阅读说明：本技术 用于检测阿片样物质的电化学测定 (Electrochemical assay for the detection of opioids ) 是由 N.韦斯特 E.迈恩蒂南 T.劳里拉 于 2018-03-22 设计创作，主要内容包括：根据本发明的一个示例性方面,提供了多层测试条,所述多层测试条包括基材,在所述基材上沉积有电极组件层,所述电极组件层包括碳基工作电极、碳基对电极、伪参比电极和用于使电极直接接触电压供应的触点,其中所述伪参比电极、所述工作电极和所述对电极彼此相邻布置在同一平面内,并且所述测试条还包括选择性渗透膜层,所述电极组件层的所述电极彼此电隔离并且所述电极组件层位于所述基材与所述选择性渗透膜层之间。(According to an exemplary aspect of the present invention, there is provided a multilayer test strip comprising a substrate on which is deposited an electrode assembly layer comprising a carbon-based working electrode, a carbon-based counter electrode, a pseudo-reference electrode and a contact for directly contacting an electrode with a voltage supply, wherein the pseudo-reference electrode, the working electrode and the counter electrode are arranged adjacent to each other in the same plane, and further comprising a selectively permeable membrane layer, the electrodes of the electrode assembly layer being electrically isolated from each other and the electrode assembly layer being located between the substrate and the selectively permeable membrane layer.)

1. A multilayer test strip comprising a substrate having deposited thereon an electrode assembly layer comprising

A carbon-based working electrode,

a carbon-based counter electrode,

a pseudo-reference electrode, wherein the pseudo-reference electrode, the working electrode and the counter electrode are arranged adjacent to each other in the same plane,

a contact for bringing the electrode into direct contact with a voltage supply, and

the test strip also includes a selectively permeable membrane layer,

the electrodes of the electrode assembly layer are electrically isolated from each other and the electrode assembly layer is located between a substrate and a selectively permeable membrane layer.

2. The strip according to claim 1, wherein said carbon based electrode comprises carbon selected from the group consisting of amorphous carbon such as tetrahedral amorphous carbon, diamond like carbon, graphite, carbon nanotubes and mixtures thereof.

3. The strip of claim 1 or 2, wherein said substrate is selected from the group consisting of polymers and glass.

4. The strip of any preceding claim, wherein the working electrode or counter electrode, or both the working electrode and counter electrode further comprise titanium.

5. The strip of any preceding claim, wherein said pseudo-reference electrode comprises silver.

6. The strip according to any one of claims 1 to 4, wherein the pseudo-reference electrode comprises silver-silver chloride (Ag/AgCl).

7. The strip according to any one of claims 1 to 4, wherein the pseudo-reference electrode comprises platinum.

8. The strip of any preceding claim, wherein said contacts comprise silver.

9. The strip according to any one of the preceding claims, wherein the permselective membrane layer comprises a cation permselective membrane of a polymer selected from the group consisting of Nafion, cellulose acetate, conventional dialysis membranes, polyvinyl sulfonate, carboxymethyl cellulose, polylysine, peroxypolypyrrole, and other sulfonated polymers.

10. The strip according to any one of the preceding claims, wherein the permselective membrane layer comprises Nafion.

11. The strip of any preceding claim, further comprising a filtration layer, wherein the strip is arranged such that the selectively permeable membrane layer is located between the filtration layer and the electrode assembly layer.

12. The strip of claim 11, further comprising a hydrophobic membrane/membrane layer, wherein the strip is arranged such that the filtration layer is located between the selectively permeable membrane layer and the hydrophobic membrane/membrane layer.

13. An apparatus, comprising:

-a memory configured to store reference data;

-at least one processing core configured to:

processing information from a strip according to any one of claims 1 to 12;

comparing information from a strip according to any of claims 1 to 12 with reference data, and

drawing conclusions about the processing of information from the strip according to any of claims 1 to 12.

14. A method of detecting an opioid in a sample comprising the steps of:

providing a sample, and providing a sample,

bringing the sample into electrical contact with a working electrode (2) and a counter electrode (4) of an electrode assembly of the multilayer test strip,

changing the voltage between the working electrode (2) and the counter electrode (4),

measuring the current between the working electrode (2) and the counter electrode (4) in relation to the voltage applied between the working electrode (2) and the counter electrode (4), and

detecting a change in a current characteristic of one or more opioid analytes in the sample.

