JP7819908B2 - Electrochemical sensor for drugs in urine using diamond electrodes and analytical method for the detected substances - Google Patents

Description

This disclosure relates to an analytical method for detecting drugs in urine using a conductive diamond electrode as an electrochemical sensor.

There are several needs for testing drug concentrations in urine. For example, doping among athletes can become a problem at international competitions, and a method for detecting it is needed. Also, in investigations, a means of confirming whether or not illegal drugs have been taken is needed. Other cases include the need to identify the drugs administered to unconscious emergency patients who may be suffering from drug poisoning. Furthermore, there may be cases where it is necessary to confirm whether or not drugs have been taken to confirm whether antibiotics have been forgotten or are still being administered.

Traditional drug testing methods use separation and analysis techniques such as HPLC, LC-MS, and GC-MS. However, these methods require large measuring equipment, which makes testing expensive and time-consuming. Furthermore, they have the disadvantage of not being able to be easily performed anywhere.

Other conventional drug testing methods that have been developed include electrochemical measurement methods using glassy carbon electrodes and mercury electrodes. However, these methods require pretreatment, such as diluting the urine sample with a buffer solution, and mercury electrodes have a significant environmental impact.

Non-Patent Document 1 reports the use of conductive diamond electrodes to measure bumetanide in the cochlea of guinea pigs. Conductive diamond electrodes have also been used to measure lamotrigine in rat brains. There is also a description of the measurement of doxorubicin.

Non-patent document 2 reports measuring methylcobalamin in the cochlea and muscle of guinea pigs using conductive diamond electrodes.

Non-patent document 3 reports the detection of acetaminophen using a nanometer-sized graphite/boron-doped diamond electrode. The acetaminophen measured is pure (page 6332, left column, 4.1 Materials).

Non-Patent Document 4 reports the electrochemical measurement of triamterene in urine samples diluted 5-fold with BRP using a graphite-epoxy composite electrode modified with magnetic particles. This method uses optimized square-wave voltammetry.

Non-patent document 5 describes the measurement of losartan and triamterene using a mercury dropping electrode as the working electrode.

Non-patent documents 6 to 9 describe the measurement of triamterene using a glassy carbon electrode modified with special carbon nanotubes.

There is a need for a simple, rapid, and inexpensive method for screening for drugs in urine.

Ogata et al., Nat Biomed Eng. 2017 Aug;1(8):654-666 Hanawa et al., Anal. Chem. 2020, 92, 20, 13742-13749 Wang et al., ACS Omega 2021, 6, 9, 6326-6334 Hudari, F. et al. J Solid State Electrochem. 2016, 20, 2491-2501 Ensafi, A. et al. Analytical Sciences, 2008, 24, 1449-1454 Nisha and Kumar, Journal of Electroanalytical Chemistry, 2019, 839, 214-223 Karimi, Journal of Electroanalytical Chemistry, 2019, 847, 113176 Nezhadali and Mojarrab, Journal of Electroanalytical Chemistry, 2015, 744, 85-94 Hudari et al. Talanta, 2018, 179, 652-657

The objective of this disclosure is to provide a method for detecting drugs in urine and a sensor therefor that at least partially solves the problems of the prior art.

The inventors conducted extensive research to solve the above-mentioned problems, and when they attempted to detect drugs in urine using a boron-doped conductive diamond electrode, they surprisingly found that drugs in urine could be detected simply and quickly by directly using the urine sample in an electrochemical measurement method without pretreatment, and completed the present invention, which incorporates this as one embodiment. Therefore, the present disclosure provides a method for simply and quickly detecting drugs in urine by an electrochemical measurement method using a boron-doped conductive diamond electrode, as well as a sensor and the like used therefor.

The present disclosure includes the following embodiments:
[1] An electrochemical measurement method for detecting drugs in urine using a conductive diamond electrode, comprising:
The urine is a real urine sample that has not been pretreated,
i) applying a potential to the untreated real urine sample using a conductive diamond electrode; and
ii) measuring the oxidation current and/or reduction current of the drug in the silent region of urine;
The method comprising:
[2] The method according to embodiment 1, wherein the electrochemical measurement is performed in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl) as the silent region of urine.
[3] i) applying a potential to an unpretreated real urine sample using a conductive diamond electrode,
applying a first potential that reduces the drug in the urine of interest; and
applying a second potential to oxidize the reduced drug in the urine;
ii) the step of measuring the oxidation current and/or reduction current of the drug in the silent region of urine comprises measuring the oxidation current of the drug in the silent region of urine when the second potential is applied.
[4] i) applying a potential to an untreated real urine sample using a conductive diamond electrode,
applying a first potential that oxidizes the drug in the urine of interest; and then
applying a second potential that reduces the reduced drug in the urine;
ii) measuring the oxidation current and/or reduction current of the drug in the silent region of urine;
3. The method of embodiment 1 or 2, comprising measuring the reduction current of the drug in a silent region of urine when the second potential is applied.
[5] The method of any one of embodiments 1 to 4, wherein the drug in the urine is selected from the group consisting of triamterene, gentamicin, dihydrostreptomycin, vancomycin, cephalexin, ampicillin, ceftriaxone, amoxicillin, teicoplanin, rifampicin, theophylline, furosemide, bumetanide, quinine hydrochloride, lamotrigine, carbamazepine, topiramate, doxorubicin, pazopanib, lenvatinib, imatinib, nortriptyline, acetaminophen, ascorbic acid, methylcobalamin, and cyanocobalamin.
[6] The drug in the urine is triamterene,
i) applying a first potential at −1.5 V to reduce triamterene, and then applying a second potential at +0.2 V to oxidize the reduced triamterene;
ii) the step of measuring the oxidation and/or reduction current of the drug in the silent region of urine comprises measuring the oxidation current when a second potential is applied at +0.2 V.
[7] A conductive diamond electrode, a counter electrode, and a reference electrode, and
A storage medium storing software or a program that implements a protocol or algorithm for executing the electrochemical measurement method according to any one of embodiments 1 to 6;
An electrochemical measurement device for detecting drugs in urine, comprising:
[8] A screening method for determining whether a drug in urine can be detected without pretreatment by electrochemical measurement using a conductive diamond electrode, comprising:
(i) providing a real urine sample containing a drug candidate that has not been pretreated;
(ii) obtaining a cyclic voltammogram of the unpretreated real urine sample using a conductive diamond electrode;
(iii) preparing a cyclic voltammogram obtained using a conductive diamond electrode on a real urine sample from a healthy subject that is not pretreated and does not contain a candidate drug;
(iv) comparing the cyclic voltammogram of (ii) with the cyclic voltammogram of (iii) to determine whether or not a discernible oxidation current and/or reduction current of the candidate drug is observed in the silent region of urine in the cyclic voltammogram of (ii) compared with the cyclic voltammogram of (iii);
(v) in the step (iv), when an identifiable oxidation current and/or reduction current of the candidate drug in the cyclic voltammogram of (ii) is observed in the silent region of urine in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl), determining the potential at which the oxidation current and/or reduction current is observed as the potential at which electrochemical measurement is performed;
The method comprising:
[9] A method for detecting drugs in urine by electrochemical measurement without pretreatment using a conductive diamond electrode, comprising: performing a screening method for determining whether drugs in urine can be detected by electrochemical measurement using a conductive diamond electrode without pretreatment; and performing electrochemical measurement at the potential determined by the screening method,
The screening method comprises:
(i) providing a real urine sample containing a drug candidate that has not been pretreated;
(ii) obtaining a cyclic voltammogram of the unpretreated real urine sample using a conductive diamond electrode;
(iii) preparing a cyclic voltammogram obtained using a conductive diamond electrode on a real urine sample from a healthy subject that is not pretreated and does not contain a candidate drug;
(iv) comparing the cyclic voltammogram of (ii) with the cyclic voltammogram of (iii) to determine whether or not a discernible oxidation current and/or reduction current of the candidate drug is observed in the silent region of urine in the cyclic voltammogram of (ii) compared with the cyclic voltammogram of (iii);
(v) in the step (iv), when an identifiable oxidation current and/or reduction current of the candidate drug in the cyclic voltammogram of (ii) is observed in the silent region of urine in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl), determining the potential at which the oxidation current and/or reduction current is observed as the potential at which electrochemical measurement is performed;
Including,
The method for detecting drugs in urine by electrochemical measurement using the conductive diamond electrode without pretreatment comprises the steps of:
1) applying a potential to the unpretreated real urine sample using a conductive diamond electrode, wherein the potential applied to the unpretreated real urine sample is the potential at which electrochemical measurements are performed determined in step (v) of the screening method; and
2) measuring the oxidation current and/or reduction current of the drug in the silent region of urine in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl);
The method comprising:

