Electrochemical detection of cannabinoids

Abstract

The effect of a cationic hydrophobic polymer on improving sensitivity and selectivity toward the electrochemical detection of cannabinoid analytes, e.g. Δ9-tetrahydrocannabinol (Δ⁹-THC), has been investigated. Herein we report a rapid, inexpensive and stable method for the detection of 10 – 1000 ng / mL of cannabinoid analytes, e.g. Δ9-tetrahydrocannabinol (Δ⁹-THC), 5 in buffer solutions and in human saliva.

Description

Electrochemical Detection of Cannabinoids

[0001] This invention relates to the field of electrochemical sensors, particularly the detection of cannabinoids (e.g. A9-THC) in a buffer solution.BACKGROUND

[0002] Cannabis, commonly known as marijuana, is increasing in use worldwide and several countries worldwide have legalised the use of cannabis for recreational purposes. Driving under the influence of cannabis is a major concern. A recent meta-analysis has found that cannabis increases impairment for lane control and reaction time. In the UK, among the deceased drivers between 2014 to 2021 , cannabis was the most common illegal drug leading to road fatality.

[0003] A9-Tetrahydrocannabinol (A9-THC) is the psychoactive component in cannabis, because of its high lipophilicity it can easily cross over the blood-brain barrier and interact with the central nervous systems’ (CNS) cannabinoid receptors. This leads to several psychological effects such as euphoria, relaxation, decrease concentration, somnolence, temporal distortions, and hallucinations.

[0004] Therefore, multiple jurisdictions have attempted to set per se A9-THC limits in blood and saliva. In Canada, a roadside threshold of 5 ng / ml when analysed in blood and 25 ng / ml when measured in saliva has been adopted (P. Di Ciano, B. Brands, A. Fares, M. Wright, G. Stoduto, P. Byrne, M. McGrath, O. S. M. Hasan, B. Le Foil and C. M. Wickens, Cannabis and Cannabinoid Research, 2023, 8, 408-413). In England and Wales, a per se threshold of 2 ng / ml in blood has been established (Department for Transport UK , Changes to drug driving law,the US the perse limits or cutoffs values vary among different states but range from THC blood levels of 1 to 5 ng / ml A9- THC (T. R. Arkell, T. R. Spindle, R. C. Kevin, R. Vandrey and I. S. McGregor, Traffic Injury Prevention, 2021 , 22, 102-107). On site testing for THC levels has proved problematic, hence there is a need for rapid roadside detection of A9-THC that can be used to accurately screen drivers.

[0005] A9-THC can be tested in urine, blood, breath, and saliva. Urine tests, a commonly employed method for drug testing, can only detect A9-THC metabolites, which are only traceable in urine several hours after the intake of cannabis, far past the window of intoxication and impairment. Blood testing is not necessarily a good approach for A9-THC detection either due to its high logP and enterohepatic recirculation: A9-THC usually accumulates in the tissuesfor long period of times and it can be detected in the blood up to 30 days post consumption. A9-THC is retained in the breath for short period of times, no longer than 3h, making it difficult to determine if the driver is still under the influence. Oral fluid appears to be preferable for A9- THC roadside monitoring, being quick and less invasive. Preliminary evidence exists that indicates the concentration of A9-THC in saliva can be correlated with impairment (J.-R. Lee, J. Choi, T. O. Shultz and S. X. Wang, Analytical Chemistry, 2016, 88, 7457-7461).

[0006] The A9-THC concentration in saliva has been found to be affected by the method of consumption. Via smoking, it is reported to take 10 mins to detect THC’s maximum concentration. The A9-THC level in saliva of frequent smokers is on average 9 pM (2.83 pg / mL) and 3 pM (944 ng / mL) for occasional users. A9-THC consumed orally, takes 20-25 mins to detect the maximum salivary concentration presenting concentrations of 944 nM (297 ng / mL) frequent users and 642 nM (202 ng / mL) for occasional users (M. J. Swortwood, M. N. Newmeyer, M. Andersson, O. A. Abulseoud, K. B. Scheidweiler and M. A. Huestis, Drug Test. Analysis, 2017, 9, 905-915). Hence, it is necessary to develop a detection method able to comply with current jurisdictions and capable of detecting a range of salivary A9-THC concentration from 25 to 1000 ng / mL.

[0007] In recent years, multiple techniques for detecting A9-THC in saliva have been researched including the use of liquid chromatography with UV-vis or mass spectrometry detection, immunoassays, and surface enhanced Raman scattering. Although these techniques can detect low levels of A9-THC, they rely on relatively costly, non-portable, or non- scalable instrumentation. For example, the current on-market roadside test device, Drager DrugTest 5000, employs an immunoassay approach to detect A9-THC with a detection limit of 5 ng / mL but at high cost (~5500 USD). Another device is Druglizer LE5 Drug Testing System, based on a competitive lateral flow immunoassay with a A9-THC cut-off of 15 ng / mL, provides qualitative test results costs in excess of ~3195 USD plus cartridges. Other commercial roadside test devices are based on colour test strips, but do not provide quantitative information (Rapid STAT®, DrugWipe® S), which is key requirement in jurisdictions where there is a A9-THC threshold.

[0008] Electrochemical sensors, on the other hand, offer the possibility of low cost, rapid, high selectivity and sensitivity, and low sample volume. Electrochemical sensors are simple and portable devices for the detection of A9-THC in biological fluids such as saliva.