15. A method of detecting an opioid in a sample comprising the steps of:

providing a sample, and providing a sample,

bringing a sample into electrical contact with a working electrode (2) and a counter electrode (4) of an electrode assembly of a multilayer test strip according to any one of claims 1 to 11,

changing the voltage between the working electrode (2) and the counter electrode (4),

measuring the current between the working electrode (2) and the counter electrode (4) in relation to the voltage applied between the working electrode (2) and the counter electrode (4), and

detecting a change in a current characteristic of one or more opioid analytes in the sample.

16. The method according to claim 14 or 15, wherein the voltage between the working electrode (2) and the counter electrode (4) is swept from-0.6V to 0.2V.

17. The method according to claim 14 or 15, wherein the voltage between the working electrode (2) and the counter electrode (4) is swept from-0.5V to 1.5V.

18. The method of any one of claims 14 to 16, wherein the scan rate is in the range of 2.5-40 mV/s.

Technical Field

The present invention relates to a multilayer test strip, in particular for the detection of opioids and their metabolites in a sample, and to a method for manufacturing such a multilayer test strip. In addition, the present invention relates to a system for detecting opioids and metabolites thereof, comprising a multilayer test strip and a measurement circuit. Furthermore, the present invention relates to a method of measuring an opioid in a sample.

Background

Morphine (MO), Codeine (CO), Tramadol (TR), Oxycodone (OXY), and Fentanyl (FEN) are widely used opioids for the control of severe pain. These opioids are widely used and very effective analgesics for the treatment of acute and chronic pain. However, establishing therapeutic efficacy while ensuring patient safety is challenging due to individual pharmacokinetic and pharmacogenetic factors associated with opioid use (fig. 24).

These factors particularly affect the use of prodrugs such as CO and TR, which are partially or completely inactive when administered, but chemically converted to their active forms in vivo. CO is first metabolized to Norcodeine (NC) by N-demethylation and further metabolized to its active form MO by O-demethylation, a pharmacologically active analgesic. MO and 6-acetylmorphine are also the major metabolites tested in the heroin drug test. Similarly, TR is metabolized to its major active metabolite, O-desmethyltramadol (ODMT). The metabolic activity of the enzyme responsible for the metabolism of both CO and TR (liver enzyme CYP2D6) is highly individualized, and thus the analgesic effect of CO and TR ranges from no effect to highly sensitive. Furthermore, the pharmacokinetic parameters (such as the rate of excretion) of opioids that are active at the time of administration are also highly individualized.

Determination of the concentration of opioids in a sample is currently performed using High Performance Liquid Chromatography (HPLC) and liquid chromatography in combination with mass spectrometry (LC-MS). Using these methods, inter-individual variability of opioid metabolism in humans, and in particular, prodrug activation, can be detected and quantified. However, these methods are expensive and time consuming and are therefore impractical in terms of pain control and differential diagnosis of opioid intoxication. Furthermore, highly skilled experts are required to perform the protocol and analyze the results.

Electrochemical detection methods have been found to be inexpensive, rapid, and highly sensitive, and to be relatively simple to operate. Such methods have been investigated to detect opioids in samples. However, since therapeutic concentrations of opioids are very low (e.g., therapeutic concentrations of CO and MO in the tens of nM to hundreds of nM range depending on the dose; typically, therapeutic concentrations are about 100 nM and lower), and due to high concentrations (100-. While there are several groups reporting the detection of MO (Li 2010, Rezaei 2015, dehdashtie 2016) and CO (Li 2013, Piech 2015), few groups report the simultaneous detection of MO and CO in the presence of interferents such as AA and UA (Li 2014, Ensafi 2015, Taei 2016). However, in these studies, tolerance levels have been reached at lower AA and UA levels than expected to be seen in, for example, blood samples.

Recently, carbon-based materials, such as amorphous carbon, Carbon Nanotubes (CNTs), and various other forms of graphite, have attracted considerable attention, particularly as novel electrode materials. Carbon materials have unique structures and unusual properties such as large surface area, high mechanical strength, high electrical conductivity, and electrocatalytic activity. Although the electrocatalytic properties and surface treatment of these new electrode materials contribute greatly to the selectivity of voltammetric detection, the electrocatalytic properties and surface treatment of this carbon material alone are not sufficient to completely eliminate the above and other possible interferents in the electrochemical detection and quantification of opioids.