According to the present disclosure, by using a conductive diamond electrode, drugs in urine can be directly measured by electrochemical measurement without pretreatment.

FIG. 1 is a schematic diagram of a three-electrode cell used for electrochemical measurements. Voltammograms of 0 and 50 μM bumetanide in PB are shown. Starting potential: 0 V, scan range: -1.0 to 1.5 V, scan rate: 0.1 V s ⁻¹ , positive scan. Voltammograms of 0 and 100 μM furosemide in PB are shown. Starting potential: 0 V, scan range: -1.0 to 1.5 V, scan rate: 0.1 V s ⁻¹ , negative scan. Voltammograms of 0 and 20 μM triamterene in PB are shown. Starting potential: 0 V, scan range: -1.0 to 1.5 V, scan rate: 0.1 V s ⁻¹ , negative scan. Voltammograms of 0 and 100 μM triamterene in PB are shown. Starting potential: 0 V, scan range: -1.5 to 0.5 V, scan rate: 0.1 V s ⁻¹ , negative scan. Voltammograms of 0 and 200 μM triamterene in PB are shown. Starting potential: 0 V, scan range: -1.0 to 0.5 V, scan rate: 0.1 V s ⁻¹ , negative scan. Voltammograms of 10 μM triamterene in PB, showing the scan rate dependence. Starting potential: 0 V, scan range: -1.0 to 0.5 V, scan rates: 0.01 (a), 0.02 (b), 0.04 (c), 0.06 (d), and 0.1 (e) V s ^-1 , scanned in the negative direction. Calibration curve of peak oxidation current vs. square root of scan rate (n = 1). The potential application conditions for CA measurement in PB are as follows: -1.5 V was applied for -2 mC using the CA method, followed by +0.1 V for 10 seconds. The graph shows the change in current over time (concentration dependence) when an oxidation potential of +0.1 V for the reduced product of triamterene (0 (a), 10 (b), 25 (c), 50 (d), 75 (e), 100 (f) nM) in PB is applied using the CA method. A calibration curve (n = 3) of the current versus the square root of the triamterene concentration in PB 4.5 seconds after application of an oxidation potential of +0.1 V is shown. Voltammograms of triamterene in urine samples (0 (a), 10 (b), 25 (c), 50 (d), 75 (e), and 100 (f) nM) are shown. Starting potential: 0 V, scan range: -1.5 to 0.5 V, scan rate: 0.1 V s ^-1 , negative scan. Voltammograms of the PB solution and the urine sample are shown. Starting potential: 0 V, scan range: -1.5 to 0.5 V, scan rate: 0.1 V s ^-1 , scanned in the negative direction. Voltammograms of 10 μM triamterene in PB and urine samples are shown. Starting potential: 0 V, scan range: -1.5 to 0.5 V, scan rate: 0.1 V s ⁻¹ , negative scan. The urinary voltammogram obtained using a GC electrode is shown below. The starting potential was 0 V, the scan range was -1.5 to 0.5 V, and the scan rate was 0.1 V s ^-1 , with the scan in the negative direction. The voltammogram in urine using a Pt electrode is shown below. The starting potential was 0 V, the scan range was -1.5 to 0.5 V, and the scan rate was 0.1 V s ^-1 , with the scan in the negative direction. Voltammograms of 0 and 10 μM triamterene in urine using a GC electrode are shown. Starting potential: 0 V, scan range: -1.5 to 0.5 V, scan rate: 0.1 V s ^-1 , negative scan. Voltammograms of 0 and 10 μM triamterene in urine using a Pt electrode are shown. Starting potential: 0 V, scan range: -1.5 to 0.5 V, scan rate: 0.1 V s ^-1 , negative scan. The potential application conditions for measuring urinary CA are as follows: -1.5 V was applied for -2 mC using the CA method, followed by +0.2 V for 20 seconds. The graph shows the change in current over time (concentration dependence) when an oxidation potential of +0.2 V for the reduced product of urinary triamterene (0 (a), 10 (b), 25 (c), 50 (d), 75 (e), 100 (f) nM) is applied using the CA method. A calibration curve of the current vs. the square root of the urinary triamterene concentration after 2.5 seconds of application of an oxidation potential of +0.2 V is shown (n = 3).

In one embodiment, the present disclosure provides an electrochemical measurement method for detecting drugs in urine using a conductive diamond electrode. In this method, the urine is a real urine sample that has not been pretreated. This method may include the steps of: i) applying a potential to the real urine sample that has not been pretreated using a conductive diamond electrode; and ii) measuring the oxidation current and/or reduction current of the drug in the silent region of urine. In one embodiment, electrochemical measurement can be performed in the potential range of -1.5 V to +0.5 V (vs. Ag/AgCl), which is the silent region of urine.

The inventors have discovered that for a specific drug, first applying a first potential to reduce the drug to a reduced form, and then applying a second potential to oxidize the reduced drug to an oxidized form, results in an increase in the current value when the second potential is applied depending on the concentration of the drug. Therefore, in one embodiment, the present disclosure provides an electrochemical measurement method in which: i) the step of applying a potential to an unpretreated actual urine sample using a conductive diamond electrode includes applying a first potential that reduces the target drug in the urine, and then applying a second potential that oxidizes the reduced drug in the urine; and ii) the step of measuring the oxidation current and/or reduction current of the drug in the silent zone of urine includes measuring the oxidation current of the drug when the second potential is applied in the silent zone of urine.

In another embodiment, the present disclosure provides an electrochemical measurement method, in which: i) the step of applying a potential to an unpretreated real urine sample using a conductive diamond electrode includes applying a first potential that oxidizes a target drug in the urine and then applying a second potential that reduces the reduced drug in the urine; and ii) the step of measuring the oxidation current and/or reduction current of the drug in the silent region of the urine includes measuring the reduction current of the drug when the second potential is applied in the silent region of the urine.

The disclosed method and device can detect a variety of drugs in urine. In certain embodiments, the drug in urine is selected from the group consisting of triamterene, gentamicin, dihydrostreptomycin, vancomycin, cephalexin, ampicillin, ceftriaxone, amoxicillin, teicoplanin, rifampicin, theophylline, furosemide, bumetanide, quinine hydrochloride, lamotrigine, carbamazepine, topiramate, doxorubicin, pazopanib, lenvatinib, imatinib, nortriptyline, acetaminophen, ascorbic acid, methylcobalamin, and cyanocobalamin. Equivalents of these drugs can also be measured.

