Investigation On The Application Of TiO₂ Nanoparticles And Nanocompoites In Bioelectrochemistry

Titanium dioxide (TiO₂) is n-type semiconductor and has unique photocatalytic properties. Its principle is as follows: (1) When TiO₂ photocatalysts were illuminated under UV light with wavelengths of less than 385 nm, electrons in the valence band of the TiO₂ semiconductor nanoparticles are excited to jump to the conduction band, and create photo-induced electron-hole pairs at the surface of the TiO₂; (2) Under the action of electric field in space charge layer, the photoinduced electrons and holes separated; (3) The photogenerated holes can react with adsorbed hydroxyl ions (OH^-) or water (H₂O) to produce the highly reactive oxygen species (ROS) such as the radicals OH and HO₂; (4) The photoinduced electrons can react with oxygen vacancies to form superoxide ions (O₂^-). These ROS will react with organic substance, bacteria, virus, cancer cells, which results in the decomposition and damage of organism structure through a series of oxidation reaction. TiO₂ nanoparticle has important applications in catalysis, environmental protection, medicine and health, electronic industry because of its unique characteristics such as safety, little noxious and side effect, minor wound, excellent chemical stability, and low price.Cancer has become severe harm to people's health in our country. The rising incidence of cancer in the world demands an increase in effort towards the development of novel and effective therapy for killing cancer cells. TiO₂ photocatalytic oxidation killing cancer cells is an exploring therapy for cancer. As one kind of photosensitize, TiO₂ nanoparticle is an attractive minimal-invasive treatment reagent for cancers because of the localized phototoxic effect upon irradiation and has been successfully used to treat gastric cancer, colon carcinoma, and so on. It is important and worthwhile to explore further the photocatalytic killing effect of TiO₂．However, up till now, such researches on TiO₂ photocatalytic killing cancer cells have still been few. Furthermore, TiO₂ nanoparticles have some drawbacks in clinical use: (1) The high degree of recombination between photogenerated electrons and holes is a major limiting factor controlling the photocatalytic efficiency; (2) TiO₂ has insufficient selectivity and low efficiency resulted from lack of cell-specific accumulation of TiO₂ on cancer cells. In order to improve the selectivity and photocatalytic efficiency of TiO₂ on cancer cells, we took chemical modification methods. In addition, we studied other application of TiO₂ and Au/TiO₂ in bioelectrochemical area. The main results and conclusions are summarized as follows:1．Gold-capped TiO₂ (AU/TiO₂) nanocomposites with different Au ratio (1-4 wt%) were successfully prepared by gold deposition on the surface of TiO₂ nanoparticles (P25 or anatase TiO₂) using deposition-precipitation (DP) and chemical reduction methods. The synthesized Au/TiO₂ nanocomposites were characterized by X-ray reflection diffraction (XRD), transmission electron microscopy (TEM), inductively coupled plasma atom emission spectroscopy (ICP-AES) and UV-vis spectroscopy. The photocatalytic killing effect of TiO₂ nanoparticles and Au/TiO₂ nanocomposites on cancer cells was investigated quantitatively and compared using human colon carcinoma LoVo cells as a model. The experimental results show that in comparison with photoexcited pure TiO₂ nanoparticles, the modification of gold nanoparticles on the surface of the TiO₂ nanoparticles (P25 or anatase TiO₂) can greatly improve the photocatalytic killing effect on LoVo cancer cells. In the case of using culture medium containing 2 wt% Au/P25 TiO₂ nanocomposites, the LoVo cancer cells were totally photokilled within 100 min under the irradiation of UV light (λ=365nm, 1．8 mW/cm²) , while only 40% cancer cells were photokilled in the case using P25 TiO₂ nanoparticles. Furthermore, the noble metal amount on the TiO₂ surface influence the efficiency of the photocatalytic process and the maximum photocatalytic killing effect was obtained with about 2 wt% Au on TiO₂ sample.2．