Test strip for detecting neutral analytes in a sample - Patent Application 20070122997

Abstract

Disposable multilayer test strips are provided, each comprising a substrate having deposited thereon an electrode assembly including a carbon-based working electrode, a carbon-based counter electrode, and a pseudo-reference electrode, the pseudo-reference electrode, the working electrode, and the counter electrode being disposed adjacent to one another in the same plane, the test strip including contacts for directly contacting the electrodes to a voltage source, and the test strip further including a permselective membrane layer. The electrodes of the electrode assembly layer are electrically isolated from one another, the electrode assembly layer being disposed between the substrate and the permselective membrane layer. The permselective membrane has a structure adapted to allow one or more electrically neutral analytes in a sample to pass through the permselective membrane to the electrode assembly.

Description

[Technical Field]

[0001] The present invention relates to a multi-layer test strip, in particular a multi-layer test strip for detecting a neutral analyte such as paracetamol in a sample, and a method for manufacturing such a multi-layer test strip. Furthermore, the present invention relates to a system for detecting a neutral analyte, comprising a multi-layer test strip and a measurement circuit. Furthermore, the present invention relates to a method for measuring a neutral analyte in a sample. Furthermore, the present invention relates to a method for diagnosing an overdose and / or toxic level of a neutral analyte or substance, such as paracetamol, in a patient. Still further, the present invention relates to determining individual pharmacokinetic parameters for the purpose of personalized medicine. [Background technology]

[0002] Paracetamol, also known as acetaminophen, has antipyretic properties and is one of the most widely used analgesics. It is readily available, inexpensive, and better tolerated than NSAIDs, making it widely recommended as a first-line treatment for a variety of pain conditions. Unlike NSAIDs, large doses of paracetamol can cause liver toxicity. Paracetamol is one of the most commonly used drugs in overdose, and paracetamol poisoning is now the leading cause of acute liver failure in the United States and Europe. In the United States alone, more than 111,000 exposures are reported to poison centers, with 40,000 related emergency department cases annually. Both intentional and unintentional exposure to toxic levels of paracetamol is common.

[0003] Paracetamol toxicity is due to the highly reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI). A toxic dose of paracetamol causes excessive amounts of the drug to be metabolized by the CYP2E1 enzyme to NAPQI. At therapeutic doses, this toxic metabolite is readily inactivated by conjugation with glutathione and excreted in the urine. However, at toxic concentrations, this detoxification pathway is exhausted. NAPQI can covalently bind to cellular proteins, forming toxic adducts that can cause mitochondrial dysfunction and early oxidative stress. This can ultimately lead to hepatocyte necrosis and acute liver failure. Cellular damage has been shown to be directly related to the paracetamol dose.

[0004] Paracetamol poisoning can be effectively treated with N-acetylcysteine, a glutathione precursor. Unfortunately, paracetamol poisoning presents with few nonspecific symptoms during the first 24 hours. Furthermore, N-acetylcysteine ​​treatment is most effective when initiated within 8–12 hours of exposure, as the effectiveness of the antidote rapidly declines after 15 hours. For these reasons, the National Academy of Clinical Biochemistry recommends screening for paracetamol in all emergency department patients presenting with intentional drug ingestion. Diagnosis of paracetamol overdose is typically made by measuring paracetamol serum concentrations. The Rumack-Matthew nomogram, which plots paracetamol concentrations as a function of time after ingestion, is useful for determining the likelihood of hepatotoxicity. Serum levels of 200 μg / mL (1.323 mM) or greater at 4 hours after ingestion and 6.25 μg / mL (43.1 μM) or greater at 24 hours after ingestion have been found to consistently predict hepatotoxicity. The line between these points is called the probabilistic toxicity line. The FDA recently required an additional line 25% below the original line to incorporate additional safety. Summary of the Invention [Problem to be solved by the invention]

[0005] In clinical settings, rapid testing is typically performed using spectrophotometry due to its relative simplicity and low cost. Despite these advantages, this method is still limited to specialized laboratories and is not well suited for point-of-care testing. Furthermore, interferences that can cause both falsely high and falsely low results have been reported with these methods. Additionally, competitive lateral flow immunoassays can also be used for the qualitative measurement of paracetamol. However, these tests are not quantitative and have high cutoff concentrations, resulting in reported false negatives. Therefore, the development of a simple, quantitative, point-of-care assay with high portability for screening for paracetamol poisoning is particularly desirable. [Means for solving the problem]

[0006] The invention is defined by the features of the independent claims. Some particular embodiments are defined in the dependent claims. According to a first aspect of the present invention, a disposable multilayer test strip is provided, comprising a substrate having deposited thereon an electrode assembly including a carbon-based working electrode, a carbon-based counter electrode, and a pseudo-reference electrode. The pseudo-reference electrode, working electrode, and counter electrode are disposed adjacent to one another in the same plane. The strip further comprises contacts for directly connecting the electrodes to a voltage source and a permselective membrane layer. The electrodes of the electrode assembly layer are electrically isolated from one another, and the electrode assembly layer is disposed between the substrate and the permselective membrane layer. The permselective membrane has a structure adapted to allow one or more electrically neutral analytes in a sample to pass through the permselective membrane to the electrode assembly.

[0007] According to a second aspect of the present invention, there is provided an apparatus comprising: a memory configured to store reference data; and at least one processing core configured to process information from a multi-layer test strip described herein, compare information from the test strip described herein with the reference data, and draw conclusions based on the processed information from the strip described herein.

[0008] According to a third aspect of the present invention, there is provided a method for detecting electrically neutral analytes in a sample, the method comprising the steps of providing a sample, electrically contacting the sample with a working electrode and a counter electrode of an electrode assembly of a multi-layer test strip, varying a voltage between the working and counter electrodes, measuring a current between the working and counter electrodes relative to the voltage applied between the working and counter electrodes, and detecting a change in a current characteristic of one or more analytes in the sample.

