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