Permselective membranes, such as the sulfonated copolymer Nafion, are known in the art and are widely used due to antifouling and cation exchange properties, which provide an increase in selectivity and long-term signal stability in electrochemical measurements. Nafion membranes have been shown to support rapid electron transfer, especially at reasonable scan rates. Hydrophilic negatively charged sulfonate groups enable preconcentration of positively charged analytes and selective detection of cationic analytes. Since several interferents such as AA and UA exist as anionic molecules in solution (at neutral pH), their interference with the target analyte can be significantly reduced by Nafion membranes, as has been shown in numerous studies (Rocha 2006, Hou 2010, Ahn 2012). Nafion membranes also exhibit size exclusion due to the nanoscale hydrophilic channels, thereby filtering out macromolecules.

In addition to biomolecules, other interfering anionic drug molecules also coexist with opioids in biological samples (at physiological pH). In particular non-steroidal anti-inflammatory drugs are present in high concentrations. Nafion can eliminate the interference of these molecules. In addition, Nafion membranes also provide a diffusion barrier that selectively enriches cations. Thus, selectivity to cations is also increased in the presence of neutral species such as acetaminophen, xanthine and hypoxanthine.

Summary of The Invention

The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

According to a first aspect of the present invention there is provided a multilayer test strip comprising a substrate on which is deposited an electrode assembly layer comprising: a carbon-based working electrode, a carbon-based counter electrode, wherein the working electrode and the counter electrode comprise the same carbon-based material; a pseudo-reference electrode, wherein the pseudo-reference electrode, the working electrode, and the counter electrode are disposed adjacent to each other in the same plane; a contact for directly contacting the electrode to a voltage supply; and a selectively permeable membrane layer, the electrode assembly layer being located between the substrate and the selectively permeable membrane layer.

According to a second aspect of the present invention, there is provided an apparatus comprising a memory configured to store reference data, at least one processing core configured to process information from a strip as described herein, compare the information from a strip as described herein with the reference data, and draw a conclusion about the processed information from a strip as described herein.

According to a third aspect of the present invention there is provided a method of detecting an opioid in a sample, the method comprising the steps of: providing a sample, placing the sample in electrical contact with a working electrode (2) and a counter electrode (4) of an electrode assembly of a multi-layer test strip, varying a voltage between the working electrode (2) and the counter electrode (4), measuring a current between the working electrode (2) and the counter electrode (4) related to the voltage applied between the working electrode (2) and the counter electrode (4), and detecting a change in a current characteristic of one or more opioid analytes in the sample.

Brief Description of Drawings

Fig. 1 illustrates a method of fabricating an electrode according to at least some embodiments of the invention.

Fig. 2 shows a plan view and cross section of a pressure-transferred CNT network on a glass substrate and coated with Nafion.

Fig. 3 illustrates an exemplary device capable of supporting at least some embodiments of the present invention.

Figure 4 shows cyclic voltammograms of CNT and CNT + Nafion electrodes in: a) fe (CN) in 1M KCl₆ ^4-/3-B) IrCl in 1M KCl₆ ^2-C) FcMeOH in 1M KCl, d) FcMeOH in PBS, e) Ru (NH) in 1M KCl₃)₆ ^2+/3+And f) Ru (NH) in PBS₃)₆ ^2+/3+The scanning rate is 100 mV/s or 500 mV/s.

FIG. 5 shows differential pulse voltammograms of CNT and CNT + Nafion electrodes in a) 500 μ M AA and UA and b) 50 μ M MO and CO.

FIG. 6 shows differential pulse voltammograms of virgin and Nafion coated SWCNTN electrodes at 500 μ M AA, 500 μ M UA, and c) increasing concentrations of MO from 10 μ M CO + 10 nM to 2.5 μ M and d) increasing concentrations of CO from 10 μ M MO + 10 nM to 2.5 μ M at a scan rate of 50 mV/s.

Fig. 7 shows a) thickness profile of a dip-coated Nafion membrane as measured from cross-sectional SEM images (y-axis thickness in microns, x-axis measurement points over the entire cross-section, arbitrary distance). The cyclic voltammetry peak current (oxidation peak and reduction) was measured forPeak) as a function of the square root of the scan rate: b) 1mM IrCl in 1M KCl₆C) with bare SWCNT electrode, 1mM FcMeOH in PBS, d) with SWCNT electrode coated with Nafion, 1mM MFcMeOH in PBS, e) with bare SWCNT electrode, 1mM Ru (NH) in 1M KCl₃)₆F) SWCNT electrode coated with Nafion, 1mM Ru (NH) in 1M KCl₃)₆G) SWCNT electrode coated with bare SWCNT electrode and Nafion, 1mM Ru (NH) in PBD₃)₆The cyclic voltammetry measurements performed in (1).