[0009] According to a fourth aspect of the present invention, there is provided a method of diagnosing an overdose in a patient, the method comprising obtaining a sample from a subject, bringing the sample into electrical contact with a working electrode and a counter electrode of an electrode assembly of a multi-layer test strip, varying a voltage between the working and counter electrodes, measuring a current between the working and counter electrodes relative to the voltage applied between the working and counter electrodes, detecting a change in the current characteristics of one or more analytes in the sample, and determining the amount of analyte in the sample in a device according to the second aspect of the present invention. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 illustrates an example method for manufacturing a multi-layer test strip in accordance with at least some embodiments of the present invention. [Figure 2] 1A-1B show scanning electron micrographs of cross sections of a Nafion-coated working electrode (A) and a Nafion-coated reference electrode (B) according to at least some embodiments of the present invention. [Figure 3A] 1A is a graph showing the potential of an uncoated and Nafion-coated pseudo-reference electrode versus Ag / AgCl (saturated) in 0.1 M PBS solution, according to at least some embodiments of the present invention. All measurements were performed in a conventional 50 ml electrochemical cell. [Figure 3B] (B) Graph showing potential as a function of Cl concentration in KCl solution, in accordance with at least some embodiments of the present invention. All measurements were performed in a conventional 50 ml electrochemical cell. [Figure 3C](C) Cyclic voltammogram of 1 mM Ru(NH3)6 in 1 M KCl, according to at least some embodiments of the present invention. All measurements were performed in a conventional 50 ml electrochemical cell. [Figure 4A] FIG. 10 shows a comparison of DPV measurements of 50 μM PA performed in a 50 mL cell and in a 40 μL droplet. [Figure 4B] FIG. 10 shows optimization of DPV pulse amplitude for 50 μM PA in 40 μL of diluted human plasma. [Figure 5A] Figure 1 shows DPV of increasing concentrations of paracetamol in PBS. Error bars indicate the standard deviation of four measurements using different electrodes. [Figure 5B] Figure 1 shows the DPV of increasing concentrations of paracetamol in human plasma. (D) Linearization of the results for all measured matrices. Error bars indicate the standard deviation of four measurements using different electrodes. [Figure 5C] Figure 1 shows DPV of increasing concentrations of paracetamol in whole blood. Error bars indicate the standard deviation of four measurements using different electrodes. [Figure 5D] Graph showing the linearization of the results for all measured matrices in Figures 5A-5C. Error bars indicate the standard deviation of four measurements using different electrodes. [Figure 6A] FIG. 1 shows the CV of 1 mM Ru(NH3)6 in PBS and plasma. [Figure 6B] 1 is a graph showing DPV peak current as a function of scan number in 1 mM PA in whole blood and plasma. [Figure 7A] Figure 1 shows an interference study. DPV scans with blank PBS (black line), interferent only (blue line), and interferent + 50 μM PA (red line). Error bars represent 5% error, defined as the acceptable limit. DPV scans have been offset for clarity. [Figure 7B]Figure 1 shows interference studies. Background subtracted peak currents of 50 μM PA alone (red) and in the presence of interferents (blue). Error bars represent 5% error, defined as the tolerance limit. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a disposable electrochemical test strip for quantitative point-of-care measurement of target molecules, i.e., neutral analytes, overdosed or otherwise administered or accumulated in a subject at toxic or therapeutic levels. The present invention also relates to a method for producing such a test strip. The fabrication or manufacturing method according to the present invention results in highly conductive, electrically well-isolated, patterned carbon-based electrodes printed on a substrate. The present invention produces a screen-printed silver pseudo-reference electrode with excellent shelf life, long-term stability, and short hydration time. The test strip provides a sufficiently low detection limit and wide linear range for measuring the concentration of target molecules, e.g., paracetamol, in suspected paracetamol poisoning. Surprisingly, it has been found that detection and quantitative determination of target molecules, e.g., paracetamol, can be performed using assays according to embodiments of the present invention with sample volumes as low as 20 μL. Such samples include blood samples obtained by finger prick, optionally diluted with up to 20 μL of PBS, venous blood, urine, or venous blood, optionally diluted with PBS, or even saliva. No further sample processing is required, and rapid results are obtained. Assay times of less than 5 minutes are achieved, which is crucial in cases of overdose or toxicity. Furthermore, selectivity is achieved even in the presence of several interfering substances.

[0012] Figure 1 illustrates the fabrication of the sensor strip. In this exemplary embodiment, SWCNTs were first grown by aerosol CVD and collected on a filter. The SWCNT network was then pressed onto an A4 PET sheet, compressed by spraying IPA from a spray bottle, and dried with nitrogen, e.g., blow-dried or compressed nitrogen. To achieve patterned electrodes, the lines separating the electrodes were removed. To achieve a reference electrode, a silver line was screen-printed directly on the SWCNT layer (see Figure 1, step 3). A silver contact pad was also fabricated in the same way. Finally, according to at least some embodiments of the present invention, the entire A4 PET sheet was coated with Nafion.

[0013] Figure 2 shows cross-sectional images taken from cut regions of A) the working electrode and B) the reference electrode according to at least some embodiments of the present invention. The overall thickness of the SWCNT / Nafion layer on the working electrode is found to be approximately 170 nm thick. A dark layer, 65–75 nm thick, can also be observed between the SWCNT / Nafion layer and the Au coating, likely due to Nafion. This result is consistent with previous studies and suggests that the SWCNTs are at least partially coated with Nafion. The cross-section of the Ag reference electrode shows flat, elongated Ag particles with sizes in the range of several microns. The cross-section of the reference electrode yielded a thickness of 5.9–7.2 μm. Several measurements of the silver wire, also performed with a contact profilometer, yielded thicknesses in the range of 5.5–7 μm. Due to the high roughness, no clear layer of Nafion can be found, even on top of the Ag particles.

[0014] Figure 3A shows the potential of both an uncoated and a Nafion-coated screen-printed Ag reference electrode in 0.1 M PBS solution relative to an Ag / AgCl [saturated] electrode, supporting at least some embodiments of the present invention. Both types of electrodes start at a potential of 84 ± 1 mV. However, it is clear from Figure 3A that the potential of the uncoated electrode drifts during the potential measurement.

[0015] Figure 3B shows the potential of the Ag reference electrode versus Cl. -The potential of the Nafion-coated electrode is shown as a function of the logarithm of the Cl concentration. - ] and the Cl of the electrolyte - The potential of an uncoated Ag electrode also depends linearly on the logarithm of Cl concentration. - It depends on the concentration, but the linearity is low. - Despite the sensitivity to concentration, the Nafion coated electrode exhibits an immediate stable potential at all concentrations with no run-in time.

[0016] Figure 3C shows CV measurements at various scan rates for 1 mM Ru(NH) in 1 M KCl. The peak potential separation (ΔE) of 68.8 mV (scan rate: 100 mV / s) was observed. p ) was obtained, indicating near-reversible electron transfer.

