Rational Design of Carbon Nanotube Sensors for Real-Time Monitoring of Cholinesterase Activity and Inhibition in Biological Fluids

Cholinesterase is a family of enzymes found in the central nervous system, responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid. This reaction is essential for allowing cholinergic neurons to return to their resting state after activation. Cholinesterase is considered one of the many critical enzymes necessary for the proper functioning of the human nervous system and they include acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE). If these enzymes malfunction, it may lead to neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or other nerve-related disorders. They are also targets for certain toxins, like pesticides or even chemical warfare agents, which can block their activity entirely and pose a life-threatening risk. Thus, the activity and levels of these enzymes are such a big deal, both for health and environmental safety. However, it has been really challenging to monitor the cholinesterase enzyme activity in the blood, and traditional methods, like the Ellman assay, which rely on chemical reactions to create a product with a certain color, face challenges in real-world samples because of interference from blood components like hemoglobin or proteins. On top of that, these methods are indirect, meaning they depend on secondary reactions, which can be a source of inaccuracies and added complexity. Moreover, when enzyme levels are super low, as they often are in biological samples, these older techniques may struggle to pick them up at all.

To solve this, a team of researchers led by Professor Gili Bisker from the Faculty of Engineering at Tel Aviv University, with Dr. Srestha Basu and Dr. Adi Hendler-Neumark, took a fresh approach. They used single-walled carbon nanotubes (SWCNTs)—tiny, tube-like structures that are incredibly stable and emit light in the near-infrared (NIR) range. These nanotubes were coated with myristoylcholine (MC), a molecule that is a substrate of cholinesterase enzymes, with which they can interact directly. When the enzymes break down the MC into choline, the nanotubes’ fluorescence changes (it fades), and this can provide a real-time signal that the enzyme is active. Unlike conventional methods, this sensor overcomes the usual interference from blood plasma and works directly without the need for extra steps or reactions. The use of NIR fluorescence enables the sensors to overcome interference from blood and tissue, delivering clear and reliable results even in complex biological fluids like plasma, giving the sensors a clear, superior signal to work with. Their study, published in Small, shows how this technology fills a big gap in enzyme monitoring. It’s faster, more accurate, and works even in complex samples like blood plasma—potentially a game-changer for diagnosing diseases, testing treatments, or monitoring environmental toxins.

To validate that these sensors were specific and precise, the researcher tested their specificity and precision with both types of cholinesterase: ACHE and BCHE. Sure enough, the nanotubes’ fluorescence dimmed every time these enzymes broke down the MC. To double-check, they used a separate technique, mass spectrometry, to confirm that choline was being released. These results made it clear: the sensors were reacting specifically to CHE activity, not just to random chemical noise. The sensors demonstrated exceptional sensitivity, with the ability to detect cholinesterase activity at a level of sensitivity comparable to, if not better than, the widely used Ellman assay. For instance, the authors reported the limit of detection (LOD) for ACHE as 0.0626 U/L, while for BCHE, the LOD was even lower at 0.0129 U/L. In plasma samples, where BCHE is the predominant enzyme, the system achieved a similar level of sensitivity, detecting activity in biologically relevant ranges. Indeed, this high sensitivity is one of the study’s most significant achievements because it allows for the detection of very low enzyme activity in complex environments like blood plasma. It also positions this sensor platform as a powerful tool for applications that require precise measurements, such as early diagnostics and environmental monitoring. Next, the researchers explored whether the sensors could also detect when the enzymes were blocked. They tested inhibitors like neostigmine bromide and organophosphates, which block the cholinesterase activity. In the presence of these blocked enzymes, the nanotubes’ fluorescence stayed steady with no fading, indicating the lack of enzymatic activity. This clear difference between active and inhibited enzymes made the sensors perfect for applications like drug testing or monitoring pesticide exposure.

In conclusion, using cutting-edge nanotechnology, Professor Gili Bisker and her team successfully developed biosensors that can measure enzyme activity, particularly CHE, in real time, and in complex fluids like blood plasma. We believe this innovation has a huge potential across multiple fields. For instance, in healthcare, it could change how we diagnose and monitor conditions like Alzheimer’s or Parkinson’s, where cholinesterase levels are key biomarkers. It’s also a game-changer for detecting exposure to dangerous chemicals, such as pesticides or nerve agents, where a quick detection can make all the difference. Because the sensors are so sensitive, they could even pick up changes in enzyme activity early, offering a way to catch health problems before they become serious. But the applications don’t stop at medicine. These sensors could also be used to monitor pesticide contamination in agriculture for assessing environmental health risks. The fact that they work in such complicated samples means they’re versatile and practical for real-world challenges. What’s even more exciting is the potential for the future. This research shows how customizable and scalable SWCNT technology can be. These sensors could be adapted for other enzymes, creating portable diagnostic devices, wearable health monitors, or even tools for monitoring processes inside the body.