In certain embodiments, triamterene can be detected by the methods of the present disclosure, comprising the steps of: i) applying a first potential at −1.5 V to reduce triamterene, and then applying a second potential at +0.2 V to oxidize the reduced triamterene;
ii) The step of measuring the oxidation current and/or reduction current of the drug in the silent region of urine comprises measuring the oxidation current when a second potential of +0.2 V is applied.

When a real urine sample is used without pretreatment and a potential is applied to it using a conductive diamond electrode, a region appears where no or almost no reduction or oxidation current is detected, or where only a weak response current that does not interfere with the detection of the target compound is detected. The region where almost no reduction or oxidation current is detected, or where only a weak response current that does not interfere with the detection of the target compound is detected when a potential is applied to an unpretreated real urine sample using a conductive diamond electrode, is referred to herein as the "silent region." In one embodiment, the silent region is a potential range of -1.5 V to +0.5 V (vs. Ag/AgCl). The real urine sample here is an unpretreated urine sample from a typical healthy individual who is not taking drugs.

In certain embodiments, electrochemical measurements of drugs in urine can be performed in a silent region within a potential range of -1.5 V to +0.45 V (vs. Ag/AgCl). In certain embodiments, electrochemical measurements of drugs in urine can be performed in a silent region within a potential range of -1.5 V to +0.40 V (vs. Ag/AgCl). In certain embodiments, electrochemical measurements of drugs in urine can be performed in a silent region within a potential range of -1.5 V to +0.35 V (vs. Ag/AgCl).

Conductive diamond electrodes are defined as thin-film or bulk diamond electrodes imparted with electrical conductivity. While there are no particular limitations on the method for imparting electrical conductivity to diamond, diamond can be doped with trace amounts of impurities. Impurities include boron (B), sulfur (S), nitrogen (N), oxygen (O), and silicon (Si). For example, thin-film or bulk diamond can be obtained by vapor-phase synthesis. To dope boron, diborane, trimethylborane, or boron oxide can be added to a carbon source-containing source gas. Sulfur oxide or hydrogen sulfide can be added to dope sulfur, oxygen or carbon dioxide can be added to dope oxygen, ammonia or nitrogen can be added to dope nitrogen, and silane can be added to dope silicon. Boron-doped conductive diamond electrodes are particularly advantageous because of their wide potential window and low background current compared to other electrode materials. Hereinafter, conductive diamond electrodes will sometimes be referred to simply as diamond electrodes, and boron-doped diamond electrodes will sometimes be referred to as boron-doped diamond electrodes or BDD electrodes.

In one embodiment, the conductive diamond electrode may be in the form of a thin film formed on a substrate. Because the substrate often becomes very hot when forming a conductive diamond film on it, the substrate can be made of a semiconductor such as silicon or a high-melting-point metal such as tungsten, niobium, molybdenum, or titanium. A known method can be used to form a thin film of the conductive diamond electrode on the substrate, such as chemical vapor deposition (CVD), as described in the examples below.

In some embodiments, the counter electrode can be a metal electrode such as platinum or nickel, a common carbon electrode such as glassy carbon or graphite, or a conductive diamond electrode.

In some embodiments, electrochemical measurements may include a reference electrode, which allows for precise control of the potential. Examples of reference electrodes include well-known electrodes such as a standard hydrogen electrode, a silver-silver chloride electrode, and a saturated calomel electrode. Unless otherwise specified, electrode potentials in this specification refer to potentials relative to a silver-silver chloride electrode (vs. Ag/AgCl).

In certain embodiments, a voltage can be applied between the working electrode and counter electrode using an external power supply mechanism. The external power supply mechanism is not particularly limited, but a potentio-galvanostat can be used. A potentio-galvanostat controls the electrochemical reaction of the working electrode in solution and measures the resulting potential and current. To achieve a specific range of counter electrode potential and current density relative to the reference electrode using a potentio-galvanostat, the potential can be controlled to obtain the desired current density using constant-potential electrolysis, or the current value can be controlled to obtain the desired current value using constant-current electrolysis. A potentiostat or galvanostat can be used as the external power supply mechanism.

In certain embodiments, the potential applied to the working electrode can be, for example, -1.5V or higher, -1.4V or higher, -1.3V or higher, -1.2V or higher, -1.1V or higher, -1.0V or higher, -0.9V or higher, -0.8V or higher, -0.7V or higher, -0.6V or higher, -0.5V or higher, -0.4V or higher, -0.3V or higher, -0.2V or higher, -0.1V or higher, 0V, or, for example, +0.1V or lower, +0.2V or lower, +0.3V or lower, +0.4V or lower, +0.5V or lower, +0.6V or lower, +0.7V or lower, +0.8V or lower, +0.9V or lower, +1.0V or lower, +1.1V or lower, +1.2V or lower, +1.3V or lower, +1.4V or lower, for example, +1.5V or lower. 0V is the potential relative to the reference electrode.

In the present disclosure, the application of potential may be a constant potential method, in which the working electrode potential relative to the reference electrode is kept constant, a constant voltage method, in which the voltage between the working electrode and counter electrode is kept constant, or a constant current method, in which the current density at the working electrode is kept constant. In some embodiments, the current may be direct current.

When using the constant potential, constant voltage, or constant current method, the electrochemical measurement device can have a three-electrode configuration with a working electrode, a counter electrode, and a reference electrode.

In one embodiment, the current of the conductive diamond electrode during electrochemical measurement of drugs in urine can be, but is not limited to, -500 μA to +500 μA, -400 μA to +400 μA, -300 μA to +300 μA, -200 μA to +200 μA, -100 μA to +300 μA, -100 μA to +200 μA, for example, -100 μA to +100 μA.

In one embodiment, the current density of the conductive diamond electrode during electrochemical measurement of drugs in urine can be -0.5mA/ ^cm2 to -5mA/ ^cm2 , -0.6mA/ ^cm2 to -4.5mA/ ^cm2 , -0.7mA/ ^cm2 to -4.0mA/ ^cm2 , -0.8mA/ ^cm2 to -3.5mA/ ^cm2 , -0.9mA/ ^cm2 to -3.0mA/ ^cm2 , -1.0mA/ ^cm2 to -2.5mA/ ^cm2 , -1.5mA/ ^cm2 to -2.0mA/ ^cm2 , for example, -0.5mA/ ^cm2 , -1.0mA/ ^cm2 , -2.0mA/ ^cm2 , -3mA/ ^cm2 , -4mA/ ^cm2 , or -5mA/ ^cm2 .

In one embodiment, the BDD electrode has a diamond layer formed by vapor-depositing diamond containing 0.01 to 8% w/w boron raw material on the substrate surface. The substrate can be a silicon substrate, a glass substrate such as _SiO2 , _a quartz substrate, a ceramic substrate such as _Al2O3 , silicon carbide, or silicon nitride, or a metal such as tungsten, molybdenum, niobium, or titanium. All or part of the substrate surface can be a diamond layer. In another embodiment, the electrode portion of the BDD electrode can have bulk diamond.

