₂ films is only referring to laboratory use, and not for practical use.2. The bovine serum albumin (BSA) and urease imprinted TiO₂ films were prepared via surface sol-gel process using nano-sized TiO₂ sol as imprinted matrix. QCM study indicated that the imprinted TiO₂ film possessed good stability, the adsorption behavior of imprinted molecules onto non-imprinted and imprinted TiO₂ films fitted into Langmuir and Allosteric model respectively. The adsorption amount of imprinted molecules onto imprinted film increased with the increasing concentration and pH, while decreased with the increase of ionic strength. By immobilizing urease to imprinted TiO₂ film modified at the surface of titanium silk, a cheap and miniaturized urea biosensor was developed. Potential measurements indicated that the obtained urea biosensor had a good stability, and exhibited shorter response time (25 s) and wider linear range (8μM-3 mM).3. A novel piezoelectric urea biosensor has been developed for urea determination, based on the immobilization of urease to nanoporous alumina membranes prepared by two-step anodization. The ESPS/FIA monitoring indicated that the enzymes immobilized into porous alumina possessed high activity. Factors affecting urease immobilization were discussed and the optimized immobilization conditions obtained were pH of 7.5, urease concentration of 2.0 mg/mL, temperature of 25℃, immobilization time of 2.5 hours and relatively big pore dimension. In addition, it was observed that the urea biosensor prepared by glutaraldehyde reticulation for 60 min and followed by chitosan coating exhibited shorter response time (30 s), lower detection limit (0.2μM), wider linear range (0.5μM-3 mM), high selectivity (0.92-1.03), better reproducibility (S.D. = 0.02, n = 6) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The practical application of the urea biosensor not only demonstrated the feasibility of urea detection in urine sample, but also meant that a urea biosensor with low cost and anti-jamming was obtained in our study. Such sensors might be widely applied to medical and environmental fields in the future.4. Magnetic single-enzyme nanoparticles (SENs) encapsulated within a composite inorganic/organic polymer network were fabricated via the surface modification and in situ aqueous polymerization of separate enzyme molecule. The analyses of TEM, FTIR and XRD indicated that the synthesized SENs with about 50 nm in diameter were spherical in shape, quite polydisperse and the nanoshell entrapping enzyme was composed of Fe₃O₄/poly(pyrrole-N-propylsulfonic acid) composites. Electrical and magnetic measurements revealed that the magnetic SENs had a conductivity of 2.7×10^-3S.cm^-1, and were superparamagnetic with a saturation magnetization of 14.5 emu.g^-1 and a coercive force of 60 Oe. Compared with free enzyme, encapsulated enzyme exhibited a strong tolerance to the variation of solution pH and temperature, organic solvent and long-term storage, thus showing significantly enhanced enzyme performance and stability. The magnetic SENs with high activity and stability would find potential applications in many fields, such as biological detection and sensing, enzymatic catalysis and so on.5. The adsorption and photochemical reduction process of Cu(â…¡) and Hg(â…¡) at the surface of nanometer TiO₂ were investigated using in situ quartz crystal microbalance (QCM). It was found that the adsorption of Cu(â…¡) onto active sites of nanocrystalline fit the pseudo-second-order reaction reaction, and that the rate constant of the reaction was estimated about 0.09 g·mmol^-1·min^-1; whereas the adsorption equilibrium constant of Hg(â…¡) was about 3.9×10⁵ L.mol^-1 based on the pseudo-first-order kinetic model. The adsorption amount of Cu(â…¡) and Hg(â…¡) depended on pH,concentration and coexisting anions, and the saturated amounts of adsorbed Cu(â…¡) and Hg(â…¡) were approximately 1.5 and 0.85 mmol·g^-1 at pH 4, respectively. During UV illumination, the frequency of QCM decreased gradually, which meaned the photoreduction deposition of Cu(â…¡) from the solution; whereas as for Hg(â…¡), at the initial stage of UV illumination, the protons produced in photodegradative reactions of water could cause the desorption of adsorbed Hg(â…¡) from the surface of TiO₂, and the degree of desorption increased with the increase of both the concentration of Hg(â…¡) and pH value of solution, then the frequency decreased due to the strength of photochemical deposit reaction of Hg(â…¡). In addition, the photodeposition rates of Cu(â…¡) and Hg(â…¡) increased with increasing pH of solution, and the rate of photoreduction enhanced significantly in the presence of the organisms.6. Based on the study of physical and chemical process of Cu(â…¡), Hg(â…¡) ions at the interface, the nucleation, growth and crystal of apatite induced by negatively charged nanometer TiO₂ coatings soaked in simulated body fluid (SBF) were investigated using SEM, FTIR, XRD, EDX and QCM. Two different stages were clearly observed in the process of apatite formation, indicating two different kinetic processes. At the first stage, the calcium ions in SBF were initially attracted to the negatively charged TiO₂ surface, and then the calcium titanate formed at the interface combined with phosphate ions, consequently forming apatite nuclei. After the nucleation, the TiO₂ surface did not act as the center of nucleation, and the apatite formed at the first stage became the new center of nucleation and growth; the calcium ions, phosphate ions and other minor ions (i.e., CO₃^2- and Mg²⁺) in supersaturated SBF deposited spontaneously on the original apatite coatings to form apatite precipitates. In terms of the in situ frequency shifts, the growth rate constants of apatite (K₁ and K₂) were estimated respectively at two different stages. It was found that the reaction rate at the first stage was obviously higher than that at the second stage.