A system and method for real-time monitoring of enzymatic activity using functionalized near-infrared fluorescent single-walled carbon nanotubes

Abstract

A sensor and method for measuring enzymatic activity, such as cholinesterase (CHE) enzymatic activity. The sensor comprises a near-infrared (NIR) fluorescent single-walled carbon nanotube (SWCNT) functionalized with a substrate such as myristoylcholine (MC). Quantitative analysis correlates changes in fluorescence of the SWCNTs with enzymatic activity. A decrease in NIR fluorescence intensity of SWCNTs upon interaction of the substrate with the target enzyme allows real-time monitoring and is positively correlated with enzymatic activity. An absence of modulation of fluorescence of the SWCNTs is indicative of enzymatic inhibition and the presence of enzyme inhibitors.

Description

A SYSTEM AND METHOD FOR REAL-TIME MONITORING OF ENZYMATIC ACTIVITY USING FUNCTIONALIZED NEAR-INFRARED FLUORESCENT SINGLEWALLED CARBON NANOTUBESSTATEMENT OF PRIORITY

[0001] This application claims priority to U.S. Provisional App. No. 63 / 624,431 for a System and Method for Real-Time Monitoring of Enzymatic Activity Using Functionalized Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.EUROPEAN RESEARCH COUNCIL FUNDING

[0002] The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. 101039127).BACKGROUND

[0003] Real-time monitoring of enzyme activity in biological samples is a fundamental pillar of point-of-care diagnostics. Enzymes, prevalent in biological fluids like blood plasma, play pivotal roles in numerous physiological functions, and any deviations in their activity are intricately linked to the emergence of various diseases. Furthermore, a multitude of chemical agents have been identified as interacting, either reversibly or irreversibly, with essential enzymes, leading to their inactivation and thereby impeding their vital biological functionality. Consequently, monitoring of enzyme inhibition arises as an equally essential challenge as tracking their activity. Nevertheless, the discernment of enzymatic activity and inhibition within blood plasma poses a far greater challenge compared to monitoring enzymes in a clean, simple, and homogenous environment, primarily due to two critical factors. Firstly, blood plasma contains enzymes at notably low concentrations, and secondly, the complex environment of plasma is rife with chemical species that interfere with the detection process. To address these challenges, sensors utilized for monitoring enzyme activity in plasma must have high sensitivity capable of detecting the activity at low enzyme concentrations and high selectivity to ensure the exclusive response to the enzyme of interest despite the presence of interfering substances.

[0004] To date, a variety of sensors have been developed to monitor the activity and inhibition of enzymes, employing diverse methodologies, including radiometry, electrochemistry, mass spectrometry, capillary electrophoresis, colorimetric analysis, and fluorescence -based techniques. However, many of these sensors have been primarily used for monitoring the activity of enzymes in isolated and uncontaminated environments rather than within complex blood samples.

[0005] Still, efforts to probe enzymatic activity within blood serum have been undertaken. For example, probing the activity of the protease enzyme renin was demonstrated using nanopore analytics, where peptides bound to resin within a single spin-column were enzymatically cleaved by renin and detected through electrical nanopore recordings. Furthermore, a strategy termed “single enzyme activity-based protein profiling (SEAP)” was formulated to identify multiple enzymes and determine their activity at a single molecule level. Moreover, alanine aminotransferase, a crucial indicator of liver function, has been successfully detected using a compact blood enzyme analyzer, through a colorimetric enzyme assay based on reflection photometry. Additionally, aptamer conformation-cooperated enzyme-assisted surface- enhanced Raman scattering has been demonstrated as a novel principle for ultrasensitive detection of tyrosine kinase-7 in blood samples.

[0006] Nevertheless, it remains essential to develop innovative sensors that operate within spectral regions where biological components are mostly transparent, with the ability to provide real-time data and spatiotemporal information. Near-infrared (NIR) fluorescent probes present a significant benefit in this context, as biological samples, including blood, plasma, and tissue, are mostly transparent in this region. A compelling avenue involves the utilization of single-walled carbon nanotubes (SWCNTs), which offer several advantageous features such as remarkable photostable fluorescence emission in the NIR range, biocompatibility, and ease of surface functionalization. SWCNTs have been used for fluorometric detection of important analytes, including small molecules, oncometalbolites, proteins, neurotransmitters, pathogens, metal ions, endolysosomal pH, disease biomarkers, plant hormones, sugars, quaternary ammonium compounds, microRNA, cytokines, volatiles, hormones, and bacterial siderophores, to name a few.

[0007] In addition, SWCNTs have been utilized as a probe for the inactivation of tyrosinase enzyme by observing a bathochromic shift in their emission wavelength, induced by the generation of singlet oxygen during the enzyme inactivation process. Notably, attempts have also been undertaken to monitor the enzymatic degradation of Impranil, a type of polyester polyurethane nanoparticle, using SWCNTs as NIR fluorescent sensors. The SWCNTs fluorescence underwent sequential modulation upon the degradation of Impranil, followed by the binding of the degradation product to the SWCNTs surface. Furthermore, the strategic functionalization of SWCNTs with synthetic dendrons incorporating ester and amide bonds was used for monitoring esterase and amidase enzymes, respectively, in a buffer medium, and peptide-functionalized SWCNTs were used to detect protease activity.

[0008] According to the present disclosure, monitoring of cholinesterase (CHE) activity of acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE) has been demonstrated using DNA-functionalized SWCNTs in buffer and serum environments. The underlying principle behind the sensing mechanism involved the utilization of a synthetic substrate of ACHE and BCHE, namely, acetylthiocholine (ATC), which, upon this enzymatic reaction, released thiocholine that subsequently interacted with the DNA-SWCNTs, leading to a significant enhancement of the SWCNTs fluorescence intensity. (Figure 27). This approach not only enables monitoring of the activity of ACHE and BCHE but also facilitated the detection of their inhibition. Nevertheless, the sensing mechanism relied on the indirect detection of the product of the CHE activity rather than the direct detection of the enzymatic hydrolysis process itself. Also, the approach was not utilized for monitoring enzyme activity in blood plasma samples.

[0009] Utilizing SWCNTs as fluorescent probes for CHE offers a promising solution to address some of the challenges associated with the conventional Ellman assay for tracking CHE activity by absorption-based approach. In the Ellman assay, thiocholine, the product of CHE hydrolysis of acetyl thiocholine, interacts with 5,5'-dithio-bis-[2-nitrobenzoic acid] (DTNB), generating a yellow product that is quantified by its absorption at 436 nm. However, the Ellman reagent, DTNB, reacts with any sulfhydryl groups, and it could be susceptible to reduction by other thiol molecules present in blood plasma, potentially leading to interference with the assay results. Additional background interference in plasma samples occurs due to the Soret band of hemoglobin, which closely aligns with the absorption maximum of the product of the Ellmanassay. Consequently, the utilization of fluorometric means for gauging CHE activity, particularly within the near-infrared (NIR) spectrum, emerges as an advantageous and preferable choice.

[0010] The present disclosure introduces a novel method to directly assess enzymatic activity, such as CHE activity in blood plasma. This strategy involves the rational design of the SWCNTs corona phase by suspending SWCNT with a substrate such as myristoylcholine (MC), the natural substrate of CHE. Given the established correlation between the fluorescence properties of SWCNTs and their surface composition, it was hypothesized that the CHE- mediated hydrolysis of MC would induce changes in the fluorescence emissions of SWCNTs, consequently offering a direct means to quantify CHE activity. The proposed mechanism centers on the CHE hydrolysis of MC present in blood plasma or its isolated form resulting in the release of choline molecules.

[0011] Indeed, according to the present disclosure, it was found that upon the interaction with CHE present within plasma, or with clean ACHE or BCHE in a buffer, the fluorescence intensity of SWCNTs decreases over time in a concentration-dependent manner, with a limit of detection in the sub U L1range. The fluorescence intensity decrease was found to be correlated to the amount of choline released, verified by an independent choline assay. Moreover, upon inhibiting the enzymes with chemical agents like neostigmine bromide used as a drug for an autoimmune neuromuscular disease, or organophosphates found in pesticides, the fluorescence of SWCNTs was barely altered, thereby allowing for detection of CHE inhibition. The presently disclosed sensor platform and method is expected to propel significant advancements in real-time tracking of enzymatic activity within biological fluids, particularly in the NIR region. Additionally, it holds the potential to fuel the development of strategic functionalization of nanosensors for tailored applications.SUMMARY OF THE INVENTION

[0012] 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 present disclosure presents a system and method to fluorometrically monitor the activity and inhibition of cholinesterase (CHE) enzymes in blood plasma using near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) as probes, strategicallyfunctionalized with myristoylcholine (MC) - the substrate of CHE. A significant decrease in the fluorescence intensity of MC-suspended SWCNTs upon interaction with CHE was observed which was 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-SWCNTs fluorescence are observed, allowing effective detection of CHE inhibition. The presently disclosed rationally designed SWCNT sensor 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.

[0013] According to some embodiments, the disclosure relates to a sensitive fluorescent nanosensor for cholinesterase (CHE) activity that can be utilized for an extensive range of military and scientific applications. Acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE) are the two major cholinesterase enzymes. The reaction rate of ChE is swift and close to diffusion limited. The strategy involves the functionalization of SWCNT by a substrate of ChE (an acetylcholine-like molecule) so that the activity of the CHE would directly affect the SWCNT corona phase. Such a change would result in a modulation of the SWCNT near-infrared fluorescence that can be manifested in either an intensity change or a shift in the peak emission wavelength. The SWCNT-sensor platform benefits from real-time optical feedback, with signals in the biologically transparent spectral window. This approach is expected to be highly sensitive and robust, opening numerous possibilities for new applications.

[0014] Subsequently, a novel methodology has been introduced to assess CHE activity in blood plasma directly. The strategy involves the rational design of the SWCNT corona phase by suspending SWCNT with myristoylcholine (MC), the natural substrate of CHE. Given the established correlation between the fluorescence properties of SWCNTs and their surface composition, it is believed that the CHE-mediated hydrolysis of MC induces changes in thefluorescence emissions of SWCNTs, consequently offering a direct means to quantify CHE activity.