The size of the electrode part of the conductive diamond electrode is not particularly limited, but can be 0.1 cm ² or more, 0.2 cm ² or more, 0.3 cm ² or more, 0.4 cm ² or more, 0.5 cm ² or more, 1 cm ² or more, 5 cm ² or more, 10 cm ² or more, or 50 cm ² or more. All or part of the diamond layer can be contacted with a solution containing carbon dioxide and used in the formic acid production reaction. The area and shape of the electrode part can be appropriately determined depending on the configuration of the device.

In one embodiment, the BDD electrode has a diamond layer formed by depositing diamond with a high boron content (0.01 to 8% w/w boron content) on the surface of a Si substrate. The boron content is, for example, 0.01 to 5% w/w, 0.02 to 4% w/w, 0.03 to 3% w/w, 0.04 to 2% w/w, or 0.05 to 1% w/w, e.g., approximately 0.1 to 1.0% w/w.

The deposition process of the boron-mixed diamond onto the substrate can be carried out, for example, at 700-900°C for 2-12 hours. Conductive diamond thin films can be produced by chemical vapor deposition (CVD), such as microwave plasma chemical vapor deposition (MPCVD). A substrate, such as a silicon single crystal (100), is placed in a deposition apparatus, and a deposition gas containing high-purity hydrogen gas as a carrier gas is introduced. The deposition gas contains gaseous components including carbon and boron. When microwaves are applied to the deposition apparatus through which the deposition gas is flowing, a plasma discharge occurs. Carbon radicals are generated from the carbon source in the deposition gas, and the carbon radicals are deposited on the silicon single crystal while maintaining the ^sp3 structure and incorporating boron, forming a diamond thin film. Unless otherwise specified, the conductive diamonds disclosed herein, particularly boron-doped conductive diamonds, have an ^sp3 structure. In certain embodiments, the conductive diamonds disclosed herein, particularly boron-doped conductive diamonds, do not have an ^sp2 structure.

The thickness of the diamond thin film can be controlled by adjusting the deposition time. The thickness of the diamond thin film can be, for example, 100 nm to 1 mm, 1 μm to 0.1 mm, 1 μm to 50 μm, or 2 μm to 20 μm.

The conditions for the deposition process of boron-doped diamond onto the substrate surface can be determined according to the substrate material. As an example, the plasma power can be 500 to 7000 W, e.g., 3 kW to 5 kW, and preferably 5 kW. If the plasma power is within this range, synthesis proceeds efficiently, resulting in the formation of a high-quality conductive diamond thin film with few by-products.

All known methods can be used to manufacture BDD electrodes, including CVD methods (including those using the hot filament method), as well as vacuum deposition, ion plating, ion implantation, and other methods.

In certain embodiments, the BDD electrode may be hydrogen-terminated or cathodically reduced. In certain embodiments, the BDD electrode may be oxygen-terminated or anodized. Specific methods for hydrogen termination include annealing (heating) or hydrogen plasma treatment of the conductive diamond electrode in a hydrogen atmosphere. Specific methods for cathodic reduction include, for example, applying a potential of -3 V in a 0.1 M sodium perchlorate solution for 5 to 10 minutes to continuously generate hydrogen. Specific methods for oxygen termination include annealing (heating) or oxygen plasma treatment of the conductive diamond electrode in an oxygen atmosphere (in air). Specific methods for anodization include, for example, applying a potential of +3 V in a 0.1 M sodium perchlorate solution for 5 to 10 minutes to continuously generate oxygen.

The above electrodes are disclosed in JP 2006-98281 A, JP 2007-139725 A, JP 2011-152324 A, JP 2015-172401 A, JP 2018-141220 A, etc., and can be prepared according to the descriptions in these publications.

The conductive diamond electrodes disclosed herein have high thermal conductivity, high hardness, are chemically inert, have a wide potential window, low background current, and excellent electrochemical stability.

An example of the device disclosed herein is shown in Figure 1. The electrochemical measurement device 1 includes a working electrode 2, a counter electrode 3, a reference electrode 4, and a control unit 6. A sample 5 is added to the device, and a potential is applied via the working electrode 2. In Figure 1, the working electrode 2 is a boron-doped conductive diamond electrode. The counter electrode 3 is a metal electrode, and platinum, silver, gold, carbon, stainless steel, iridium, palladium, osmium, rhodium, ruthenium, etc. may be used. In Figure 1, the counter electrode 3 is a platinum electrode. The reference electrode 4 may be a silver-silver chloride electrode. In certain embodiments, the control unit may include a means for maintaining a constant potential (a potentiostat). The potentiostat can control the potential to a constant value during electrochemical measurement. In another embodiment, the control unit may include a means for controlling the current to a constant value (a galvanostat, also known as an amperostat). The galvanostat or the like can control the current to a constant value during electrochemical measurement.

In certain embodiments, the device of the present disclosure may include instructions for use. The instructions may include instructions specifying the conditions for applying a potential to the conductive diamond electrode and performing electrochemical measurement as described herein. The device of the present disclosure may include a program or software implementing the program that controls the application of a potential to the conductive diamond electrode and the performance of electrochemical measurement under such conditions. That is, in certain embodiments, the present disclosure provides a control program for applying a potential to the conductive diamond electrode and performing electrochemical measurement, or software implementing the program. In certain embodiments, the present disclosure also provides an electrochemical measurement device equipped with a control program for applying a potential to the conductive diamond electrode and performing electrochemical measurement, or software implementing the program. In certain embodiments, the program or software may be stored on a recording medium. In certain embodiments, the program or software may be a cloud application. In other embodiments, the program or software may be stored on the cloud and control the device of the present disclosure using appropriate implementation means. In these embodiments, the program or software may be temporarily stored in the hardware, for example, memory, of the device of the present disclosure and control the device of the present disclosure. Therefore, these embodiments also apply to cases where the device of the present disclosure includes the program or software.

In one embodiment, the present disclosure provides a screening method for determining whether drugs in urine can be detected without pretreatment by electrochemical measurement using a conductive diamond electrode.
(i) providing a real urine sample containing a drug candidate that has not been pretreated;
(ii) obtaining a cyclic voltammogram of the unpretreated real urine sample using a conductive diamond electrode;
(iii) preparing a cyclic voltammogram obtained using a conductive diamond electrode on a real urine sample from a healthy subject that is not pretreated and does not contain a candidate drug;
(iv) comparing the cyclic voltammogram of (ii) with the cyclic voltammogram of (iii) to determine whether or not a discernible oxidation current and/or reduction current of the candidate drug is observed in the silent region of urine in the cyclic voltammogram of (ii) compared with the cyclic voltammogram of (iii);
(v) in the step (iv), when an identifiable oxidation current and/or reduction current of the candidate drug is observed in the silent region of urine in the cyclic voltammogram of (ii), determining the potential at which the oxidation current and/or reduction current is observed as the potential at which electrochemical measurement is performed;
Furthermore, electrochemical measurements can be performed in the silent region of urine in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl).

In one embodiment, the above screening method may involve preparing multiple untreated urine samples containing the candidate drug at different concentrations in step (i). Using these samples, a calibration curve may be created by acquiring cyclic voltammograms in step (ii) for the urine samples containing various concentrations of the candidate drug, applying the potential for electrochemical measurement determined in step (v), and measuring the response current at the applied potential. In one embodiment, the calibration curve may be, for example, a calibration curve plotting the square root of concentration against the response current, or a calibration curve plotting concentration against the response current, but is not limited to these. In a further embodiment, the detection limit of the electrochemical measurement method may be determined from the created calibration curve. If the detection limit meets the detection limit requirements required, for example, in drug doping tests or clinical tests, this method may be used as a drug testing method.

