Rapid fentanyl sensing via biochemical assay

Abstract

The present disclosure pertains to a method of detecting an opioid in a sample by (1) determining a butyrylcholinesterase inhibition activity of the sample; and (2) correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample, where no relative inhibition of butyrylcholinesterase activity is correlated to the absence of opioid in the sample, and where a relative inhibition of butyrylcholinesterase activity is correlated to the presence of opioid in the sample. The present disclosure also pertains to assays for detecting opioid in a sample in accordance with the methods of the present disclosure.

Description

PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 TITLE RAPID FENTANYL SENSING VIA BIOCHEMICAL ASSAY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 556,480, filed on February 22, 2024. The entirety of the aforementioned application is incorporated herein by reference. BACKGROUND

[0002] A need exists for more effective methods and systems for detecting fentanyl in various settings at shorter periods of time. Numerous embodiments of the present disclosure aim to address the aforementioned need. SUMMARY

[0003] In some embodiments, the present disclosure pertains to a method of detecting an opioid in a sample. In some embodiments, such methods include: (1) determining a butyrylcholinesterase inhibition activity of the sample; and (2) correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample. In some embodiments, no relative inhibition of butyrylcholinesterase activity is correlated to the absence of opioid in the sample. In some embodiments, a relative inhibition of butyrylcholinesterase activity is correlated to the presence of opioid in the sample.

[0004] In some embodiments, the step of determining the butyrylcholinesterase inhibition activity of a sample includes: (1) adding the sample to a medium that includes butyrylcholinesterase and a butyrylcholinesterase substrate; and (2) determining a relative decrease of the conversion of the butyrylcholinesterase substrate to a product after the addition of the sample to the medium. In some embodiments, the relative decrease of the conversion of the butyrylcholinesterase substrate to the product after the addition of the sample to the medium is determined by a relative change in 1 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 absorbance of the medium. In some embodiments, the relative decrease of the conversion of the butyrylcholinesterase substrate to the product after the addition of the sample to the medium is determined by a relative change in color of the medium.

[0005] In some embodiments, the step of determining the butyrylcholinesterase inhibition activity of a sample includes: (1) adding the sample to a medium that includes butyrylcholinesterase, a butyrylcholinesterase substrate, and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product; and (2) determining a relative change in color of the medium. In some of such embodiments, the step of correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample includes correlating no relative change in color of the medium to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample. In some of such embodiments, the step of correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample may also include correlating a relative change in color of the medium to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample.

[0006] Additional embodiments of the present disclosure pertain to assays for detecting opioid in a sample in accordance with the methods of the present disclosure. In some embodiments, the assay includes butyrylcholinesterase. In some embodiments, the opioid detection assay also includes a medium that includes butyrylcholinesterase and a butyrylcholinesterase substrate. In some embodiments, the assay medium includes butyrylcholinesterase, a butyrylcholinesterase substrate, and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS.1A-1C illustrate methods of detecting opioid in a sample in accordance with various embodiments of the present disclosure.

[0008] FIG. 1D illustrates an assay for detecting opioid in a sample in accordance with various embodiments of the present disclosure.

[0009] FIGS.1E-1G show results and illustrations of an Ellman’s Assay for detecting Fentanyl. FIG. 1E shows a calibration curve generated from variable BtCh concentrations with constant 2 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 substrate concentrations. Absorbance values at 405 nm range from approximately 0.1–1.7 with 10 μM BtCh producing the lowest response (0.1) and 150 μM BtCh producing the highest response (1.7) at 600s. FIG. 1F shows that butyrylcholinesterase (BChE) activity dependence shows a strong linear correlation of 10 μM–150 μM BtCh with 25 mU BChE. FIG. 1G illustrates the addition of fentanyl analogues to an Ellman’s Assay, where the fentanyl analogues interact with BChE and lower its reaction rate. In particular, the presence of fentanyl analogues reduces the availability of BChE for BtCh to bind, resulting in the decreased hydrolysis of BtCh and a corresponding change in color produced compared to the assay conducted without fentanyl analogues.

[0010] FIG. 2 shows the activity of BChE exposed to Desopropionyl meta-methylfentanyl. Response values at 405 nm range from approximately 0.6–1.6 at 600s. The highest concentration (1 × 10–7M) produced the lowest response (0.6), and the lowest concentrations (1 × 10–10M to 1 × 10–12M) produced the highest response (1.4). At 600s, 1 × 10–10M to 1 × 10–12M caused a decrease in BChE activity by 0.1 compared to the control samples.