[0009] A9-THC is detectable electrochemically due to the direct oxidation of the phenol moiety. In order for the direct oxidation of the phenol moiety to occur it must be deprotonated to form the phenoxide anion that can be oxidised under low potential values. The oxidation ofthe A9-THC has been proven to be favoured at carbon-based surfaces. However, the instability of phenoxy radical produced can lead to subsequent chemical reactions:

[0010] Several electrochemical approaches for A9-THC detection are known. However, some of the known designs require the use of expensive metal electrodes, are not portable, involve a lengthy electrode or sample pre-treatment step, or use modified electrodes which have a short shelf-life.

[0011] It would be advantageous to provide a roadside testing method of cannabinoid analytes, e.g. A9-THC. It would be advantageous to provide a low-cost testing method of cannabinoid analytes, e.g. A9-THC.BRIEF SUMMARY OF THE DISCLOSURE

[0012] In accordance with the present invention there is provided an electrochemical method of quantitatively detecting a cannabinoid analyte in a sample, wherein the method involves: a. solvating the cannabinoid analyte in a buffer solution to provide a buffered electrostatically charged cannabinoid analyte; b. exposing the buffered electrostatically charged cannabinoid analyte to an electrostatically charged, hydrophobic polymer, wherein the electrostatically charged, hydrophobic polymer is in contact with a carbon-based working electrode and has the opposite charge to the electrostatically charged cannabinoid analyte; c. preconcentrating the buffered electrostatically charged cannabinoid analyte at the working electrode; and d. measuring the electrochemical response to quantitatively detect the cannabinoid analyte in the sample.

[0013] In an embodiment, the electrostatically charged, hydrophobic polymer is an ionically charged, hydrophobic polymer, for example a cationic hydrophobic polymer. In an embodiment, the electrostatically charged, hydrophobic polymer is a cationic hydrophobicpolymer. Since a cationic polymer will exchange anions (due to the presence of the opposing electrostatic charges), it is also known as an “anion exchange polymer”. The terms “cationic polymer” and “anion exchange polymer” may therefore be used interchangeably.

[0014] In an embodiment, the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise: (1) hydrocarbon moieties, such as alkyl chains and aromatic rings (which have no significant dipole moment and therefore result in large non-polar surfaces); and (2) at least one heteroatom that is capable of ionisation. The presence of the at least one heteroatom that is capable of ionisation thus allows the electrostatically charged, hydrophobic polymer to be an ionically charged, hydrophobic polymer. In an embodiment the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise at least one cationic moiety selected from the group consisting of: an benzimidazolium cation, an imidazolium cation, an ammonium cation, a sulfonium cation, a pyridinium cation, a benzpyrrolium cation and a pyrrolium cation. In an embodiment the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise at least one cationic moiety selected from the group consisting of: an imidazolium cation, an ammonium cation, a sulfonium cation, a pyridinium cation and a pyrrolium cation. In an embodiment the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise at least one cationic moiety selected from the group consisting of: a benzimidazolium cation, an imidazolium cation, an ammonium cation, a pyridinium cation, a benzpyrrolium cation and a pyrrolium cation. In an embodiment the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise at least one cationic moiety selected from the group consisting of: an imidazolium cation, an ammonium cation, a pyridinium cation and a pyrrolium cation. Preferably, the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise an imidazolium cationic moiety. Preferably, the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise a benzimidazolium cationic moiety.

[0015] In an embodiment, the electrostatically charged, hydrophobic polymer has a solubility in water of less than 0.01 mg / ml. For example, the electrostatically charged, hydrophobic polymer may have a solubility in water of less than 0.009 mg / ml, 0.008 mg / ml, 0.007 mg / ml, 0.006 mg / ml or 0.005 mg / ml.

[0016] In an embodiment, the electrostatically charged, hydrophobic polymer is a polymer selected from group consisting of: poly(ionic liquids), poly(4-vinylpyridinium) (P4VP) salts, polypyrrolium (PPy) salts, poly(benzimidazolium) salts, poly(imidazolium) salts,poly(alkylammonium) salts, poly(1-ethyl-3-methylimidazolium) salts and poly(quaternary ammonium) salts. In an embodiment, the electrostatically charged, hydrophobic polymer is a polymer selected from group consisting of: poly(ionic liquids), polypyrrolium (PPy) salts, poly(benzimidazolium) salts, poly(imidazolium) salts, poly(1 -ethyl-3-methylimidazolium) salts and poly(quaternary ammonium) salts. In an embodiment, the electrostatically charged, hydrophobic polymer is a polymer selected from group consisting of: poly(ionic liquids), poly(benzimidazolium) salts, poly(1 -ethyl-3-methylimidazolium) salts and poly(quaternary ammonium) salts. Preferably, the electrostatically charged, hydrophobic polymer is a poly(benzimidazolium) salt.

[0017] In an embodiment, the counterion for the cationic moiety of the electrostatically charged, hydrophobic polymer is selected from the group consisting of: TFSF, PF6“, BF4“, CT, Br“, Ts“, SO3“ and CIO4“. In an embodiment, the counterion for the cationic moiety of the electrostatically charged, hydrophobic polymer is selected from the group consisting of: CT and Br“. Preferably, the counterion for the cationic moiety of the electrostatically charged, hydrophobic polymer is CT.

