We employ a fluorometric approach to monitor the activity and inhibition of cholinesterase (CHE) enzymes in blood plasma. We utilize near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) as probes strategically functionalized with myristoylcholine (MC), which is a substrate for CHE. The fluorescence intensity of MC-suspended SWCNTs undergoes a significant decrease upon interaction with CHE, attributed to CHE hydrolysis of the MC molecules on the SWCNT surface. Therefore, SWCNTs are rendered optical sensors for CHE activity, providing real-time feedback in the biologically transparent NIR spectral range. Furthermore, when synthetic and naturally occurring inhibitors are applied to impede the CHE enzymes in blood plasma, no significant alterations in the MC-SWCNT fluorescence are detected, enabling effective detection of CHE inhibition.

Figure 1: Schematic illustration of (A) hydrolysis of myristoylcholine (MC) into myristic acid and choline by cholinesterase (CHE). (B) The rationale behind monitoring the hydrolysis of MC through the fluorescence emission of the SWCNTs. The SWCNTs, functionalized by MC, exhibit a decrease in their fluorescence intensity upon CHE activity. In the presence of a CHE inhibitor, there is no fluorescence response.

Advantages over existing technologies:
Traditionally, enzyme activity is tracked using chemically interactive probes operating in the UV-visible region. However, there are clear advantages to developing spectroscopic probes designed for NIR operation. This is particularly advantageous because biological components exhibit high transparency in the NIR region. The use of NIR fluorescent probes allows for background-free monitoring of enzymatic activity. SWCNTs, known for their fluorescence in the NIR region, present a compelling option for such probes, especially when evaluating enzymatic activity in complex biological fluids like blood plasma. We have strategically functionalized SWCNTs with substrates for enzyme activity or functional groups susceptible to enzyme activity. This enables real-time enzyme activity monitoring in complex biofluids in the NIR region. This approach is essential to achieve successful tracking of enzyme activity in the presence of interfering species that could pose challenges in the UV-visible spectral region.

Future Applications:
The technologies described above rely on fundamental principles of SWCNT functionalization using the substrate of an enzyme of interest. These developed technologies are general, allowing them to be applied to monitor the activity of a diverse array of enzymes. This versatility stems from the ability to rationally design the surface coatings of SWCNTs, enabling targeted and adaptable approaches for monitoring enzyme activity and detecting their inhibition.

Intellectual Property
US patent pending

Reference:
Basu, S.; Hendler-Neumark, A.; Bisker, G., Rationally Designed Functionalization of Single-Walled Carbon Nanotubes for Real-Time Monitoring of Cholinesterase Activity and Inhibition in Plasma. Small 2024, 2309481.