[0011] FIG. 3 shows the activity of BChE exposed to Benzyl acrylfentanyl (hydrochloride). Response values at 405 nm range from approximately 0.7–1.1 at 600s. The highest concentration (1 × 10–7M) produced the lowest response (0.7), and the lowest concentrations (1 × 10–10M to 1 × 10–12M) produced the highest response (0.9). At 600s, 1 × 10–9M to 1 × 10–12M caused a decrease in BChE activity by 0.2 compared to the control samples.

[0012] FIG. 4 shows the activity of BChE exposed to N-benzyl furanyl norfentanyl (hydrochloride). Response values at 405 nm range from approximately 0.9–1.7 at 600s. The highest concentration (1 × 10–7M) produced the lowest response (0.9), and the lowest concentrations (1 × 10–8M to 1 × 10–12M) produced the highest responses (1.2–1.4). At 600s, 1 × 10–8M to 1 × 10–12M caused a decrease in BChE activity by 0.2–0.4 compared to the control samples.

[0013] FIG. 5 shows the activity of BChE exposed to Benzyl carfentanil (hydrochloride). Response values at 405 nm range from approximately 1.0–1.6 at 600s. The highest concentration (1 × 10–7M) produced the lowest response (1.0), and the lowest concentrations (1 × 10–8M to 1 × 3 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 10–12M) produced the highest responses (1.2–1.5). At 600s, 1 × 10–8M to 1 × 10–12M caused a decrease in BChE activity by 0.1–0.4 compared to the control samples.

[0014] FIG.6 shows the activity of BChE exposed to Norsufentanil. Response values at 405 nm range from approximately 0.8–1.1 at 600s. The highest concentration (1 × 10–7M) produced the lowest response (0.8), and the lowest concentrations (1 × 10–8M to 1 × 10–12M) produced by the highest responses (1.0–1.1). At 600s, 1 × 10–8M to 1 × 10–12M caused a decrease in BChE activity by 0.0–0.1 when compared to the control samples.

[0015] FIG. 7 shows BChE inhibition from all fentanyl analogues at 90s. BChE inhibition is expressed in percentages derived from control sample comparison when exposed to 1 × 10–7M to 1 × 10–12M levels of respective fentanyl analogues at 90s.

[0016] FIG.8 shows BChE Inhibition from exposure to fentanyl analogues. BChE inhibition is expressed in percentages derived from control sample comparison when exposed to all levels of respective fentanyl analogues at 600s. The highest inhibitory pattern correlates to the highest concentration (1 × 10–7M), and the lowest inhibition correlates to the lowest concentrations (1 × 10–12M) of fentanyl analogues tested. DETAILED DESCRIPTION

[0017] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components that includes one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0018] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and 4 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0019] Fentanyl is a synthetic opioid developed as an intravenous anesthetic, which gained FDA approval for human medical use in 1968. Fentanyl is also used as an anesthetic in veterinary medicine for large animals. The U.S. Drug Enforcement Agency (DEA) classifies fentanyl and fentanyl analogues or Fentanyl-Related Substances (FRS) as Schedule II substances based on high abuse potential. The DEA states that fentanyl and FRS are considered dangerous since it is known to be up to 10,000 times more potent than morphine, dependent on the specific analogue.

[0020] Trafficking, distribution, and consumption of fentanyl and FRS have contributed to the nationwide opioid epidemic. In 2017, the Centers for Disease Control and Prevention (CDC) declared a public health emergency that arose from a 5-fold increase in opioid overdoses from 2012 to 2016. Fentanyl was determined to be associated with 57,834 deaths in 2020 and 71,238 deaths in 2021. According to the CDC, fentanyl and FRS were involved in 66% of all drug overdose fatalities in 2021.

[0021] The precursor for fentanyl is predominantly sourced from China and imported from Mexico, where it is produced and distributed across the U.S. from Mexico’s borders. In 2023, the U.S. Border Patrol and Office of Field Operations collaboratively seized 27,000 lbs of fentanyl, which is a 40% increase since 2021. Drug traffickers mix fentanyl and FRS with other illicit drugs such as cocaine, heroin, and methamphetamine to increase the potency and financial profit from products. This practice leads to users and victims unknowingly ingesting fentanyl and FRS. In 2023, the DEA seized 77 million fentanyl pills along with 12,000 lbs of fentanyl powder.

[0022] Fentanyl is not visually distinguishable from other illicit drugs in powder form. However, fentanyl and FRS are far more potent than other illicit substances. Therefore, a need exists for a rapid detection method for law enforcement and first responders to determine the specific substance(s) directly handled at the scene. Knowledge of fentanyl surface contamination is a key factor in minimizing accidental overdose risk. 5 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012

[0023] The Lateral Flow Immunoassay (LFI)-fentanyl test strips (FTS) and Enzyme-linked Immunosorbent Assay (ELISA) tests are currently used for fentanyl detection. FTS requires a 2– 5 min waiting period that is crucial to the safety of first responders, law enforcement, and other individuals who may have been exposed to fentanyl or FRS as overdoses can occur within this time frame. The presence of additional illicit substances and heterogenicity within the sample can also affect the FTS results.

