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Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond
Kuang-Kai Liu1, Mei-Fang Chen, Po-Yi Chen
1Institute 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.
Carboxylated nanodiamond (cND) conjugated with alpha-bungarotoxin (α-BTX) effectively binds to the alpha7-nicotinic acetylcholine receptor (α7-nAChR). This cND-α-BTX complex retains biological activity, blocking receptor function and enabling visualization of binding.
Area of Science:
- Bioconjugation chemistry
- Nanomaterials science
- Neuroscience
Background:
- Biological molecule conjugation with nanoparticles is crucial for bio-imaging, bio-detection, and bio-sensing.
- Nanodiamonds offer excellent properties for bio-conjugation due to their nanometer size and surface chemistry.
- Alpha-bungarotoxin (α-BTX) is a neurotoxin that specifically blocks the alpha7-nicotinic acetylcholine receptor (α7-nAChR).
Purpose of the Study:
- To conjugate alpha-bungarotoxin (α-BTX) with carboxylated nanodiamond (cND) particles.
- To investigate the binding of cND-α-BTX complexes to α7-nAChR on cell membranes.
- To assess the biological activity and visualize the interaction of cND-α-BTX with α7-nAChR.
Main Methods:
- Carboxylation of nanodiamonds to create cND.
- Electrostatic conjugation of α-BTX to cND via non-covalent interactions.
- Characterization using SDS-PAGE, MALDI-TOF-MS, fluorescence microscopy, laser scanning confocal microscopy, two-electrode voltage-clamp recording, and flow cytometry.
Main Results:
- Successful non-covalent conjugation of α-BTX to cND was confirmed through various analytical techniques.
- cND-α-BTX complexes demonstrated binding to α7-nAChR expressed on Xenopus laevis oocytes and A549 cells.
- The cND-α-BTX complex effectively blocked choline-evoked α7-nAChR currents in a concentration-dependent manner.
- Fluorescence imaging and flow cytometry provided visual evidence and quantification of cND-α-BTX binding to cells.
Conclusions:
- Carboxylated nanodiamonds can be successfully conjugated with α-BTX while preserving its biological activity.
- The cND-α-BTX conjugate serves as a viable tool for studying α7-nAChR function and localization.
- This approach offers a visual system for observing the interaction between α-BTX and its target receptor, α7-nAChR.