Fig. 8a) shows the composition of an exemplary sample for testing. The sample consists of whole blood comprising plasma, white blood cells, platelets, and red blood cells. The plasma fraction in turn contains a challenge matrix with analytes including AA (50-200. mu. mol/l), UA (100-. Fig. 8b) shows passive filtration of a whole blood sample, filtering out e.g. red blood cells, white blood cells and platelets, allowing protein, anionic and cationic analytes to pass through the filter, the cationic analytes then passing through a permselective membrane, which prevents neutral and anionic components from passing through. This results in the cationic analyte contacting only the working electrode of the test strip.

Fig. 9 is a scanning electron micrograph of a cross section of an electrode according to at least some embodiments of the present invention. SWCNTN is shown deposited on a glass substrate and a Nafion layer (permselective membrane) coats the SWCNTN.

FIG. 10 shows a) differential pulse voltammograms of acetaminophen (PA) at different concentrations in the presence of 500 uA AA and 500 uM UA measured with SWCNT electrodes coated with 5% Nafion solution (dip-coated for 5 seconds in solution), b) differential pulse voltammograms of Morphine (MO) and Codeine (CO) at different concentrations in Phosphate Buffered Saline (PBS) measured with SWCNT electrodes coated with 5% Nafion solution (dip-coated for 5 seconds in solution), c) differential pulse voltammograms of MO at different concentrations in the presence of 500 uM AA, 500 uM UA and 10 uM CO, and two linear ranges of peak current as a function of MO concentration, d) a magnification of the smaller MO concentration in FIG. 10c), e) differential pulse voltammograms of MO at different concentrations measured in undiluted pooled plasma, and an enlarged view of the smaller concentration.

Fig. 11 illustrates a test strip according to at least some embodiments of the invention and the electrochemical reaction of the analyte (oxidation of the MO), which is a result of the passage of an electric current through the analyte, which in turn produces a signal in a voltammogram for the analyte (MO). The test strip shown comprises an electrode assembly (1) on which a cation exchange membrane (11) is deposited, said cation exchange membrane (11) being a permselective membrane, such as Nafion; a filter (10) for passively filtering a sample to be analyzed; and a hydrophobic protective film (9), such as a Teflon film.

Fig. 12 depicts an electrode assembly (1) for a test strip according to at least some embodiments of the present invention. The electrode assembly (1) comprises a working electrode (2), a counter electrode (4) and a pseudo-reference electrode (3). The working electrode (2) is a titanium/tetrahedral amorphous carbon (Ti/taC) electrode. The pseudo reference electrode (3) and the counter electrode (4) are formed of silver. The electrodes are electrically isolated (8) from each other on the same plane, and the working electrode (2) is located between the pseudo-reference electrode (3) and the counter electrode (4). Each electrode (2, 3, 4) is provided with a contact (5, 6, 7) for direct connection to a voltage supply. The contacts (5, 6, 7) are typically made of silver, such as silver paint.

Figure 13 shows the results of differential pulse voltammetry measurements of some opioids and common interferents with Ti/taC electrodes. The schematic depicts the oxidation peak position and the measured current is not to scale.

Figure 14 shows the results of differential pulse voltammetry measurements of some opioids with SWCNT electrodes.

FIG. 15 shows differential pulse voltammograms of a) MO and b) CO with a conventional SWCNT electrode and a Nafion coated SWCNT electrode. The use of Nafion membrane improves the selectivity and sensitivity of SWCNT electrodes to both MO and CO.

FIG. 16 shows the DPV signal measured in a 10 μ M MO and CO solution as a function of retention time.

Figure 17 shows DPV scans of morphine-3-glucuronide (M-3-G) obtained with a) a common SWCNT electrode and b) a Nafion coated SWCNT electrode.