[0024] Although FTS can detect nanogram levels of fentanyl and FRS with a minimum of 96% accuracy in numerous forms (pills, powder, and injectables), it is useful to evaluate additional techniques. A minimum of 10 mg or 5 mL, dependent on the form obtained, is recommended for FTS to accurately and precisely provide positive or negative results.

[0025] Other technological advancements have allowed law enforcement to detect fentanyl via hand-held Fourier Transform Infrared spectrometers and Raman spectrometers. Using these hand- held devices provides a safe, efficient, and intuitive method for individuals to analyze samples accurately without contact. Routine sample analyses with spectroscopic devices may not be available for agencies that do not have the required funding since the hand-held FT-IR and Raman analyzers cost thousands of dollars. Given the cost efficiency, accuracy, and precision, FTS technology can be explored and enhanced to require a smaller sample volume and decreased response time while still providing accuracy at ng levels. LFI-FTS is deemed to be a useful detection tool. However, knowledge of biochemical interactions may provide another mode for FTS analysis.

[0026] As such, a need exists for more effective methods and systems for detecting fentanyl in various settings at shorter periods of time. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0027] Methods of detecting an opioid in a sample

[0028] In some embodiments, the present disclosure pertains to a method of detecting an opioid in a sample. In some embodiments illustrated in FIG.1A, such methods include: determining a butyrylcholinesterase (BChE) inhibition activity of the sample (step 10); and correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the 6 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 sample (step 12). In some embodiments, no relative inhibition of butyrylcholinesterase activity (step 14) is correlated to the absence of opioid in the sample (step 16). In some embodiments, a relative inhibition of butyrylcholinesterase activity (step 18) is correlated to the presence of opioid in the sample (step 20). As set forth in more detail herein, the opioid detection methods of the present disclosure can have numerous embodiments.

[0029] Various methods may be utilized to determine the butyrylcholinesterase inhibition activity of a sample. For instance, in some embodiments, the step of determining the butyrylcholinesterase inhibition activity of the sample includes: (1) adding the sample to a medium that includes butyrylcholinesterase and a butyrylcholinesterase substrate; and (2) determining a relative decrease of the conversion of the butyrylcholinesterase substrate to a product after the addition of the sample to the medium. In some embodiments, the relative decrease of the conversion of the butyrylcholinesterase substrate to the product after the addition of the sample to the medium is determined by a relative change in absorbance of the medium. In some embodiments, the relative decrease of the conversion of the butyrylcholinesterase substrate to the product after the addition of the sample to the medium is determined by a relative change in color of the medium.

[0030] In some embodiments illustrated in FIGS. 1B and 1C, the step of determining the butyrylcholinesterase inhibition activity of the sample includes: adding the sample to a medium that includes butyrylcholinesterase, a butyrylcholinesterase substrate, and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product (step 30). In some of such embodiments, the step of correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample includes correlating no relative change in color of the medium (step 32) to no relative inhibition of butyrylcholinesterase activity (step 34) and the absence of opioid in the sample (step 36). In some of such embodiments, the step of correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample may include correlating a relative change in color of the medium (step 38) to relative inhibition of butyrylcholinesterase activity (step 40) and the presence of opioid in the sample (step 42). In some embodiments, the relative change in color of a medium is correlated to 7 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 relative inhibition or no relative inhibition of butyrylcholinesterase activity by comparing the relative change in color of the medium to a set of known colors.

[0031] Opioid detection assays

[0032] Additional embodiments of the present disclosure pertain to assays for detecting opioid in a sample. In some embodiments, the assay includes butyrylcholinesterase. In some embodiments, the assay is operable for determining a butyrylcholinesterase inhibition activity of the sample and correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample. In some embodiments, no relative inhibition of butyrylcholinesterase activity is correlated to the absence of opioid in the sample. In some embodiments, a relative inhibition of butyrylcholinesterase activity is correlated to the presence of opioid in the sample.

[0033] In some embodiments, the opioid detection assay also includes a medium that includes butyrylcholinesterase and a butyrylcholinesterase substrate. In some embodiments, the assay is operable for determining a relative decrease of the conversion of the butyrylcholinesterase substrate to a product after the addition of the sample to the medium.

[0034] In some embodiments, the assay medium includes butyrylcholinesterase, a butyrylcholinesterase substrate, and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product. In some embodiments, the assay is operable for determining a relative change in color of the medium, correlating no relative change in color of the medium to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample, and correlating a relative change in color of the medium to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample.