[0015] The proposed mechanism centers on the CHE hydrolysis of MC, present in blood plasma or its isolated form, resulting in the release of choline molecules. Indeed, it was found that upon interaction with CHE present within plasma, or with clean ACHE or BCHE in a buffer, the fluorescence intensity of SWCNTs decreases over time in a concentration -dependent manner, with a limit of detection in the sub U L1range. The fluorescence intensity decrease was found to be correlated to the amount of choline released, verified by an independent choline assay.

[0016] Moreover, upon inhibiting the enzymes with chemical agents like neostigmine bromide, used as a drug for an autoimmune neuromuscular disease, or organophosphates, found in pesticides, the fluorescence of SWCNTs was barely altered, thereby allowing the detection of CHE inhibition.

[0017] The sensor platform is expected to propel significant advancements in real-time tracking of enzymatic activity within biological fluids, particularly in the NIR region. Additionally, it holds the potential to fuel the development of strategic functionalization of nanosensors for tailored applications.

[0018] Several non-limiting features of the present disclosure include:

[0019] Sensor Design: The nanosensors are based on near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs), which are functionalized with myristoylcholine (MC), a natural substrate of CHE enzymes. Critical features of some embodiments of the MC molecule include its long carbon chain that can bind the SWCNT surface through hydrophobic interactions and its charged choline head group, which facilitates suspension in an aqueous environment and colloidal stability. This understanding can be adapted to attach different substrates that are specific to other enzymes of interest by adding, if necessary, the relevant chemical moieties to allow for both the binding to the SWCNT surface and the suspension in water, rending the platform highly versatile.

[0020] Signal Transduction: When CHE interacts with these MC-suspended SWCNTs, there is a noticeable decrease in their NIR fluorescence intensity due to the hydrolysis of the MC corona phase. The transduction of the enzymatic activity to fluoresce modulation is crucial forrendering the MC-SWCNT optical nanosensors for the CHE activity. Operating in the NIR range, with minimal interference from biological samples, is an important feature for designing optical nanosensors for various enzymatic activities.

[0021] Quantitative Analysis: By measuring the amount of choline, the hydrolysis product of MC, the present disclosure establishes a correlation between the decrease in fluorescence intensity and the quantity of choline produced, providing a quantitative analysis of the CHE activity. Correlating fluorescence changes with product concentration is an important point, underscoring the direct effect of the enzyme activity on the SWCNT fluorescence emission.

[0022] Inhibitor Detection: The sensors can also detect the inhibition of CHE enzymes. When CHE inhibitors (both synthetic and naturally abundant) are present in blood plasma, there are no significant changes in the fluorescence of the MC-SWCNTs. Crucial for the effective detection of CHE inhibition is that the inhibitors themselves do not interfere with the nanosensors’ functionality.

[0023] Clinical and Research Applications: The designed SWCNT sensors are promising for advancing clinical diagnostics and provide valuable insights into enzyme -related processes in complex biological fluids, owing to the demonstration in clinically relevant plasma samples.

[0024] Therefore, based on the foregoing and continuing description, the subject invention in its various embodiments may comprise one or more of the following features in any non-mutually-exclusive combination:

[0025] A sensor for monitoring enzymatic activity in a biological sample, comprising a near-infrared (NIR) fluorescent single-walled carbon nanotube (SWCNT) functionalized with a substrate hydrolysable by an enzyme;

[0026] A sensor for monitoring enzymatic activity in a biological sample, wherein the hydrolysis of the substrate by the enzyme results in a decrease in NIR fluorescence intensity of the SWCNT;

[0027] A sensor for monitoring enzymatic activity in a biological sample, wherein a magnitude of the decrease in NIR fluorescence intensity is quantitatively correlated with an amount of the substrate hydrolyzed by the enzyme;

[0028] A sensor for monitoring enzymatic activity in a biological sample wherein the enzyme is a cholinesterase (CHE);

[0029] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is selected from the group consisting of plasma-cholinesterase (P-CHE), acetylcholinesterase (ACHE), and butrylcholinesterase (BCHE);

[0030] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is CHE;

[0031] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is P-CHE;

[0032] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is ACHE;

[0033] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is BCHE;

[0034] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is inhibited by an enzyme inhibitor selected from the group consisting of neostigmine bromide, organophosphate, and carbamate;

[0035] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme inhibitor prevents a decrease in the NIR fluorescence intensity of the SWCNT upon binding the enzyme by preventing hydrolysis of the substrate by the enzyme, and wherein the NIR fluorescence intensity is correlated with a concentration of the enzyme inhibitor present, allowing detection of enzyme inhibition;

[0036] A sensor for monitoring enzymatic activity in a biological sample, wherein the substrate comprises a hydrophobic carbon chain configured to interact with the surface of the SWCNT through hydrophobic interactions;

[0037] A sensor for monitoring enzymatic activity in a biological sample, wherein the substrate comprises a hydrophilic head group to enhance dispersion of the SWCNT in an aqueous medium;

[0038] A sensor for monitoring enzymatic activity in a biological sample, wherein the substrate is selected from the group consisting of myristoylcholine, acetylthiocholine, butrylcholine, and propionylcholine;

[0039] A sensor for monitoring enzymatic activity in a biological sample, wherein the substrate is myristoylcholine;

[0040] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is P-CHE; and wherein a correlation coefficient between a decrease in NIR fluorescence of the SWCNT upon hydrolysis of myristocholine by P-CHE and a release of choline is approximately -0.95;

[0041] A sensor for monitoring enzymatic activity in a biological sample, wherein the substrate is acetyl thiocholine;

[0042] A sensor for monitoring enzymatic activity in a biological sample, wherein the substrate is butrylcholine;

[0043] A sensor for monitoring enzymatic activity in a biological sample, wherein the substrate is propionylcholine;

[0044] A sensor for monitoring enzymatic activity in a biological sample, wherein the enzyme is CHE;

[0045] A sensor for monitoring enzymatic activity in a biological sample, wherein a limit of detection (LOD) for detecting CHE enzyme activity is experimentally determined to be less than 1 U L- 1 ;

[0046] A sensor for monitoring enzymatic activity in a biological sample, wherein a LOD for detecting enzyme activity is approximately 0.0626 ± 0.0002 U L- 1 for ACHE, corresponding to a concentration of approximately 0.208 nM;

[0047] A sensor for monitoring enzymatic activity in a biological sample, wherein a LOD for detecting enzyme activity is approximately 0.0129 ± 0.001 U L-l for BCHE, corresponding to a concentration of approximately 7.14 nM;

[0048] A sensor for monitoring enzymatic activity in a biological sample, wherein the limit of detection (LOD) for enzymatic activity is approximately 0.0168 ± 0.00005 U L-l for P- CHE, corresponding to a concentration of approximately 7.14 nM;

[0049] A method for measuring enzymatic activity in a biological sample, comprising introducing into a biological sample, a sensor comprising a NIR fluorescent SWCNT functionalized with a substrate hydrolyzable by an enzyme;

[0050] A method for measuring enzymatic activity in a biological sample, comprising measuring a change in NIR fluorescence intensity of the SWCNT, wherein the change in fluorescence intensity is indicative of enzymatic activity;

[0051] A method for measuring enzymatic activity in a biological sample, comprising correlating the change in fluorescence intensity to an amount of enzyme activity present in the biological sample;

[0052] A method for measuring enzymatic activity in a biological sample, wherein the substrate comprises a hydrophobic carbon chain configured to interact with the surface of the SWCNT through hydrophobic interactions, and a hydrophilic head group to enhance dispersion of the SWCNT in an aqueous medium;

[0053] A method for measuring enzymatic activity in a biological sample, wherein the enzyme is CHE;

[0054] A method for measuring enzymatic activity in a biological sample, wherein the enzyme is selected from the group consisting of P-CHE, ACHE, and BCHE;

[0055] A method for measuring enzymatic activity in a biological sample, wherein the substrate is selected from the group consisting of myristoylcholine, acetylthiocholine, butyrylcholine, and propionylcholine;

[0056] A method for measuring enzymatic activity in a biological sample, wherein the enzyme is CHE;

[0057] A method for measuring enzymatic activity in a biological sample, wherein a limit of detection (LOD) for detecting CHE enzyme activity is experimentally determined to be less than 1 U L- 1 ;

[0058] A method for measuring enzymatic activity in a biological sample, wherein a magnitude of a decrease in NIR fluorescence intensity is quantitatively correlated with the amount of enzymatic activity in the biological sample;

[0059] A method for measuring enzymatic activity in a biological sample, wherein the biological sample is a blood sample;

[0060] A method for measuring enzymatic activity in a biological sample, wherein the biological sample is a plasma sample.

[0061] A method for measuring enzymatic inhibition, comprising introducing into a biological sample, a sensor comprising a NIR fluorescent SWCNT functionalized with a substrate hydrolysable by an enzyme, the enzyme inhibitable by an enzyme inhibitor;

[0062] A method for measuring enzymatic inhibition, comprising measuring a change in NIR fluorescence intensity of the SWCNT, wherein the change in fluorescence intensity is indicative of enzymatic activity;

[0063] A method for measuring enzymatic inhibition, comprising comparing a measured fluorescence intensity to a baseline fluorescence intensity obtained from a control sample lacking the enzyme inhibitor;

[0064] A method for measuring enzymatic inhibition, comprising correlating the change in NIR fluorescence intensity to an amount of enzyme inhibitor present in the biological sample;

[0065] A method for measuring enzymatic inhibition, wherein the substrate comprises a hydrophobic carbon chain configured to interact with the surface of the SWCNT through hydrophobic interactions, and a hydrophilic head group to enhance dispersion of the SWCNT in an aqueous medium;

[0066] A method for measuring enzymatic inhibition, wherein the enzyme is selected from the group consisting of CHE, P-CHE, ACHE, and BCHE;

[0067] A method for measuring enzymatic inhibition, wherein the enzyme inhibitor is selected from the group consisting of neostigmine bromide, organophosphates, and carbamates;

[0068] A method for measuring enzymatic inhibition, wherein the enzyme is CHE, and the LOD for detecting enzyme activity is less than 1 U L1;

[0069] A method for measuring enzymatic inhibition, wherein the enzyme inhibitor prevents hydrolysis of the substrate by the enzyme;

[0070] A method for measuring enzymatic inhibition, wherein inhibition of hydrolysis of the substrate by the enzyme results in no reduction in NIR fluorescence intensity of the biological sample.DESCRIPTION OF THE DRAWINGS

[0071] Figure 1. Schematic illustration of (A) hydrolysis of myristoylcholine into myristic acid and choline by cholinesterase and (B) the rationale behind monitoring the hydrolysis of myristoylcholine through the fluorescence emission of the SWCNTs. The SWCNTs, functionalized by myristoylcholine, exhibit a decrease in their fluorescence intensity upon cholinesterase activity. In the presence of a cholinesterase inhibitor, there is no fluorescence response.