[0017] Figure 4A shows DPV measurements performed using a sensor strip in a conventional 50 ml electrochemical cell with a 40 μL droplet placed directly on the sensor. Background-subtracted oxidation peaks of 1.178 and 1.159 μA (average 1.07 μA for the PBS concentration series) were measured for 50 μM PA in the 50 mL cell and 40 μL droplet, respectively.

[0018] Figure 4B shows DPV measurements at different pulse amplitudes using a 40 µL droplet of diluted human plasma. It is expected that a larger pulse amplitude will result in greater sensitivity to PA. Despite this, only a slight increase in the small peaks around 150 mV and 550 mV is observed with increasing pulse amplitude.

[0019] Figure 5 shows the DPV measurements with increasing PA concentration. It can be seen that the current changes proportionally to the concentration in the concentration range of 1 μM to 2 mM.

[0020] Figure 5D shows that recoveries of 79% and 74% were obtained in plasma and whole blood, respectively.

[0021] FIG. 6 shows that there is no passivation of the electrode when 1 mM Ru(NH3)6 is measured in PBS and in diluted human plasma.

[0022] FIG. 6B shows the measured oxidation current as a function of scan number.

[0023] FIG. 7 shows the results of DPV scans in the absence and presence of 100 μM NSAID mixture of ibuprofen, naproxen, and aspirin, 1 mM salicylic acid (a metabolite of aspirin), 1 mM nicotine, 1 mM amoxicillin, and 1 mM caffeine, as well as 2.5 μM morphine and 10 μM o-desmethyltramadol.

[0024] As mentioned above, the present invention relates to a multilayer test strip. In one embodiment, a disposable multilayer test strip is described that includes a substrate on which an electrode assembly is deposited. The electrode assembly includes a carbon-based working electrode, a carbon-based counter electrode, and a pseudo-reference electrode, where the pseudo-reference electrode, working electrode, and counter electrode are disposed adjacent to one another in the same plane. The multilayer test strip includes contacts for directly connecting the electrodes to a voltage source, and the test strip further includes a permselective membrane layer. The electrodes of the electrode assembly layer are electrically isolated from one another, and the electrode assembly layer is disposed between the substrate and the permselective membrane layer. Surprisingly, it has been discovered that, in some embodiments, the passage of analytes across the permselective membrane can be controlled by adapting the structure of the permselective membrane. Thus, in one embodiment, the permselective membrane has a structure adapted to allow one or more electrically neutral analytes in the sample to be analyzed to pass through the permselective membrane to the electrode assembly. For purposes of the embodiments, an electrically neutral analyte refers to an analyte that is neutral under physiological conditions. Physiological conditions refer to a pH of about 7.4; for example, the normal pH of human blood is typically in the range of 7.35 to 7.45. Zwitterions, which have an equal number of positive and negative charges, are also included in the definition of a neutral analyte.

[0025] In one embodiment, the substrate of the strip is selected from the group consisting of polymers and glass. The substrate is selected based on disposability. In one embodiment, the substrate is a polymer such as polycarbonate or PET. Most preferably, the substrate is polycarbonate because polycarbonate is biodegradable by the action of enzymes or by whole bacterial cells.

[0026] As described in the above embodiment, the strip includes carbon-based electrodes. In one embodiment, one or both of the carbon-based electrodes includes carbon selected from the group consisting of amorphous carbon, such as tetrahedral amorphous carbon, diamond-like carbon, graphite, carbon nanotubes, graphene, and mixtures thereof. In a preferred embodiment, one or both of the carbon-based electrodes includes carbon nanotubes, particularly single-walled carbon nanotubes. While each of the aforementioned forms of carbon is suitable for embodiments of the present invention, single-walled carbon nanotubes offer the additional advantages of high surface area, strong mechanical strength, high electrical conductivity, electrocatalytic activity, low charging current, and enhanced mass transport, enabling high signal-to-noise ratios in electrochemical detection when networks / thin films are deposited on insulating substrates, for example. Large-area porous SWCNT electrodes with high conductivity and surface area can be fabricated by aerosol chemical vapor deposition. This method allows for the collection of patterned networks that can be easily press-transferred to fabricate electrodes without the need to modify conventional carbon electrodes. This enables the fabrication of inexpensive, disposable SWCNT electrodes on a wide range of substrates, including polymers. SWCNT films can be patterned by standard lithography or laser-patterned down to 10 μm by laser ablation without damaging polymer substrates such as polycarbonate or PET. The method can be performed at high throughput and is fully roll-to-roll compatible.

[0027] In a further embodiment, the pseudo-reference electrode comprises silver. Ag / AgCl electrodes perform adequately. Thus, in one embodiment, the pseudo-reference electrode comprises Ag / AgCl. However, it has surprisingly been discovered that a permselective membrane coating stabilizes the potential of the pseudo-reference electrode, allowing the pseudo-reference electrode to be fabricated in the same process as the conductive silver wire, eliminating the need for a second screen-printing step using AgCl ink. Thus, in a preferred embodiment, the pseudo-reference electrode is comprised of silver.

[0028] The permselective membrane material can be selected from a variety of materials. In one embodiment, the permselective membrane comprises a membrane material selected from the group of polymers consisting of Nafion, cellulose acetate, polyvinyl sulfonate, carboxymethyl cellulose, polylysine, peroxidized polypyrrole, and other sulfonated polymers. In one embodiment, the permselective membrane comprises a conventional dialysis membrane material. The sulfonic acid groups of the sulfonated polymers repel / exclude negatively charged anions that interfere with the quantitative detection of neutral analytes such as paracetamol, allowing neutral molecules to diffuse through the permselective membrane. Therefore, sulfonated polymers are particularly desirable in embodiments of the disposable multilayer test strip of the present invention. Nafion has a particularly high concentration of sulfonic acid groups throughout the polymer. Therefore, in a preferred embodiment, the permselective membrane comprises Nafion. Nafion also has an affinity for cations such as morphine and tramadol and their metabolites, which are often present in samples and may interfere with the measurement of neutral molecules such as paracetamol. In accordance with embodiments, it has been found that the Nafion membrane enables the electrode to function such that opioid interferents, such as morphine, do not cause interference in the measurement of neutral analytes, such as paracetamol.

[0029] In a further embodiment, the structure of the permselective membrane is formed from one or more layers of membrane material applied to a strip, whereby a stack of membrane material layers forms the permselective membrane. Thus, depending on the embodiment, the thickness of the permselective membrane can be tailored.

