Monitoring cholinesterase enzyme activity and inhibition in plasma using single-walled carbon nanotubes

Once acetylcholine has relayed its message by binding to the target receptors on nerve or muscle cell membranes, it must be swiftly cleaved by an enzyme called acetylcholinesterase to avoid the risk of overstimulation. This biological breakdown is as critical as the transmission itself, ensuring that our muscular responses are precisely timed and regulated.

Enzymes are proteins that, amongst other roles, help break down different molecules in our body and thus play a crucial role in numerous biological processes. Cholinesterase enzymes, in particular, break down acetylcholine and release choline molecules, which are then recycled to produce new acetylcholine molecules. However, when the activity of cholinesterase is slowed down or inhibited, the resulting acetylcholine buildup can trigger a cascade of health issues—from reduced heart rates to paralysis and, in severe cases, even death.

Inhibitors of cholinesterase are found in chemical weapons and nerve gases, like sarin, as well as certain pesticides used in farming. If pests and other animals, including humans, are exposed to these pesticides, this inhibits their cholinesterase, which can harm them. On the other hand, therapeutic doses of cholinesterase inhibitors are used for treatments of Alzheimer’s disease, Parkinson’s disease, and Myasthenia gravis. Therefore, carefully monitoring the activity and inhibition of cholinesterase enzymes is of great importance in medicine.

In a recent study published in Small, researchers from the group of Prof. Gili Bisker demonstrated the monitoring of the activity and inhibition of cholinesterase enzymes using single-walled carbon nanotubes (SWCNTs). SWCNTs are hollow cylinders of 1-2 nanometers in diameter (100,000 times smaller than the width of the human hair!) and hundreds of nanometers in length, and they fluoresce (i.e., emit light) in the near-infrared spectral range, where biological samples are mostly transparent. Importantly, the SWCNTs are biocompatible and can be easily functionalized by coating their surface with different molecules tailored for the desired application. In this research, the SWCNTs were functionalized by myristoylcholine (MC) molecules, which also contain the target site for cholinesterase enzymes, to mimic the natural acetylcholine. The MC acts like a type of decoy molecule to the natural one. Therefore, MC, just like acetylcholine, can also be cut by cholinesterase into two molecules – one of them being choline. When the MC-SWCNTs complexes were exposed to cholinesterase found in clinical plasma samples, the activity of cholinesterase led to significant changes in the near-infrared fluorescence of the SWCNTs, providing a tool to directly monitor the activity of cholinesterase. The fluorescence modulation could be quantified by a fluorescence microscope and a near-infrared detector, which can be correlated to the concentration of the active cholinesterase in the sample. Furthermore, the inhibition of cholinesterase by organophosphate pesticides could also be inferred from the lack of fluorescence modulation of the SWCNTs.

A key feature of this study is that SWCNTs could be used to effectively monitor cholinesterase activity and inhibition even in complex biological samples, like blood plasma, which contain other interfering molecules. This was enabled owing to the unique optical properties of the SWCNTs that fluoresce in the near-infrared region and the strategic functionalization of the SWCNTs with acetylcholine-mimicking molecules, ensuring the specificity and sensitivity of the SWCNT sensors.

In the scheme: The designed functionalization of SWCNTs for monitoring the activity and inhibition of cholinesterase through fluorescence modulation.

This study, led by Prof. Gili Bisker, is the first to use near-infrared emitting SWCNTs as unique tools to track enzyme activity and inhibition in complicated biological fluids such as plasma. This biomedical breakthrough opens the door to developing novel optical nanosensors using strategically modified SWCNTs for essential biomarkers in clinically relevant samples.

In the image: the team of researchers who led the research, from left to right: Dr. Adi Hendler-Neumark, Dr. Srestha Basu, and Prof. Gili Bisker.

The research was funded by the Israeli Ministry of Defense – CBRN Defense Division, the Israel Science Foundation, the Ministry of Science, Technology, and Space, Israel, the European Research Council, and the Zuckerman STEM Leadership Program. Moreover, the research was supported by several centers at Tel Aviv University, including the Marian Gertner Institute for Medical Nanosystems, the Center for Combatting Pandemics, the Zimin Institute for Engineering Solutions Advancing Better Lives, and the Nicholas and Elizabeth Slezak Super Center for Cardiac Research and Biomedical Engineering.