[0035] The opioid detection assays of the present disclosure may be in various forms. For instance, in some embodiments, the opioid detection assay may be in the form of lateral flow assays, vibrational spectroscopy assays (e.g., mid-infrared and Raman assays), gas chromatography–mass spectrometry assays, ion mobility spectrometry assays, high-pressure mass spectrometry assays, or combinations thereof. In some embodiments, the opioid detection assay is in the form of a lateral flow assay. In some embodiments illustrated in FIG.1D, the lateral flow assay includes a test strip 50 that includes at least one region 52 associated with 8 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 butyrylcholinesterase 53. In some embodiments, butyrylcholinesterase 53 may be cross-linked to region 52. In some embodiments, the assay further includes a medium 56 that includes a butyrylcholinesterase substrate 57 and a colorogenic compound 58 that reacts with the substrate’s product to form a colorogenic by-product.

[0036] In some embodiments, medium 56 is operable for flowing through test strip 50. In some embodiments, no relative change in color of region 52 is correlated to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample. In some embodiments, a relative change in color of region 52 is correlated to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample. In some embodiments, the opioid detection assay further includes a color chart 60. In some embodiments, color chart 60 includes a set of known color standards that correlate with the absence (-) or presence (+) of the opioid.

[0037] In some embodiments, the assay further includes a receptacle 54 positioned below region 52. In some embodiments, receptacle 54 is operable for receiving medium 56 and flowing medium 56 through test strip 50.

[0038] Opioid detection through a lateral flow assay

[0039] In some embodiments also illustrated in FIG. 1D, the opioid detection methods of the present disclosure may detect opioid through a lateral flow assay. In some embodiments, such methods may include: (1) adding a sample to a medium 56 that includes a butyrylcholinesterase substrate 57 and a colorogenic compound 58 that reacts with the substrate’s product to form a colorogenic by-product; (2) flowing the medium through the test strip 50; and (3A) correlating no relative change in color of region 52 to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample, and (3B) correlating a relative change in color of region 52 to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample.

[0040] In some embodiments, flowing the medium through the test strip 50 includes placing the medium 56 containing the sample in a receptacle 54 positioned below the region 52. In some embodiments, the relative change in color of the region is determined by a color chart 60 that includes a set of known standards. In some embodiments, colorimetric change can be quantitatively measured by a software, such as a software in a smartphone device. 9 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012

[0041] Opioid detection reagents

[0042] The opioid detection methods and assays of the present disclosure may utilize various reagents. For instance, in some embodiments, the medium is in the form of a solution. In some embodiments, solution includes a buffer. In some embodiments, the butyrylcholinesterase substrate includes butyrylthiocholine (BtCh). In some embodiments, the formed product includes thiocholine. In some embodiments, the colorogenic compound includes 5,5′-dithiobis-(2- nitrobenzoic acid) (DTNB) (i.e., Ellman’s reagent) and the colorogenic by-product includes 2- nitro-5-thiobenzoate (TNB). In some embodiments, DTNB cleaves a disulfide bond from the product to yield TNB, which has a yellow color.

[0043] Samples

[0044] The opioid detection methods and assays of the present disclosure may detect opioids from various samples. For instance, in some embodiments, the sample includes, without limitation, a water sample, a soil sample, a food product sample, a biological sample, a pill, a packaging sample, a surface sample, a paraphernalia sample, or combinations thereof. In some embodiments, the sample is in solid form. In some embodiments, the sample is in liquid form.

[0045] Opioids

[0046] The opioid detection methods and assays of the present disclosure may detect various opioids. For instance, in some embodiments, the opioid includes, without limitation, heroin, oxycodone, hydrocodone, morphine, codeine, opium, oxymorphone, fentanyl, fentanyl analogs, fentanyl derivatives, or combinations thereof. In some embodiments, the opioid includes fentanyl. In some embodiments, the opioid includes one or more fentanyl analogs. In some embodiments, the one or more fentanyl analogs include, without limitation, desopropionyl meta-methylfentanyl, benzyl acrylfentanyl (hydrochloride), n-benzyl furanyl norfentanyl (hydrochloride), benzyl carfentanil (hydrochloride), norsufentanil, tetrahydrofuranylfentanyl (THFF), furanylfentanyl, acetylfentanyl, ocfentanil, butyrfentanyl, cyclopropylfentanyl, methoxyacetylfentanyl, acrylfentanyl, para-fluoroisobutyrfentanyl, para-fluoroisobutyrylfentanyl, carfentanil, a- methylfentanyl, or combinations thereof.

[0047] Speed 10 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012

[0048] The opioid detection methods and assays of the present disclosure may detect opioids at various speeds. For instance, in some embodiments, the opioid detection methods and assays of the present disclosure detect opioid in less than 30 minutes. In some embodiments, the opioid detection methods and assays of the present disclosure detect opioid in less than 120 seconds. In some embodiments, the opioid detection methods and assays of the present disclosure detect opioid in less than 90 seconds.