[0030] Selectively permeable membranes, such as sulfonate-containing polymers like Nafion membranes, have been shown to form coatings that concentrate cations through ion exchange reactions. Negatively charged channels within the coating, measuring a few nanometers, prevent anions from passing through. Neutral analytes may pass through the membrane by passive diffusion. Different neutral molecules also exhibit different permeabilities due to different interactions between different analytes and permselectively permeable membranes, such as sulfonic acid-containing polymer membranes like Nafion membranes. Therefore, in one embodiment, by carefully controlling deposition parameters such as deposition method, coating time, sulfonic acid group concentration in the membrane, e.g., Nafion membrane, and number of layers, multilayer test strips can be provided that allow for control of neutral permeability and the degree of surface functionalization of the SWCNT electrode. We have previously shown that multilayer electrodes can be fabricated that optimize the concentration of cations by blocking anions and most neutrals from reaching the electrode, thereby enabling the selective detection of opioids in the presence of neutrals like paracetamol. The current multilayer electrode has been optimized by controlling deposition parameters to allow the passage of neutral analytes without compromising selectivity in complex matrices containing high concentrations of anions, such as blood, urine, and saliva. When measuring paracetamol, the SWCNT electrode layer is also partially functionalized with a permselective membrane, so that the cations morphine and o-desmethyltramadol do not interfere with the measurement at clinically relevant levels. In one embodiment, the stack of membrane materials has a thickness ranging from 10 nm to 4000 nm. The stacking has a dual effect: as the number of layers increases, the amount of sulfonic acid groups increases, making it increasingly difficult for cationic groups to pass through the membrane, and because the layers are so thin, defects are found in each layer that allow neutral analytes to pass easily. In one embodiment, the permselective membrane has a thickness ranging from 50 to 3000 nm, preferably from 75 to 2500 nm, and suitably from 100 to 2000 nm. In a further embodiment, the permselective membrane has a thickness in the range of 50 to 400 nm, preferably 75 to 250 nm, suitably 100 to 200 nm.

[0031] Further embodiments relate to an apparatus for analyzing data from a multi-layer test strip. In one embodiment, the apparatus comprises a memory configured to store reference data and at least one processing core configured to process information from a multi-layer test strip according to embodiments described herein, compare information from the strip according to embodiments described herein to the reference data, and draw conclusions based on the processed information from the strip according to embodiments described herein.

[0032] Further embodiments relate to methods for detecting electrically neutral analytes in a sample. In one embodiment, the method includes the steps of providing a sample, electrically contacting the sample with a working electrode and a counter electrode of an electrode assembly of a multi-layer test strip, varying a voltage between the working electrode and the counter electrode, measuring a current between the working electrode and the counter electrode relative to the voltage applied between the working electrode and the counter electrode, and detecting a change in the current characteristics of one or more analytes in the sample. In one embodiment, the method detects a free or unbound fraction of neutral analytes in the sample. The free or unbound fraction is the fraction that is not bound to blood and / or serum proteins. In a further embodiment, the detection of the free or unbound fraction of neutral analytes is performed without the use of equilibrium dialysis. In other words, in certain embodiments, the detection of the free or unbound fraction of neutral analytes is performed using a detection method that does not use equilibrium dialysis.

[0033] A further embodiment describes a method for detecting electrically neutral analytes in a sample. In one embodiment, the method includes the steps of preparing a sample, typically a blood sample obtained, for example, by finger prick, electrically contacting the sample with the working and counter electrodes of the electrode assembly of the multilayer test strip described above, varying the voltage between the working and counter electrodes, measuring the current between the working and counter electrodes relative to the voltage applied between the working and counter electrodes, and detecting changes in the current characteristics of one or more analytes in the sample. In a further embodiment, the electrically neutral analyte being detected is selected from the group consisting of anesthetics such as paracetamol, tetrahydrocannabinol (THC), alprazolam, lorazepam, and propofol. In one embodiment, the sample is diluted with a buffer solution, preferably PBS. Preferably, the sample is not diluted at all. In one embodiment, the volume of the sample contacting the working and counter electrodes amounts to approximately 3.5 to 20 μl, preferably 5 to 15 μl, and suitably 10 μl.

[0034] The voltage between the working electrode and the counter electrode is scanned depending on the analyte to be detected. For example, in one embodiment, the voltage between the working electrode and the counter electrode is scanned at a certain scan rate from −0.2 V to 0.8 V, preferably from 0.1 V to 0.6 V, which is a range suitable for detecting paracetamol.

[0035] Similarly, in one embodiment, the scan rate is adjusted according to the analyte being detected. In one embodiment, the scan rate is in the range of 5 to 1000 mV / sec, preferably 10 to 400 mV / sec.

[0036] A further embodiment describes a method for fabricating a multilayer test strip. In one embodiment, the method includes providing an SWCNT network, pressing the SWCNT network onto a substrate to form carbon-based electrodes, isolating the electrodes by laser patterning, screen-printing silver to form a carbon-based working electrode and a silver pseudo-reference electrode adjacent to the carbon-based counter electrode, screen-printing silver contact pads on each electrode, and coating the electrodes with a permselective membrane layer. In one embodiment, the coating step is adapted to coat the electrodes with a permselective membrane of a predetermined thickness. In another embodiment, the carbon-based electrodes are formed on the substrate from amorphous carbon. The amorphous carbon is applied to the substrate by physical vapor deposition using a shadow mask or standard photolithography. One embodiment of this method produces the multilayer test strip described above.

[0037] Also disclosed are embodiments in which an overdose is diagnosed in a patient or subject. In one embodiment, the diagnostic method comprises obtaining a sample from the subject, electrically contacting the sample with a working electrode and a counter electrode of an electrode assembly of a multi-layer test strip, varying a voltage between the working electrode and the counter electrode, measuring a current between the working electrode and the counter electrode relative to the voltage applied between the working electrode and the counter electrode, detecting a change in the current characteristics of one or more analytes in the sample, and determining the amount of analyte in the sample in a device according to the second aspect of the invention.