[0018] In an embodiment, the electrostatically charged, hydrophobic polymer is selected from the group consisting of: a halide salt of hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI) and a halide salt of tris(2,4,6-trimethoxyphenyl)polysulfone-methylene quaternary phosphonium (TPQP). In an embodiment, the electrostatically charged, hydrophobic polymer is a halide salt of hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI). Preferably, the electrostatically charged, hydrophobic polymer is an iodide or chloride salt of HMT-PMBI or TPQP. Preferably, the electrostatically charged, hydrophobic polymer is an iodide or chloride salt of HMT-PMBI. More preferably, the electrostatically charged, hydrophobic polymer is a chloride salt of HMT-PMBI.

[0019] In an embodiment, the electrostatically charged, hydrophobic polymer is HMT-PMBI and has the formula:2X", wherein X is halide, n s 5 and each R is independently selected from the group consisting of: H and Me. Preferably, X is Cl. This polymer is commercially available under the trade name Aemion™.

[0020] In an embodiment, the electrostatically charged, hydrophobic polymer is HMT-PMBI and has the formula:2X', wherein X is halide and n £ 5. Preferably, X is Cl.

[0021] In an embodiment, the electrostatically charged, hydrophobic polymer is HMT-PMBI as described in A. G. Wright, J. Fan, B. Britton, T. Weissbach, H.-F. Lee, E. A. Kitching, T. J. Peckham and S. Holdcroft, Energy Environ. Sci., 2016, 9, 2130-2142. HMT-PMBI polymer was commercially available under the trade name Aemion™.

[0022] In an embodiment, the degree of methylation (dm %) for the HMT-PMBI polymer is £90% dm, £91% dm or £92% dm.

[0023] In an embodiment, the electrostatically charged, hydrophobic polymer has the formula:2X_, wherein X is halide, n £ 5 and each R is independently selected from the group consisting of: H and Me. Preferably, X is Cl. This polymer is commercially available under the trade name Aemion+™.

[0024] In an embodiment, the degree of methylation (dm %) for this polymer is £75% dm. For example, the degree of methylation (dm %) for this polymer may be £76% dm, >77% dm, £78% dm, >79% dm or £80% dm. For example, the degree of methylation (dm %) forthis polymer may be £81 % dm, £82% dm or £83% dm. Preferably, the degree of methylation (dm %) for this polymer is £84% dm. The degree of methylation (dm %) for this polymer might be higher thanthis, however. For example, the degree of methylation (dm %) for this polymer might be £85% dm, £86% dm, £87% dm, £88% dm, £89% dm, £90% dm, £91% dm or £92% dm.

[0025] In an embodiment, the electrostatically charged, hydrophobic polymer is deposited onto the carbon-based working electrode. For example, the electrostatically charged, hydrophobic polymer can be drop casted or dip coated onto the carbon-based working electrode. Preferably, the electrostatically charged, hydrophobic polymer is drop casted onto the carbon-based working electrode as this allows control over the thickness of the polymer coating over the carbon-based working electrode.

[0026] In an embodiment, the concentration of the electrostatically charged, hydrophobic polymer that is drop casted onto the electrode is up to 0.5%, 1%, 1 .5%, 2%, 2.5%, 3%, 3.5%, 4% (w / v).

[0027] In an embodiment, the concentration of polymer that is drop casted onto the electrode is up to 0.5%, 1%, 1 .5%, 2%, 2.5%, 3%, 3.5%, 4% (w / v).

[0028] In an embodiment, the concentration of polymer that is drop casted onto the electrode is up to 1 % - 2% (w / v).

[0029] In an embodiment, the concentration of polymer that is drop casted onto the electrode is 2% (w / v).

[0030] In an embodiment, the sample is a biological sample.

[0031] In an embodiment, the biological sample is saliva.

[0032] In an embodiment, the analyte is a buffered anionic cannabinoid analyte.

[0033] In a n embodiment, the analyte is selected from the group consisting of: cannabigerol, cannabinol, tetrahydrocannabinol acid, tetrahydrocannabivarin, cannabinol and tetrahydrocannabinol. In an embodiment, the analyte is tetrahydrocannabinol. In an embodiment, the analyte is A9-THC.

[0034] In an embodiment, the carbon-based working electrode is selected from the group consisting of: an amorphous carbon-based working electrode, a graphite-based working electrode, a graphene-based working electrode and a glassy carbon-based working electrode. In an embodiment, the carbon-based working electrode is selected from the group consisting of: an amorphous carbon-based working electrode, a graphite-based working electrode and a graphene-based working electrode. In an embodiment, the carbon-based working electrode is selected from the group consisting of: an amorphous carbon-based working electrode and a graphene-based working electrode. In an embodiment, the carbon-based working electrode isa graphene-based working electrode. Preferably, the carbon-based working electrode is an amorphous carbon-based working electrode.

[0035] In an embodiment, the carbon-based working electrode is a component of a screen printed electrode. In an embodiment, the screen printed electrode further comprises a silverbased reference electrode (e.g. Ag or Ag / AgCl). In an embodiment, the screen printed electrode further comprises a carbon-based counter electrode. In an embodiment, the screen printed electrode further comprises a silver-based reference electrode (e.g. Agor Ag / AgCl) and a carbon-based counter electrode.