[0072] Figure 2. Time-dependent fluorescence spectra of MC-SWCNTs before and following the addition of ACHE. (B) Normalized fluorescence intensity response of the MC- SWCNTs (9,4) chirality to ACHE as a function of time (black squares) and concentration of choline released as a function of time (green circles) (C) Time-dependent fluorescence spectra of MC-SWCNTs before and following the addition of BCHE. (D) Normalized fluorescence intensity response of the MC-SWCNTs (9,4) chirality to BCHE as a function of time (black squares) and concentration of choline released as a function of time (orange circles). (E) Correlation between the decrease in fluorescence of MC-SWCNTs and the concentration of the choline released upon interaction with ACHE and (F) BCHE. The error bars in all figures represent the standard deviation of experimental replicates (n = 3).

[0073] Figure 3. Fluorescence spectra and MC-SWCNTs before and after the addition of ACHE, acquired after 3h of incubation (B) Normalized fluorescence response of (9,4) peak ofMC-SWCNTs as a function of the activity of ACHE (C) Fluorescence spectra and MC-SWCNTs before and after the addition of BCHE, acquired after 3h of incubation (D) Normalized fluorescence response of (9,4) peak of MC-SWCNTs as a function of the activity of BCHE. The error bars in all figures represent the standard deviation of experimental replicates (n = 3).

[0074] Figure 4. (A) Fluorescence spectra and (B) normalized fluorescence response of MC-SWCNTs in the presence of uninhibited ACHE (purple), inhibited ACHE (ACHE-NE, cyan), and NE (brown), acquired after 3h of incubation. (C) Fluorescence spectra and (D) normalized fluorescence response of MC-SWCNTs in the presence of uninhibited BCHE (purple), inhibited BCHE (BCHE-NE, cyan), and NE (brown), acquired after 3h of incubation. The error bars in all figures represent the standard deviation of experimental replicates (n = 3).

[0075] Figure 5. (A) Time-dependent fluorescence spectra of MC-SWCNTs before and following the addition of P-CHE (plasma). (B) Normalized fluorescence intensity response of the MC-SWCNTs (9,4) chirality to P-CHE as a function of time (black squares) and concentration of choline released on the reaction as a function of time (yellow circles). (C) Correlation between choline released and decrease in fluorescence of MC-SWCNTs upon interaction with P-CHE. The error bars in all figures represent the standard deviation of experimental replicates (n = 3).

[0076] Figure 6. (A) Fluorescence spectra and MC-SWCNTs before and after the addition of P-CHE acquired after 3h of incubation and (B) Normalized fluorescence response of (9,4) peak of MC-SWCNTs as a function of the CHE activity of P-CHE. The error bars represent the standard deviation of experimental replicates (n = 3).

[0077] Figure 7. (A) Fluorescence spectra and (B) extent of fluorescence intensity decrease in MC-SWCNTs in response to uninhibited and inhibited P-CHE using NE, acquired after 3h of incubation. (C) Fluorescence spectra and (D) extent of fluorescence intensity decrease in MC-SWCNTs in response to uninhibited and inhibited P-CHE using OP, acquired after 3h of incubation. The error bars in all figures represent the standard deviation of experimental replicates (n = 3).

[0078] Figure 8. Optical properties of MC-SWCNTs. (A) Absorption spectrum, (B) Fluorescence emission spectrum under 730 nm excitation, showing distinct emission peaksattributed to the (6,5), (10,2), (9,4) (8,6), and (8,7) SWCNT chiralities, and (C) Excitationemission map.

[0079] Figure 9. TEM image of MC-SWCNTs showing an individual nanotube with a diameter of approximately 2 nm.

[0080] Figure 10. Ellman assay of (A) ACHE and (B) BCHE, using ATC as the substrate.

[0081] Figure 11. Fluorescence spectrum of MC-SWCNTs before and after the addition of (A) choline and (B) myristic acid.

[0082] Figure 12. ESI-MS spectra of (A) the product of the reaction between MC- SWCNTs and ACHE and (B) only MC-SWCNTs.

[0083] Figure 13. Time-dependent fluorescence spectra of washed MC-SWCNTs following the addition of (A) ACHE, and (B) BCHE.

[0084] Figure 14. Fluorescence spectra of MC SWCNTs before and after the addition of (A) proteinase k, (B) amidase, and (C) esterase, acquired after 3h of incubation. (D) Bar diagram showing the extent of decrease in fluorescence of MC-SWCNTs upon the addition of esterase (green), proteinase (sky blue), and amidase (orange). The error bars represent the standard deviation of experimental replicates (n = 3).

[0085] Figure 15. Fluorescence spectra of (GT)is-SWCNTs before and after the addition of (A) ACHE and (B) BCHE, acquired after 3h of incubation. Fluorescence spectra of SC- SWCNTs before and after the addition of (C) ACHE and (D) BCHE, acquired after 3h of incubation.

[0086] Figure 16. Ellman assay of P-CHE, using ATC as the substrate.

[0087] Figure 17. ESI-MS spectra of (A) the product of the reaction between MC- SWCNTs and P-CHE and (B) only P-CHE.

[0088] Figure 18. Time-dependent fluorescence spectra of washed MC-SWCNTs and that following the addition of P-CHE.

[0089] Figure 19. Fluorescence spectrum of MC-SWCNTs before (black) and after the addition of cysteine (dark blue), glutamic acid (cyan), glucose (light green), iron (II) sulfate (grass green), zinc chloride (orange), copper chloride (lavender), and P-CHE (red), acquired after 3h of incubation.

[0090] Figure 20. Fluorescence spectrum of MC-SWCNTs before (black) and after 3h (red) the addition of BSA.

[0091] Figure 21. Fluorescence spectrum of (GT)is-SWCNTs before and after 3h the addition of P-CHE.

[0092] Figure 22. Absorbance spectra confirming inhibition of P-CHE via interaction with OP. The black curve shows the absorbance spectrum of DTNB (Ellman reagent). The red curve shows the absorbance spectrum of DTNB following the addition of P-CHE. The blue curve shows the absorbance curve of DTNB added with P-CHE and ATC, where the absorption peak at 436 nm indicates the interaction of DTNB with the product of P-CHE hydrolysis of ATC. The green curve shows the absorbance spectrum of DTNB added with P-CHE, ATC, and OP. The inset shows digital photographs of cuvettes containing uninhibited P-CHE (corresponding to the blue curve) and OP-inhibited PCHE (corresponding to the green curve).

[0093] Figure 23. Normalized fluorescence intensity response of MC-SWCNTs upon interaction with uninhibited and inhibited ACHE, BCHE, and P-CHE. The error bars represent the standard deviation of experimental replicates (n = 3).

[0094] Figure 24. Zeta potential distribution curves of MC-SWCNTs (A) before and after 3h reactions with (B) ACHE, (C) BCHE, and (D) P-CHE, respectively.

[0095] Figure 25. UV-vis -NIR absorbance spectrum of MC-SWCNTs before and 3 h after the addition of (A) ACHE, (B) BCHE, and (C) P-CHE.

[0096] Figure 26. Fluorescence spectra of MC- SWCNTs before and after the additions of P-CHE incubated with 0, 10, 50, and 100 pL of OP.

[0097] Figure 27. AChE activity sensing mechanism. AChE hydrolyses acetylthiocholine to acetic acid and thiocholine; thiocholine is then recognized by a DNA- SWCNT sensor, resulting in increased near-IR fluorescence intensity.

[0098] Figure 28. Fluorescence spectra of (GT)15-SWCNTs before (black curve) and after the additions of plasma (red curve), and plasma incubated with ATC (blue curve).

[0099] Figure 29. Fluorescence spectra of T30 SWCNTs before (black curve) and after the additions of plasma (red curve), and plasma incubated with ATC (blue curve).DETAILED DESCRIPTION

[0100] SWCNTs, synthesized by a high-pressure carbon monoxide (HiPCO) process, were suspended by myristoylcholine (MC), a substrate of cholinesterase (CHE). The molecular structure of MC involves a hydrophobic chain constituting fourteen carbons and a hydrophilic choline head group. This molecular structure renders MC an ideal candidate for stabilizing SWCNTs in an aqueous solution, given the hydrophilic interactions between the choline part of MC and water molecules and the hydrophobic interaction between the SWCNTs and the nonpolar carbon chain of MC. Upon the interaction with CHE, MC undergoes hydrolysis to myristic acid and choline (Scheme 1A). In essence, the SWCNTs MC corona phase is a substrate of the CHE, providing a rationally designed nanosensor for CHE activity, as the hydrolysis of the MC corona results in a modulation of the SWCNTs fluorescence emission (Scheme IB). Consequently, if CHE is inhibited, no fluorescence response is observed.

[0101] Successful suspension of SWCNTs by MC (hereafter referred to as MC- SWCNTs) is apparent from the UV-vis-NIR absorption spectrum, which features distinct peaks in the range of 300-1380 nm (Figure 8A). Moreover, the fluorescence spectrum of MC- SWCNTs, upon laser excitation at 730 nm (Figure 8B), and the excitation-emission map of MC- SWCNTs (Figure 8C) exhibits well-defined emission peaks attributable to the various SWCNT chiralities. In addition to the optical characterization of the MC-SWCNTs suspension, transmission electron microscopic (TEM) confirms the formation of individual SWCNTs of approximately 2 nm in diameter (Figure 9).

