FITC-Functionalized TiO 2 Nanoparticles for Simultaneous Neuron Imaging and in Cell Photocatalysis
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FITC-Functionalized TiO2 Nanoparticles for Simultaneous Neuron Imaging and in Cell Photocatalysis Tina Zhang1, Mary Ann Go2, Christian Stricker2, Vincent Daria2 and Antonio Tricoli1 1
Nanotechnology Research Laboratory, Research School of Engineering, College of Engineering and Computer Sciences, The Australian National University, Canberra, Australia 2
Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University, Canberra, Australia ABSTRACT Crystalline TiO2 nanoparticles were produced by scalable flame spray pyrolysis of organometallic solutions. A protocol is presented for the optimized functionalization of these particles with fluorescein isothiocyanate (FITC), an important biomedical dye via a lysine linker. The pH, stoichiometry and time for lysine reaction were determined for highest dye loading and minimized degree of polylysine formation. Acidic reaction conditions, low lysine concentration and short reaction times were found to meet this aim. The resulting particles were used for imaging single neurons, showing high fluorescence emission and ability for the particles to diffuse into small neuron structures such as dendrites. INTRODUCTION In recent years, the use of stimuli-responsive nanocomposites with biological end applications has greatly advanced due to progresses in the synthesis of novel functional nanocomposites[1]. Semiconductor nanoparticles such as TiO2 are particularly useful due to their photo and electro chemistry. TiO2 is the most efficient photocatalyst and is already synthesized by low cost, large-scale flame reactors[2]. Electron/hole separation in TiO2 can be triggered by ultraviolet light[3] and X-ray[4] having energies above the material band gap (3.0 eV and 3.2 eV for rutile and anatase, respectively). TiO2 surfaces are amphoteric[5], readily adsorbing both carboxylates[6] and amines,[7] and may therefore undergo diverse bonding configurations with linking molecules. Here, for the first time, we have injected TiO2-based nanocomposites into neuron cells for simultaneous imaging and photostimulation. The neuron is a convenient system in which to study this phenomenon due to the measurable potential difference across the cell membrane. A neuron has a resting potential of -70mV with a greater abundance of negative ions within the intracellular fluid. The ability to artificially change this potential and observe the neuron’s responses is of importance in neuroscience research. Currently, membrane depolarization can be achieved by direct current injection through intracellular pipette or extracellular application neurotransmitters (such as glutamate)[8].
EXPERIMENTAL Flame Synthesis and Characterization of TiO2 Nanoparticles A flame spray pyrolysis (FSP) reactor was used to synthesize the TiO2 nanoparticles using a liquid precursor containing 0.5 mol L−1 of titanium (IV) isopropoxide (TTIP, Aldrich, purity > 97%) in xylene (Fluka, purity > 98.5%), as previously discussed in details.[9] This solution was fed at 5 ml min−1 through the FSP du
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