FIG. 18 shows the DPV of a) Tramadol (TR) and b) O-desmethyltramadol (ODMT) at several concentrations in different solutions, C) the DPV of 50 μ M TR and 50 μ MODMT in the same solution measured with a Ti/ta-C electrode without Nafion, and d) the DPV of 50 μ M TR and 50 μ M ODMT in the same solution measured with a Ti/ta-C electrode coated with Nafion.

Figure 19 shows DPV of AA and UA obtained with a normal SWCNT electrode and a Nafion coated SWCNT electrode.

FIG. 20 shows DPV of 50 μ M of a) xanthine (Xn) and b) hypoxanthine (HXn) obtained with a conventional Ti/taC electrode and a 2.5% coated Ti/taC electrode.

Figure 21 shows the results of DPV measurements on undiluted plasma with a normal SWCNT electrode (black) and a Nafion coated SWCNT electrode (grey).

Figure 22 shows DPV obtained with Nafion coated SWCNT electrodes against undiluted human plasma supplemented with increasing concentrations of morphine.

Figure 23 shows DPV measurements of 50 μ M ketamine.

Fig. 24 shows the variation in blood concentration of a given opioid from dose to dose.

Fig. 25 shows a plurality of electrode assemblies according to at least some embodiments of the invention. Each electrode assembly (1) comprises a working electrode (3), a reference electrode (4) and a counter electrode (2). Each electrode is provided with three contacts (5, 6, 7) for direct connection to an external voltage supply.

Fig. 26 shows a test strip according to at least some embodiments of the present invention that includes a working electrode (2) made of a carbon-based material, a counter electrode (4) made of a carbon-based material, a pseudo-reference electrode (3) made of silver, and contacts (5, 6, 7) for directly connecting the electrodes (2, 3, 4) to an external voltage supply.

Fig. 27 shows a test strip electrode assembly according to at least some embodiments of the present invention that includes a working electrode (3) made from a carbon-based material, a counter electrode (2) made from a carbon-based material, a pseudo-reference electrode (4) made from silver, and contacts (5, 6, 7) for directly connecting the electrodes to an external voltage supply. Also shown is an electrode assembly, the dimensions of which are shown in mm.

Fig. 28 shows differential pulse voltammetry measurements performed on 50 uM MO (a) and 50 uM CO (b) with bare SWCNT and Nafion coated SWCNT electrodes. The figure shows how the Nafion membrane reduces the number of peaks of the opioid analyte, thereby further improving selectivity.

FIG. 29 shows differential pulse voltammetry measurements of 50 uM morphine-3-glucuronide (M3G) and 100 uM 3G in PBS with a) bare SWCNT electrode and b) SWCNT with Nafion. Nafion membranes efficiently filter out inactive metabolites of MO.

FIG. 30 effect of cathode modulation of working electrode on fentanyl detection.

Figure 31 shows differential pulse voltammograms measured with SWCNT electrodes coated with 5% Nafion solution (dip-coated for 5 seconds in solution) versus different concentrations of Morphine (MO) and Codeine (CO) in Phosphate Buffered Saline (PBS). Peak current of MO removalvs.Outside the linear range of concentrations, the peak current of CO is also shownvs.Linear range of concentration.

FIG. 32 shows differential pulse voltammograms of MO at different concentrations in the presence of 500 uM AA, 500 uM UA, and 10 uM CO, as well as two linear ranges of peak current as a function of MO and CO concentration.

Detailed description of the preferred embodiments

In order to establish individual pharmacokinetic and pharmacogenetic factors, it is important to be able to simultaneously measure quantitatively the blood concentration of an opioid in a patient. In the case of determining MO produced by CO and heroin metabolism, in particular, morphine must be accurately measured. It can be seen that the electrodes used in this work can repeatedly measure the current of 50 nM morphine in the presence of AA, UA and CO, with the peak current of MO yielding two linear ranges. The lower range is well within the therapeutic concentrations of pain and most cases of intoxication (oxygenation) and intoxication (poisoning).

It is therefore an object of embodiments to overcome at least some of the above disadvantages and to provide a multilayer test strip for detecting opioids in a sample. In one embodiment, the multilayer test strip includes a substrate on which are deposited electrode assembly layers including a carbon-based working electrode, a carbon-based counter electrode, a pseudo-reference electrode, contacts for directly contacting the electrodes with a voltage supply, and a permselective membrane. In one embodiment, the pseudo-reference electrode, the working electrode, and the counter electrode are disposed adjacent to each other in the same plane. In one embodiment, the electrodes forming the electrode assembly layers are electrically isolated from each other. In another embodiment, the working electrode and the counter electrode comprise the same carbon-based material. In yet another embodiment, the counter electrode is formed of the same material as the reference electrode. In a preferred embodiment, the counter and reference electrodes are formed from a material different from the material forming the working electrode. In one embodiment, the carbon-based material included in the working electrode is different from the carbon-based material included in the counter electrode. In one embodiment, the electrode assembly layer is located between the substrate and the selectively permeable membrane layer.

