title: Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond.
abstract: Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.
authors.affiliation: Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien 970, Taiwan. Biomedical Nanotechnology Laboratory, Tzu Chi University, Hualien 970, Taiwan. Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu 300, Taiwan.
authors.forename: Kuang-Kai
authors.initials: KK
authors.lastname: Liu
authors.forename: Mei-Fang
authors.initials: MF
authors.lastname: Chen
authors.forename: Po-Yi
authors.initials: PY
authors.lastname: Chen
authors.forename: Tony J F
authors.initials: TJ
authors.lastname: Lee
authors.forename: Chia-Liang
authors.initials: CL
authors.lastname: Cheng
authors.forename: Chia-Ching
authors.initials: CC
authors.lastname: Chang
authors.forename: Yen-Peng
authors.initials: YP
authors.lastname: Ho
authors.forename: Jui-I
authors.initials: JI
authors.lastname: Chao