[0036] In an embodiment, the carbon-based counter electrode is selected from the group consisting of: an amorphous carbon-based counter electrode, a graphite-based counter electrode, a graphene-based counter electrode and a glassy carbon-based counter electrode. In an embodiment, the carbon-based counter electrode is selected from the group consisting of: an amorphous carbon-based counter electrode, a graphite-based counter electrode and a graphene-based counter electrode. In an embodiment, the carbon-based counter electrode is selected from the group consisting of: an amorphous carbon-based counter electrode and a graphene-based counter electrode. In an embodiment, the carbon-based counter electrode is a graphene-based counter electrode. Preferably, the carbon-based counter electrode is an amorphous carbon-based counter electrode.

[0037] Any electrode will have a capacitive current. It is preferable to minimise the capacitive current. Increasing the surface area of an electrode (e.g. by enhancing porosity or surface roughness) will increase its capacitive current. Increasing the capacitive current can interfere with analytical measurements. Judicious selection of the electrolyte and / or scan rate can overcome the limitation of high capacitive currents and improve the sensitivity in analytical measurements.

[0038] In an embodiment, the screen printed electrode is selected from the group consisting of: Metrohm SPE and Gii-Sense SPE.

[0039] In an embodiment, the cannabinoid analyte is present in the sample at a concentration of at least 10 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of at least 15 ng / ml, at least 20 ng / ml, at least 25 ng / ml, at least 50 ng / ml, or at least 100 ng / ml.

[0040] In an embodiment, the cannabinoid analyte is present in the sample at a concentration of less than 5000 ng / ml. In an embodiment, the cannabinoid analyte is present in the sample at a concentration of less than 3000 ng / ml. In an embodiment, the cannabinoidanalyte is present in the sample at a concentration of less than 2000 ng / ml. In an embodiment, the cannabinoid analyte is present in the sample at a concentration of less than 1500 ng / ml. In an embodiment, the cannabinoid analyte is present in the sample at a concentration of less than 1000 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of less than 750 ng / ml, less than 500 ng / ml, less than 250 ng / ml, less than 125 ng / ml, or less than 100 ng / ml.

[0041] In an embodiment, the cannabinoid analyte is present in the sample at a concentration of from 10 ng / ml to 1000 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of 25 ng / ml to 1000 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of 50 ng / ml to 750 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of 100 ng / ml to 500 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of 10 ng / ml to 1000 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of 10 ng / ml to 750 ng / ml. For example, the cannabinoid analyte is present in the sample at a concentration of 10 ng / ml to 500 ng / ml. In an embodiment, step (a) further dilutes the cannabinoid analyte in the sample between 1 and 100 fold. For example, step (a) further dilutes the cannabinoid analyte in the sample 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 fold. In an embodiment, step (a) further dilutes the cannabinoid analyte in the sample between 1 and 50 fold. In an embodiment, step (a) further dilutes the cannabinoid analyte in the sample between 1 and 25 fold. In an embodiment, step (a) further dilutes the cannabinoid analyte in the sample between 1 and 10 fold.

[0042] In an embodiment, step (a) involves buffering between pH s 7 and pH s 12. In an embodiment, step (a) involves buffering between pH s 7 and pH s 11 .

[0043] In an embodiment, step (a) involves is buffering between pH s 8 and pH s 12. In an embodiment, step (a) involves is buffering between pH s 8 and pH s 11 . in an embodiment, step (a) involves is buffering between pH s 9.5 and pH s 10.5, e.g. about pH 10. Buffering at the higher pH range of 8-11 is preferable to ensure full ionisation of the phenolic moiety on the cannabinoid analyte.

[0044] In an embodiment, the buffer solution is selected from the group consisting of: TRIS, PBS and Borax. In an embodiment, the buffer solution is TRIS.

[0045] In an embodiment, the buffer solution (i.e. TRIS, PBS or Borax) additionally includes a metal hydroxide, e.g. NaOH or KOH.

[0046] In an embodiment, the buffer solution further comprises an electrolyte to increase conductivity of the buffer solution. For example, the electrolyte may be a metal halide. Preferably, the electrolyte may be a metal chloride.

[0047] In an embodiment, the electrolyte is selected from the group consisting of: NaF, NaCl, NaBr, Nal, KF, KCl, KBr and KI. In an embodiment, the electrolyte is selected from the group consisting of: NaCl and KCl.

[0048] In an embodiment, step (b) involves exposing between 0.1 and 25 ml of buffer solution to the electrostatically charged, hydrophobic polymer. In an embodiment, step (b) involves exposing between 0.5 and 15 ml of buffer solution to the electrostatically charged, hydrophobic polymer. In an embodiment, step (b) involves exposing between 1 and 10 ml of buffer solution to the electrostatically charged, hydrophobic polymer.

[0049] In an embodiment, step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 45 minutes. In an embodiment, step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40 or 45 minutes. In an embodiment, step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 30 minutes. In an embodiment, step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 20 minutes. In an embodiment, step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 15 minutes. In an embodiment, step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 10 minutes. In an embodiment, step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 5 minutes.

[0050] In an embodiment, the electrochemical response is measured using cyclic voltammetry. Cyclic voltammetry is advantageous when measuring higher concentrations of analyte.

[0051] In an embodiment, the electrochemical response is measured usingdifferential pulse voltammetry.