[0049] Sensitivity

[0050] The opioid detection methods and assays of the present disclosure can have various sensitivities. For instance, in some embodiments, the opioid detection methods and assays of the present disclosure are operable to detect the opioid at concentrations ranging from about 1×10–7M to about 1×10–12M.

[0051] Additional Embodiments

[0052] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0053] Example 1. Evaluation of fentanyl exposure effects on butyrylcholinesterase activity as a tool for future on-site detection methods

[0054] Prior studies concur there is an interaction between butyrylcholinesterase (BChE) and choline-based esters, such as organophosphates, carbamate, cocaine, and heroin. It is suggested that BChE hydrolyzes methyl ester and benzoyl ester sites to form inactive metabolites. Heroin is also metabolized by BChE to produce morphine. In a previous study, the effect of fentanyl and remifentanil on neuron damage and oxidative stress during neurotoxicity induction was evaluated using whole-cell specimens. It was suggested that BChE activity decreased while acetylcholinesterase (AChE) activity remained relatively constant providing insight into the concept that fentanyl specifically interacts with BChE.

[0055] However, the interaction between BChE and fentanyl has remained unknown due to the study of complex specimens, which may contribute to the conclusion that BChE inactivity is a 11 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 direct result of fentanyl exposure as opposed to unknown mechanisms influencing decreased enzymatic activity.

[0056] This Example aims to provide proof of concept that an interaction between BChE and fentanyl analogues occurs by direct molecule exposure in solution versus evaluation of complex systems to fill the gap in existing research. This Example assessed BChE function from exposure to 5 different fentanyl analogues at variable concentrations with Ellman’s Assay.

[0057] Results demonstrate inhibition of BChE from all fentanyl analogues tested in concentrations from 1 × 10–7M to 1 × 10–12M. These findings can faciliate developments of technology that can detect fentanyl-related substances (FRS) in addition to current techniques. For instance, the Ellman’s assay implemented onto a single-use portable kit-like device would immensely impact law enforcement, first responders, and the general public by providing an accurate technique for the evaluation of surface contamination and illicit substances on-site.

[0058] Example 1.1. Materials

[0059] The following enzymes and reagents utilized during this Example were purchased from Millipore Sigma (Burlington, MA): butyrylcholinesterase from equine serum (BChE; EC 3.1.1.8), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), s-butyrylthiocholine iodide (butyrylthiocholine or BtCh), potassium phosphate monobasic, potassium phosphate dibasic, and 100% methanol. The fentanyl analogues were chosen based on availability and purchased from Cayman Chemical Company (Ann Arbor, MI): benzyl acrylfentanyl (hydrochloride), benzyl carfentanil (hydrochloride), N-benzyl furanyl norfentanyl (hydrochloride), norsufentanil, and desopropionyl meta-methyl fentanyl. A Barnstead Nano-Pure purification system from Thermo-Scientific (Waltham, MA) was used to obtain ultrapure (18.2 MΩ·cm) water. All analyses were performed at 37 °C using the BioTek Synergy H1 microplate reader from Agilent with 96-well microtiter polystyrene plates purchased from Avantor-VWR (Radnor, PA).

[0060] Example 1.2. Optimization of Ellman’s Assay

[0061] Before BChE exposure to all fentanyl analogues, optimization of Ellman’s assay was completed with the following reaction components: 50 mM potassium phosphate buffer, pH 7.4, 25 mU BChE, 500 μM DTNB, and variable BtCh concentrations at 10 μM, 25 μM, 50 μM, 75 μM, 12 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 100 μM, 125 μM, and 150 μM. Each concentration was measured in triplicate at 405 nm for 600s at 37 °C using the BioTek Synergy H1 microplate reader,

[0062] Example 1.3. Ellman’s Assay with Fentanyl Analogues

[0063] Following protocol optimization, the components were added to a 96-well microtiter polystyrene plate in the following order: 50 mM potassium phosphate buffer, pH 7.4, 25 mU BChE, 1 × 10–7to 1 × 10–12M fentanyl analogue, 500 μM DTNB, and 100 μM BtCh. Each FRS concentration was measured in triplicate at 405 nm for 600s at 37 °C using the BioTek Synergy H1 microplate reader.

[0064] Example 1.4. Statistical Analysis

[0065] Excel was used to determine the change in absorbance, triplicate response averages, standard deviations, and percent inhibition values. Enzyme inhibition was calculated by (Absblank – Abssample) / Absblank × 100. All graphs were generated using OriginPro 9.1.

