Rationally Designed Functionalization of Single‐Walled Carbon Nanotubes for Real‐Time Monitoring of Cholinesterase Activity and Inhibition in Plasma

Abstract

Enzymes play a pivotal role in regulating numerous bodily functions. Thus, there is a growing need for developing sensors enabling real‐time monitoring of enzymatic activity and inhibition. The activity and inhibition of cholinesterase (CHE) enzymes in blood plasma are fluorometrically monitored using near‐infrared (NIR) fluorescent single‐walled carbon nanotubes (SWCNTs) as probes, strategically functionalized with myristoylcholine (MC)– the substrate of CHE. A significant decrease in the fluorescence intensity of MC‐suspended SWCNTs upon interaction with CHE is observed, attributed to the hydrolysis of the MC corona phase of the SWCNTs by CHE. Complementary measurements for quantifying choline, the product of MC hydrolysis, reveal a correlation between the fluorescence intensity decrease and the amount of released choline, rendering the SWCNTs optical sensors with real‐time feedback in the NIR biologically transparent spectral range. Moreover, when synthetic and naturally abundant inhibitors inhibit the CHE enzymes present in blood plasma, no significant modulations of the MC‐SWCNT fluorescence are observed, allowing effective detection of CHE inhibition. The rationally designed SWCNT sensors platform for monitoring of enzymatic activity and inhibition in clinically relevant samples is envisioned to not only advance the field of clinical diagnostics but also deepen further understanding of enzyme‐related processes in complex biological fluids.

Monitoring Enzyme Activity Using Near-Infrared Fluorescent Single-Walled Carbon Nanotubes

“…In this regard, it has been reported that MC-SWCNTs could be used to detect the inhibition of CHE enzymes in addition to monitoring their activity 107 SWCNTs were instrumental in monitoring CHE inhibition in buffer and plasma fluids, representing a vital strategy to be used in clinically relevant samples. When incubated with CHE enzymes previously exposed to synthetic inhibitors such as neostigmine bromide (NE), MC-SWCNTs did not show a significant decrease in fluorescence.…”

“…Further, our laboratory has exemplified such an approach, wherein the surface of SWCNTs was strategically functionalized with myristoylcholine (MC), serving as the substrate for cholinesterase (CHE) enzymes. 107 MC is a condensation product of myristic acid and choline, comprising a lengthy hydrocarbon chain with 14 carbon atoms coupled with a positively charged choline group. Importantly, upon hydrolysis by CHE, MC molecules undergo cleavage into myristic acid and choline.…”

“…(C) Correlation between the time-dependent normalized fluorescence intensity of MC-SWCNTs upon exposure to P-CHE, and the amount of choline released as a function of P-CHE activity. Reproduced with permission from ref ( 107 ). Available under a Creative Commons CC BY license.…”

“…Importantly, in certain studies, it has been explicitly demonstrated that SWCNTs-based detection of enzyme activity is comparable to, or even superior to, conventional assays in terms of sensitivity. Furthermore, it has been emphasized that SWCNTs-based optical transducers outperform conventional probes for monitoring enzymatic assays due to their optical and temporal stability in biological media, consistent dynamic range of response, reduced susceptibility to interfering molecules, and minimized background interference. ,,,, Thus, recognizing the broad significance of enzymes and the progress made in utilizing SWCNTs in this context, it is imperative to delineate the essential features of studies conducted to monitor enzyme activity using NIR luminescent SWCNTs.…”

“… 47 − 56 Moreover, SWCNTs exhibit remarkable photo and chemical stability, rendering them a compelling choice as sensors for monitoring essential biomarkers, including proteins, 57 − 66 oncometabolites, 67 pathogens, 68 , 69 various small molecules, 70 − 77 hormones, 58 , 78 , 79 volatiles, 80 , 81 lipids, 44 , 82 , 83 sugars, 84 − 89 neurotransmitters, 30 , 34 , 90 − 94 microRNA, 95 , 96 metal ions, 97 lysosomal pH, 98 and enzymatic activity and inhibition. 99 − 107 …”