[0038] The following non-limiting examples illustrate at least some embodiments of the present invention. [Example]

[0039] The SWCNTs are first grown by aerosol CVD, as discussed in detail in Kaskela, A. et al., “Aerosol-Synthesized SWCNT Networks with Tunable Conductivity and Transparency by a Dry Transfer Technique.” Nano Lett. 2010, 10 (11), pp. 4349-4355, https: / / doi.org / 10.1021 / nl101680s. and Moisala, A. et al., “Single-Walled Carbon Nanotube Synthesis Using Ferrocene and Iron Pentacarbonyl in a Laminar Flow Reactor.” Chem. Eng. Sci. 2006, 61 (13), pp. 4393-4402, https: / / doi.org / 10.1016 / j.ces.2006.02.020., both methods are incorporated herein by reference, and collected on a filter. Next, an 18 × 26 cm SWCNT network was press-transferred onto an A4 PET sheet, compressed by spraying IPA from a spray bottle, and dried with nitrogen.SWCNT electrodes fabricated in the same manner have previously been characterized in detail in Wester, N. et al., "Simultaneous Detection of Morphine and Codeine in the Presence of Ascorbic Acid and Uric Acid and in Human Plasma at Nafion Single-Walled Carbon Nanotube Thin-Film Electrode." ACS Omega 2019, 4 (18), pp. 17726-17734, https: / / doi.org / 10.1021 / acsomega.9b02147. and Wester, N. et al., "Single-Walled Carbon Nanotube Network Electrodes for the Detection of Fentanyl Citrate." ACS Appl. Nano Mater. 2020, acsanm.9b01951, https: / / doi.org / 10.1021 / acsanm.9b01951, the teachings of which are incorporated herein by reference. The optical transmittance of the press-transferred SWCNTs was 71.6% (550 nm), and the sheet resistance was 73 Ω / sq. To achieve patterned electrodes, the lines separating the electrodes were removed by pulsed laser ablation.

[0040] To realize the reference electrode and reduce the wire resistance between the active electrode area and the contact pad, a silver wire was screen-printed directly on the SWCNT layer (see Figure 1, step 3). The silver contact pad was also fabricated in the same way. Finally, the entire A4 PET sheet was coated with Nafion at room temperature using a slot die coater (Schneider Electric). For this method, a 5% Nafion solution (Sigma-Aldrich) was diluted to 2.5% with ethanol (94.5 wt%, Altia, Finland) before coating. The following slot die coating parameters were used: coating width: 200.0 mm, syringe diameter: 22.0 mm, pump speed: 1.2 ml / min, wet film thickness: 15.0 μm, speed: 40 cm / min. The PET sheet was placed in the slot die coater so that the electrode was coated first and the contact pad was coated last. Prior to measurement, the electrodes were covered with a PTFE film (Saint-Gobain Performance Plastics CHR 2255-2) with a 6 mm pre-drilled hole. However, for a single measurement, this mask was not necessary because the laser-ablated area around the electrode was hydrophobic, sufficient to keep the 40 μL droplet in place during the measurement. After slot-die coating with Nafion, the electrical insulation of the electrodes was tested with a multimeter for each test strip.

[0041] The thickness of the Ag reference electrode and SWCNT / Nafion layer was measured using a scanning electron microscope (SEM). Prior to imaging, cross-section samples were prepared by focused ion beam (FIB) milling. Both FIB milling and SEM imaging were performed on an FEI Helios NanoLab 600 Dual Beam System. Prior to milling, the samples were coated with 100 nm of gold by evaporation to serve as a conductive coating to protect against beam damage during ion milling and SEM imaging. Cross-sections were milled at an accelerating voltage of 16 kV and a current of 280 / 460 pA for coarse milling. SEM imaging was performed at lower currents of 5–30 kV and 43–170 pA. The thickness of the silver wire was also measured using a contact profilometer (Dektak 6M) on several locations on the silver wire and on the reference electrode.

[0042] Cyclic voltammetry (CV) using Ru(NH3)6 in KCl and potential measurements of a screen-printed Ag pseudo-reference electrode were performed in a conventional 50 ml glass electrochemical cell using a Gamry Reference 600 potentiostat. A three-electrode setup was used, with a Pt wire counter electrode placed in a Luggin capillary and an Ag / AgCl [saturated] (+0.199 V vs. SHE, radiometric analysis) reference electrode, coupled to the Ag electrode as the working electrode. For CV measurements performed in the 50 ml cell, the integrated electrodes of the test strip were connected. For these measurements, a modified serial ATA cable was used as the connector.

[0043] All differential pulse voltammetry (DPV) and CV experiments using 40 μL droplets were performed using a PalmSens4 portable potentiostat. Test strips were directly connected to connectors purchased from PalmSens, with 2 mm banana clips attached to any electrode on the connector. - To investigate the sensitivity of the Ag reference electrode to concentration, KCl solutions of different concentrations were prepared by dissolving KCl (Merck Suprapur) in deionized water (18.2 MOhm-cm).

[0044] Morphine hydrochloride was obtained from University Pharmacy, Helsinki, Finland. All other chemicals were obtained from Sigma-Aldrich. To study electron transfer, a 1 mM solution of the outer-sphere redox probe Ru(NH3)6 was prepared in 1 M KCl and PBS. Paracetamol and interference solutions were prepared in phosphate-buffered saline (PBS) at pH 7.4. Fresh stock solutions were prepared on each day of measurement.

[0045] For plasma measurements, expired human plasma (Octaplas AB, Sweden) was used. Plasma samples were diluted 1:1 by adding 1 ml of plasma to 1 ml of pH 7.4 PBS in an Eppendorf flask. Whole blood was obtained from healthy volunteers by finger prick and collected in a 20 μL calibrated microcapillary tube (Drummond Scientific, USA). The blood sample was then placed in a 2 ml Eppendorf flask and diluted with 20 μL of PBS. Plasma and whole blood samples containing paracetamol were prepared by mixing a sample with twice the target PA concentration with the PBS used for dilution. To avoid clotting of whole blood, a new sample was taken for each measurement. For each measurement, a 40 μL droplet was placed on the test strip using a micropipette. A 2.5-minute accumulation time was used because a gradual increase in the PA signal was observed with increasing accumulation time. Between each measurement, the measured droplet was wiped with tissue paper and rinsed with a PBS droplet for 2.5 minutes before the next droplet was placed on the test strip.

[0046] The total thickness of the SWCNT / Nafion layer on the working electrode is found to be approximately 170 nm. A dark layer, 65–75 nm thick, between the SWCNT / Nafion layer and the Au coating (deposited by electron beam evaporation to protect the Nafion layer from beam damage during ion milling and SEM imaging) is also observed, likely due to Nafion. This result is consistent with our previous studies, e.g., Wester, N. et al., "Simultaneous Detection of Morphine and Codeine in the Presence of Ascorbic Acid and Uric Acid and in Human Plasma at Nafion Single-Walled Carbon Nanotube Thin-Film Electrode." ACS Omega 2019, 4 (18), pp. 17726–17734. https: / / doi.org / 10.1021 / acsomega.9b02147, and studies by other groups, suggesting that the SWCNTs are at least partially coated by Nafion. A cross-section of the Ag reference electrode shows flat, elongated Ag particles with sizes in the range of a few micrometers. A cross-section of the reference electrode yielded a thickness of 5.9–7.2 μm. Several measurements of the silver wire were also performed with a contact profilometer and found to have thicknesses in the range of 5.5–7 μm. Due to the high roughness, no transparent layer of Nafion can be found on top of the Ag particles.