[0102] Fluorescence modulation of MC-SWCNTs following MC hydrolysis by acetylcholinesterase and butyrylcholinesterase

[0103] Since MC, the choline ester of myristic acid (MA), is hydrolyzed to MA and choline by cholinesterase (CHE) enzymes, the disclosers tested whether the activity of CHE on MC-SWCNTs would alter the SWCNTs surface functionalization, leading to a modulation of thefluorescence characteristics of SWCNTs. Such a platform can be used as a NIR probe for monitoring CHE activity.

[0104] To test whether the activity of CHE on MC-SWCNTs would modulate the fluorescence characteristics of SWCNTs, ACHE and BCHE were used as model enzymes. Firstly, the activity of ACHE and BCHE was found to be 5.05 ± 0.72 and 6.2 ± 0.55 U L1, respectively, via the Ellman assay (Figure 10A-B). This assay consists of measuring the timedependent absorption of the sample in the presence of acetylthiocholine (ATC), an alternative substrate of CHE that contains a thiol group, and Ellman’s reagent, 5,5'-dithio-bis-[2- nitrobenzoic acid] (DTNB), which absorbs at 436 nm upon binding free thiols produced following ATC hydrolysis by CHE.

[0105] Next, an aqueous dispersion of MC-SWCNTs (2 mg L1) was exposed to 5.05 U L-l of ACHE, and the fluorescence of the MC-SWCNTs was monitored at 30 min intervals for a total duration of 3 h. Interestingly, the fluorescence emission of the MC-SWCNTs significantly decreased upon the incubation with ACHE (Figure 2A). Monitoring the fluorescence intensity of the (9,4) chirality of the MC-SWCNTs, a characteristic decay time of 26 ± 3.8 min (Figure 2B) was found. To confirm the hydrolysis of MC, the concentration of choline in the reaction mixture was independently quantified over time using a choline assay. This assay relies on the oxidation of choline to oxidized choline by an oxidizing enzyme, resulting in the formation of a colored product whose absorption maximum at 570 nm is proportional to the concentration of choline. The increase in the concentration of choline accumulated in the solution showed a similar trend to the decrease in the fluorescence intensity (Figure 2B). The fluorescence response of the MC- SWCNTs could, therefore, be attributed to the ACHE hydrolysis of the MC thereby affecting the surface functionalization of the SWCNTs.

[0106] Similar experiments were conducted with BCHE, where the fluorescence intensity of the MC-SWCNTs decreased over time upon the interaction with 6.2 U L1BCHE (Figure 2C), showing a characteristic decay time of 66 ± 14.7 min for the intensity of the (9,4) chirality and a matching trend as the choline produced in the solution quantified by the choline assay (Figure 2D). The correlation coefficient between the decrease in fluorescence of the MC- SWCNTs and the amount of choline produced as a result of the MC hydrolysis was calculated to be -0.91 and -0.97 for ACHE and BCHE, respectively (Figures 2E and 2F).

[0107] The direct addition of MA or choline, the direct products of the MC, to MC- SWCNTs did not induce a decrease in the MC-SWCNTs fluorescence following 3 h incubation (Figure 11). This supports the conclusion that the fluorescence decrease is a consequence of the enzymatic hydrolysis process and not an outcome of the interactions between MC-SWCNTs and the hydrolyzed products themselves.

[0108] To further validate the generation of choline as a result of the hydrolysis of MC in the MC-SWCNTs suspension by the CHE enzymes, electrospray ionization mass spectrometric (ESI-MS) analysis was performed. Comparing the ESI-MS spectra of the MC- SWCNTs suspension before and after the addition of ACHE, a peak at a mass / charge (m / z) ratio of 104.1, which can be assigned to choline clearly appeared in the sample in the presence of ACHE (Figure 12A). Conversely, in the control sample of MC-SWCNTs, without the enzyme, multiple peaks emerged within noise levels, including two possibly associated with choline and MA (Figure 12B). These peaks were challenging to discern distinctly due to their extremely low intensities, indicating that the initial presence of choline and MA likely stemmed from impurities in the MC sample. In contrast, following the reaction of ACHE and MC-SWCNTs, the so- formed choline product featured a distinct and intense peak in the ESI-MS, which could unambiguously be correlated to the formation of choline. These findings, in conjunction with the choline assay confirming the release of choline from the reaction mixture of MC-SWCNTs and CHE enzymes, firmly underscored the successful hydrolysis of MC molecules, which constitute the corona phase of the SWCNTs. Moreover, the results demonstrate that modulation of the MC- SWCNTs fluorescence can probe enzymatic activity to provide a real-time optical signal.

[0109] Furthermore, to demonstrate that the MC molecules on the MC-SWCNTs surfaces were susceptible to hydrolysis by ACHE and BCHE, the MC-SWCNTs were thoroughly washed to remove any unbound MC molecules present in the solution. These cleaned MC-SWCNTs were then re-dispersed in a buffer and subjected to interactions with ACHE and BCHE. Intriguingly, it was observed that the extent of fluorescence decrease in MC-SWCNTs, following the elimination of free MC molecules in the solution, closely resembled the behavior of unwashed MC-SWCNTs (Figure 13). This finding implies that the MC molecules present on the surface of the MC-SWCNTs were indeed hydrolyzed by CHE enzymes, subsequently leading to alterations in the fluorescence of the SWCNTs.

[0110] In order to test the specificity of the MC-SWCNT sensors, the fluorescence response to other enzymes, specifically amidase, proteinase k, and esterase was monitored. Following 3 h incubation, proteinase k, and amidase induced an approximate 16±10% and 8±13% increase in the MC-SWCNTs fluorescence intensity, respectively, which were not statistically differentiated from zero. In contrast, esterase was observed to reduce the fluorescence intensity of MC-SWCNTs by approximately 8±1%, which was significantly differentiated from 0 (Figure 14). Since the proteinase k and amidase led to an insignificant fluorescence increase, their effect could not be confused with the CHE that resulted in a fluorescence decrease. The effect of the esterase, on the other hand, was expected as it could hydrolyze ester bonds similar to CHE, thus serving as a positive control. Generally, esterases can hydrolyze ester bonds into their respective acid and alcohol components. CHE enzymes represent a distinct subset of esterases where the alcohol component is choline. Consequently, the observed MC-SWCNTs fluorescence decrease following the treatment with esterase not only supports the conclusion of the susceptibility of the MC molecules to enzymatic ester hydrolysis but also further supports that the hydrolysis of MC in the MC-SWCNTs suspension is likely to induce fluorescence intensity decrease of the MC-SWCNTs.

[0111] Subsequently, the disclosers sought to validate the role played by the MC corona phase of the SWCNTs. To this end, the disclosers employed SWCNTs suspended by DNA, (GT)is-SWCNTs, and by sodium cholate (SC), SC-SWCNTs, and exposed them to ACHE and BCHE for a 3 h duration (Figure 15). Following the incubation, there was negligible to no fluorescence response of the SWCNTs, further validating that the significant fluorescence intensity decrease observed for MC-SWCNTs was an exclusive consequence of the hydrolysis of the MC molecules by the CHE enzymes.

[0112] Calibration of MC-SWCNTs sensors for ACHE and BCHE:

[0113] Having demonstrated the successful hydrolysis of MC in the MC-SWCNTs suspension upon the interaction with ACHE and BCHE, the concentration-dependent fluorescence response of MC-SWCNTs for varying ACHE and BCHE concentrations was studied. The fluorescence response of the MC-SWCNTs was recorded following 3 h of incubation with ACHE or BCHE, after which the response of the sensors was nearly saturated (Figures 3B and 3D). Interestingly, the fluorescence intensity of the (9,4) chirality of MC-SWCNTs showed a concentration-dependent decrease for increasing concentrations of ACHE (Figure 3A) and BCHE (Figure 3B). The ACHE and BCHE enzyme concentrations were selected within the range of 10’3to 101U L1, falling within the accepted range for monitoring enzyme activity in biological samples. The normalized fluorescence response of the (9,4) chirality of the MC-SWCNTs as a function of the ACHE concentration was fitted with a four- parameter logistic function with a zero baseline, ultimately resulting in a three -parameter fit. The CHE activities resulting in half of the maximal response were found to be 0.0685 ± 0.008 U L1and 1.25 ± 0.58 U L1, for ACHE and BCHE, respectively. Furthermore, the limit of detection of enzymatic activity of ACHE and BCHE was found to be 0.0626 ± 0.0002 U L1and 0.0129 ± 0.001 U L1for ACHE and BCHE, respectively. The calculated concentrations of ACHE and BCHE corresponding to the Limit of Detection (LOD) in terms of activity using the presently disclosed method were in the range of 0.208 nM and 7.14 nM, respectively. On the other hand, the LOD of CHE activity based on the Ellman assay has been reported to be in the range of nM. This indicates that the method for determining CHE activity according to the present disclosure are comparable to the gold standard Ellman assay, particularly in terms of sensitivity.

[0114] Monitoring the inhibition of ACHE and BCHE

[0115] Anticholinesterase, or CHE inhibitors, are compounds that impede the hydrolysis of choline-based esters by the CHE enzymes. These inhibitors are widely used in treating neurogenerative disorders like Alzheimer’s disease, Parkinson’s disease, and Lewy body dementia. These conditions are characterized by physiological processes that reduce the cellular production of acetylcholine, thereby hampering the cholinergic transmission in the brain. In such contexts, anticholinesterases serve to hinder acetylcholine hydrolysis, thereby maintaining optimal acetylcholine levels by preventing its breakdown. Given this clinical perspective, it is imperative to monitor not only CHE activity but also its inhibition.