Permselective layers provide the inherent permselective property that anionic interferents such as UA and AA and neutral interferents such as xanthine (Xn) and hypoxanthine (HXn) are blocked from allowing transfer from the sample to the electrodes. With such test strips, opioids can be detected electrochemically using Cyclic Voltammetry (CV), Linear Sweep Voltammetry (LSV), conventional pulse voltammetry, square wave voltammetry, Differential Pulse Voltammetry (DPV), adsorption stripping voltammetry, chronocoulometry, and chronoamperometry.

In one embodiment, the carbon-based electrode comprises carbon selected from the group consisting of amorphous carbon such as tetrahedral amorphous carbon, diamond-like carbon, graphite, carbon nanotubes, and mixtures thereof. In another embodiment, the carbon-based electrode comprises a single-walled carbon nanotube network (SWCNTN). SWCNTN has high conductivity, can be used to make wires, and can be in direct contact with a voltage supply. For example, the film may be patterned to make wires and electrodes (which may be wires).

Opioids, as well as most other biological and drug molecules, are so-called inner shell (inner sphere) analytes, which means that they are sensitive to the surface chemistry of the electrode material. Thus, the oxidation potential and sensitivity can be fine tuned by changing the carbon-carbon bond and surface functional groups. Similarly, surface metal catalysts used in the synthesis of carbon nanomaterials also affect electrochemical properties. Controlling the surface loading of these catalyst metals and their oxidation states can also be used to increase selectivity. Thus, in one embodiment, one or more carbon-based electrodes further comprise one or more catalytic metals. In a preferred embodiment, one or more carbon-based electrodes comprise titanium.

As described above, the electrode assembly is deposited on the substrate. In one embodiment, the substrate is selected from the group consisting of polymers and glass. The polymer/glass substrate provides an inexpensive disposable test strip.

In addition to the working and counter electrodes, the test strip also includes a pseudo-reference electrode, sometimes referred to as a quasi-reference electrode. The working electrode is the electrode in the electrochemical system where the reaction of interest takes place. The counter electrode is the electrode that is used only to carry the current flowing through the electrochemical cell. The pseudo-reference electrode is an electrode that does not allow significant current to flow and is used to observe or control the potential on the working electrode. In one embodiment, the pseudo-reference electrode comprises silver. In a preferred embodiment, the pseudo-reference electrode comprises silver-silver chloride (Ag/AgCl). In a particular embodiment, the pseudo-reference electrode comprises platinum.

In embodiments, the permselective membrane layer comprises a permselective membrane of a polymer selected from the group consisting of Nafion, cellulose acetate, conventional dialysis membranes, polyvinylsulfonate, carboxymethylcellulose, polylysine, peroxypolypyrrole, and other sulfonated polymers. Commonly used polymer membranes such as Nafion exhibit size exclusion, charge exclusion, ion exchange, complexation, catalytic and conductive properties. In a preferred embodiment, the permselective membrane comprises Nafion.

Extensive Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) results were obtained with electrodes coated with Nafion membrane. CV results for various redox probes with both positive and negative charges can be found in the manuscript attached to the provisional patent. The results show that the Nafion coating excludes negatively charged ferricyanide Fe (CN)₆And iridium chloride IrCl₆And the cation ruthenium hexamine Ru (NH)₃)₆And ferrocene methanol FcMeOH under the membraneAnd (4) enriching. These results confirm the known permselectivity properties of Nafion.

DPV experiments with Nafion coated SWCNT electrodes in morphine solution (fig. 15a) and codeine solution (fig. 15b) showed that fewer peaks were seen for both morphine and codeine Nafion coated electrodes, increasing the selectivity of the electrodes. The selectivity towards morphine is increased in particular by a significant reduction in the current or the complete disappearance of the higher potential peak, ensuring the simultaneous detection of morphine and codeine. It can further be seen that the Nafion coating enhances the signal of morphine, in particular codeine. This may be due to an increase in sub-membrane concentration caused by the Gibbs-Donnan (Gibbs-Donnan) effect. The manuscript in this provisional patent shows that both nanomolar concentrations of morphine and codeine can be detected simultaneously.