In certain embodiments, the screening method may include determining a first potential at which the candidate drug is reduced. The first potential at which the candidate drug is reduced may be determined by starting cyclic voltammetry at, for example, 0 V, scanning in the negative direction, varying the minimum potential of the cyclic voltammetry scan range, and confirming whether a response current peak corresponding to the reduction product of the candidate drug is observed when the reduction potential is applied. For example, the minimum potential of the CV scan range may be, but is not limited to, -0.5 V, -0.6 V, -0.7 V, -0.8 V, -0.9 V, -1.0 V, -1.1 V, -1.1 V, -1.2 V, -1.3 V, -1.4 V, or -1.5 V. The cyclic voltammograms at the minimum potential of each CV scan range may be compared to confirm whether a reduction product is produced. Furthermore, the CV may be scanned in the positive direction, and the magnitude of the oxidation current peak may be compared. If an oxidation current peak of the reduced product is observed, the lowest potential in the CV scan range can be used as the first potential for reducing the candidate drug. The first potential can then be applied to the candidate drug, and the reduced candidate drug can be subjected to steps (i) to (v) of the screening method. In this case, step (iv) can be performed to determine whether an oxidation current of an identifiable candidate drug is observed in the silent region of urine. Furthermore, if an oxidation current of an identifiable candidate drug is observed in the silent region of urine in step (v), the potential at which the oxidation current is observed can be used as the potential for electrochemical measurement (the second potential for oxidizing the candidate drug). However, the electrochemical measurement performed by applying the second potential is performed in the silent region of urine. The first potential for reducing the candidate drug does not necessarily have to be a potential within the silent region of urine.

In another embodiment, the screening method may include determining a first potential at which the candidate drug is oxidized. The first potential at which the candidate drug is oxidized may be determined by starting cyclic voltammetry at, for example, 0 V, scanning in a positive direction, varying the maximum potential of the cyclic voltammetry scan range, and confirming whether a response current peak corresponding to the oxidized form of the candidate drug is observed when the oxidizing potential is applied. For example, the maximum potential of the CV scan range may be +0.4 V, +0.5 V, +0.6 V, +0.7 V, +0.8 V, +0.9 V, +1.0 V, +1.1 V, +1.1 V, +1.2 V, +1.3 V, +1.4 V, or +1.5 V, but is not limited to this. The cyclic voltammograms at the maximum potentials of each CV scan range may be compared to confirm whether an oxidized form is formed. Furthermore, the CV may be scanned in a positive direction, and the magnitude of the reduction current peak may be compared. If a reduction current peak of the oxidant is observed, the highest potential in the CV scan range can be used as the first potential for oxidizing the candidate drug. The first potential can then be applied to the candidate drug, and the oxidized candidate drug can be subjected to steps (i) to (v) of the screening method. In this case, step (iv) can be performed to determine whether a reduction current of an identifiable candidate drug is observed in the silent region of urine. Furthermore, if a reduction current of an identifiable candidate drug is observed in the silent region of urine in step (v), the potential at which the reduction current is observed can be used as the potential for electrochemical measurement (the second potential for reducing the candidate drug). However, the electrochemical measurement performed by applying the second potential is performed in the silent region of urine. The first potential for oxidizing the candidate drug does not necessarily have to be a potential within the silent region of urine.

Drug doping has become more sophisticated in recent years, and it is expected that derivatives or analogs with structures similar to prohibited drugs will be used instead of the prohibited drugs themselves. It is also expected that derivatives and analogs that do not currently exist will be developed in the future. The above screening method can be applied not only to existing known drugs, but also to their equivalents. Equivalents may include drug derivatives and analogs that do not currently exist but will be developed in the future.

The electrochemical measurement method disclosed herein does not require pretreatment of the urine sample. In certain embodiments, the electrochemical measurement method disclosed herein can directly detect drugs from 1 mL or less of urine. In certain embodiments, the electrochemical measurement method disclosed herein can detect drugs in urine in a measurement time of approximately 2 minutes.

It is common technical knowledge that urine contains many types of contaminants, including ascorbic acid and amino acid residues derived from proteins. Furthermore, due to these endogenous contaminants, it has previously been thought that using urine samples directly in electrochemical assays was impossible or impractical. However, the method disclosed herein makes it possible to measure drugs in urine directly, without pre-treating the urine sample. This is quite surprising, given the presence of various contaminants with different redox potentials in urine. This disclosure makes it possible to realize inexpensive, simple, and rapid measurement of drugs in urine, and the method disclosed herein can be used, for example, in primary screening tests for doping.

The present invention will be explained in more detail below using examples. However, the technical scope of the present invention is not limited in any way by these examples.

[Example 1]
Experimental Reagents: Sodium dihydrogen phosphate and disodium hydrogen phosphate used in phosphate buffer (PB) were purchased from Wako. Triamterene was purchased from Sigma-Aldrich. No further purification of the reagents was performed. 0.1 M PB was adjusted to pH 6.4 and used as the electrolyte. Ultrapure water (electrical resistivity: 18.2 MΩ cm) purified using a DIRECT-Q 3 UV (Merck Millipore Corporation) was used to prepare the reagents. Pooled urine samples (pH 6.542) from 15 individuals were purchased from Lee BioSolutions. Immediately after thawing, the samples were vacuum-filtered through a 0.45 μm pore size PES (polyethersulfone) membrane (Thermo Fisher Scientific) and stored refrigerated.

BDD deposition
For the BDD planar electrode, a film was deposited on a Si<100> substrate by microwave plasma-assisted chemical vapor deposition (MPCVD) using a CVD system (AsTex). The substrate was pretreated by polishing with a diamond powder-containing polishing pad for 10 minutes, followed by ultrasonic cleaning with ethanol for 5 minutes. The ethanol was replaced with fresh ethanol and ultrasonic cleaning was continued for another 5 minutes. The microwave intensity during deposition was 5000 W, and the deposition time was 4 hours. BDD films were deposited with a boron/carbon ratio (B/C) of 1% using 50 mL of acetone and 4 mL of trimethylborane as carbon sources. The surface structure of the resulting BDD was characterized using a Raman spectrometer (Acton SP2500, CORNES Technologies). Raman spectra were measured at room temperature in air using a green laser beam with a wavelength of 532 nm. The peak at 1332 cm ^-1 in the Raman spectrum indicates ^sp3 carbon, and the peaks at 500 and 1200 cm ^{- 1} indicate boron, confirming that the diamond structure is doped with boron. Furthermore, the absence of a peak near 1500 cm-1 due to ^sp2 carbon confirmed the formation of a BDD film. The diamond crystal structure on the electrode surface was also confirmed using a scanning electron microscope (SEM). This confirmed that a polycrystalline film with a grain size of approximately 5 μm covered the substrate surface. Furthermore, cyclic voltammetry (CV) measurements of PBS were performed using the fabricated BDD, and the waveform of the voltammogram was evaluated.

Electrochemical measurements All measurements were performed at room temperature (approximately 25 °C). In the electrochemical experiments, a potentiostat (Autolab) was used for cyclic voltammetry (CV) and chronoamperometry (CA) measurements. Before use, the cell and BDD plate electrode were ultrasonically cleaned in ultrapure water for 10 minutes. Before starting the measurements, the electrode surface state was initialized by scanning CV in PBS from 0 to +3.0 V in the positive direction for 10 cycles at a scan rate of 0.5 V s ^-1 .