[0116] The presently disclosed rationally designed MC-SWCNT sensors, featuring a surface-bound CHE substrate, were strategically crafted with this dual purpose in mind. In cases where CHE is inhibited, only negligible fluorescence changes of the MC-SWCNTs was anticipated. To validate this hypothesis, ACHE and BCHE were incubated with neostigmine bromide (NE), a prominent CHE inhibitor, for 3 h. Subsequently, the neostigmine-inhibited ACHE (ACHE-NE) and BCHE (BCHE-NE) were added to MC-SWCNTs, and the SWCNTsfluorescence was recorded after 3 h. While uninhibited ACHE and BCHE, under similar conditions, led to a significant decrease in the MC-SWCNTs fluorescence, the fluorescence of MC-SWCNTs showed a slight increase upon interaction with inhibited ACHE and BCHE (Figure 4A-D). The observed net increase in the fluorescence of MC-SWCNTs upon interaction with inhibited CHE enzymes could be attributed to non-specific interactions between the inhibited CHE enzymes and MC-SWCNTs, as there was no enzyme activity in the presence of the inhibitors. Further, pristine NE, i.e., without the CHE enzymes, had a negligible effect on the MC-SWCNT's fluorescence. This outcome not only underscored the capability of MC-SWCNTs to detect the inhibition of ACHE and BCHE but also affirmed that the fluorescence decrease of MC-SWCNTs observed in the presence of uninhibited ACHE and BCHE was distinctly attributed to MC hydrolysis by these enzymes.

[0117] Monitoring CHE activity in blood plasma:

[0118] Having successfully demonstrated the capability to monitor both the activity and inhibition of CHE using pure ACHE and BCHE, the disclosers tested the viability of MC- SWCNT sensors in detecting CHE activity present in biological fluid. Specifically, blood plasma was chosen which is known to contain CHE predominantly in the form of BCHE. In plasma, therefore, the preferred substrate for Plasma cholinesterase (P-CHE) is butyrylcholine and / or propionylcholine. Nevertheless, since pure BCHE can hydrolyze MC in an MC-SWCNTs suspension and result in discernible SWCNTs fluorescence modulation, according to certain embodiments of the present disclosure, MC-SWCNT sensors can be used to monitor the P-CHE activity. This endeavor holds promise in expanding the application of the presently disclosed sensors to real- world biological contexts.

[0119] As a starting point, appropriately diluted P-CHE were subjected to the Ellman assay, resulting in a calculated P-CHE activity of 0.36 ± 0.03 U L1(Figure 16). Owing to the inherent coloration of plasma that may interfere with the 436 nm absorption monitored in the Ellman assay or interfering thiol groups present in plasma regardless of ATC hydrolysis, a control experiment was conducted without ATC, and this baseline absorption was subtracted from the results. Consequently, the derived net absorbance values exclusively represented the P- CHE activity within the plasma.

[0120] Subsequently, diluted P-CHE having a calculated activity of U L1was incubated with MC-SWCNTs and the disclosers monitored the SWCNTs fluorescence at 30 min intervals for 3 h. Similar to the case of ACHE and BCHE, P-CHE also resulted in a gradual decrease in the MC-SWCNT's fluorescence intensity (Figure 5A). Moreover, in this instance, a wavelength shift in the fluorescence of MC-SWCNTs was observed following interaction with P-CHE. However, as no such wavelength shift was observed in the fluorescence spectra of MC-SWCNTs following interaction with pure ACHE and BCHE, it is possible that these shifts resulted from chemical interactions with other components present within the plasma. Monitoring the fluorescence intensity of the (9,4) chirality of the MC-SWCNTs, a characteristic decay time of 48.7 ± 12.4 min was found (Figure 5B). It is worth noting that the characteristic decay time of the fluorescence decrease in the case of P-CHE is similar to that of BCHE, which is the predominant CHE present in plasma. To validate the hydrolysis of MC, the concentration of choline in the reaction mixture over time was independently monitored using the choline assay. The increase in choline concentration within the solution exhibited a comparable trend to the decrease in fluorescence intensity of MC-SWCNTs (Figure 5B). Hence, the fluorescence decrease of the MC-SWCNTs could be attributed to the hydrolysis of MC by P-CHE, which in turn influenced the surface functionalization of the SWCNTs. Moreover, the correlation coefficient between the decrease in fluorescence of MC-SWCNTs upon hydrolysis by P-CHE and the release of choline was found to be -0.95 (Figure 5C).

[0121] To corroborate the generation of choline resulting from MC hydrolysis in the MC-SWCNTs suspension by P-CHE, an ESI-MS analysis was performed which showed the choline peak following the addition of P-CHE, a feature that was clearly absent in the control sample comprising only plasma (Figure 17). These findings, combined with the choline assay verifying the choline release from the MC-SWCNTs and P-CHE reaction mixture, affirmed the successful hydrolysis of MC molecules constituting the corona phase of the SWCNTs. Furthermore, this modulation of MC-SWCNTs fluorescence served as a real-time optical tool to probe enzymatic reactions in biological fluids.

[0122] To provide empirical evidence supporting the hydrolysis of the MC molecules constituting the corona phase of SWCNTs by P-CHE, a purification step involving the washing of MC-SWCNTs was executed, followed by their subsequent interaction with P-CHE.Remarkably, the extent of the reduction in fluorescence observed in the washed MC-SWCNTs upon interaction with P-CHE closely paralleled that of the unwashed MC-SWCNTs. This observation serves to emphasize the high susceptibility of the MC moieties residing on the SWCNT surfaces to hydrolysis by P-CHE (Figure 18).

[0123] Calibration of MC-SWCNTs sensors in plasma

[0124] After successfully demonstrating the hydrolysis of MC in the MC-SWCNTs suspension through interactions with P-CHE, the disclosers examined the concentrationdependent fluorescence response of MC-SWCNTs to varying P-CHE concentrations and recorded the fluorescence response of the MC-SWCNTs after 3 h of incubation (Figure 6A). Intriguingly, a consistent decrease in the fluorescence intensity of (9,4) chirality of the MC- SWCNTs was observed with increasing concentrations of P-CHE. The chosen P-CHE concentrations fell within the accepted range for monitoring enzyme activity in biological samples. To analyze the normalized fluorescence response of (9,4) chirality of MC-SWCNTs as a function of P-CHE concentration, the data was fitted with a logistic function. The CHE activities resulting in half of the maximal response were found to be 0.017 ± 0.002 U L1. The limit of detection was found to be 0.0168 ± 0.00005 U L1. The calculated concentration of P- CHE corresponding to the LOD determined using the disclosed method is in the range of 7.14 nM. As previously mentioned, the LOD for determining CHE activity typically falls within the nM range. This reaffirms that the disclosed method exhibits comparable sensitivity compared to the Ellman assay, particularly when applied to monitoring enzymatic activity in blood plasma.

[0125] One aim of the present disclosure is to demonstrate monitoring the activity of cholinergic enzymes within blood plasma. Thus, in order to further attribute the MC-SWCNTs fluorescence changes observed upon the treatment with P-CHE to MC hydrolysis by P-CHE, the influence of various interfering analytes was tested. To accomplish this, a range of cations, anions, sugar molecules, and amino acids were introduced into an aqueous dispersion of MC- SWCNTs alongside P-CHE. These analytes were allowed to interact with MC-SWCNTs for a duration of 3 hours, following which the dicslosers monitored the fluorescence emission of MC- SWCNTs. Interestingly, under analogous conditions, P-CHE induced substantial reduction in the fluorescence of MC-SWCNTs. In contrast, the control analytes either increased the fluorescence or had minimal impact on the MC-SWCNTs fluorescence (Figure 19). This observationconfirmed that the decrease in fluorescence intensity of MC-SWCNTs when interacting with plasma primarily arises from the hydrolysis of MC by P-CHE, with negligible contributions from other constituents present in blood plasma.

[0126] In order to rule out nonspecific interactions, the effect of albumin, the most abundant protein in plasma, was tested on the MC-SWCNT sensors. To this end, bovine serum albumin (BSA) was incubated with MC-SWCNTs for a duration of 3 h. Following the incubation, the fluorescence of the MC-SWCNTs was unaffected (Figure 20), confirming that the presence of BSA in blood plasma did not contribute to the observed reduction in MC- SWCNT fluorescence.

[0127] Subsequently, P-CHE was added to (GT)15-SWCNTs, and observed only a minor increase of the fluorescence (Figure 21), substantiating the pivotal role of the MC corona phase of the SWCNTs. This outcome further demonstrates that the fluorescence decrease observed in MC-SWCNTs could be exclusively attributed to the hydrolysis of MC molecules by P-CHE enzymes. Thus, the presently disclosed sensors are versatile tools allowing for the comprehensive monitoring of CHE enzyme activity and inhibition in biological fluids.

[0128] Inhibition of P-CHE:

[0129] Monitoring the inhibition of CHE enzymes holds great significance in drug discovery and the screening of nerve agents that irreversibly bind to these enzymes and inhibit their activity. While the present disclosure successfully probed the inhibition in pure ACHE and BCHE enzyme samples, the disclosure aimed to expand the utility of MC-SWCNTs for monitoring CHE inhibition in P-CHE. Since detecting inhibition in biological fluids like plasma is considerably more challenging than with pure enzymes, the development of sensors that exhibit selectivity toward the target enzyme activity is crucial in this context. Nonetheless, since MC-SWCNTs were designed with the substrate functionalizing the SWCNTs surface, it was envisioned that inhibition of P-CHE would prevent the hydrolysis of the MC molecules, resulting in minimal changes in the MC-SWCNTs fluorescence.

[0130] To test this concept, 10 mM of neostigmine (NE) was incubated with P-CHE for 3 h. The inhibited P-CHE referred to as P-CHE-NE, was then exposed to MC-SWCNTs for 3 h, and the resulting fluorescence was monitored. Intriguingly, the addition of P-CHE-NE led to adecrease of less than 10% in the fluorescence of the MC-SWCNTs. In contrast, under similar conditions, uninhibited P-CHE, i.e., without NE treatment, resulted in a more than 55% reduction in the MC-SWCNTs fluorescence intensity. Importantly, as previously demonstrated with ACHE and BCHE, NE alone had no discernible effect on the fluorescence (Figure 7A-B). These results clearly demonstrate that MC-SWCNTs are not only capable of monitoring the enzymatic hydrolysis of P-CHE but also its inhibition.