The enrichment as a function of retention time (time between bringing the electrode into contact with the solution and the start of the measurement) was further investigated in solutions of morphine and codeine at a concentration of 10 μ M. Figure 16 shows the measured current as a function of retention time and clearly shows that the signal current for both morphine and codeine increases with retention time.

It is predicted that Nafion membranes can also be used to inhibit interference from certain opioid metabolites present in authentic samples. Some measurements have been made using metabolites of morphine, and others are planned using metabolites of oxycodone.

The major metabolite of morphine is glucuronide, which is produced by coupling glucuronide to carbon 3 or 6. Morphine-6-glucuronide (M-6-G) is the major active metabolite of morphine, whereas morphine-3-glucuronide (M-3-G) is not an active opioid agonist. FIG. 17 shows the results of the measurement of M-3-G with and without a Nafion coating. It can be seen that M-3-G did not penetrate the Nafion membrane. Morphine glucuronides and glucuronides are expected to be generally impermeable to membranes, resulting in increased selectivity to morphine.

The role of Nafion coating in the selective and sensitive detection of opioids can also be seen in experiments using Tramadol (TR) and its major metabolite O-desmethyl tramadol (ODMT). In fig. 18, both analytes were measured using tetrahedral amorphous carbon (ta-C) electrodes with and without Nafion coating. While the normal ta-C electrodes were able to see TR and ODMT separately (in fig. 18a and 18b, respectively), they both exhibited several oxidation peaks and therefore were not measurable from the same solution (fig. 18C).

In contrast, by coating the electrode with Nafion membrane, only one peak per analyte was recorded, so TR and ODMT could be selectively detected from the same solution (fig. 18 d). Currently, such results are not found in the literature. However, the oxidation potential of tramadol varies significantly from electrode material to electrode material. For example, according to some preliminary results, with SWCNT electrodes, the signals of TR and ODMT overlap. Thus, some studies that might measure true biological samples in the form of tramadol might actually measure the superposition of tramadol and O-desmethyl tramadol.

The Nafion coating, which is a cation exchange membrane, further increases selectivity by blocking negatively charged species such as Ascorbic Acid (AA) and Uric Acid (UA) from reaching the electrodes. Figure 19 shows DPV of plain and Nafion coated SWCNT electrodes in AA and UA solutions.

Interference caused by other biomolecules with neutral charge, such as xanthines and hypoxanthine, at physiological pH values has also been studied using ta-C electrodes (fig. 20). The Nafion coating also appears to reduce the interference of these molecules.

Experiments have also been performed with real human plasma samples. Initial experiments, shown in fig. 21, show that Nafion coatings can effectively limit interference by interfering substances in plasma samples. Figure 22 further shows that morphine could be detected in undiluted human plasma samples after the addition of different concentrations of morphine.

In other embodiments, the strip further comprises a filtration layer. A filter layer is provided to passively filter blood forming elements (blood cells) from a whole blood sample provided for detection (fig. 8). In one embodiment, the strips are arranged such that the selectively permeable membrane layer is located between the filtration layer and the electrode assembly layer.

Other embodiments of the strip further comprise a hydrophobic film/membrane layer. In one embodiment, the strips are arranged such that the filtration layer is located between the selectively permeable membrane layer and the hydrophobic membrane/membrane layer. In another embodiment, the hydrophobic membrane/membrane layer comprises Teflon. The hydrophobic film/membrane layer is present as a protective layer.

In one embodiment, a multi-layered electrode is provided that includes a filter capable of passively filtering blood forming elements (blood cells), a cation exchange membrane and a carbon electrode, a selectively permeable membrane that exhibits both size exclusion and charge exclusion, a carbon-based electrode (such as carbon nanotubes, amorphous carbon, or graphite). Opioids, as well as most other biological and drug molecules, are so-called inner layer analytes, which means that they are sensitive to the surface chemistry of the electrode material. Thus, the oxidation potential and sensitivity can be fine tuned by changing the carbon-carbon bond and surface functional groups. Similarly, surface metal catalysts used in the synthesis of carbon nanomaterials also affect electrochemical properties. Controlling the surface loading of these catalyst metals and their oxidation states can also be used to increase selectivity and selectivity. In the case of opioids which are predominantly positively charged (i.e., cationic) under physiological conditions, the permselective membrane layer consists of a cationic permselective membrane such as Nafion. Selectivity is increased due to the enrichment of opioids under the membrane, and the membrane blocks negatively charged anions like ascorbic acid and uric acid, which are present in high concentrations in biological fluids (see fig. 11 and 12).