[0047] Typically, quasi-reference electrodes fabricated from silver suffer from potential drift during measurement, short lifespan, long adjustment times until the potential stabilizes, and a relatively short shelf life. While disposable test strips do not necessarily require long-term stability, the adjustment time and potential drift during measurement can potentially cause problems. Figure 3A shows the OCP potential of both uncoated and Nafion-coated screen-printed Ag reference electrodes in 0.1 M PBS solution relative to the Ag / AgCl [saturated] electrode. Both types of electrodes start at a potential of 84 ± 1 mV. However, it is clear from Figure 3A that the potential of the uncoated electrode drifts during potential measurement. Despite this potential drift, the uncoated electrode also reached a stable potential after approximately 1 hour. In contrast, the Nafion-coated electrode immediately exhibited a stable potential, requiring no adjustment time. One of the four Nafion-coated electrodes was also measured for 7.5 hours and showed an average potential of 84.78 mV ± 0.35. At no time during the measurements did the minimum and maximum potentials measured change more than ±1 mV, as they were 84.07 mV and 85.39 mV, respectively. A long-term stability study was also conducted, and a potential drop of less than 10 mV (9.85 mV) was observed after 7 days of immersion in PBS. This potential stability and drift rate are comparable to a screen-printed Ag / AgCl electrode with a much more complex design, featuring a protective layer incorporating a salt matrix (KCl). The electrode in this study quickly produced a stable potential and remained stable for up to 7 days. These measurements clearly demonstrate that Nafion-coated electrodes can be used for voltammetric measurements in point-of-care applications without pretreatment. Furthermore, one of the four electrode strips measured was from a different batch that had been stored under ambient conditions for approximately 1.5 years before measurement. This electrode also exhibited a stable potential of 84.42 ± 0.47 mV during a 3-hour measurement, demonstrating the excellent shelf life of the reference electrode without electrode packaging.

[0048] By measuring the potential of a conventional Ag / AgCl electrode against an Ag reference electrode fabricated in KCl solutions of different concentrations, Cl - The sensitivity to Cl concentration was investigated. -The potential of the Ag reference electrode is shown as a function of the logarithm of the Cl concentration. - The Nafion-coated Ag electrode is sensitive to Cl in the electrolyte. - It is linearly dependent on the concentration, with a slope of -33.9 mV / log[Cl - The uncoated electrode was - It showed low dependence on Cl concentration. - Despite the sensitivity to concentration, the Nafion-coated electrode showed an immediate stable potential at all concentrations without any conditioning time. However, these results suggest that the ionic strength of the electrolyte solution needs to be controlled.

[0049] The outer-sphere redox probe Ru(NH3)6 was used to study electron transport. Figure 3C shows CV measurements at different scan rates for 1 mM Ru(NH3)6 in 1 M KCl. The peak potential separation (ΔE) of 68.8 mV (scan rate: 100 mV / s) was observed. p ) was obtained, indicating near-reversible electron transfer. However, the increase in peak potential separation with increasing scan rate (110 mV at 400 mV / s) indicates quasi-reversible electron transfer. Uncompensated resistance values ​​of 164.1 ± 25.6 Ω were also measured for six electrodes in PBS solution.

[0050] For linearization of paracetamol concentration measurements, R 2 =0.9959, R 2 = 0.9999, and R 2A correlation coefficient of 0.9984 was obtained for PBS, plasma, and whole blood, respectively, indicating that the resulting signal linearly depends on the paracetamol concentration over a wide linear range from 1 μM to 2 mM, covering the entire physiologically relevant concentration range. The limit of detection (LOD) was calculated as LOD = (3 * σ) / S), where σ is the standard deviation of triplicate measurements in empty PBS, and S is the sensitivity across the entire linear range. The LOD was determined independently for four electrodes, with a mean value of 0.819 ± 0.265 μM. The highest LOD was 1.06 μM, still well below the cutoff concentration required for paracetamol intoxication. Most clinical laboratories use a cutoff of approximately 66.15 μM (10 mg / L). Considering the low recovery rates of plasma and whole blood, even after dilution in a 1:1 ratio with PBS, these results demonstrate that the developed test strip can easily quantify paracetamol in blood at these levels.

[0051] The average relative standard deviations (RSDs) of the oxidation current across the entire linear range were 4.3, 7.0, and 10.0% for PBS, plasma, and whole blood, respectively. It should be noted that the plasma and whole blood samples used were from different individuals. It should be further noted that the whole blood measurements were performed at different times of day over three days. Due to the high variability of the plasma and whole blood measurements in Figure 5, a single measurement was performed using three electrodes in plasma spiked with 1 mM PA. These measurements yielded a relative standard deviation of 4.0% and a recovery rate of 75.7 ± 0.22%. This suggests that electrode passivation may occur during prolonged measurements in protein-containing solutions. Because the electrode is intended for single point-of-care measurements, a recovery study using whole blood samples spiked at three different concentrations was also performed with three electrodes at each concentration. The results of this recovery study are shown in Table 1. The recovery rate was approximately 74%.

[0052] [Table 1]

[0053] A protein-bound fraction of 20-25% has previously been reported for paracetamol. The unbound fraction was also found to be concentration-independent across clinically relevant concentration ranges. Similar results were obtained in a recent report, where recoveries of 60% and 40% were found for morphine and codeine, respectively, using a Nafion-coated SWCNT electrode. All of these recoveries are in close agreement with previously reported unbound fraction recoveries. Banis et al. also used a chitosan-based composite-coated electrode to conclude that only the free fraction of the benzodiazepine clozapine contributed to the electrochemical signal measured in a BSA-containing analyte solution. These results suggest that the unbound PA fraction can be directly measured using a polymer membrane-coated electrode without the need for time-consuming equilibrium dialysis.

