Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond

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 (?-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (?7-nAChR). The electrostatic binding of cND??-BTX was mediated by the negative charge of the cND and the positive charge of the ?-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 ?-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 ?-BTX presented a yellow color at the same location, which indicated that ?-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human ?7-nAChR proteins by microinjection with ?7-nAChR mRNA. The cND??-BTX complexes were bound to ?7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked ?7-nAChR-mediated inward currents of the oocytes were blocked by cND??-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND??-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated ?-BTX still preserves its biological activity in blocking the function of ?7-nAChR, and provide a visual system showing the binding of ?-BTX to ?7-nAChR.