We firstly conjugated TiO₂ nanoparticles with a specific antibody against the carcinoembryonic antigen (CEA) of human LoVo cancer cells, which is useful for target accumulation of TiO₂ nanoparticles on LoVo cancer cells. Furthermore, we utilized electroporation to improve the delivery of antibody-TiO₂ bioconjugates into the cancer cells. The combination of electroporation and synthesized antibody-TiO₂ bioconjugates can improve the photokilling selectivity and efficiency of photoexcited TiO₂ on cancer cells. The experimental results demonstrate that highly cell-specific antibody-TiO₂ bioconjugates were achieved and delivered into human LoVo cancer cells using electroporation technique. Under UV light (365 nm) irradiation, 100% of the cancer cells were photokilled within 90 min, while in control, only 39% of the normal cells were killed. This combination method shows high cell-specificity and efficiency in photokilling cancer cells, indicating the potential of this bioconjugates as photosensitizes for photodynamic therapy. Furthermore, this method may be used to photokill various kinds of caner cells by using similar procedure, just need to change the corresponding antibodies.3．We synthesized ordered mesoporous TiO₂ using block copolymer EO₂₀ PO₇₀ EO₂₀ (P123) as the template. The mesoporous material presents high surface area (BET surface area: 208m²/g) and high ordered structure. The photocatalytic bactericidal behaviors of this mesoporous TiO₂ are firstly studied. Experimental results show that E.coli can be efficiently killed by the mesoporous TiO₂ under UV irradiation and the cell viability is only 10% of initial cell amount after 60min illumination. After 120min irradiation, E.coli can be completely killed. The effect of different TiO₂ loading on the inactivation of E.coli is investigated, revealing that the optimum concentration of TiO₂ is 1 mg/mL. Compared with other TiO₂ materials, ordered mesoporous TiO₂ shows its high photocatalytic bactericidal capability. After 60min irradiation, the ordered mesoporous TiO₂ can photokill 90% of E.coli, but the bactericidal efficiency of commercial bulk TiO₂ and nano-TiO₂ is 25% and 70%, respectively. The possible bactericidal mechanism is also discussed.4．Gold nanoparticles-modified TiO₂ nanocomposite (AuNP-TiO₂) was used to immobilize horseradish peroxidase (HRP) on a glassy carbon electrode surface for the construction of an amperometric hydrogen peroxide (H₂O₂) biosensor. The properties of HRP immobilized in the AuNP-TiO₂ film were characterized by the electrochemical methods. The HRP immobilized in the AuNP-TiO₂/GC electrode retained its bioactivity and exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at about-0．330 V versus saturated calomel electrode (SCE) in pH 7．0 buffers. Moreover, the HRP immobilized in AuNP-TiO₂/GC electrode exhibited a rapid electrocatalytical response (less than 2 s), a linear calibration range from 2．0μM to 280μM and a sensitivity of 16μA mM^-1 for monitoring of H₂O₂．The apparent Michaelis-Menten constant (K_m^app) of the biosensor was calculated to be 234μM. The good direct electrochemical behavior of HRP and electrocatalytical response to H₂O₂ reduction was due to the enhancement of specific surface area and the reduction of electron transfer resistance by the uniform deposition of gold nanoparticle on TiO₂ surface.5．Au/TiO₂ nanocomposites have been prepared by using high-pressure mercury lamp as the light source to illuminate the HAuCl₄/nano-TiO₂ solution. SEM, XRD and UV-Vis absorption spectrum confirmed the existence of the Au nanoparticles in the composite material. The formation mechanism of Au nanoparticles is proposed. The nano-Au/TiO₂ composite film modified ITO presented higher photocurrent than the pure TiO₂ film modified ITO. The improvement of this photoelectrochemical performance can be explained as the inhibition in charge recombination of photo-induced electrons and holes, and the improvement in interfacial charge-transfer kinetics at nano-Au/TiO₂ composite film. The nano-Au/semiconductor composite films have potential applications in photocatalytic system and photoelectrochemical solar cells.