[0131] Additionally, the inhibition of P-CHE by organophosphorus (OP) compounds was explored. These compounds are prevalent in pesticides and can accumulate in human physiological systems, posing significant health risks. OP was incubated with P-CHE (P-CHE- OP) for 3 hours to induce inhibition. To validate the inhibition of P-CHE, an Ellman assay was conducted. When the Ellman reagent DTNB was incubated with P-CHE and ATC, a prominent yellow coloration with an absorption peak at 436 nm was observed, indicating the presence of active P-CHE. In contrast, upon the introduction of OP compounds into the mixture of P-CHE, ATC, and DTNB, no noticeable absorbance at 436 nm was detected (Figure 22), demonstrating the inhibition of P-CHE activity. Following 3 h incubation of MC-SWCNTs in the presence of inhibited P-CHE, P-CHE-OP, the fluorescence intensity of the MC-SWCNTs showed a small increase of around 5%, while the fluorescence of the MC-SWCNTs incubated with uninhibited P-CHE was substantially decreased by approximately 70%. Pristine OP, however, led to a decrease in the fluorescence intensity of MC-SWCNTs by roughly 12±11%, which was not statistically differentiated from zero (Figures 7C-D).

[0132] Furthermore, to underscore the varying sensitivity of the presently disclosed sensors to enzymatic hydrolysis in different media, namely buffer and blood plasma, the extent of the decrease in fluorescence of MC-SWCNTs during enzymatic hydrolysis with ACHE and BCHE (in buffer) and P-CHE (in blood plasma) was compared. Additionally, the extent of changes in fluorescence, if any, of MC-SWCNTs upon interaction with inhibited ACHE, BCHE, and P-CHE (Figure 23) was also compared. This comparison further highlighted the differing selectivity of the MC-SWCNTs to CHE hydrolysis in varying mediums.

[0133] These findings confirm that MC-SWCNTs sensors are capable of effectively monitoring the inhibition of cholinergic enzymes within biological samples induced by NE andOP compounds. This highlights the broad applicability of the presently disclosed sensor platform for sensing real-world analytes.

[0134] Monitoring the inhibition ofP-CHE using varying amounts of inhibitors

[0135] The fluorescence spectra of 2 mg / L MC-SWCNTs (substrate-suspended SWCNTs) were recorded (Figure 26). The fluorescence spectra of MC-S WCNTs added with plasma (1: 10 dilution) were recorded (Figure 26). In three separate vials, P-CHE (1: 10 dilution) was added with 10 pL, 50 pL, and 100 pL of 10 mM organophosphate (inhibitor of cholinesterase, hereafter referred to as OP), and the solutions were incubated for 3 h. The net volume in all three vials was maintained to be the same. Thereafter, 3 pL of P-CHE (added with 10 pL of 10 mM OP) was added to MC-SWCNTs, and the fluorescence spectra were recorded (Figure 26). Further, 3 pL of P-CHE (added with 50 pL of 10 mM OP) was added to MC-SWCNTs, and the fluorescence spectra were recorded (Figure 26). Finally, 3 pL of P-CHE (added with 100 pL of 10 mM OP) was added to MC-SWCNTs, and the fluorescence spectra were recorded (magenta curve, Figure 3). Importantly, with increasing amounts of OP added to P-CHE, the extent of fluorescence quenching of MC-SWCNTs by P-CHE was found to decrease, suggesting an increased extent of inhibition of P-CHE by OP.

[0136] Monitoring cholinesterase activity and inhibition using DNA-functionalized single-walled carbon nanotubes

[0137] In an initial approach, the disclosers used DNA-functionalized SWCNTs for monitoring the activity and inhibition of ChE. The sensor was based on selective recognition of thiocholine, the cholinesterase-hydrolysis product of acetylthiocholine, triggering a NIR fluorescence intensity increase of DNA-functionalized SWCNTs. (Figure 27). The DNA- functionalized SWCNTs are prepared by wrapping single-walled carbon nanotubes with DNA, which stabilize the nanotubes and modulate their optical properties. Upon production of thiocholine, the thiol group of thiocholine interacts selectively with the DNA-functionalized SWCNTs, triggering a detectable change in the near-infrared (NIR) fluorescence intensity of the SWCNTs. Thiocholine is recognized by a DNA-SWCNT sensor, resulting in increased near-IR fluorescence intensity. This fluorescence modulation provides a signal that directly correlates with the cholinesterase activity, enabling real-time, label-free monitoring of enzyme activity. Furthermore, the system allows for detection of cholinesterase inhibitors, such asorganophosphates and carbamates, which block the hydrolysis of ATC and suppress the production of thiocholine. The presence of such inhibitors is indicated by a reduction or absence of the NIR fluorescence signal. An optical output in the 900-1400 nm range was obtained with a sub U L1LOD, demonstrating the ability to infer BChE inhibition in serum. This approach provides a sensitive, non-Ft, and reversible method for dynamically assessing cholinesterase activity and inhibition, offering significant utility in medical diagnostics, environmental monitoring, and inhibitor screening.

[0138] However, this approach is associated with certain disadvantages, namely, plasma itself causes a response allied to the enzymatic activity, thus baseline correction is involved; it is demonstrated to work on substrate-analogues of cholinesterase; and there is an indirect correlation of the degree of hydrolysis of cholinesterase with the optical response of the sensors. In contrast, monitoring direct substrate-enzyme interaction creates an optical response exclusive to cholinesterace hydrolysis; no baseline correction is needed; it works on real substrates for cholinesterase enzymes; and provides the possibility of quantifying the product of cholinesterase hydrolysis and correlating the reaction with the optical response of the sensors.

[0139] Applicability of DNA-SWCNTs in monitoring cholinesterase activity in blood plasma

[0140] Case 1: (GT)15 SWCNTs: The fluorescence spectra of 2 mg / L (GT)15 SWCNTs were recorded (black curve, Figure 4). The fluorescence spectra of (GT) 15 SWCNTs added with plasma (1:10 dilution were recorded (Figure 28). It showed a slight increase. Next, 2 mg of ATC (the substrate of cholinesterase) was solubilized in plasma (1: 10 dilution). This ATC solution (in plasma) was added to 2 mg / L of (GT) 15 SWCNTs and incubated for 45 min. The corresponding fluorescence spectra were recorded (Figure 28). Akin to the buffer solution, ATC reacted with cholinesterase in plasma, leading to the formation of thiocholine, which further interacted with (GT) 15 SWCNTs, resulting in an enhancement in the fluorescence.

[0141] Case 2: T30 SWCNTs: The fluorescence spectra of 2 mg / L T30 SWCNTs were recorded (Figure 29). The fluorescence spectra of T30 SWCNTs added with plasma (1:10 dilution were recorded (Figure 29). It showed a slight increase. Next, 2 mg of ATC (the substrate of cholinesterase) was solubilized in plasma (1: 10 dilution). This ATC solution (in plasma) was added to 2 mg / L of T30 SWCNTs and incubated for 45 min. The corresponding fluorescencespectra were recorded (Figure 29). Akin to the buffer solution, ATC reacted with cholinesterase in plasma, leading to the formation of thiocholine, which further interacted with T30 SWCNTs, resulting in an enhancement in the fluorescence.

[0142] Stability of MC-SWCNTs suspension following the interaction with CHE enzymes

[0143] Based on the preceding results, it is evident that MC molecules, which compose the corona phase of SWCNTs, are susceptible to hydrolysis by CHE enzymes, whether in their pure form or sourced from plasma. This enzymatic hydrolysis process can effectively be monitored through the modulation of the MC-SWCNTs fluorescence. Additional zeta potential measurements were conducted to further establish the connection between the fluorescence modulation of MC-SWCNTs and the hydrolysis of the cationic MC molecules into the corresponding anionic salt of myristic acid and choline, as confirmed herein through choline assay and mass spectrometry. Pristine MC-SWCNTs exhibited a zeta potential value of 24 ± 0.9 mV, attributed to the presence of cationic MC molecules constituting the corona phase of the SWCNTs. Upon reaction with ACHE, BCHE, and P-CHE, the zeta potential of the SWCNTs changed to -2.9 ± 0.23 mV, -1.8± 0.22 mV, and -0.76 ± 0.135 mV, respectively (Figure 24). This shift from positive to negative zeta potential values clearly indicates the alteration in the surface charge of MC-SWCNTs, attributed to the hydrolysis of cationic MC species to the corresponding anionic salt of myristic acid and choline.

[0144] Since the CHE substrate, MC, was used to directly suspend SWCNTs, the hydrolysis of the MC molecules in the suspension could compromise the colloidal stability of the MC-SWCNTs, thereby leading to SWCNT aggregation and subsequent decrease in the fluorescence intensity. To rule out SWCNT aggregation, MC-SWCNTs were incubated with ACHE, BCHE, and P-CHE for 3 hours and found no significant impact on the UV-vis-NIR absorption spectra of the MC-SWCNTs (Figure 25). Since SWCNT aggregation is directly reflected in their absorption, the lack of substantial changes in these spectra ensured that the hydrolysis of MC did not induce SWCNT aggregation. Moreover, there was no visible evidence of aggregation, as demonstrated by incubation of MC-SWCNTs with ACHE, BCHE, and P- CHE. SWCNTs are believed to remain dispersed despite chemical transformations occurring in their corona phase. Thus, the absence of alterations in the absorption of MC-SWCNTssubsequent to their reaction with ACHE, BCHE, and P-CHE, despite notable changes in fluorescence, implied an overall change in their quantum yield.

[0145] In order to determine whether the products of the MC hydrolysis, MA and choline, played a role in maintaining the dispersion of SWCNTs after MC hydrolysis, a suspension was attempted of SWCNT suspended directly with MA and a combination of MA and choline, both of which failed to lead to a stable SWCNT colloidal suspension. This finding effectively rules out the possibility of MA and choline solely stabilizing the SWCNTs subsequent to MC molecule hydrolysis. Therefore, it is conclude that the SWCNTs remain dispersed after enzymatic MC cleavage by a combination of unaffected MC molecules on the SWCNT surface and fresh MC molecules from the solution replacing the hydrolyzed ones.