[0066] Example 1.5. Optimization of Ellman’s Assay

[0067] Concentrations of BtCh were measured at 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, and 150 μM to assess optimal concentration performance, which was determined to be 100 μM based on ideal signal responses shown in FIG. 1E. Determination of the 4:1 substrate-to- enzyme ratio was essential to yield the most accurate comparison between control samples and fentanyl-treated samples. It should be noted that control sample values are within the optimal 1– 2 absorbance range for all experiments and demonstrate maximum efficacy of BtCh hydrolysis via BChE.

[0068] The calibration curve for Ellman’s assay depicted in FIG.1F shows the linear correlation between variable concentrations of BtCh with 25 mU BChE at 405 nm for 600s. The R2value was 0.997, and the Pearson’s R value was 0.999. These values serve to highlight a strong substrate concentration dependence of BtCh hydrolysis by 25 mU of BChE. FIG. 1G provides an illustration of the Ellman’s Assay.

[0069] Example 1.6. Determination of BChE Activity with Fentanyl Analogues

[0070] The first fentanyl analogue assessed was desopropionyl meta-methylfentanyl. This FRS demonstrated the inhibition of BChE activity at all concentrations tested, as shown in FIG. 2. 13 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 Significant BChE inhibition was observed at higher concentrations of 1 × 10–7M to 1 × 10–9M. Lower concentrations of 1 × 10–10M to 1 × 10–12M produced indistinguishable values due to lack of inhibitory difference. Significant BChE inhibition was further expressed as concentrations of desopropionyl meta-methyl fentanyl increased.

[0071] The second fentanyl analogue acquired was benzyl acrylfentanyl (hydrochloride). This FRS demonstrated inhibition of BChE activity at all concentrations tested, as shown in FIG. 3. The lowest concentrations of 1 × 10–10M to 1 × 10–12M strongly inhibited BChE. It was observed that 1 × 10–10M exhibited greater inhibition initially as compared to 1 × 10–9M. Significant BChE inhibition was further expressed as concentrations of benzyl acrylfentanyl (hydrochloride) increased.

[0072] The third fentanyl analogue assessed was n-benzyl furanyl norfentanyl (hydrochloride). This FRS demonstrated inhibition of BChE activity at all concentrations tested, as shown in FIG. 4. Indistinguishability was observed between 1 × 10–8M and 1 × 10–12M at 600s due to lack of inhibitory difference when these concentrations were evaluated at this interval. However, 1 × 10–10M up to 500s does show a lower response compared to 1 × 10–9M, 1 × 10–11, and 1 × 10–12, but this response still does not provide distinguishability. Significant BChE inhibition was further expressed with 1 × 10–7M n-benzyl furanyl norfentanyl (hydrochloride).

[0073] The fourth fentanyl analogue assessed was benzyl carfentanil (hydrochloride). This FRS demonstrated insignificant inhibition of BChE activity between 1 × 10–8M to 1 × 10–12M, as shown in FIG.5. Distinguishability was observed with 1 × 10–7M, but concentration dependence cannot be observed at lower levels. However, there is distinguishability between 1 × 10–8M to 1 × 10–12M and the control sample, which suggests there is an effect on BChE activity when exposed to benzyl carfentanil (hydrochloride).

[0074] The fifth fentanyl analogue assessed was norsufentanil. This FRS demonstrated insignificant and indistinguishable inhibition of BChE activity between 1 × 10–11M and 1 × 10–12M after 400s shown in FIG. 6. Significant BChE inhibition was further expressed as the concentrations increased. Norsufentanil levels at 1 × 10–7M and 1 × 10–8M produced 14 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 indistinguishable response values as well, which suggests norsufentanil does not exhibit differential effects on BChE activity between these values similar to 1 × 10–11M and 1 × 10–12M.

[0075] The primary aspects to consider for the evaluation of FRS-induced inhibition of BChE is the sensitivity and speed of the modified Ellman’s assay system. Therefore, BChE activity was evaluated at 90 and 600s. FIG. 7 demonstrates the inhibition of BChE activity at all concentrations of each fentanyl analogue tested at 90s. The highest inhibition of BChE activity was observed with norsufentanyl at 41%. Negative percentages are caused by instrumentation background at the beginning of the analysis, but all fentanyl analogues exhibited inhibition within this time frame. When BChE was exposed to the highest concentration, 25%–41% inhibition was observed.

[0076] The inhibition pattern of BChE from each fentanyl analogue at 600s is shown in FIG.8. The gradual decrease in BChE activity is observed from exposure to all 5 fentanyl analogues as concentrations increased. Each FRS demonstrated the highest BChE inhibition at 1 × 10–7M which resulted in 43%–36% inhibition. Exposure to FRS at 1 × 10–8M resulted in BChE inhibition of 38%–12%. At 1 × 10–9M, FRS demonstrated BChE inhibition between 10%–29% while 1 × 10–10M caused 29% to 8% BChE inhibition. Lastly, 1 × 10–11M and 1 × 10–12M demonstrated the lowest inhibition from 25%–6%.

