Study On Tb³⁺-doped Nano-hydroxyapatite Bioluminescence Probe Materials

Biological probe materials could be widely used in cell analysis and drug analysis, and so on. In recent years, nano hydroxyapatite (abbreviated HA) with rare earth became a hot spot of research because of its labeled tracer effects and good biocompatibility. In this paper, spherical or short rod-like nano-HA fluorescent probe material with good dispersity was prepared. The main content and results were shown as follow:(1) Screened amino acid additives. HA nanomaterials were synthesized used hydrothermal method with hydrothermal temperature 150 ℃, hydrothermal time 20 h, pH 11, molar ratio Ca/P/amino acid=10/6/1 and adding different L-amino acids. In 20 kinds of L-amino acids, polar amino acids made HA nanomaterials smaller size of short rod-like particles and L-lysine helped to regulate and control anabolic spherical HA nanomaterials.(2) Used L-lysine as an additive, the influence of hydrothermal temperature, hydrothermal time, and the dose of L-lysine on the morphology and size of HA nanomaterials was tested. It was found that, as water temperature rose and time prolonged, the crystallinity of HA nanoparticles tended to rise, and its long rod-like morphology became shorter rod or sphere. The does of L-lysine had more obvious effected on the crystallinity, morphology and particle size of HA nanomaterials. When the addition amount of L-lysine is 2.11 mol/L, the shape of HA nanomaterials was basically sphere. Its crystallinity was high, and with even particle size of around 30 nm, which fit the particle size requirement of entering biological cell.(3) Used Tb3+ as doping ions, the influence of Tb3+ on the composition, structure, morphology, size and fluorescence properties of synthetic nanomaterial Tb-HA was inspected when the doping amount was 0～4 mol%. Results showed that undoped HA nanomaterial was sphere, and those four doped samples were mainly short rod, moreover, their crystallinities were lower. Fluorescence analysis showed that undoped HA nanomaterial had no fluorescence, while those four samples had green fluorescence with a strong emission peak at 491 nm and 545 nm. When the doping amount was 2 mol%, Tb-HA nanomaterial had the strongest fluorescence.(4) Co-cultured Tb-HA suspension whose Tb3+-dopted amount were 0 mol%,2 mol%,4 mol% with L929 mouse fibroblast cells, the cytotoxicity was examined. When the suspension concentration were 250 μg/mL,500 μg/mL and 1000 μg/mL and co-culture time was 12 h, cell viability was good, and cell inhibition rate was small, which in line with national biology medical devices standards. When the suspension concentration were 250 μg/mL,500 μg/mL,1000 μg/mL,1500μg/mL and 2000 μg/mL, and co-culture time was 24 h, cell viability was good, and cell inhibition rate was small, which was basically avirulent.(5) Co-cultured different amounts 2 mol% Tb-HA nanomaterials with Caco-2 colon cancer cells, cell endocytosis was inspected. Cell micrographs showed that in blank group and experimental group, Caco-2 cells were morphologically normal, and their elongation was good. Fluorescence micrographs showed no fluorescence in blank group, while there was fluorescence in experimental group. There was fluorescence after Tb-HA nanomaterials were swallowed by Caco-2 cells. Therefore, Tb-HA was hopeful to be fluorescent bio-probe.