[0146] Conclusion

[0147] The present disclosure presents a strategic approach for functionalizing SWCNTs to monitor the activity and inhibition of CHE enzymes within blood plasma. This represents the first-ever report on monitoring CHE enzymatic activity in clinical plasma samples in the NIR range using fluorometric techniques. The methodology involves the functionalization of SWCNTs with myristoylcholine (MC), a substrate for hydrolysis by CHE enzymes. The MC molecules, which constituted the corona phase of SWCNTs, underwent hydrolysis by CHE enzymes, resulting in a decrease in the SWCNT's fluorescence. The significance of the MC corona phase of the MC-SWCNTs in tracking CHE activity was further confirmed by the minimal fluorescence change of SC-SWCNTs and (GT)15-SWCNTs in the presence of CHE enzymes. Moreover, negligible change in the fluorescence of MC-SWCNTs was observed following the interaction with a diverse array of fundamental constituents found in blood plasma, including a range of cations, anions, sugar molecules, serum albumin, and amino acids. This highlights the selectivity of the response of the presently disclosed sensors towards CHE activity.

[0148] Conversely, when the CHE enzymes were exposed to inhibitor molecules such as neostigmine bromide, used as a therapeutic drug, and organophosphates, commonly found in pesticides, the enzymes were effectively inhibited, resulting in no discernible change in the fluorescence of the MC-SWCNTs. This enabled effective detection of the inhibition of CHE enzymes. Additionally, a choline assay was conducted to quantitatively assess the amount of choline released as a consequence of MC hydrolysis. Interestingly, the amount of cholinereleased exhibited a clear correlation with the observed decrease in fluorescence intensity of the MC-SWCNTs. The release of choline as a result of MC hydrolysis by CHE was further substantiated by ESI-MS analysis. The LOD for monitoring CHE activity achieved through MC- SWCNTs was observed to be on par with the conventional Ellman assay method.

[0149] The presently disclosed SWCNTs sensor platform for detection of CHE activity and inhibition, with optical feedback in the NIR biologically transparent spectral range, will not only advance diagnostic techniques for blood samples but also pave the way for design principles in sensor functionalization, enabling customized applications with significant potential.

[0150] Experimental Section / Methods

[0151] Materials: Hipco SWCNTs were purchased from Nano Integris. The Israel Institute for Biological Research supplied ACHE. BCHE, choline chloride, 5,5-dithio-bis(2- nitrobenzoic acid) (DTNB), myristic acid, glutamic acid, iron (II) sulfate heptahydrate, copper (II) sulfate, zinc chloride, sodium cholate, esterase, amidase and bis-[2-(methacryloyloxy)ethyl] phosphate were purchased from Sigma- Aldrich (Israel). L-cysteine and Neostigmine bromide were purchased from Holland Moran (Israel). Myristoylcholine was purchased from 1 ClickChemistry. (GT) 15 DNA oligomer was sourced from Integrated DNA Technologies. Blood plasma was provided by the blood bank in Sheba Hospital (Israel). Prior consents were received from the relevant subjects (approval number: 0005977-8). All chemicals were used as obtained without further purification.

[0152] Suspension of MC-SWCNTs: 1.5 mg of HipCo SWCNTs were to 15 mM MC- chloride in water and bath sonicated for 20 min. The resultant mixture was tip-sonicated in 2 cycles, each lasting 30 minutes, on ice. The so-formed dispersion of MC-SWCNTs was ultracentrifuged (Beckman Coultur) for 4 h at a speed of 40,000 rpm. The pellet comprising aggregated SWCNTs was discarded, and the supernatant was used for further experiments. The concentration of MC-SWCNTs was determined using UV-vis-NIR absorption spectroscopy with an extinction coefficient of 0.036 L • mg-1 • cm-1 at 632 nm. For an attempt to suspend the SWCNTs with myristic acid (MA) and MA in combination with choline, 15 mM of MA and choline were used.

[0153] Absorption: Absorption spectra were acquired using a UV-vis-NIR spectrophotometer (Shimadzu UV-3600 Plus) spanning within the wavelength range of 300 to 1400 nm.

[0154] Fluorescence: Fluorescence emission spectra were captured from samples within a 96-well plate placed on the stage of an inverted microscope (Olympus 1X73). The excitation source employed was a 730 nm CW laser (MDL-MD-730-1.5W, Changchun New Industries). Fluorescence emission spectra were resolved by a spectrograph (Spectra Pro HRS -300, Princeton Instruments), with a slit- width measuring 500 pm and a grating with a density of 150 g mm-1. Subsequent recording of the fluorescence intensity spectrum was executed utilizing a ID InGaAs array detector (PylonIR, Teledyne Princeton Instruments), with 3 s exposure time. Excitation-emission maps were acquired by sweeping excitation wavelength range spanning from 500 to 840 nm, incrementing in 2 nm steps, using a supercontinuum white-light laser (NKT-photonics, Super-K Extreme). All spectra were background subtracted against blank PBS. The intensity values for all the samples were acquired at the peak maximum for each spectrum. The laser power used in the experiments varied from 40 mW to 120 mW. All fluorescence responses were normalized to the initial fluorescence intensity, thereby negating the effect of absolute fluorescence counts on the quantitative results.

[0155] Transmission electron microscopy: Transmission Electron Microscopy (TEM) analysis was performed using a Talos F200i (S)TEM instrument, which is equipped with a Schottky Field Emission Gun (S-FEG) and TWIN-Lens configuration and manufactured by Thermo Fisher Scientific. The TEM was operated at an accelerating voltage of 200kV. Image acquisition was facilitated using a Ceta-M detector, also manufactured by Thermo Fisher Scientific. The TEM measurements were done following drop casting of 7 pL of MC-SWCNTs (2 mg L- 1 ) on the TEM grid and drying overnight.

[0156] Mass spectrometry: Mass spectra were acquired using an LCMS Xevo-TQD instrument, and subsequent analysis was carried out utilizing the Agilent 1260 system, which incorporated a single quadrupole Mass Spectrometric Detector (MSD) equipped with a multimode ionization chamber capable of both Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI). Mass spectra were acquired using ESI positive mode.

[0157] Zeta potential measurements: Zeta potential measurements were done using Malvern - Zetasizer nano Z instrument within the range of ±200 mV at 25 °C. The concentration of MC-SWCNTs used for zeta potential measurements was 2 mg L-L The activities of ACHE, BCHE, and P-CHE used for zeta potential measurements were 5.05, 6.2, and 0.36 U L-l, respectively.

[0158] Fluorometric response of MC-SWCNTs to ACHE, BCHE, and P-CHE: In order to probe the fluorometric response of MC-SWCNTs upon the hydrolysis of MC by ACHE, BCHE, and P-CHE, 147 pL of MC-SWCNTs (2 mg L-l) in PBS solution was placed in 96 well plates. Subsequently, aliquots of 3 pL of ACHE (5.05 U L-l), BCHE (6.2 U L-l), and P-CHE (0.18 U L-l) were introduced to the MC-SWCNT dispersions, and the fluorescence spectra of the resultant dispersions were recorded as a function of time. For monitoring the variation in fluorescence of MC-SWCNTs, upon exposure to differing quantities of ACHE, BCHE, and P- CHE, a volume of 3 pL for each respective enzyme across various concentrations was added to 147 pL of a PBS solution containing 2 mg L-l MC-SWCNTs. The fluorescence spectra of the resultant dispersions were recorded following incubation of the enzymes with MC-SWCNTs for 3 h with a 30 min interval. The interaction between MC-SWCNTs and the enzymes was probed using variation in the intensity of (9,4) chirality of the MC-SWCNTs. The enzyme concentration is quantified in units of their enzymatic activity, denoted as “U”, representing the concentration of enzyme capable of catalyzing the hydrolysis of 1 pM of the substrate within a minute under ambient conditions, specifically at room temperature and a pH value of 7.4. The error bars depicted in the figures correspond to the standard deviation derived from replicating the experiments in triplicates.

[0159] Probing enzyme inhibition: To probe inhibition of ACHE, BCHE, and P-CHE using neostigmine bromide, 100 pL of 50 mM neostigmine bromide solution was incubated with 100 pL of 5.05, 6.2, and 0.018 U L-l of ACHE, BCHE, and P-CHE, respectively, and the resultant mixture was incubated for 3 h. A total of 3 pL of the aforementioned blends were added to 147 pL of MC-SWCNTs (2 mg L-l), and the fluorescence of the latter was monitored following a 3 h incubation. Further, to probe the inhibition of P-CHE with organophosphates (OP), 100 pL of 100 pM OP solution was incubated with 0.36 U L-l of P-CHE for 3h. Aliquotsof 3 pL from the aforementioned mixture were added to 147 pL of MC-SWCNTs, incubated for 3 h, and the MC-SWCNT fluorescence modulation was monitored.

[0160] Ellman assay: The assay was performed using previously established protocols. Precisely, 10 pL of DTNB (10 mM) and 5 pL ATC (28 mM) were sequentially added to 200 pL of PBS. Next, 100 pL of ACHE, BCHE, and P-CHE were added independently, and the absorbance kinetics of the solutions were measured in 96-well plates at 436 nm for 5 min using a plate reader (Fusion Optics Reader Platform SPARK). The observed linear absorption curve over the course of time served as a direct representation of the quantity of 3-carboxy-4- generated nitrobenzene thiolate anion (with a molar absorptivity of s = 10.6 * 103 M-l cm-1). This anion formation is directly proportional to the concentration of thiocholine produced within the solution, consequently reflecting the activity of cholinesterase enzymes. A blank measurement was performed in similar conditions without adding the enzymes. The minimum values of absorbance observed in the blank samples were subtracted from the time-dependent linear absorbance curves obtained following the addition of CHE enzymes.

[0161] Choline assay: The choline assay (Sigma Aldrich) was conducted according to established procedures. Initially, 990 pL of choline assay buffer was added to 10 pL of 50 mM choline, resulting in the formulation of a 0.5 mM choline solution. Then, varying volumes of the prepared choline solution (0, 2, 4, 6, 8, 10, 12, 15, 30 pL) were combined with the choline assay buffer in a 96-well plate, culminating in a final volume of 50 pL within each well. The absorption peak of these solutions was subsequently measured at 570 nm using a plate reader, and a standard curve was constructed to facilitate choline quantification. The absorbance values at 570 nm were directly proportional to the quantity of choline present.