[0054] As can be seen in Table 2, lower detection limits have been reported previously by several groups. Similarly, a relatively wide linear range has also been reported in previous studies. However, as evident from the treatment nomogram, extreme sensitivity is not required. Furthermore, all studies in Table 2 rely on time-consuming sample processing, such as protein precipitation or substantial dilution, to reduce matrix effects. It should also be noted that studies starting from serum or plasma also involve pretreatment of the blood sample. In contrast, the assay developed in this study can be used to measure PA concentrations from whole blood in less than 5 minutes, without protein precipitation, and simply by diluting with an equal volume of PBS. Therefore, the results of this study demonstrate a much simpler system with significantly reduced sample processing requirements and, therefore, shorter assay times.

[0055] These results demonstrate the suitability of the developed sensor strip and proposed assay for screening of PA poisoning, but further studies are needed to demonstrate its suitability for actual patient samples. Pharmacokinetic parameters need to be evaluated from both intravenous and capillary finger-prick blood samples. Further development could achieve higher sensitivity or further miniaturization of the electrode, further reducing the required sample volume.

[0056] [Table 2A]

[0057] [Table 2B]

[0058] [Table 2C]

[0059] To confirm that the reduced recovery in plasma and whole blood was not due to protein contamination, electrode passivation was investigated. First, 1 mM Ru(NH3)6 was measured in both PBS and human plasma. Figure 6 shows that the electrode was not significantly passivated when 1 mM Ru(NH3)6 was measured in PBS and diluted human plasma. This result is consistent with a similar passivation study performed using a Nafion-coated SWCNT electrode in a previous report. Passivation was further investigated at higher PA concentrations by performing 10 consecutive DPV scans in plasma and whole blood containing 1 mM PA. These measurements yielded RSDs of 3.6% in whole blood and 4.3% in plasma. These RSDs are comparable to the single-measurement reproducibility and reproducibility for PA in PBS shown in Table 1. Furthermore, the electrode used for measuring 50 μM PA in whole blood (see Table 1) was also used to measure 50 μM PA in PBS. After wiping off the whole blood, washing with a 40 μL droplet of PBS, and ensuring that the background returned to that of empty PBS, an average peak current of 1.83 ± 0.09 μM was obtained with 50 μM PA, representing a recovery rate of 101.7%, indicating no permanent contamination after whole blood measurements.

[0060] The absence of matrix effects in the background current in Figure 4 indicates that endogenous substances present in the plasma and whole blood samples do not cause significant interference. Nevertheless, several other drugs may cause interference in paracetamol measurements. For this reason, several drugs frequently ingested in co-administration of paracetamol overdoses were tested. Drugs were tested at concentrations much higher than expected in blood samples. If a test substance was found to cause interference, the tolerance limit was defined as the maximum concentration of the interfering substance that caused less than a 5% error in the PA measurement. Figure 7 shows DPV scans in the absence and presence of an NSAID mixture containing 100 μM ibuprofen, naproxen, and aspirin, 1 mM salicylic acid (a metabolite of aspirin), 1 mM nicotine, 1 mM amoxicillin, and 1 mM caffeine. PA is often co-administered with opioids such as tramadol and morphine. Opioids are also one of the most frequently ingested drug groups in co-administration of paracetamol overdoses. Additionally, Nafion has been shown to accumulate cations, including interfering opioids, such as morphine and the active metabolites of codeine and heroin, two common opioids. The active metabolite of tramadol, o-desmethyltramadol (ODMT), has also been studied. Both morphine and ODMT are cationic under physiological conditions and contain phenolic functional groups. Morphine, in particular, has been shown to oxidize at approximately the same potential as PA. Therefore, interference from these two opioids was also tested.

[0061] Figure 7 reveals that the NSAID mixture, 1 mM salicylic acid, 1 mM amoxicillin, 1 mM nicotine, and 1 mM caffeine, did not cause more than 5% interference with the 50 μM PA signal. Much lower tolerance limits were obtained for 2.5 μM morphine and 10 μM o-desmethyltramadol. Despite the relatively low tolerance limits, these concentrations represent high concentrations compared to therapeutic concentrations. Even in fatal cases of morphine and tramadol intoxication, the concentrations were below the test concentrations, approximately 1.75 μM and 3.8 μM, respectively.

[0062] As can be seen in Table 1, the reproducibility of the assay was evaluated by measuring three electrodes at three different concentrations in the physiologically relevant concentration range. Relative standard deviations of 7.4%, 5.5%, and 1.9% were obtained at concentrations of 50 μM, 100 μM, and 500 μM, respectively. It should be noted that these results were obtained by drawing 20 μL of whole blood via finger prick, diluting it with PBS solution spiked with PA, and transferring the sample onto a test strip with a micropipette. Therefore, the RSD values ​​represent the cumulative error from all these steps.

[0063] The shelf life of the electrode was also tested after 4 months of storage under ambient conditions. The same sensitivity was achieved, indicating excellent stability. Similarly, a similar behavior of the Ag reference electrode was observed after 1.5 years of storage.

[0064] It should be understood that the disclosed embodiments of the invention are not limited to the particular structures, manufacturing processes, or materials disclosed herein, but extend to equivalents thereof as will be recognized by those skilled in the art. It should also be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0065] Reference throughout this specification to an embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with that embodiment is contained in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. For example, when a numerical value is referred to using terms such as "about" or "substantially," the exact numerical value is also disclosed.

[0066] As used herein, for convenience, a plurality of items, structural elements, components, and / or materials may be presented in common lists. However, these lists should be construed as though each member of the list were individually identified as a separate and unique member. Accordingly, individual members of such lists should not be construed as de facto equivalents to other members of the same list solely based on description in a common group, unless indicated to the contrary. In addition, various embodiments and examples of the present invention may be referenced herein, along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives should not be construed as de facto equivalents to one another, but should be considered separate and autonomous representations of the present invention.

[0067] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details, such as length, width, shape, etc., are presented to provide a thorough understanding of embodiments of the invention. However, one skilled in the art will recognize that the invention can be practiced without one or more of the specific details, or using other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0068] While the foregoing examples illustrate the principles of the present invention in one or more specific applications, it will be apparent to those skilled in the art that, without the faculty of invention, numerous changes in form, use, and details of implementation can be made without departing from the principles and concepts of the invention. Accordingly, it is not intended that the present invention be limited except as by the scope of the following claims.

[0069] The verbs "comprise" and "contain" are used in this document as open limitations that do not exclude or require the presence of unrecited features. Features recited in dependent claims may be freely combined with one another, unless expressly stated otherwise. Furthermore, it is to be understood that the use of "a" or "an", i.e. the use of the singular, does not exclude the plural throughout this text. [Industrial Applicability]

[0070] At least some embodiments of the present invention find industrial application in the medical profession. A fabrication method suitable for mass production of disposable electrochemical test strips for use in the quantitative point-of-care measurement of neutral analytes, such as paracetamol, in cases of suspected overdose is described. The method results in highly conductive, electrically insulating, patterned carbon-based electrodes printed onto a substrate. Furthermore, a screen-printed silver pseudo-reference electrode is produced with excellent shelf life, long-term stability, and short hydration times. The test strips achieve a sufficiently low detection limit and a sufficiently wide linear range to determine the concentration of neutral analytes suspected of poisoning. The test strips are particularly useful for detecting and measuring paracetamol concentrations in cases of suspected paracetamol overdose and / or poisoning.

[0071] The developed test strip is very portable and can be used for rapid point-of-care assays for screening of paracetamol poisoning.

[0072] Acronym List PA Paracetamol UA uric acid AA Ascorbic Acid MO Morphine CO Codeine PBS Phosphate-buffered saline DPV Differential Pulse Voltammetry PET polyethylene terephthalate

Claims

1. a carbon-based working electrode; a carbon-based counter electrode; Pseudo reference electrode and 1. A disposable test strip comprising a substrate having deposited thereon an electrode assembly comprising: the pseudo reference electrode, the carbon-based working electrode, and the carbon-based counter electrode are disposed adjacent to one another in the same plane; the test strip includes contacts for directly contacting the carbon-based working electrode with a voltage source, contacts for directly contacting the carbon-based counter electrode with the voltage source, and contacts for directly contacting the pseudo-reference electrode with the voltage source; the test strip further comprises a permselective membrane layer; the carbon-based working electrode, the carbon-based counter electrode, and the pseudo-reference electrode of the electrode assembly are electrically isolated from one another, and the electrode assembly is disposed between the substrate and the permselective membrane layer; the permselective membrane layer has a structure adapted to allow one or more electrically neutral analytes in a sample to be analyzed to pass through the permselective membrane layer to the electrode assembly, and the permselective membrane layer has a thickness in the range of 50 to 400 nm; strip.

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

3. The strip of claim 2 wherein the substrate is a polymer.

4. 4. The strip of claim 2 or 3, wherein the polymer is polycarbonate or PET.

5. 5. The strip of claim 1, wherein one or both of the carbon-based working electrode and the carbon-based counter electrode comprises carbon selected from the group consisting of amorphous carbon, diamond-like carbon, graphite, graphene, carbon nanotubes, and mixtures thereof.

6. 6. The strip of claim 5, wherein the amorphous carbon is tetrahedral amorphous carbon.

7. The strip of claim 1 , wherein the pseudo reference electrode comprises silver.

8. 8. The strip of claim 1, wherein the pseudo reference electrode is made of silver.

9. 9. The strip of claim 1, wherein the permselective membrane layer comprises a membrane material selected from the group of polymers consisting of sulfonated polymers including Nafion and polyvinyl sulfonate, cellulose acetate, carboxymethyl cellulose, polylysine, and peroxidized polypyrrole.

10. 10. The strip of claim 9, wherein the permselective membrane layer comprises Nafion.

11. 11. The strip of claim 1, wherein the structure of the permselective membrane layer is formed from one or more layers of membrane material applied to the strip, whereby a stack of membrane material layers forms the permselective membrane layer.

12. The strip according to any one of claims 1 to 11, wherein the permselective membrane layer has a thickness in the range of 75 to 250 nm.

13. The strip according to any one of claims 1 to 12, wherein the permselective membrane layer has a thickness in the range of 100 to 200 nm.

14. a memory configured to store reference data; at least one processing core; An apparatus comprising: the processing core: Processing information from a strip according to any one of claims 1 to 13. Comparing the information from the strip according to any one of claims 1 to 13 with the reference data; and Based on the processed information from the strip according to any one of claims 1 to 13, a conclusion is drawn as to whether the concentration of said neutral analyte is within an acceptable range according to said reference data. It is configured as follows: Device.

15. 1. A method for detecting an electrically neutral analyte in a sample, comprising: Preparing a sample; bringing the sample into electrical contact with the carbon-based working electrode and the carbon-based counter electrode of the electrode assembly of the test strip; varying the voltage between the carbon-based working electrode and the carbon-based counter electrode; measuring a current between the carbon-based working electrode and the carbon-based counter electrode in relation to a voltage applied between the carbon-based working electrode and the carbon-based counter electrode; detecting a change in the current characteristics of one or more analytes in the sample; Including, The method wherein the test strip further comprises a permselective membrane layer.

16. 1. A method for detecting an electrically neutral analyte in a sample, comprising: Preparing a sample; bringing a sample into electrical contact with the carbon-based working electrode and the carbon-based counter electrode of the electrode assembly of the strip of any one of claims 1 to 13; varying the voltage between the carbon-based working electrode and the carbon-based counter electrode; measuring a current between the carbon-based working electrode and the carbon-based counter electrode in relation to a voltage applied between the carbon-based working electrode and the carbon-based counter electrode; detecting a change in the current characteristics of one or more analytes in the sample; A method comprising:

17. 17. The method of claim 15 or 16, wherein the voltage between the carbon-based working electrode and the carbon-based counter electrode is scanned from −0.2 V to 0.8 V at a scan rate.

18. 18. The method of claim 17, wherein the voltage between the carbon-based working electrode and the carbon-based counter electrode is scanned from 0.1 V to 0.6 V at a scan rate.

19. 19. The method of any one of claims 15 to 18, wherein the scan rate is in the range of 5 to 1000 mV / sec.

20. 1. A method of manufacturing a test strip, comprising: providing a SWCNT network; pressing the SWCNT network against a substrate to form a carbon-based electrode; separating the carbon-based electrodes by laser patterning; screen printing silver to form a silver pseudo-reference electrode adjacent to a carbon-based working electrode and a carbon-based counter electrode; screen printing a silver contact pad on each electrode; coating the carbon-based working electrode, the carbon-based counter electrode, and the silver pseudo-reference electrode with a permselective membrane layer; Including, the coating step is adapted to coat the carbon-based working electrode, the carbon-based counter electrode, and the silver pseudo-reference electrode with a permselective membrane layer of a predetermined thickness. method.

21. 21. A method according to claim 20 for producing a strip according to any one of claims 1 to 13.

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