Electrochemical measurements for the basic electrochemical evaluation of the diuretics to be measured were performed using a three-electrode cell (Figure 1). An Ag/AgCl electrode was used as the reference electrode, a platinum (Pt) electrode as the counter electrode, and a BDD electrode as the working electrode. The working electrode was placed at the bottom of the cell using a silicon O-ring (JIS standard, P-7). The area of the working electrode was estimated to be 0.363 ^cm2 . The various diuretics used were dissolved in dimethyl sulfoxide (DMSO), and a dilution series was prepared from a highly concentrated stock solution.

Termination of the electrode surface When changing the termination of the electrode surface, a BDD electrode was also used as the counter electrode. The cell was designed so that the counter electrode area was equal to the working electrode area. Hydrogen termination of the BDD electrode surface was performed by applying +3.0 V at +1 μC and -3.5 V at -5 C in 0.1 M sulfuric acid. The BDD obtained in this way was called hydrogen-terminated BDD (hereinafter sometimes referred to as H-BDD).

Cyclic voltammetry measurements The electrochemical behavior of high-concentration diuretics was evaluated by CV measurements, starting from a potential of 0 V and scanning in the positive or negative direction at a scan rate of 0.1 V s ⁻¹ .

Potential Step Measurements: To measure low-concentration solutions of triamterene, potential step measurements using chronoamperometry (CA) were used. In basic experiments using PB, CA measurements were performed using two potential steps: a reduction potential (-1.5 V, -2 mC) followed by an oxidation potential (+0.1 V, 10 seconds). In urine samples, CA measurements were performed using two potential steps: a reduction potential (-1.5 V, -2 mC) followed by an oxidation potential (+0.2 V, 20 seconds). Using the above CA measurements, triamterene was reduced by applying a constant charge at the reduction potential, and then an oxidation potential was applied to measure the increase in current due to the oxidation reaction of the reduced product. A concentration calibration curve was created from the relationship between concentration and current obtained by CA.

Electrochemical Measurement of Diuretics CV Measurement Results for Diuretic Bumetanide The electrochemical behavior of bumetanide in the low potential range was evaluated by CV measurement. When the potential was scanned in the positive direction, the voltammogram shown in Figure 2 was obtained. In addition to an oxidation current peak near the oxidation potential of +1.0 V, current peaks were also observed near 0 and +0.2 V.

The electrochemical behavior of furosemide at low potentials was evaluated by CV measurements. Figure 3 shows the cyclic voltammogram of 100 μM furosemide in 0.1 M PB (pH 6.4) scanned in the negative direction at a scan rate of 0.1 V s ^-1 . A large oxidation current peak for furosemide was observed at an oxidation potential of +1.0 V, but a current larger than the background current was observed around -0.5 V.

The electrochemical behavior of triamterene at low potentials was evaluated by CV measurements. Figure 4 shows the cyclic voltammogram of 20 μM triamterene in 0.1 M PB (pH 6.4) scanned negatively at a scan rate of 0.1 V s ^-1 . In addition to a large current peak near -0.8 V, which is presumably due to oxygen reduction, a sharp reduction current peak appeared at -1.0 V. Furthermore, in addition to an oxidation current peak near +1.0 V, an oxidation current peak appeared at +0.1 V. These results suggest that triamterene is a diuretic whose electrochemical behavior can be observed without being affected by endogenous substances in urine.

Triamterene is a rapid-acting oral diuretic that exhibits diuretic effects within 1 hour of administration. Table 1 below shows the urinary and fecal excretion rates (ratio of dose to dose) of unchanged triamterene and metabolites in rats following subcutaneous administration of 2 mg kg ^{of 14C} -triamterene (Reference 1). Following a single oral dose of 100 mg of triamterene to six healthy adults, triamterene and its metabolites were excreted in urine, with 35-63% of the dose excreted within 24 hours. It has also been reported that after intravenous administration of 10 mg of triamterene, 4.4% of the dose was excreted without metabolism. Furthermore, following a single oral dose of 200 mg of triamterene to 15 healthy adults, excretion peaked within 2-3 hours, with 80% of the dose excreted within 8 hours (Reference 1). From these findings, it can be said that triamterene is a diuretic that is sufficiently excreted after administration, and that doping offenders can be detected by urine sensing.

The World Anti-Doping Agency (WADA) has stipulated that athletes with urinary triamterene concentrations ^above 20 ng mL (79 nM) are considered suspected of doping violations due to triamterene administration. Next, we used the BDD as an electrochemical sensor to detect low concentrations of triamterene in urine.

Electrochemical measurements of triamterene
The electrochemical behavior of triamterene reduction products was evaluated by CV measurements of triamterene. Figure 5 shows the cyclic voltammograms of 100 μM triamterene in 0.1 M PB (pH 6.4) using BDD. The potential range was from -1.5 V to +0.5 V, and the scan rate was 0.1 V s ^-1 in the negative direction from a starting potential of 0 V. In addition to a reduction current peak near -1.0 V, an oxidation current peak was observed at +0.1 V. Since no increase in current was observed in the oxidation potential range below +0.5 V in CV measurements at zero concentration, this oxidation current peak is presumed to be due to the oxidation of triamterene reduction products.

To investigate the reactivity of triamterene, measurements were performed by varying the minimum potential of the CV scan range (-0.5, -0.8, -1.0 V). All measurements were performed starting at 0 V and scanning in the negative direction. When the scan range was narrower toward the -0.5 or -0.8 V reduction potential region, no rise in oxidation current occurred. On the other hand, when scanning from -1.0 to +0.5 V, a significant increase in oxidation current due to the oxidation of triamterene reduction product was observed in the potential range of +0.1 to +0.2 V (Figure 6). This indicates that triamterene reduction product is produced when a reduction potential less noble than -1.0 V is applied. Figure 5 shows that the oxidation current peak when scanning up to -1.5 V is larger than the current peak when scanning from -1.0 to +0.5 V. This suggests that a more pronounced oxidation current peak of the reduction product would occur when a reduction potential less noble than -1.0 V is applied. In the following examples, the reduction reaction of triamterene was promoted by applying -1.5 V.

Estimation of the number of reaction electrons from the relationship between current and scan rate In a voltammogram showing a redox reaction in which electron transfer is sufficiently fast, the following relationship holds (at 25°C):
[In the formula, I is the peak current value (A), n is the number of reaction electrons, F is the Faraday constant (s A mol ^-1 ), A is the electrode area, R is the gas constant (JK ^-1 mol ^-1 ), v is the scan rate (V s ^-1 ), and D is the diffusion coefficient (cm ² s ^-1 ). c is the triamterene concentration (mol cm ^-3 ), and the absolute temperature T is 298 K.]

Measurements were performed at different CV scan rates (10, 20, 40, 60, and 100 mV s ^-1 ) (Figure 7). All measurements were performed starting at 0 V and scanning in the negative direction. The oxidation current and the square root of the scan rate showed a proportional relationship, as shown in the above formula (Figure 8). The electrode area A of the electrode used was 0.36 ^cm2 .

The number of reaction electrons is estimated from the slope of the calibration curve. Here, the diffusion coefficient D of triamterene is calculated using the Stokes-Einstein equation.
[where k _B is the Boltzmann constant (JK ⁻¹ ), η is the viscosity (Pa·s), and r is the radius of the spherical particle (m)]

Triamterene was plotted using Chem3D and the molecular radius r was estimated to be 6.3×10 ^-10 m. The literature value of 8.9×10 ^-4 Pa·s was used for the viscosity η of pure water. From these, the diffusion coefficient D of triamterene was calculated to be 3.89×10 ^-6 cm ² s ^-1 . The slope of the calibration curve was 0.237 μA s ^1/2 mV ^-1/2 . Substituting these values into the above equation, the number of reaction electrons n was calculated to be 2.48. The oxidation mechanism of the reduced triamterene is thought to be two-electron oxidation, and this value is nearly consistent with this mechanism.