[0077] In summary, the availability of BChE to hydrolyze BtCh decreased when exposed to all fentanyl analogues compared to triplicates not exposed to these FRS. Thus, a decreased rate of BtCh hydrolysis by BChE resulted in lower concentrations of butyrate and thiocholine, which decreased the availability of interactions between DTNB and the thiol group in thiocholine. This was directly observable from the decreased color intensity development measured at 405 nm.

[0078] Overall concentration dependence was observed, which is highlighted in the BChE inhibitory patterns from FIG. 8. As the concentrations of FRS increased, BChE inhibition also increased and resulted in lower responses from the color intensity depicted in FIG.1G.

[0079] Considering the sensitivity and speed of the developed concept, all fentanyl analogues were evaluated at 90s and 600s. At the 90s interval, each fentanyl analogue caused significant BChE inhibition at 1 × 10–7M. At 600s, BChE inhibition was observed with all fentanyl analogues at 1 15 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 × 10–7M to 1 × 10–12M with overall concentration dependence. Given various levels of BChE inhibition was observed, selectivity preventing detection of all FRS may cause current FTS to provide false negative results. A previous study revealed that the potency of fentanyl analogues varies based on molecular variations at para, ortho, and meta positions. These results confirm this concept reflected in the data presented as specific levels of BChE inhibition occurred when exposed to different fentanyl analogues, which is highlighted in the inhibitory patterns represented in FIG.8.

[0080] For example, it was discovered that benzyl carfentanil (hydrochloride) inhibited BChE activity at 30% during the 90s interval, but benzyl acrylfentanyl (hydrochloride) caused BChE inhibition at 41% during the 90s interval, which indicates the sensitivity of Ellman’s assay, but also introduces a nonselective method based solely on BChE inhibition for detection of FRS. However, analysis of these analogues at 600s provided increased distinguishability of inhibitory patterns observed for all FRS concentrations indicating the potential for more consistent and reliable detection of fentanyl analogues within 600s, but it does not hinder the ability of the developed concept to detect any fentanyl analogue within 90s.

[0081] New developments provide a further understanding of fentanyl toxicity that can be used for robust detection of these extremely toxic compounds in field conditions. Results demonstrate significant inhibition of BChE when exposed to all fentanyl analogues and indicate the potential to utilize BChE activity as a basis for detecting FRS at 1 × 10–7M within 90s, which highlights the sensitivity and efficiency of the developed method. The potential for a portable single-use kit to be developed for law enforcement and first responders would greatly impact the safety of public health nationwide as fentanyl can be detected from any surface at ng levels in less than 2 min with this method.

[0082] This research also suggests that BChE inhibition levels can provide distinguishability between other illicit substances based on enzyme specificity. This kit can be utilized in any environment that allows confirmatory testing without an uncompromised speed and accuracy. While this method does not solve the problem of fentanyl exposure in the U.S., it can directly prevent overdoses from accidental exposure. 16 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012

[0083] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein. 17 4823-4406-5785v.313368-42

Claims

PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 WHAT IS CLAIMED IS:

1. A method of detecting an opioid in a sample, said method comprising: determining a butyrylcholinesterase inhibition activity of the sample; and correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample, wherein no relative inhibition of butyrylcholinesterase activity is correlated to the absence of opioid in the sample, and wherein a relative inhibition of butyrylcholinesterase activity is correlated to the presence of opioid in the sample.

2. The method of claim 1, wherein the step of determining the butyrylcholinesterase inhibition activity of the sample comprises: adding the sample to a medium comprising butyrylcholinesterase and a butyrylcholinesterase substrate; and determining a relative decrease of the conversion of the butyrylcholinesterase substrate to a product after the addition of the sample to the medium.

3. The method of claim 2, wherein the butyrylcholinesterase substrate comprises butyrylthiocholine (BtCh), and wherein the product comprises thiocholine.

4. The method of claim 2, wherein the relative decrease of the conversion of the butyrylcholinesterase substrate to the product after the addition of the sample to the medium is determined by a relative change in absorbance of the medium. 18 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 5. The method of claim 2, wherein the relative decrease of the conversion of the butyrylcholinesterase substrate to the product after the addition of the sample to the medium is determined by a relative change in color of the medium.

6. The method of claim 1, wherein the step of determining the butyrylcholinesterase inhibition activity of the sample comprises: adding the sample to a medium comprising butyrylcholinesterase, a butyrylcholinesterase substrate, and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product; and determining a relative change in color of the medium.

7. The method of claim 6, wherein the step of correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample comprises: correlating no relative change in color of the medium to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample, and correlating a relative change in color of the medium to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample.

8. The method of claim 6, wherein the butyrylcholinesterase substrate comprises butyrylthiocholine (BtCh), the product comprises thiocholine, the colorogenic compound comprises 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and the colorogenic by-product comprises 2-nitro-5-thiobenzoate (TNB). 19 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 9. The method of claim 1, wherein the opioid is detected through a lateral flow assay, wherein the lateral flow assay comprises a test strip comprising at least one region associated with butyrylcholinesterase, and wherein the method comprises: adding the sample to a medium comprising a butyrylcholinesterase substrate and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product, flowing the medium through the test strip, correlating no relative change in color of the region to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample, and correlating a relative change in color of the region to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample.

10. The method of claim 1, wherein the opioid is selected from the group consisting of heroin, oxycodone, hydrocodone, morphine, codeine, opium, oxymorphone, fentanyl, fentanyl analogs, fentanyl derivatives, or combinations thereof.

11. The method of claim 1, wherein the opioid comprises fentanyl.

12. The method of claim 1, wherein the opioid comprises one or more fentanyl analogs.

13. The method of claim 12, wherein the one or more fentanyl analogs is selected from the group consisting of desopropionyl meta-methylfentanyl, benzyl acrylfentanyl (hydrochloride), n- benzyl furanyl norfentanyl (hydrochloride), benzyl carfentanil (hydrochloride), norsufentanil, tetrahydrofuranylfentanyl (THFF), furanylfentanyl, acetylfentanyl, ocfentanil, butyrfentanyl, cyclopropylfentanyl, methoxyacetylfentanyl, acrylfentanyl, para-fluoroisobutyrfentanyl, para- fluoroisobutyrylfentanyl, carfentanil, a-methylfentanyl, or combinations thereof. 20 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 14. The method of claim 1, wherein the sample comprises a water sample, a soil sample, a food product sample, a biological sample, a pill, a packaging sample, a surface sample, a paraphernalia sample, or combinations thereof.

15. The method of claim 1, wherein the method occurs in less than 120 seconds.

16. The method of claim 1, wherein the method is operable to detect the opioid at concentrations ranging from about 1×10–7M to about 1×10–12M.

17. An assay for detecting opioid in a sample, wherein the assay comprises butyrylcholinesterase, and wherein the assay is operable for determining a butyrylcholinesterase inhibition activity of the sample and correlating the butyrylcholinesterase inhibition activity of the sample to the presence or absence of opioid in the sample, wherein no relative inhibition of butyrylcholinesterase activity is correlated to the absence of opioid in the sample, and wherein a relative inhibition of butyrylcholinesterase activity is correlated to the presence of opioid in the sample.

18. The assay of claim 17, wherein the assay comprises a medium comprising butyrylcholinesterase and a butyrylcholinesterase substrate, and wherein the assay is operable for determining a relative decrease of the conversion of the butyrylcholinesterase substrate to a product after the addition of the sample to the medium.

19. The assay of claim 18, wherein the butyrylcholinesterase substrate comprises butyrylthiocholine (BtCh), and wherein the product comprises thiocholine. 21 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 20. The assay of claim 17, wherein the assay comprises a medium comprising butyrylcholinesterase, a butyrylcholinesterase substrate, and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product.

21. The assay of claim 20, wherein the assay is operable for determining a relative change in color of the medium, correlating no relative change in color of the medium to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample, and correlating a relative change in color of the medium to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample.

22. The assay of claim 20, wherein the butyrylcholinesterase substrate comprises butyrylthiocholine (BtCh), the product comprises thiocholine, the colorogenic compound comprises 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and the colorogenic by-product comprises 2-nitro-5-thiobenzoate (TNB).

23. The assay of claim 20, wherein the assay comprises a lateral flow assay, wherein the lateral flow assay comprises a test strip comprising at least one region associated with butyrylcholinesterase.

24. The assay of claim 23, wherein the assay further comprises a medium comprising a butyrylcholinesterase substrate and a colorogenic compound that reacts with the substrate’s product to form a colorogenic by-product.

25. The assay of claim 24, wherein the medium is operable for flowing through the test strip, wherein no relative change in color of the region is correlated to no relative inhibition of butyrylcholinesterase activity and the absence of opioid in the sample, and wherein a relative change in color of the region is correlated to relative inhibition of butyrylcholinesterase activity and the presence of opioid in the sample. 22 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P065WO Texas Tech No.2024-012 26. The assay of claim 23, wherein the assay further comprises a receptacle positioned below the region, wherein the receptacle is operable for receiving the medium and flowing the medium through the test strip.

27. The assay of claim 20, wherein the assay further comprises a color chart, wherein the color chart comprises a set of known color standards that correlate with the absence or presence of the opioid. 23 4823-4406-5785v.313368-42

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