[0052] In an embodiment, the electrochemical response is measured using square wave voltammetry. Square wave voltammetry is advantageous in capacitive current suppression and is therefore advantageous when the working electrode has a high capacitive current, e.g. the foamed graphene working electrode of the Gii-sense SPE.

[0053] In an embodiment, the parameters for the differential pulse voltammetry are: E Step: 0.01V, E pulse: 0.2V, T pulse: 0.02s, Scan rate: 0.01 V / s.BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:Figure 1 is A9-THC signals from triplicate experiments performed using 2% (w / v) of HMT-PMBI coated on Metrohm C11 L screen printed electrodes in TRIS buffer solution at pH 10 (a) and the linear fit of the A9-THC data presenting the corresponding error bars (b), each concentration was performed in triplicate.Figure 2 is Comparison of the selectivity of (a) unmodified Metrohm C11 L screen printed electrode (b) 2% (w / v) of HMT-PMBI coated on Metrohm C11 L screen printed electrode in the presence of ascorbic acid (AA) and caffeine (CF) as interferences (1 pg / mL A9-THC, TRIS buffer pH 10).Figure 3 is Differential pulse voltammetry of A9-THC in spiked saliva using 2% (w / v) of HMT-PMBI coated on Metrohm C11 L screen printed electrode. 1 mL of saliva was spiked with 5pg / mL of A9-THC and diluted to a 9 mL solution before testing (550ng / mL).Figure 4 is a Comparison of Metrohm C11 L and Gii-Sense (Carbon WE, Ag / AgCl RE, Carbon CE) screen printed electrodes in the presence of 10pg / mlTHC pH 10 TRIS buffer. Doted graphs correspond to unmodified or bare screen printed electrode and the continuous lines to 2% (w / v) HMT-PMBI drop casted screen printed electrode.Figure 5 is a Comparison of Metrohm C11 L and Gii-Sense (Carbon WE, Ag / AgCl RE, Carbon CE) 2% (w / v) HMT-PMBI drop casted screen printed electrodes in the presence of 50ng / ml THC pH 10 TRIS buffer.Figure 6 is the Investigation of pH effect on THC signal using TRIS buffer ranging from pH 2 to 12 with 1 pg / mlTHC. Metrohm C11 L screen printed electrode with 2% (w / v) HMT-PMBI drop casted onto the carbon working electrode.Figure 7 is the Effect of increasing the concentration of HMT-PMBI drop casted into the screen printed electrode. Metrohm C11 L screen printed electrode with 2% (w / v) HMT-PMBI was drop casted (0.5pL) onto the carbon working electrode. All the electrochemical analysis was performed by Differential Pulse Voltammetry E Step: 0.01v, E pulse: 0.2v, T pulse: 0.02s, Scan rate: 0.01 v / s. The concentration of THC employed was 1000ng / mL in TRIS buffer pH 10.Figure 8 is the Comparison of how preconcentration time effects THC absorption onto the screen printed electrode. Metrohm C11 L screen printed electrode with 2% (w / v) HMT-PMBI was drop casted (0.5pL) onto the carbon working electrode. All the electrochemical analysis was performed by Differential Pulse Voltammetry E Step: 0.01v, E pulse: 0.2v, T pulse: 0.02s, Scan rate: 0.01 v / s. The concentration of THC employed was 1000ng / mL in TRIS buffer pH 10.Figure 9a is a black and white version of the Repeatability study of 11 different Metrohm C11 L screen printed electrode with 2% (w / v) HMT-PMBI was drop casted (0.5pL) onto the carbon working electrode. Figure 9b is a greyscale version of the Repeatability study of 11 different Metrohm C11 L screen printed electrode with 2% (w / v) HMT-PMBI was drop casted (0.5pL) onto the carbon working electrode. All the electrochemical analysis was performed by Differential Pulse Voltammetry E Step: 0.01v, E pulse: 0.2v, T pulse: 0.02s, Scan rate: 0.01 v / s. The concentration of THC employed was 1000ng / mL in TRIS buffer pH 10, 40 min preconcentrating time.DETAILED DESCRIPTIONDefinitions

[0055] Throughout this specification these abbreviations have the following meanings:A9-THC A9-TetrahydrocannabinolAA Ascorbic AcidCE Counter ElectrodeCF CaffeineCNS Central Nervous SystemDVP Differential Pulse VoltammetryHMT-PMBI Hexamethyl-p-terphenyl poly(benzimidazolium)LCD Limits of DetectionLOQ Limit of QuantificationRE Reference ElectrodeSPCE Screen Printed Carbon ElectrodeSPE Screen Printed ElectrodeTRIS Tris(hydroxymethyl)aminomethaneWE Working Electrode

[0056] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0057] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0058] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.EXPERIMENTAL

[0059] All chemicals used were of analytical grade and used as received without any further purification. These included sodium chloride (>99.5%, Sigma-Aldrich), Sodium Hydroxide (>97%, Sigma-Aldrich), TRIS buffer solid (>99%, Sigma-Aldrich) methanol (>99.9%, Sigma- Aldrich) and A9-tetrahydrocannabinol in methanol (1 mg / ml, Sigma-Aldrich, stored at 0°C). All solutions were prepared using 100% ultra-pure steam distilled water from Lucemill Ltd. Hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI) was synthesized according to the procedure developed by Holdcroft et al. (Wright et al., 2016; Wright & Holdcroft, 2014). The as-prepared HMT-PMBI has a high degree of selectivity over the degree of methylation (dm %). The HMT-PMBI samples in this work are all 92% dm.