Thus, in an embodiment, a test strip is provided having a working electrode, a counter electrode, and a pseudo-reference electrode for analyzing small (10-60 μ l) blood samples drawn with a finger-stick kit. Figure 11 shows how this electrode detects morphine by electrochemical oxidation. FIG. 12 shows a test strip with a Ti/ta-C working electrode and a silver counter and reference electrode.

The test strip can be used to detect free morphine in undiluted plasma/blood. The test strip can be designed to detect only hydroxyl groups or to detect hydroxyl and amine, allowing for the selective detection of multiple opioids with some selectivity, such as the simultaneous selective detection of morphine and codeine. In addition, the metabolically produced active metabolites morphine (from codeine) and ortho-demethyltramadol (from tramadol) can also be detected. And as described below, the test strip provides for the identification of the glucoside. As seen from the difference between ta-C electrodes and SWCNTs, the electrochemical oxidation potential is highly dependent on the surface chemistry. Previous properties indicate that SWCNTs are graphitic, have low concentrations of defects and oxygen-containing groups, while ta-C has diamond-like bodies and an amorphous sp-rich 2 surface layer. These types of differences can be used by the selection of electrode materials or surface functionalization processes to tailor the selectivity and sensitivity of the test strip.

The test strip provides information about the contents of the sample being tested. Accordingly, embodiments of the present invention are directed to a device for analyzing information provided by a test strip. Accordingly, in one embodiment, there is provided an apparatus comprising a memory configured to store reference data, at least one processing core configured to process information from a strip according to any of the above embodiments, compare the information from a strip according to any of the above embodiments with the reference data, and draw conclusions regarding the processed information from a strip according to any of the above embodiments.

As noted above, the test strip is particularly useful for the detection of opioids. Several opioids were measured in Phosphate Buffered Saline (PBS) with the multilayer electrode described above and shown in figure 1. The carbon materials used in these measurements were tetrahedral amorphous carbon (Ti/ta-C) and single-walled carbon nanotubes (SWCNT) deposited on titanium. The results show some variation in both the sensitivity and position of the oxidation potential. Most of the measured opioids also showed several oxidation peaks due to the oxidation of hydroxyl and amine groups. Figure 13 shows the results of measurements of several opioids with Ti/ta-C electrodes and some common interferents. The results of measurements on the same opioid with SWCNT electrodes are shown in fig. 14.

Accordingly, embodiments of the present invention relate to a method of detecting an opioid in a sample. In one embodiment, the method comprises the steps of: providing a sample, placing the sample in electrical contact with a working electrode (2) and a counter electrode (4) of an electrode assembly of a multi-layer test strip, varying a voltage between the working electrode (2) and the counter electrode (4), measuring a current between the working electrode (2) and the counter electrode (4) in relation to the voltage applied between the working electrode (2) and the counter electrode (4), and detecting a change in a current characteristic of one or both opioid analytes in the sample.

In another embodiment, the method comprises the steps of: providing a sample, placing the sample in electrical contact with a working electrode (2) and a counter electrode (4) of an electrode assembly of a multi-layered test strip according to any of the above embodiments, varying the voltage between the working electrode (2) and the counter electrode (4), measuring the current between the working electrode (2) and the counter electrode (4) in relation to the voltage applied between the working electrode (2) and the counter electrode (4), and detecting a change in a current characteristic of one or both opioid analytes in the sample.

In one embodiment, the voltage between the working electrode (2) and the counter electrode (4) is swept from-0.6V to 0.2V. In a preferred embodiment, the voltage between the working electrode (2) and the counter electrode (4) is swept from-0.5V to 1.5V.

In another embodiment, the scan rate is in the range of 2.5-40 mV/s.

In another embodiment, the method comprises the steps of: providing a sample, contacting a test strip according to any of the above embodiments with the provided sample, passing an electrical current through the test strip and detecting a change in an electrical current characteristic of one or more opioid analytes in the sample.