Next, we investigated the difference in reactivity when the BDD termination was changed. In this example, CV measurements of 10 μM triamterene were performed using cathodic reduced BDD (H-BDD) and anodized BDD (sometimes referred to as O-BDD) to confirm the reactivity at each electrode termination state. Furthermore, the ratio of signal current to background current (S/B ratio) was calculated for each electrode surface state. The S/B ratio for H-BDD was 15.83, and the S/B ratio for O-BDD was 2.024. This indicates that triamterene can be measured with higher sensitivity when the surface is hydrogen-terminated. In the following examples, H-BDD was used to detect triamterene.

When using other electrodes
For comparison with the BDD electrode, CV measurements were performed using a glassy carbon (GC) electrode (Comparative Example 1) or a platinum (Pt) electrode (Comparative Example 2) as the working electrode in the negative potential range from 0 V to -1.5 to 0.5 V. The potential scan was performed in PBS in the positive potential range from 0 V to ^-1.0 to 1.0 V at a scan rate of 0.3 V s for 20 cycles, ensuring that the electrode surface condition remained constant for each measurement.

As a result, CV measurements showed that reduced triamterene produced an oxidation current peak at 0.0928 V at the BDD electrode, 0.0195 V at the GC electrode, and -0.322 V at the Pt electrode. At each potential, the S/B ratio, calculated by dividing the oxidation current value of 10 μM triamterene by the background current value, was 15.83 for BDD, 2.191 for GC, and 1.552 for Pt.

The BDD electrode exhibited a larger S/B ratio than the comparative GC and Pt electrodes. This is due to the small background current of the BDD electrode and the low reactivity with water due to the surface inertness of the ^sp3 structure of BDD. This indicates that BDD is more suitable for detecting triamterene in solution than other electrodes.

To demonstrate the concentration-dependent change in the oxidation current of triamterene reduction, we devised a protocol in which a constant potential of -1.5 V, at which triamterene undergoes reduction, was applied, followed by a potential of +0.1 V, at which the oxidation current peaks, for 10 seconds (Figure 9). When applying the reduction potential, the charge applied to the working electrode was fixed at -2 mC so that the charge applied to the electrode itself would remain constant even if hydrogen was generated from the electrode.

The reduced triamterene was oxidized by applying an oxidation potential of +0.1 V. Figure 10 shows the change in oxidation current over time. The oxidation current increased in a concentration-dependent manner. Figure 11 shows a calibration curve, with the vertical axis representing the oxidation current of the reduced product 4.5 seconds after application of the oxidation potential and the horizontal axis representing the square root of the concentration. The detection limit (LOD) was calculated by squaring the value obtained by dividing three times the standard deviation of the background current by the slope of the calibration curve. The LOD obtained was 3.15 nM. A calibration curve can also be created by plotting concentration versus current.

At a constant scan rate, theoretically, there is a linear relationship between concentration and current value, as shown in equation 1. While not wishing to be bound by any particular theory, the good proportional relationship between the oxidation current value of triamterene reduction product and the square root of concentration is presumed to be related to the diffusion form of triamterene on the BDD electrode, interactions between triamterenes, or interactions between triamterene and substances in the solution.

Electrochemical Measurement of Triamterene in Urine: Urinary CV Measurements To confirm whether the electrochemical behavior of triamterene in urine in the reduction potential range is similar to that in PB, we first performed CV measurements. Figure 12 shows cyclic voltammograms of triamterene in urine at concentrations of 0, 10, 25, 50, 75, and 100 nM. The potential was scanned from 0 V to -1.5 V in the negative direction at a scan rate of 0.1 V s ^-1 . A broad reduction current peak rising near -0.4 V and a sharp peak at -1.5 V, which were not observed in PB, were observed (Figure 13). Because the height of the current peaks at these potentials was independent of triamterene concentration, this is likely due to the reduction of urinary components. Next, as in the basic experiments in PB, we focused on the increase in current due to the oxidation of triamterene reduction products. In urine, an oxidation current was observed that increased in a triamterene concentration-dependent manner at an oxidation potential of 0.2 V. The waveforms of the cyclic voltammograms in PB and urine were different (Figures 13 and 14). This is presumably because, during the measurement protocol that reduces and oxidizes triamterene, components contained in urine that interfere with the reaction, such as proteins, uric acid, ascorbic acid, creatinine, and urea, also undergo electrochemical reactions.

Comparison with other electrodes
To compare with the cyclic voltammogram at the BDD electrode, CV measurements were performed in urine using a glassy carbon (GC) electrode (Fig. 15) (comparison example) or a platinum (Pt) electrode (Fig. 16) (comparison example) as the working electrode, in the negative potential range from 0 V to -1.5 to 0.5 V. It was confirmed that the background current at the BDD electrode was smaller than that at the other electrodes.

Next, 10 μM triamterene was dissolved in urine, and CV measurements were performed using a GC electrode (Figure 17) (comparison) or a Pt electrode (Figure 18) (comparison) as the working electrode, following the same procedure. Reduced triamterene produced oxidation current peaks at 0.215 V for the BDD electrode, 0.151 V for the GC electrode, and -0.444 V for the Pt electrode. The S/B ratios, calculated by dividing the oxidation current value of 10 μM triamterene at each potential by the background current value, were 5.005 for BDD, 1.904 for GC, and 2.016 for Pt. Even in urine, a larger S/B ratio was obtained with the BDD electrode than with the GC and Pt electrodes.

From the above, it was demonstrated that the BDD electrode is a highly sensitive material that exhibits a small background current and a large S/B ratio even in urinary measurements, and therefore the BDD electrode is suitable for use as a urinary triamterene sensor.

To demonstrate the concentration-dependent change in the oxidation current of triamterene reduction, we devised a protocol in which a constant potential of −1.5 V was applied, at which triamterene undergoes reduction, followed by a potential of +0.2 V, at which the oxidation current peak occurs in urine, for 20 seconds (Figure 19). To ensure reproducibility, the charge applied to the working electrode was fixed at −2 mC during the reduction potential application.

In urine, reduced triamterene was oxidized by applying an oxidation potential of +0.2 V. Figure 20 shows the time course of the oxidation current. The oxidation current increased in a concentration-dependent manner. Each concentration was tested three times, and the linear change in concentration versus current was plotted on a calibration curve. The calibration curve showed little variation between 2 and 2.5 seconds after application of +0.2 V. Figure 21 shows the calibration curve, redrawn with the vertical axis representing the oxidation current value of the reduced product 2.5 seconds after application of the oxidation potential and the horizontal axis representing the square root of the concentration. The LOD was calculated by dividing three times the standard deviation of the background current by the slope of the calibration curve and squaring the result. The LOD obtained was 7.80 nM. A calibration curve can also be created by plotting concentration versus current.