[0162] Concurrently, the disclosers prepared seven microcentrifuge tubes for experimentation. In six of these tubes, were combined 147 pL of MC-SWCNTs dispersion (at a concentration of 2 mg L-l) with 3 pL of 5.05 U L-l of ACHE, while the seventh tube contained 147 pL of MC-SWCNTs dispersion mixed with 3 pL of PBS, serving as a blank sample. In a 96- well plate was created a solution consisting of 46 pL of choline assay buffer, 2 pL of the enzyme mixture, and 2 pL of the choline probe. To this mixture, was added 50 pL of the reaction mixture containing MC-SWCNTs and ACHE. Samples were extracted from the microcentrifuge tubes at specific time intervals (0, 30, 60, 90, 120, 150, and 180 min). Of particular importance, thereaction mixture comprising SWCNTs and ACHE was pre -treated with 50 pL of neostigmine bromide (50 mM) before being added to the choline reaction set comprising choline buffer, probe, and enzyme. This pre-treatment was designed to prevent the further production of choline by ACHE hydrolysis during the choline assay. Subsequently, the disclosers measured the absorbance of these solutions at 570 nm following 30 min of incubation using a plate reader. The concentration of the released choline within the reaction mixture was determined by referencing it to an established standard choline curve. Similar experiments were conducted with BCHE (6.2 U L-l) and P-CHE (0.18 U L-l) to estimate the amounts of choline released in their respective cases. Trace amounts of choline were detected in the MC-SWCNTs, albeit significantly lower compared to the choline content within the reaction mixture of CHE enzymes and MC-SWCNTs. Consequently, when calculating the final choline quantity, the disclosers subtracted the minuscule choline content found in MC-SWCNTs from the choline concentration released in the presence of the enzymes.

[0163] Fluorometric response of MC-SWCNTs to control analytes: Aliquots of 500 pM of copper (II) sulfate, iron (II) sulfate heptahydrate, zinc chloride, glucose, L-cysteine and glutamic acid were added to MC-SWCNTs (2 mg L-l) in PBS solution for 3 h incubation, and the consequent changes in the MC-SWCNTs fluorescence were monitored. Moreover, 3 pL myristic acid or choline chloride (15 mM each) were added to 2 mg L-l of MC-SWCNTs, and the resultant fluorescence spectra were recorded. Furthermore, P-CHE and ACHE at respective activities of 0.18 U L-l and 5.05 U L-l were incubated with 2 mg L-l of (GT)15-SWCNTs for 3 h, and the fluorescence spectra of the latter were monitored. Finally, ACHE and BCHE (at respective activities of 5.05 and 6.2 U L-l) were incubated with 2 mg L-l of sodium cholate functionalized SWCNTs for 3 h, following which the fluorescence of the latter was tracked.

[0164] Inhibitors

[0165] According to certain embodiments, the disclosure includes but is not limited to inhibitors including neostigmine bromide and related cholinesterase inhibitors, such as, for example pyridostigmine bromide, physostigmine, edrophonium chloride, and rivastigmine. According to other embodiments, the disclosure includes organophosphates, including but not limited to, paraoxon, malathion, chlorpyrifos, diazinon, parathion, dimethoate, fenthion, phosmet, fenitrothion, sarin, soman, VX, and tabun. According to further embodiments, thedisclosure includes carbamates, including carbaryl, aldicarb, propoxur, methomyl, oxamyl, fenoxycarb, carbofuran, pirimicarb, physostigmine, and rivastigmine.

[0166] Supporting Tables

[0167] Table SI:Four parameters logistic fitting with a zero baseline. — - = — - — - , I isthe final fluorescence intensity, Io is the initial fluorescence intensity, A is the proportion constant, B is the inflection point, n is the cooperativity factor, and C is the concentration of cholinesterase.

Claims

CLAIMS1. A sensor for monitoring enzymatic activity in a biological sample, comprising: a near-infrared (NIR) fluorescent single- walled carbon nanotube (SWCNT) functionalized with a substrate hydrolysable by an enzyme.

2. The sensor according to claim 1 , wherein the hydrolysis of the substrate by the enzyme results in a decrease in NIR fluorescence intensity of the SWCNT; and wherein a magnitude of the decrease in NIR fluorescence intensity is quantitatively correlated with an amount of the substrate hydrolyzed by the enzyme.

3. The sensor according to claim 1, wherein the enzyme is a cholinesterase (CHE).

4. The sensor according to claim 1 , wherein the enzyme is selected from the group consisting of plasma-cholinesterase (P-CHE), acetylcholinesterase (ACHE), and butrylcholinesterase (BCHE).

5. The sensor according to claim 1, wherein the enzyme is CHE.

6. The sensor according to claim 1 , wherein the enzyme is P-CHE.

7. The sensor according to claim 1 , wherein the enzyme is ACHE.

8. The sensor according to claim 1, wherein the enzyme is BCHE.

9. The sensor according to claim 3, wherein the enzyme is inhibited by an enzyme inhibitor selected from the group consisting of neostigmine bromide, organophosphate, and carbamate.

11. The sensor according to claim 9, wherein the enzyme inhibitor prevents a decrease in the NIR fluorescence intensity of the SWCNT upon binding the enzyme by preventing hydrolysis of the substrate by the enzyme, and wherein the NIR fluorescence intensity is correlated with a concentration of the enzyme inhibitor present, allowing detection of enzyme inhibition.

12. The sensor according to claim 1, wherein the substrate comprises: a hydrophobic carbon chain configured to interact with the surface of the SWCNT through hydrophobic interactions; and a hydrophilic head group to enhance dispersion of the SWCNT in an aqueous medium.

13. The sensor according to claim 1, wherein the substrate is selected from the group consisting of myristoylcholine, acetylthiocholine, butrylcholine, and propionylcholine.

14. The sensor according to claim 1, wherein the substrate is myristoylcholine.

15. The sensor according to claim 14, wherein the enzyme is P-CHE; and wherein a correlation coefficient between a decrease in NIR fluorescence of the SWCNT upon hydrolysis of myristocholine by P-CHE and a release of choline is approximately -0.95.

16. The sensor according to claim 1, wherein the substrate is acetylthiocholine.

17. The sensor according to claim 1, wherein the substrate is butrylcholine.

18. The sensor according to claim 1, wherein the substrate is propionylcholine.

19. The sensor according to claim 1 , wherein the enzyme is CHE; and wherein a limit of detection (LOD) for detecting CHE enzyme activity is experimentally determined to be less than 1 U L-l.

20. The sensor according to claim 1 , wherein a LOD for detecting enzyme activity is approximately 0.0626 ± 0.0002 U L-l for ACHE, corresponding to a concentration of approximately 0.208 nM.

21. The sensor according to claim 1, wherein a LOD for detecting enzyme activity is approximately 0.0129 ± 0.001 U L-l for BCHE, corresponding to a concentration of approximately 7.14 nM.

22. The sensor according to claim 1 , wherein the limit of detection (LOD) for enzymatic activity is approximately 0.0168 ± 0.00005 U L-l for P-CHE, corresponding to a concentration of approximately 7.14 nM.

23. A method for measuring enzymatic activity in a biological sample, comprising: introducing into a biological sample, a sensor comprising a NIR fluorescent SWCNT functionalized with a substrate hydrolyzable by an enzyme; measuring a change in NIR fluorescence intensity of the SWCNT, wherein the change in fluorescence intensity is indicative of enzymatic activity; and correlating the change in fluorescence intensity to an amount of enzyme activity present in the biological sample.

24. The method according to claim 22, wherein the substrate comprises a hydrophobic carbon chain configured to interact with the surface of the SWCNT through hydrophobic interactions, and a hydrophilic head group to enhance dispersion of the SWCNT in an aqueous medium.

25. The method according to claim 22, wherein the enzyme is CHE.

26. The method according to claim 22, wherein the enzyme is selected from the group consisting of P-CHE, ACHE, and BCHE.

27. The method according to claim 22, wherein the substrate is selected from the group consisting of myristoylcholine, acetylthiocholine, butyrylcholine, and propionylcholine.

28. The method according to claim 22, wherein the enzyme is CHE; and wherein a limit of detection (LOD) for detecting CHE enzyme activity is experimentally determined to be less than 1 U L-1.

29. The method according to claim 22, wherein a magnitude of a decrease in NIR fluorescence intensity is quantitatively correlated with the amount of enzymatic activity in the biological sample.

30. The method according to claim 22, wherein the biological sample is a blood sample.

31. The method according to claim 22, wherein the biological sample is a plasma sample.

32. A method for measuring enzymatic inhibition, comprising: introducing into a biological sample, a sensor comprising a NIR fluorescent SWCNT functionalized with a substrate hydrolysable by an enzyme, the enzyme inhibitable by an enzyme inhibitor; measuring a change in NIR fluorescence intensity of the SWCNT, wherein the change in fluorescence intensity is indicative of enzymatic activity; comparing a measured fluorescence intensity to a baseline fluorescence intensity obtained from a control sample lacking the enzyme inhibitor; and correlating the change in NIR fluorescence intensity to an amount of enzyme inhibitor present in the biological sample.

33. The method according to claim 30, wherein the substrate comprises a hydrophobic carbon chain configured to interact with the surface of the SWCNT through hydrophobic interactions, and a hydrophilic head group to enhance dispersion of the SWCNT in an aqueous medium.

34. The method according to claim 30, wherein the enzyme is selected from the group consisting of CHE, P-CHE, ACHE, and BCHE.

35. The method according to claim 30, wherein the enzyme inhibitor is selected from the group consisting of neostigmine bromide, organophosphates, and carbamates.

36. The method according to claim 30, wherein the enzyme is CHE, and the LOD for detecting enzyme activity is less than 1 U L'1.

37. The method according to claim 30 wherein the enzyme inhibitor prevents hydrolysis of the substrate by the enzyme; and wherein inhibition of hydrolysis of the substrate by the enzyme results in no reduction in NIR fluorescence intensity of the biological sample.

Images