[0060] Differential pulse voltametric measurements were preformed using a Metrohm Autolab PGSTAT302N controlled by the NOVA 2 software. Silver-Silver Chloride screen printed electrodes were purchased from Metrohm UK Ltd., C11 L Screen-Printed Electrode with carbon working, carbon auxiliary electrode and silver / silver chloride reference electrode. The Gii- Sense electrodes were purchased from PalmSense (UK), Gii-Sens Integrated Graphene SPE - Pure 3D Graphene sensing electrode - Graphene CE and WE, Ag / AgCl RE.

[0061] All experiments were performed at room temperature under ambient conditions. TRIS buffer solution was made using TRIS buffer solid dissolved in distilled water to make a 20mM solution and then pH adjusted to pH 10 using 2M sodium hydroxide and Jenway 3510 pH meter.

[0062] Example 1 : Preparation of polymer:

[0063] The HMT-PMBI polymer was received in a membrane form, with iodide (I ) as the counterion. In order to avoid potential interferences with the iodide form during the voltammetric studies, the counterion of HMT-PMBI (iodide) was exchanged for chloride (Cl-), by soaking the membrane in 1 M NaCl for 24 h, then transferring it into distilled water for 24 h.

[0064] Example 2: Manufacture of polymer coated electrode:

[0065] Hexamethyl-p-terphenyl poly(benzimidazolium) solid of Example 1 was dissolved in >99.9% methanol to produce a solution of 2% weight by volume of HMT-PMBI, the mixture was sonicated for 15 minutes to ensure dissolution and homogeneousness.

[0066] The modified screen-printed electrodes were prepared by drop casting 0.5pL of the methanolic solution of 2% (w / v) HMT-PMBI polymer in the working electrode of Metrohm SPE and 1 pL in the working electrode of Gii-Sense SPE. The film deposited on the electrode surface was dried at room temperature for 10 minutes.

[0067] Example 3: Preconcentration of A9-THC:

[0068] Experiments were carried out using 10ml of solution in a small 10ml glass beaker. The beaker was set up on a magnetic stirrer to stir solutions during preconcentration, the screen- printed electrode of Example 2 was submerged into these solutions for experimentation.

[0069] Prior to taking measurements the test solutions were stirred for 30 minutes to preconcentrate A9-THC on the working electrode. Stirring was then shut off and the measurement was performed as described in Example 4.

[0070] Example 4: Electrochemical detection of A9-THC:

[0071] The A9-THC current at 0.3 V was used to generate linear fit data. The average current drawn and standard deviation from the triplicate sets of experimental data was plotted with a linear fit. The differential-pulse parameters used were as follows: E Step: 0.01v, E pulse: 0.2v, T pulse: 0.02s, Scan rate: 0.01 v / s.

[0072] This example demonstrates that the anion exchange polymer (HMT-PMBI) can be used to fabricate modified SPEs, which can electrochemically detect A9-THC in concentrations in buffer solutions.

[0073] For both Gii-Sense and Metrohm electrodes the presence of HMT-PMBI increases the anodic current density of A9-THC oxidation. (Figure 4). In the presence of 10pg / mL A9-THC, a typical unmodified Gii-Sense SPCE presents a peak current density of 5.6pAcm-2and unmodified Metrohm SPE, 14.6 pAcrrr2. The modification of SPEs with HMT-PMBI leads to an increase in anodic current density. Without being bound by theory, the observed increase in anodic current density may be due to preconcentration of the negatively charged A9-THC analyte atthe electrode bythe positively charged HMT-PMBI solid polymer electrolyte. Without being bound by theory, the observed increase in anodic current density may additionally or alternatively be due to A9-THC being hydrophobic in character which is complementary with the highly hydrophobic segments of the HMT-PMBI polymer. These segmented electrostatic and hydrophobic interactions may be responsible for the preconcentration of A9-THC and an increase in sensitivity of its detection.

[0074] The HMT-PMBI polymer modified Metrohm electrodes exhibited a 4-fold increase in the anodic peak current (63.4 pAcrrr2), and the HMT-PMBI polymer modified Gii-Sense electrodes exhibited a 13-fold increase in the anodic peak current (71.2 pAcm-2). The larger increase of the latter may be due to its foam-like structure, presenting higher electrode effective area and increased porosity, thus, facilitatingthe integration of the polymerwithin the foam during the drop-cast method and easing the diffusion of the electroactive species in comparison with packet carbon working electrodes.

[0075] Example 5: Electrochemical detection of low concentration A9-THC:

[0076] This example demonstrates that the anion exchange polymer (HMT-PMBI) can be used to fabricate modified SPEs, which can electrochemically detect A9-THC in concentrations as low as 10 ng / mL in buffer solutions.

[0077] Both electrodes were examined under conditions of low A9-THC concentrations (50ng / mL) which are more akin to roadside saliva A9-THC concentrations (Figure 5). For 50ng / mL A9-THC concentrations, the polymer modified Metrohm electrodes presented aclearly visible oxidation peak, whereas modified Gii-Sense electrodes did not. No reproducible anodic peak was achievable with polymer modified Gii-Sense electrodes with A9-THC concentrations below 500 ng / mL, which may be due to the Gii-Sense electrodes having a large capacitive current masking the low current density of A9-THC oxidation.

[0078] Differential pulse voltammetry (DVP) (E Step: 0.1 V, E pulse: 0.2V, T pulse: 0.02s, Scan rate: 0.01 V / s) using Metrohm C11 L electrodes provided greater consistency and smaller background, capacitive signals, resulting in enhanced A9-THC detection at low A9-THC concentrations9.

[0079] Example 6: Effect of pH

[0080] Taking into consideration the pKa of A9-THC phenol (10.6), the effect of solution pH on electrochemical detection of A9-THC at polymer modified electrodes was examined using Metrohm electrodes (Figure 6). The electrochemical response to A9-THC was found to increase with increasing pH, which is deemed due to increasing deprotonation, rendering an anion which may diffuse into HMT-PMBI. The use of solutions of pH > 11 were avoided as the high concentration of OH- competes against A9-THC anions for polycation sites in the HMT-PMBI, lowering the concentration of electrostatically bound A9-THC. In contrast, the anodic current decreases below pH 5.0-6.0 due to protonation of A9-THC to form a cation, which is repelled by the polycationic film12>16>17. Therefore, the pH was optimized to 10 employing TRIS buffer as adapted above with the addition of NaOH.

[0081] Example 7: Effect of thickness of HMT-PMBI polymer layer

[0082] The quantity of HMT-PMBI deposited on electrodes was found to have a direct impact on the anodic oxidation of A9-THC (Figure 7). The anodic current corresponding to A9-THC oxidation increased with increasing concentrations of drop cast HMT-PMBI solutions, up to 2% (w / v). However, the oxidative peak decreased when higher concentration polymer solutions were deposited, dropping to virtually no observable current for 10% (w / v) solutions, presumably due to high ionic resistance and long diffusion lengths imposed by the film. Consequently, 2% (w / v) of polymerwas considered to be the optimal concentration forfurther study.

[0083] Example 8: Preconcentration time:

[0084] The rate of A9-THC uptake on the HMT-PMBI modified electrodes was investigated by immersing modified electrodes in a pH 10, TRIS buffer solution containing 1000ng / mL A9-THC (Figure 8). It was found that the electrochemical response of A9-THC increases with time,reaching a maximum after 45 minutes. 40 minutes was chosen as the pre-concentration period.

[0085] Example 9: Calibration curve:

[0086] With all the above parameters investigated, a A9-THC calibration curve was constructed (Figure 1 ). Fig 1a presents DVP curves of polymer modified electrodes recorded in buffer solutions of increasing A9-THC concentration, and Fig 1 b shows the plot of the anodic peak current vs. A9-THC concentration. A linear regressive response for A9-THC was found (Ipa (pA) = 0.0005 X (ng / mL) + 0.0985 (R2 = 0.996). A9-THC was detected at concentrations as low as 10 ng / mL. The calculated limits of detection (LCD) was 5.28 ng / mL obtained from the relation LCD = 3.3Sb / S18. Consequently, the calculated limit of quantification (LOQ), was 16 ng / mL (10 SD blank / slope).

[0087] Example 10: HMT-PMBI modified electrode reproducibility:

[0088] Reproducibility of the response to 1000 ng / mL A9-THC was examined by preparing 11 different modified electrodes (Figure 9). The mean of the anodic peaks was 0.614pA [std dev, 0.0587, 9.5% RSD, and 0.614 ± 0.0347 (±6.65%) margin of error at 95% confidence interval] (Figure 9).

[0089] Example 11 : HMT-PMBI modified electrode stability:

[0090] Moreover, the electrodes exhibited notable stability over time, when stored in a dry room and room temperature environment; no visible differences in their A9-THC detection capabilities were detected after several weeks of storage.

[0091] Example 12: HMT-PMBI modified electrode selectivity:

[0092] A variety of components are expected to be found in human saliva during a roadside testing. These include ascorbic acid (AA) or caffeine (CF)12. The impact of these analytes on pristine and modified electrodes was therefore investigated (Figure 2) by mixing 1 pg / mL of A9- THC with increasing concentrations of AA and CF (concentrations ranging from 10 pg / mL to 100 pg / mL) in TRIS buffer solutions (pH 10). The electrochemical responses of bare electrodes are significantly affected (up to 400% increase in anodic current) by the presence of the AA and / or CF, indicating their clear interference.

[0093] In contrast, the presence of AA and CF did not influence the electrochemical response of A9-THC even for 100-fold mass ratios of AA and / or CF.

[0094] Selectivity may be due to the hydrophobic nature of the polymer structure repelling the relatively hydrophilic ascorbic acid and promotin the preconcentration of the hydrophobic A9-THC.

[0095] Example 13: THC-doped saliva experimental:

[0096] Preliminary studies of modified electrodes were performed on human saliva (Figure 3) spiked with A9-THC. 5pg / mL of A9-THC was added to 1 mL of saliva and diluted to a 9 mL solution. Dilution of the saliva was carried out to reduce viscosity as typical human saliva possesses kinematic viscosities of 1.40mm2s-1whereas water is 1 mm2s-1. High viscosities would be expected to lower the absorption rate of electroactive species on the surface of the carbon working electrode. Additionally, the pH of the saliva sample was adjusted in light of the finding of Example 6. Saliva has a neutral pH, but detection was found in Example 6 to be optimised at pH 10. For that reason, the pH of the saliva sample was adjusted to pH 10. Voltammetry on saliva alone did not reveal any anodic peak in the voltage range of interest; whereas the spiked saliva yielded a 0.39 pA peak current.

Claims

CLAIMS1. An electrochemical method of quantitatively detecting a cannabinoid analyte in a sample, wherein the method involves: a. solvating the cannabinoid analyte in a buffer solution to provide a buffered electrostatically charged cannabinoid analyte; b. exposing the buffered electrostatically charged cannabinoid analyte to an electrostatically charged, hydrophobic polymer, wherein the electrostatically charged, hydrophobic polymer is in contact with a carbon-based working electrode and has the opposite charge to the electrostatically charged cannabinoid analyte; c. preconcentrating the buffered electrostatically charged cannabinoid analyte at the working electrode; and d. measuring the electrochemical response to quantitatively detect the cannabinoid analyte in the sample.

2. The method of claim 1 , wherein the electrostatically charged, hydrophobic polymer is a cationic hydrophobic polymer and wherein the buffered electrostatically charged cannabinoid analyte is a buffered anionic cannabinoid analyte.

3. The method of claim 2, wherein the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise at least one cationic moiety selected from the group consisting of: a benzimidazolium cation, an imidazolium cation, an ammonium cation, a sulfonium cation, a pyridinium cation, a benzpyrrolium cation and a pyrrolium cation.

4. The method of claim 3, wherein the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise an imidazolium cationic moiety.

5. The method of claim 3, wherein the electrostatically charged, hydrophobic polymer is derived from monomeric units that each comprise a benzimidazolium cationic moiety.

6. The method of claim 2, wherein the electrostatically charged, hydrophobic polymer is a polymer selected from group consisting of: poly(ionic liquids), poly(4-vinylpyridinium) (P4VP)salts, polypyrrolium (PPy) salts, poly(benzimidazolium) salts, poly(imidazolium) salts, poly(alkylammonium) salts, poly(1-ethyl-3-methylimidazolium) salts and poly(quaternary ammonium) salts.

7. The method of claim 6, wherein, the electrostatically charged, hydrophobic polymer is a poly(benzimidazolium) salt.

8. The method of any of claims 2 to 7, wherein the counterion for the cationic moiety is selected from the group consisting of: TFST, PF6“, BF4“, CT, Br“, Ts“, SO3“ and CIO4“.

9. The method of claim 8, wherein the counterion forthe cationic moiety is Cl .

10. The method of claim 2, wherein the cationic hydrophobic polymer is a chloride salt ofHMT-PMBI and has the formula:2Cl_, wherein n > 5 and each R is independently selected from the group consisting of: H andMe.11 . The method of claim 10, wherein the degree of methylation (dm %) of the polymer is£90% dm.

12. The method of claim 2, wherein the cationic hydrophobic polymer has the formula:2CT, wherein n > 5 and each R is independently selected from the group consisting of: H andMe.

13. The method of claim 12, wherein the degree of methylation (dm %) of the polymer is £75% dm.

14. The method of any preceding claim, wherein sample is a biological sample, optionally wherein the biological sample is saliva.

15. The method of any preceding claim, wherein the analyte is selected from the group consisting of: cannabigerol, cannabinol, tetrahydrocannabinol acid, tetrahydrocannabivarin, cannabinol and tetrahydrocannabinol; optionally the analyte is A9-THC.

16. The method of any preceding claim, wherein the carbon-based working electrode is a component of a screen printed electrode.

17. The method of claim 16, wherein the screen printed electrode further comprises a silver-based reference electrode and a carbon-based counter electrode.

18. The method of any preceding claim, wherein step (a) further dilutes the cannabinoid analyte in the sample between 1 and 10 fold.

19. The method of any preceding claim, wherein step (a) involves buffering to between pH > 7 and pH s 11 , optionally between pH s 8 and pH s 11 .

20. The method of claim 19, wherein the buffer solution further includes a metal hydroxide, optionally wherein the metal hydroxide is selected from the group consisting of: NaOH and KOH.

21. The method of claim 20, wherein the buffer solution is selected from the group consisting of: TRIS, PBS and Borax.

22. The method of claim 21 , wherein the buffer solution is TRIS.

23. The method of any preceding claim, wherein the buffer solution further comprises an electrolyte, optionally wherein the electrolyte is selected from the group consisting of: NaCl and KCl.

24. The method of any preceding claim, wherein step (b) involves exposing between 1 and 10 ml of buffer solution to the electrostatically charged, hydrophobic polymer.

25. The method of any preceding claim, wherein step (c) involves preconcentrating the electrostatically charged cannabinoid analyte at the working electrode for up to 45 minutes, optionally for up to 15 minutes.

26. The method of any preceding claim, wherein the electrochemical response is measured using differential pulse voltammetry.

27. The method of claim 26, wherein the parameters for the differential pulse voltammetry are E Step: 0.01V, E pulse: 0.2V, T pulse: 0.02s, Scan rate: 0.01 V / s.

28. The method of any preceding claim, wherein the cannabinoid analyte is present in the sample at a concentration of at least 10 ng / ml.

29. The method of any preceding claim, wherein the cannabinoid analyte is present in the sample at a concentration of less than 1000 ng / ml.

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