Detection of Other Drugs Supplementary Table 2 on page 40 of Non-Patent Document 1 reports the electrochemical measurement of various compounds using a BDD electrode. In recent studies, the inventors have confirmed that bumetanide and furosemide undergo electrochemical reactions in the range of −1.5 V to +0.5 V. Therefore, by appropriately setting the applied potential and other parameters according to the present disclosure, it is believed that bumetanide and furosemide can be detected by electrochemical measurement from actual urine samples without pretreatment. Non-Patent Document 2 also reports the measurement of methylcobalamin. Non-Patent Document 3 also describes the measurement of acetaminophenone, reporting measurements at around +0.4 V. Furthermore, at the 93rd Annual Meeting of the Japanese Pharmacological Society, Session ID: 93_1-YIA-18 (DOI https://doi.org/10.1254/jpssuppl.93.0_1-YIA-18), a rapid method for measuring plasma concentrations of molecularly targeted drugs using a diamond sensor was presented, and pazopanib measurement was reported. Furthermore, at the 93rd Annual Meeting of the Japanese Pharmacological Society, Session ID: 93_1-P-133 (DOI https://doi.org/10.1254/jpssuppl.93.0_1-P-133), a presentation was given on the rapid measurement of vancomycin in plasma using a diamond sensor.Furthermore, at the 92nd Annual Meeting of the Japanese Pharmacological Society, Session ID: 92_1-P-131 (DOI https://doi.org/10.1254/jpssuppl.92.0_1-P-131), a report was given on the rapid measurement of the molecular targeted drug imatinib in plasma using a diamond sensor. Furthermore, at the 92nd Annual Meeting of the Japanese Pharmacological Society, Session ID: 92_1-P-130 (DOI https://doi.org/10.1254/jpssuppl.92.0_1-P-130), the development of a rapid measurement method for molecular targeted drugs in plasma using a diamond sensor was presented, and the measurement of lenvatinib was reported. It is believed that these compounds can also be detected by electrochemical measurement from actual urine samples without pretreatment by appropriately setting the applied potential, etc., according to the present disclosure.

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The disclosed method and device allow for electrochemical measurement of drugs directly from urine samples without pretreatment.

1 Electrochemical measurement device 2 Working electrode 3 Counter electrode 4 Reference electrode 5 Sample 6 Control unit 7 Salt bridge

Claims (8)

An electrochemical measurement method for detecting drugs in urine using a conductive diamond electrode, the urine being a real urine sample that has not been pretreated;
i) applying a potential to the untreated real urine sample using a conductive diamond electrode; and
ii) measuring the oxidation current and/or reduction current of the drug in the silent region of urine;
The method comprising:
i) applying a potential to a non-pretreated real urine sample using a conductive diamond electrode;
applying a first potential that reduces the drug in the urine of interest; and
applying a second potential to oxidize the reduced drug in the urine;
The method, wherein the step ii) of measuring the oxidation current and/or reduction current of the drug in the silent region of urine comprises measuring the oxidation current of the drug in the silent region of urine when the second potential is applied . The method of claim 1, wherein electrochemical measurements are performed in the silent region of urine, within a potential range of -1.5 V to +0.5 V (vs. Ag/AgCl). An electrochemical measurement method for detecting drugs in urine using a conductive diamond electrode, the urine being a real urine sample that has not been pretreated;
i) applying a potential to the untreated real urine sample using a conductive diamond electrode; and
ii) measuring the oxidation and/or reduction current of the drug in the silent region of urine;
The method comprising:
i) applying a potential to a non-pretreated real urine sample using a conductive diamond electrode;
applying a first potential that oxidizes the drug in the urine of interest; and
applying a second potential that reduces the oxidized drug in the urine;
The method, wherein the step ii) of measuring the oxidation current and/or reduction current of the drug in the silent region of urine comprises measuring the reduction current of the drug in the silent region of urine when the second potential is applied. 4. The method of claim 1, wherein the drug in the urine is selected from the group consisting of triamterene, gentamicin, dihydrostreptomycin, vancomycin, cephalexin, ampicillin, ceftriaxone, amoxicillin, teicoplanin, rifampicin, theophylline, furosemide, bumetanide, quinine hydrochloride, lamotrigine, carbamazepine, topiramate, doxorubicin, pazopanib, lenvatinib, imatinib, nortriptyline , acetaminophen, ascorbic acid, methylcobalamin, and cyanocobalamin. The drug in the urine was triamterene,
i) applying a first potential at −1.5 V to reduce triamterene, and then applying a second potential at +0.2 V to oxidize the reduced triamterene;
ii) The method of claim 1 , wherein the step of measuring the oxidation current and/or reduction current of the drug in the silent region of urine comprises measuring the oxidation current when a second potential of +0.2 V is applied. A conductive diamond electrode, a counter electrode, and a reference electrode, and
A storage medium storing software or a program that implements a protocol or algorithm for executing the electrochemical measurement method according to any one of claims 1 to 5 ;
An electrochemical measurement device for detecting drugs in urine, comprising: A screening method for determining whether a drug in urine can be detected without pretreatment by electrochemical measurement using a conductive diamond electrode, comprising:
(i) providing a real urine sample containing a drug candidate that has not been pretreated;
(ii) obtaining a cyclic voltammogram of the unpretreated real urine sample using a conductive diamond electrode;
(iii) preparing a cyclic voltammogram obtained using a conductive diamond electrode on a real urine sample from a healthy subject that is not pretreated and does not contain a candidate drug;
(iv) comparing the cyclic voltammogram of (ii) with the cyclic voltammogram of (iii) to determine whether or not a discernible oxidation current and/or reduction current of the candidate drug is observed in the silent region of urine in the cyclic voltammogram of (ii) compared with the cyclic voltammogram of (iii);
(v) in the step (iv), when an identifiable oxidation current and/or reduction current of the candidate drug in the cyclic voltammogram of (ii) is observed in the silent region of urine in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl), determining the potential at which the oxidation current and/or reduction current is observed as the potential at which electrochemical measurement is performed;
The method comprising: A method for detecting drugs in urine by electrochemical measurement without pretreatment using a conductive diamond electrode, comprising: performing a screening method for determining whether or not drugs in urine can be detected by electrochemical measurement without pretreatment using a conductive diamond electrode; and performing electrochemical measurement at a potential determined by the screening method,
The screening method comprises:
(i) providing a real urine sample containing a drug candidate that has not been pretreated;
(ii) obtaining a cyclic voltammogram of the unpretreated real urine sample using a conductive diamond electrode;
(iii) preparing a cyclic voltammogram obtained using a conductive diamond electrode on a real urine sample from a healthy subject that is not pretreated and does not contain a candidate drug;
(iv) comparing the cyclic voltammogram of (ii) with the cyclic voltammogram of (iii) to determine whether or not a discernible oxidation current and/or reduction current of the candidate drug is observed in the silent region of urine in the cyclic voltammogram of (ii) compared with the cyclic voltammogram of (iii);
(v) in the step (iv), when an identifiable oxidation current and/or reduction current of the candidate drug in the cyclic voltammogram of (ii) is observed in the silent region of urine in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl), determining the potential at which the oxidation current and/or reduction current is observed as the potential at which electrochemical measurement is performed;
Including,
The method for detecting drugs in urine by electrochemical measurement using the conductive diamond electrode without pretreatment comprises the steps of:
1) applying a potential to the unpretreated real urine sample using a conductive diamond electrode, wherein the potential applied to the unpretreated real urine sample is the potential at which electrochemical measurements are performed determined in step (v) of the screening method; and
2) measuring the oxidation current and/or reduction current of the drug in the silent region of urine in the potential range of −1.5 V to +0.5 V (vs. Ag/AgCl);
The method comprising: