Process for detecting and then quantifying nitrites in a biological sample

Abstract

The present invention relates to an electrochemical process for detecting and then quantifying the nitrites contained in a biological fluid, using a three-electrode system which includes a boron-doped diamond working electrode.

Description

[0001] DESCRIPTION

[0002] METHOD FOR DETECTING AND QUANTIFYING NITRITES IN A BIOLOGICAL SAMPLE

[0003] TECHNICAL FIELD

[0004] The present invention belongs to the general field of medical diagnostics and in particular to the detection and possible quantification of nitrites in a biological sample such as urine, making it possible in particular to indicate the presence of microorganisms and facilitate the detection of a urinary tract infection.

[0005] More specifically, the present invention proposes a voltammetric electrochemical method for detecting the presence of nitrites in a biological fluid such as urine samples. The basic principle of this method consists of applying a potential difference between two electrodes, which can cause the reduction or oxidation of certain molecules, namely N-nitrosamines, resulting from the reaction of nitrites with secondary amines such as indole compounds. This reduction / oxidation produces a return current that can be measured.

[0006] PREVIOUS STATE OF THE ART

[0007] When a urinary tract infection (UTI) is suspected, a bacterial culture is performed to fully assess the bacterial content of urine samples. Although urine culture is the gold standard for diagnosing UTIs, it is expensive and time-consuming, as it generally takes 48 hours to obtain test results and begin antibiotic treatment when a UTI is confirmed.

[0008] Alternatively, nitrite detection is a routine procedure commonly used by healthcare professionals. The presence of nitrites in urine is due to the bacterial conversion of endogenous nitrates into nitrites. Determining the presence of nitrites in urine can be a sign of infection with these types of bacteria and, therefore, is a commonly used indirect indicator for detecting bacteriuria, that is, the presence of bacteria in the urine. Currently, the most common method for rapidly detecting nitrites uses color-changing test strips. This method relies on the Griess reaction, where nitrites react with Griess reagent, composed of an aniline derivative and a coupling agent, to form a red-pink dye, which can then be visually observed.Although this method is inexpensive, requires little time and expertise, the interpretation of the result can be subjective, its sensitivity is relatively low (approximately 10-15 pM) and it does not give an indication of the actual concentration of nitrites present.

[0009] For the quantitative determination of nitrites, the most common method is the Griess reaction, usually performed colorimetrically. This method can be used with large colorimetric imaging instruments or small portable devices, and its detection limit is approximately 1 pM. Although this method is quantitative and can be easily miniaturized, it still requires the use of chemical reagents, making it impractical outside the laboratory.

[0010] Furthermore, any method using the Griess reaction (strips or colorimetric tests) presents problems with false negatives in the presence of ascorbic acid, which is commonly found in urine. Indeed, high levels of ascorbic acid are known to produce false negatives for nitrite detection in tests using the Griess reaction. This is due to the highly reducing nature of ascorbic acid, which prevents other reactions from occurring.

[0011] Other methods for detecting nitrites include ultraviolet spectrophotometric methods, fluorometric assays, chemiluminescence, high-performance liquid chromatography, capillary electrophoresis, gas chromatography, and gas chromatography with mass spectrometry [1]. The use of capillary electrophoresis to monitor nitrites in urine has been described, but the sample processing time of over 20 minutes and the washing steps are complicated for untrained users [2]. Furthermore, these methods require significant equipment and highly trained personnel, making them unsuitable for home or pharmacy testing. Finally, other methods for detecting nitrites rely on electrochemistry. In electrochemical nitrite detection, the most common mechanism is based on the electro-oxidation of nitrite.In other words, the nitrite molecule undergoes an oxidation reaction, and the nitrate product is then generated. Since the electrical signal is proportional to the nitrite concentration within a certain concentration range, quantitative nitrite analysis is performed based on the linear relationship between the two.

[0012] Electrochemical sensors, particularly those based on nanomaterials, offer remarkable selectivity; they do not require complex sample pretreatment and can be used for detection in serum, urine, and saliva after dilution. Despite their numerous advantages, such as high sensitivity and the absence of time-consuming pretreatment, the drawback of these sensors lies in the often complex modification of the electrodes, which increases the cost of the equipment and reduces its robustness, making them unstable for long-term use. Furthermore, these electrodes are all susceptible to fouling, which precludes their use in portable devices where a fouling removal step is impractical.

[0013] In 2023, Zhang et al. published work on the detection of nitrites in urine using boron-doped diamond (BDD) electrodes [3]. According to this article, nitrites can form an intermediate species involving creatinine after a pre-oxidation process (at 1.5 V), which can then be sensitively detected by performing a sweep in the reduction portion of the spectrum (cyclic or pulsed differential voltammetry), observing a current increase at approximately -0.5 V. This method offers the ease of use and instrument simplicity of other electrochemical techniques. Furthermore, diamond is a very robust material, making it suitable for integration into a commercial device.

[0014] However, the detection method described by Zhang et al. requires a pretreatment step consisting of maintaining the electrodes at -3 V for 5 min in 0.1 M sulfuric acid between each sample. This time-consuming pretreatment involving strong acids makes this method impractical for routine analyses.

[0015] Furthermore, the detection method described by Zhang et al. lacks the sensitivity required to detect low concentrations of nitrites in urine, which are always indicative of infection (false negative results). Moreover, this method exhibits cross-sensitivity with ascorbic acid, which is often present in high concentrations in urine and therefore leads to false positive results.

[0016] The inventors therefore set themselves the goal of providing a method for detecting and possibly quantifying nitrites in urine that is sensitive, selective, fast, simple and easily miniaturized.

[0017] The inventors also set themselves the goal of providing a method for detecting and possibly quantifying nitrites in urine, without the drawbacks of prior art methods and in particular without the problems of false negatives or false positives as mentioned above.

[0018] DESCRIPTION OF THE INVENTION

[0019] The goals set by the inventors and others are achieved by the invention which proposes a method for detecting and possibly quantifying nitrites contained in urine and, in general, in any biological fluid, said method being both rapid, simple and easily miniaturized.

[0020] Furthermore, the method according to the invention can be easily used in a portable device for the development of an in situ nitrite detection sensor.

[0021] More specifically, the inventors' work has shown that the detection and quantification of nitrites in a biological fluid such as urine is possible by detecting and quantifying N-nitrosamines (NAms) formed by the reaction of nitrites with secondary amines such as indole compounds like, for example, tryptophan. Indeed, in the process according to the invention, the resulting N-nitrosamines are detected by means of a two-step procedure: an oxidized intermediate is first pre-concentrated on the surface of a working electrode of the boron-doped diamond type.This step is followed by a voltammetry sweep at a negative potential, where the oxidized intermediate is reduced at the electrode surface, resulting in a clear current peak at -0.7 V against a silver chloride (Ag / AgCl) reference electrode. This peak is proportional to the nitrite concentration in the biological fluid sample, such as urine. This exceptionally clear current peak has never been reported before and is unaffected by common contaminants, including those present in urine such as ascorbic acid, thus resolving one of the major technical problems for many techniques currently in use.

[0022] More particularly, the present invention relates to a method for detecting and possibly quantifying nitrites in a biological fluid involving a three-electrode system with a boron-doped diamond working electrode, a counter electrode, and a third electrode which is either a reference electrode or a pseudo-reference electrode, said method comprising the following steps:

[0023] a) electrochemically activate said working electrode;

[0024] b) apply and maintain a first predetermined voltage to the electrochemically activated working electrode, said working electrode and counter electrode being in contact with said biological fluid, whereby, when nitrites are present in said biological fluid, said nitrites react with secondary amines, such as indole derivatives, naturally present or added to said biological fluid to form N-nitrosamines, which oxidize and concentrate on the surface of said working electrode;

[0025] (c) subject the working electrode to a voltammetric sweep in a potential range from the first predetermined voltage to a second, more reducing voltage, whereby the reduction of said oxidized N-nitrosamines causes, at a third predetermined voltage between the first predetermined voltage and the second voltage, the appearance of a current peak indicating the presence of nitrites in said biological fluid and

[0026] d) optionally quantify the nitrites in said biological fluid as a function of the height of the current peak observed at the third predetermined voltage. As previously mentioned, the present invention applies not only to the detection and possible quantification of nitrites in urine but also to their detection and possible quantification in any biological fluid likely to contain nitrites naturally or as a result of a condition or disorder such as infection by microorganisms.

[0027] In a particular embodiment, the biological fluid in which nitrites are to be detected and possibly quantified is advantageously chosen from the group consisting of blood such as whole blood or anticoagulated whole blood, blood serum, blood plasma, lymph, tears, semen, urine, milk, cerebrospinal fluid, interstitial fluid, joint fluid, pericardial fluid, isolated bone marrow fluid, cell extract, tissue extract, organ extract and a mixture thereof.

[0028] Thus, the biological fluid in which nitrites are to be detected and possibly quantified can be any fluid naturally secreted or excreted from a human or animal body, or any fluid recovered from a human or animal body by any technique known to those skilled in the art, such as extraction, sampling, puncture, or washing. The steps of recovering and isolating these different fluids from the human or animal body are carried out prior to implementing the process according to the invention.

[0029] In a more particular embodiment, the biological fluid in which nitrites are to be detected and possibly quantified is chosen from the group consisting of blood, blood serum, blood plasma, lymph, urine and saliva.

[0030] In a further particular embodiment, the biological fluid in which nitrites are to be detected and possibly quantified is urine.

[0031] Prior to implementing the process according to the invention, the biological fluid may be subjected to a preparation step known to those skilled in the art, depending on the fluid used. This step may include, but is not limited to, dilution, concentration, filtration, or centrifugation.

[0032] Furthermore, as previously explained, the process according to the invention is based on the electrochemical detection of NAms, which are formed by the reaction of nitrites with secondary amines such as indole derivatives or indole compounds. Therefore, if the biological fluid contains few or no secondary amines such as indole derivatives compared to the nitrites it may or could contain, at least one secondary amine such as an indole derivative can be added to the biological fluid prior to implementing the process according to the invention. A person skilled in the art will be able to determine, without inventive effort, the quantity of secondary amine(s) such as one or more indole derivative(s) to be added based on the quantity of nitrites that the biological fluid may or could contain.Typically, before implementing the process according to the invention, the biological fluid must contain a concentration of secondary amines, such as indole derivatives, exceeding the maximum amount of nitrites that the fluid can contain. The secondary amine, such as an indole derivative usable for this purpose, is advantageously chosen from the group consisting of tryptophan, indole, indoleacetic acid, melatonin, serotonin, skatole, tryptamine, and any mixture thereof.

[0033] The process according to the present invention is an electrochemical process employing an electrochemical system or electrochemical cell containing the biological fluid in which one seeks to detect and possibly quantify nitrites at three electrodes: a working electrode, a counter electrode and a third electrode which is either a reference electrode or a pseudo-reference electrode.

[0034] Working electrodes conventionally used in electrochemical processes can be made from any material that facilitates the generation of oxidized and reduced species, including noble metal electrodes such as gold and platinum, or carbon-based electrodes such as graphite, glassy carbon, and boron-doped diamond (BDD). In the present invention, the working electrode used is made of boron-doped diamond.

[0035] The counter electrode is essential because it allows the flow of electric current from the working electrode and is often made of a highly conductive and stable material, such as gold, platinum, stainless steel or boron-doped diamond (BDD). In a particular embodiment, the counter electrode used in the process according to the invention is made of boron-doped diamond.

[0036] Indeed, the use of two BDD electrodes in the process according to the invention constitutes the optimal selection due to their exceptional stability and resistance to fouling, attributable to their high atomic density. The exceptional durability of diamond minimizes electrode degradation.

[0037] Furthermore, BDD electrodes have been shown to facilitate electrochemical cleaning through the application of high positive and negative currents. This is particularly crucial for nitrite detection in biological fluids such as urine, and especially human urine, as these extracts are likely to contain numerous dissolved compounds that can easily contaminate the working and counter electrode surfaces, leading to decreased electrochemical efficiency.

[0038] Finally, although not mandatory, the inclusion of a reference or pseudo-reference electrode in the electrochemical setup is highly advantageous. The reference or pseudo-reference electrode maintains a constant specific potential, which determines the precise voltage supplied to the working electrode. In the context of the present invention, the presence of a reference or pseudo-reference electrode is crucial because the two essential steps for the detection of NAms, and therefore nitrites, occur at a specified optimal voltage.

[0039] The third electrode implemented in the present invention can be a genuine reference electrode, such as a silver chloride (Ag / AgCl) electrode, a saturated calomel electrode, or a copper(II) sulfate electrode. However, a simpler electrode, also known as a "pseudo-reference electrode," such as a platinum wire, is also usable and may be preferred for practical or economic reasons.

[0040] In one particular embodiment, the third electrode of the three-electrode system used in the method according to the invention—i.e., the reference electrode or pseudo-reference electrode—is brought into contact with the biological fluid during steps b) and c). In other words, in this particular embodiment, the three electrodes—the working electrode in BDD, the counter electrode, and the reference or pseudo-reference electrode—are in contact with the biological fluid in which nitrites are to be detected and possibly quantified. Such an embodiment is particularly advantageous when the method according to the invention is used in a portable device for developing an in situ nitrite detection sensor.

[0041] In another particular embodiment, the third electrode of the three-electrode system used in the process according to the invention, i.e., the reference electrode or pseudo-reference electrode, is not in contact with the biological fluid. In this particular embodiment, the third electrode of the three-electrode system is electrically connected to the working electrode and the counter electrode in steps b) and c) by means of a salt bridge. A salt bridge is also known in electrochemistry as an "electrolytic bridge." Any salt bridge known to those skilled in the art and conventionally used in electrochemistry is suitable for use in the present invention. Advantageously, this salt bridge consists of an aqueous solution containing salts such as potassium chloride or potassium nitrate.

[0042] Step a) of the process according to the invention, also referred to in this description as "step 1," is an essential aspect in the detection and eventual quantification of nitrites. This electrochemical activation step of the working electrode in BDD can also be defined as a preconditioning step of the working electrode in BDD.

[0043] Step a) of the process according to the invention has two functions. First, it cleans the surface of the working electrode by forming reactive radicals on the electrode surface. These radicals can react, by oxidizing and / or reducing them, with any chemical species that have adsorbed or adhered to the electrode surface, a process known as fouling. This is important because electrochemical detection in biological fluids such as urine has shown that electrodes foul very quickly, thus reducing sensitivity over time. Therefore, cleaning between measurements, i.e., between each repetition of the process according to the invention, is necessary to ensure repeatability.

[0044] Furthermore, this activation step affects the chemical termination composition of the BDD electrode surface and therefore its reactivity. Thus, the BDD electrode surface termination can be refined and optimized for the detection of NAms, and therefore nitrites, and it is also kept constant between measurements for repeatability.

[0045] Compared to what has been previously reported using a BDD electrode for nitrite detection, this preconditioning step (i.e., step a) of the process according to the invention can be very rapid, i.e., its duration is less than 3 minutes, in particular less than 2 minutes, and especially less than or equal to 1 minute. As a reminder, in Zhang et al., 2023 [3], the pretreatment of the BDD electrode takes 5 minutes. This is an essential element of the process according to the present invention, as it allows for routine analysis of biological fluids such as urine outside the laboratory.

[0046] The electrochemical activation technique of the working electrode in BDD as implemented in the process according to the invention is described in particular in international application WO 2012 / 110600 A2 [4]. It consists of applying electrical pulses to the working electrode in BDD.

[0047] Typically, a pulse is a short-duration variation of a physical quantity (voltage, current, etc.) with a return to the initial state. In the context of this invention, an "electrical pulse" is defined as a brief variation in voltage or current, lasting for a total duration of 10 seconds or less, followed by a return to the initial state (generally to an amplitude of zero) of the voltage or current value.

[0048] Similarly, within the framework of the invention, "amplitude" means the maximum value of the anode or cathode voltage (or current) reached during an anode or cathode voltage (or current) pulse relative to a baseline, generally equal to zero.

[0049] Consequently, a positive impulse can be considered a wave whose amplitude increases relative to the baseline and, after a time interval td, returns to the baseline. A negative impulse, on the other hand, can be considered a wave whose amplitude decreases relative to the baseline and returns to the baseline after a time interval td.

[0050] Within the framework of the invention, it is preferable to have pulses with maximum current or voltage amplitudes and the shortest possible duration, in order to achieve the fastest possible activation of the electrode in BDD. Therefore, the electrical pulses advantageously have a maximum amplitude within an activation time t a minimal.

[0051] In a particular embodiment, step a) therefore consists of applying a series of electrical pulses to the working electrode brought into contact with a saline solution which can be the biological fluid in which one seeks to detect and possibly quantify nitrites.

[0052] Thus, in a first embodiment, step a) of the process according to the invention is carried out in a saline solution different from the biological fluid in which nitrites are to be detected and possibly quantified. This saline solution can be any aqueous solution containing an electrolyte, with or without electroactive species, as described in international application WO 2012 / 110600 A2 [4]. In this embodiment, it is evident that the saline solution will be withdrawn or removed before bringing the working electrode and the counter electrode, and possibly the reference or pseudo-reference electrode, into contact with the biological fluid and carrying out steps b) and c) of the process according to the invention.Furthermore, the saline solution used differs from a 0.1 M aqueous sulfuric acid solution as used in Zhang et al., 2023 [3]. In a second embodiment, step a) of the process according to the invention is carried out in the biological fluid in which nitrites are to be detected and possibly quantified. This embodiment is also advantageous for a rapid process and when the process according to the invention is used in a portable device for the development of an in situ nitrite detection sensor.

[0053] The electrical pulses usable during step a) of the process according to the invention can be current pulses, and in particular current pulses of - / + 1 mA.cm -2 Alternatively, electrochemical activation during step a) of the process according to the invention is also effective using voltage pulses and in particular pulses of - / + 2 V.

[0054] Advantageously, the activation time t a the electrical pulses implemented during step a) of the process according to the invention is less than or equal to 1 second, in particular less than or equal to 100 milliseconds and, in particular, less than or equal to 10 milliseconds.

[0055] Advantageously, the number of electrical pulse cycles implemented during step a) of the process according to the invention is between 30 and 70, in particular between 40 and 60 and, in particular, on the order of 50 (i.e. 50 ± 5).

[0056] Step b) of the process according to the invention, also referred to in this description as "step 2", involves an oxidative pre-concentration.

[0057] To achieve this, the working electrode is maintained at a specific potential, also referred to in this description as the "predetermined first voltage" and "pre-concentration potential," for a predetermined duration. While this potential is maintained, certain chemical species are oxidized at the surface. These oxidized species accumulate, meaning that the longer the potential is maintained, the more the species are pre-concentrated. This step allows the NAms (i.e., the compounds involved in nitrite detection) to oxidize. They can then be reduced in the third step of the process according to the invention. It also improves the selectivity of the measurement, since only a certain number of species will oxidize optimally at the specific chosen potential.

[0058] In one particular embodiment, when the biological fluid is urine and the third electrode is a silver chloride reference electrode, the first predetermined voltage applied and maintained during step b) is 1.1 V.

[0059] In a particular embodiment, the first predetermined voltage is applied for a duration of between 30 and 90 seconds, in particular between 40 and 60 seconds and, in particular, on the order of 50 seconds (i.e.

[0060] 50 seconds ± 5 seconds). Such a duration is notably that used when the biological fluid is urine. Step c) of the process according to the invention, also designated in this description as "step 3", is a voltage sweep from the pre-concentration potential to a second, more reducing voltage.

[0061] Advantageously, this second tension is lower than the reduction potential of oxidized species, i.e., oxidized forms of NAms.

[0062] In particular, when the biological fluid is urine and the third electrode is a silver chloride reference electrode, the second voltage is -1.5 V.

[0063] Any electrochemical potential scanning technique known to those skilled in the art can be used in step c) of the process according to the invention. This technique is in particular chosen from the group consisting of cyclic voltammetry, linear potential scanning voltammetry, variable pulse voltammetry and square wave voltammetry.

[0064] In one particular embodiment, the voltammetry scan implemented in step c) of the method according to the invention uses square wave voltammetry (or SWV for "Square Wave Voltammetry"). SWV is a variant of linear potential scanning voltammetry that uses a superimposed square wave and a stepped potential delivered to a stationary electrode. The square wave is known to provide an optimal signal-to-noise ratio for such a scan.

[0065] In step c) of the process according to the invention, the oxidized species, i.e., the oxidized forms of NAms, are reduced, resulting in a current peak at a third predetermined voltage that is proportional to their concentration and thus allows them to be quantified. Therefore, if a current peak is present at this third predetermined voltage, NAms have been oxidized in step b) and then reduced in step c), and their presence in the biological fluid is an indication of the presence of nitrites in that fluid. Conversely, the absence of a current peak at this third predetermined voltage indicates the absence of nitrites in the tested biological fluid.

[0066] In a particular embodiment, when the biological fluid is urine and the third electrode is a silver chloride reference electrode, the third predetermined voltage in step c) is -0.7 V. Typically, steps b) and c) of the method according to the invention in which electrical potential and current response measurements are performed can be carried out using a potentiostat.

[0067] In the optional step d) of the process according to the invention, the nitrites in the tested biological fluid are quantified. This quantification is possible, as illustrated in the experimental section below, based on the height of the current peak observed at the third voltage during step c) of the process according to the invention.

[0068] As an illustrative and non-limiting example of a technique usable for this quantification, one can consider using a biological fluid of the same nature as the one tested, but without nitrites, to which different known quantities of nitrites are added, and comparing the height of the current peak observed for the biological fluid with the height of the current peak obtained for each sample containing a known quantity of nitrites. It is evident that the procedure according to the invention, implemented between the biological fluid and the samples with a known quantity of nitrites, must be identical.

[0069] As previously mentioned, the three-electrode electrochemical system used in the implementation of the process according to the invention may include either a reference electrode which is a standard reference, such as an Ag / AgCl reference electrode, for which the reference potential is stable and known, or a pseudo-reference electrode such as a wire or pin made of any metal in the platinum group, for example, platinum or iridium, or any other noble metal, for example, gold or silver.

[0070] These electrodes are called pseudo-reference electrodes because they do not maintain a stable potential but fluctuate predictably depending on the conditions. Once the circumstances are established, the potential can be calculated, allowing the electrode to serve as a reference. Most electrodes operate within a narrow range of conditions, such as pH or temperature; beyond this range, their behavior becomes unstable. This means that when using a pseudo-reference electrode, this potential varies depending on the composition of the biological fluid being tested. In particular, it has been found to change significantly between urine samples. The existence of a fluctuating reference potential has two consequences.First, identifying / locating the current peak of interest corresponding to the reduction of NAms is more difficult because it also occurs at a fluctuating potential (depending on that of the pseudo-reference electrode), meaning it does not necessarily occur at -0.7 V in the case of a biological fluid such as urine. Furthermore, the efficiency of the reaction is highly dependent on the pre-concentration potential. If there is a shift in the reference potential, the applied pre-concentration potential is no longer optimal, resulting in a loss of efficiency and repeatability between samples.

[0071] A particularly advantageous feature of the method according to the invention is that it is compatible, by an additional step, with the use of a pseudo-reference electrode such as a platinum wire or pin instead of a stable standardized reference such as, for example, an Ag / AgCl reference electrode. Therefore, when the third electrode is a pseudo-reference electrode, the method according to the invention may include, prior to step b), a step for determining said first voltage.

[0072] To do this, a short reference oxidation or reduction scan of a biological fluid such as urine can be performed using a pseudo-reference electrode. All biological fluids, such as urine, contain a number of characteristic oxidation or reduction peaks, and the shift of one of these peaks from the expected value allows for calibration of the detection and adjustment of the pre-concentration potential. For example, when the biological fluid is urine, these characteristic peaks indicate, among other things, the presence of uric acid.

[0073] Any scanning technique can be used to perform this short reference oxidation or reduction scan of the biological fluid. This technique can be chosen, in particular, from the group consisting of cyclic voltammetry, linear voltammetry, pulsed differential voltammetry, or square wave voltammetry. The experimental section below illustrates this aspect of the invention.

[0074] By allowing the use of a pseudo-reference electrode, the process according to the present invention can be integrated into a simplified device, since the complex standard reference of the silver chloride reference electrode type can be replaced by a simple platinum electrode which is easier to integrate into a portable commercial device and less expensive than a silver chloride reference electrode.

[0075] Other features and advantages of the present invention will become apparent to the person skilled in the art upon reading the examples below, given by way of illustration and not limitation, with reference to the attached figures.

[0076] BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 shows the SWV voltammeter obtained in a 0.1 M PBS solution containing 50 pM nitrite (black dashed line), 200 pM tryptophan (grey solid line) and 50 pM nitrite + 200 pM tryptophan (black solid line).

[0078] Figure 2 shows the height of the current peak at 0.7 V from a solution containing 50 pM nitrite and 200 pM of various indole compounds.

[0079] Figure 3A shows the reduction peaks centered on -0.7 V vs Ag / AgCl in a urine sample supplemented with 50 pM nitrite, with different oxidation pre-concentration potentials applied (here also vs Ag / AgCl) for 50 seconds.

[0080] Figure [Fig. 3B] shows the height of the corresponding peak recorded at -0.7 V vs Ag / AgCl as a function of the pre-concentration potential.

[0081] Figure 4 shows SWV voltammeterograms for urine samples containing a variety of nitrite concentrations ranging from 5 pM to 25 pM.

[0082] Figure 5 shows the height of the nitrite peak at -0.7 V after the addition of different amounts of ascorbic acid ranging from 0.5 mM to 2 mM.

[0083] Figure 6A shows the SWV voltamgrams for 24 urine samples (peak of -0.7 V). Samples positive for nitrites using the test strip are in bold.

[0084] Figure 6B shows the nitrite peak heights obtained from the voltamgrams in Figure 6A. Figure 7A shows the SWV voltamgrams for urine samples containing different nitrite concentrations ranging from 5 pM to 25 pM.

[0085] Figure 7B presents the calibration curve for the detection of nitrites in urine obtained from the graphs shown in Figure 7A.

[0086] Figure [8A] presents the CV graph showing the possible reference peak of uric acid.

[0087] Figure [Fig. 8B] presents the SWV volt-amperograms showing three possible reference peaks.

[0088] Figure 9A presents the CV graph showing the oxidation region in a urine sample with the Ag / AgCl reference electrode (solid line) and the platinum wire reference electrode (dashed line).

[0089] Figure 9B shows the peak heights for nitrite detection with the Ag / AgCl reference electrode, the Pt reference electrode and the corrected Pt reference electrode.

[0090] DETAILED DESCRIPTION OF SPECIFIC METHODS OF IMPLEMENTATION

[0091] 1. Experimental setup.

[0092] The electrodes are fabricated as 10 mm x 10 mm (working electrode, WE) or 15 mm x 15 mm (counter electrode, CE) sections of boron-doped diamond (BDD) on a highly conductive silicon substrate. Electrical contact is made on the back of the silicon substrate using a copper strip. Both electrodes, along with a salt bridge connecting an Ag / AgCl reference electrode in a KCl solution, or a Pt quasi-reference wire electrode, are then placed in a glass beaker.

[0093] 2. Compounds studied during the process according to the invention.

[0094] For a current peak to be observed at -0.7 V vs Ag / AgCl, nitrite ions must be present in the sample, such as a urine sample. However, no peak is observed in a solution containing only nitrite ions, suggesting that it is not due to the direct oxidation and reduction of nitrites. When nitrite is in the presence of secondary amines, a reaction occurs between the nitrite and the amine group to form an N-nitrosamine. The carcinogenic potential of N-nitrosamines (NAms) is well documented. Human exposure to NAms can originate from the everyday environment and endogenous formation through the reaction of secondary amines with nitrites, or from bacterial infection.

[0095] Although this reaction is known to occur with a wide range of compounds containing secondary amines, only indole derivatives are present in sufficiently high concentrations in urine and their contribution is assumed to be sufficient to allow the detection / quantification of nitrites.

[0096] An example of a typical reaction mechanism between nitrite and indole is shown in Scheme 1 below, illustrating how N-nitrosamines are formed in reaction with secondary amines and typically indole compounds.

[0097] Diagram 1:

[0098]

[0099] It is assumed that the -0.7 V peak observed using the method according to the invention is due to the oxidation and subsequent reduction of this class of compounds (NAms). Although nitrite is not directly detected, NAms are formed in urine in the presence of nitrite, and the presence of NAms therefore indicates the presence of nitrite in the urine, and thus a bacterial infection.

[0100] Tryptophan, an indole chemical compound known to be relatively abundant in urine, is present in all human urine samples. The average concentration of tryptophan in urine is known to be around 200 pM, an order of magnitude higher than the elevated nitrite levels observed during urinary tract infections, typically around 50 pM. Since the reaction between nitrite and tryptophan occurs in a 1:1 ratio, tryptophan should always be in excess of nitrite, and the abundance of NAms should therefore depend on the nitrite concentration.

[0101] Urine also contains tryptophan metabolites such as kynurenic acid, xanthurenic acid, 5-hydroxyindoleacetic acid, and serotonin, which also lead to the formation of NAms in urine and most likely contribute to the nitrite detection signal. Therefore, the concentration of NAms detected using this technique should be proportional to the nitrite concentration and can thus be used to deduce the target analyte.

[0102] Figure 1 shows the peak observed from this three-step process in a phosphate-buffered solution containing both tryptophan and nitrite. For comparison, it is also shown that no peak is observed when the solution contains only tryptophan or only nitrite, suggesting that the peak is observed through the formation of NAms. Tryptophan is the most abundant indole compound in urine, but NAms can be formed by the reaction of nitrite with other secondary amines. The height of the peak generated at -0.7 V vs. Ag / AgCl from a solution containing nitrite and seven examples of indole compounds (some of which are found in urine) is shown in Figure 2.

[0103] It should be noted that the variation in peak height depending on the type of secondary amines does not significantly influence the accuracy of the assay based on the composition of the urine sample, because tryptophan is present at a concentration approximately two orders of magnitude higher than all other indole compounds. It therefore remains the major reaction intermediate.

[0104] 2. Measurements according to the method of the invention.

[0105] For the standard detection of indole N-nitrosamines formed between nitrite and secondary amines such as indole compounds like tryptophan, the procedure has been optimized and can be divided into several main steps:

[0106] Step 1 - Electrode activation - + / - 1 mA.cm -2, for 50 cycles (~50 seconds) Step 2 - Oxidation pre-concentration - holding 1.1 V for 50 seconds Step 3 - Square wave voltamperometry - sweep from 1.1 V to -1.5 V, (step = 0.01, amplitude = 0.05 and frequency = 20 Hz).

[0107] As mentioned previously, step 1 involves cleaning the electrode between each measurement, as fouling is a major problem during analyses in biological media. This step is essential before measurements can be taken. It also has the advantage of slightly terminating the electrode with hydrogen, which helps increase the electrode's reactivity and therefore its sensitivity for nitrite detection.

[0108] Step 2 is critical for the formation of an accumulation of the oxidized NAm intermediate on the electrode surface. The longer the voltage is maintained, the more oxidized species are present on the electrode surface, and the higher the expected signal. However, beyond 50 seconds, the signal increase becomes negligible. This oxidation step is highly dependent on the precise voltage applied, as shown in Figure 3A and Figure 3B, which illustrate the peak intensity recorded at -0.7 V against Ag / AgCl in the presence of 50 pM nitrite in urine after the application of different pre-concentration voltages.

[0109] The species formed during the oxidative pre-concentration step are then reduced to -0.7 V. By sweeping down to this potential, we observe a current whose height is proportional to the nitrite concentration in the urine sample.

[0110] Step 3 involves sweeping the pre-concentration current at -1.5 V against Ag / AgCl using square wave voltammetry. Figure 4 shows the voltamgram of samples spiked with a range of nitrite concentrations from 5 pM to 25 pM. The peak height observed at -0.7 V against Ag / AgCl can then be measured to deduce the nitrite concentration in the sample. The square wave voltammetry parameters have been optimized to increase the peak height at -0.7 V across a wide range of parameters, but other sweep techniques (CV, linear sweep, etc.) can also be used successfully for nitrite detection in urine. 4. Interferences.

[0111] Urine contains thousands of molecules, and the risk of interference is therefore generally high. As mentioned earlier, using a specific oxidation potential (1.1 V) means that only certain molecules are oxidized at this value. Furthermore, by focusing on the reduction peaks at -0.7 V, the number of molecules likely to cause interference has also been reduced.

[0112] Several molecules that could potentially interfere with this analysis were tested. The first was nitrate, which is known in electrochemistry to often be difficult to distinguish from nitrite. Even when adding a very high concentration of nitrate (10 mM), no effect was observed on the nitrite signal. This is probably because nitrite is not detected directly but via a byproduct of N-nitrosamine, which does not occur with nitrate.

[0113] Another common interference in nitrite detection is ascorbic acid, which is very often found in high concentrations in urine, as it is dependent on dietary intake. As explained previously, high levels of ascorbic acid are known to give false negatives for nitrite detection in both tests using the Griess reaction (strip and colorimetric tests) as well as in other electrochemical detection methods [1]. Figure 5 shows that no significant effect is observed on the nitrite peak (50 pM) detected after the addition of different concentrations of ascorbic acid (from 0.05 mM to 2 mM). Again, this is probably because the assay indirectly detects nitrites and instead examines the oxidation and reduction of NAms.

[0114] 5. Method according to the invention on human urine.

[0115] 5.1. Sample preparation.

[0116] This invention relates to a method directly applicable to urine. Due to the high salinity of human urine, it is not necessary to add any additional salts or reagents. The urine used can be tested either fresh, i.e., on the day following collection, or from samples kept chilled (+4°C), or from thawed samples, having been frozen on the day of collection.

[0117] 5.2. Analysis on human urine samples.

[0118] To validate this technique, experiments were conducted on real human urine samples containing endogenous nitrite. The validation was performed using 24 human urine samples.

[0119] The presence or absence of nitrite in the samples was first determined using a reagent strip, the detection limit of which is approximately 10-15 pM. Among these samples, 4 of the 24 samples tested positive.

[0120] The 24 urine samples were tested in parallel according to the procedure described above, with an Ag / AgCl reference electrode, and the height of the peak at -0.7 V was evaluated to determine the presence or absence of nitrite in the urine sample.

[0121] The results were then compared to those obtained with the test strips. Figure 6A shows the results. The peaks at -0.7 V are shown with their height (Figure 6B). Samples that tested positive on the strip are indicated in bold. It is clear that the peak height for each of the positive samples is significantly higher compared to the negative samples, demonstrating the good correlation between the two detection methods.

[0122] 5.3. Calibrations.

[0123] To assess the feasibility of quantifying nitrite in urine, a calibration using urine samples spiked with varying amounts of nitrite was performed, using a resulting range of nitrite concentrations of 5 pM, 10 pM, 15 pM, 20 pM, and 25 pM, and a nitrite-free control. Each sample was measured five times using the SWV method, and peak heights were plotted against known concentrations. In Figure 7A, the baseline is corrected to clearly show how the peak height depends on the nitrite concentration added to the urine. The calibration illustrated in Figure 7B shows very good linearity (R 2(=0.999), thus demonstrating the potential of this technique for determining the presence of nitrites in urine. The limit of detection (LOD) was calculated using [Signal to blank + 3*STDEV(Blank)] and was found to be 0.46 pM. The limit of quantification (LOQ) was calculated using [Signal to blank + 10*STDEV(Blank)] and was found to be 0.90 pM. The sensitivity of the technique was found to be 64.9 nA / pM, and the full linear range tested was from 1 pM to 100 pM.

[0124] 6. Method according to the invention with a pseudo-reference electrode.

[0125] One option for the present invention is to perform the measurements with a pseudo-reference electrode, such as a platinum wire, rather than with a stable reference electrode such as Ag / AgCl. When using a pseudo-reference, the voltage applied between the reference electrode and the working electrode may vary because the reference potential of the pseudo-reference electrode also varies depending on the composition of the sample being measured.

[0126] One consequence is that the position of oxidation or reduction current peaks can vary on the voltammeters along with variations in the reference potential.

[0127] Another consequence is that the optimized preconditioning potential (+1.1 V vs Ag / AgCl) applied to the electrode (step 2) can also vary. To obtain sensitive and reproducible results, this potential shift must be corrected for each sample before the measurement takes place.

[0128] The proposed solution involves performing a reference scan of the samples, for example, by cyclic voltammetry or square wave voltammetry, between steps 1 and 2 to determine the potential position of certain electrochemical peaks. These peaks are common to all urine samples and their potential position relative to Ag / AgCl (or any other reference potential) is known. Once these peaks are identified and their new positions recorded, the potential shift can be calculated, and the reference potential can be mathematically corrected. Possible candidates for reference peaks are shown in Figure 8A and Figure 8B. The oxidation of uric acid, which is always present in urine, produces a peak at approximately 0.5 V relative to Ag / AgCl, measurable by cyclic voltammetry (Figure 8A) or SWV (peak SWV1 in Figure 8B).Two other peaks are present in reduction, one of which is attributed to the reduction of uric acid (peaks SWV 2 and 3 in Figure 8B). The position of a peak or the combination of 1, 2, or 3 peaks can be used for correction.

[0129] Figures 9A and 9B provide an example. Figure 9A shows two CV scans of the same sample, one recorded using an Ag / AgCl reference electrode and the other with a Pt reference electrode. A potential shift of 0.21 V can be observed at the position of the uric acid oxidation peak. Figure 9B shows the peak heights recorded during the detection of 50 pM nitrite in urine, first with the Ag / AgCl reference electrode and then with the Pt reference electrode. The potential shift visible at both uric acid oxidation peaks also exists when the preconditioning step is applied and clearly results in reduced detection efficiency when using the Pt reference electrode. The third column shows the same measurement recorded after accounting for the reference potential shift when the preconditioning step is applied.Here, the intensity of the peak obtained is the same as when using the Ag / AgCl reference electrode, which shows that the applied correction is effective. This clearly demonstrates the ability to correct this pseudo-reference potential shift, thus opening the possibility of performing this analysis with a pseudo-reference electrode.

[0130] Blood nitrite concentration can also be measured by the method according to the invention because it reflects the overall state of nitric oxide (NO) metabolism, one of the main molecules regulating vascular homeostasis. Indeed, an alteration in NO production or availability is generally associated with functional alterations such as, for example, disturbances in the body's inflammatory response, endothelial dysfunction, or the development of atherosclerotic lesions.

[0131] Références bibliographiques[1] Tsikas, 2005, « Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluid », Free Radie. Res., vol. 39, pages 797-815.

[0132] [2] Morcos & Wiklund, 2004, « Nitrite and nitrate measurements in human urine by capillary electrophoresis », Methods Mol. Biol., vol. 279, pages 21-34.

[0133] [3] Zhang et al, 2023, « Electrochemical diagnosis of urinary tract using boron-doped diamond electrodes », ACS Sensors, vol. 8, pages 4245-4252.

[0134] [4] Demande internationale WO 2012 / 110600 A2 au nom du CEA publiée le 23 août 2012.

Claims

26 DEMANDS 1. A method for detecting and optionally quantifying nitrites in a biological fluid involving a three-electrode system with a boron-doped diamond working electrode, a counter electrode, and a third electrode which is either a reference electrode or a pseudo-reference electrode, said method comprising the following steps: a) electrochemically activate said working electrode; b) apply and maintain a first predetermined voltage to the electrochemically activated working electrode, said working electrode and counter electrode being in contact with said biological fluid, whereby, when nitrites are present in said biological fluid, said nitrites react with secondary amines naturally present or added to said biological fluid to form N-nitrosamines, which oxidize and concentrate on the surface of said working electrode; (c) subject the working electrode to a voltammetric sweep in a potential range from the first predetermined voltage to a second, more reducing voltage, whereby the reduction of said oxidized N-nitrosamines causes, at a third predetermined voltage between the first predetermined voltage and the second voltage, the appearance of a current peak indicating the presence of nitrites in said biological fluid and d) possibly quantify the nitrites in said biological fluid as a function of the height of the current peak observed at the third predetermined voltage.

2. A method according to claim 1, characterized in that said biological fluid is selected from the group consisting of blood, blood serum, blood plasma, lymph, urine, and saliva.

3. A method according to claim 1 or 2, characterized in that said biological fluid is urine.

4. A method according to any one of claims 1 to 3, characterized in that said counter electrode is a boron-doped diamond electrode.

5. A method according to any one of claims 1 to 4, characterized in that the third electrode comprising the three-electrode system is brought into contact with the biological fluid during said step b) and said step c).

6. A method according to any one of claims 1 to 4, characterized in that the third electrode comprising the three-electrode system is electrically connected with the working electrode and the counter electrode during said step b) and said step c) by means of a salt bridge.

7. A method according to any one of claims 1 to 6, characterized in that said step a) consists of applying a series of electrical pulses to the working electrode brought into contact with a saline solution which may be the biological fluid in which nitrites are to be detected and possibly quantified.

8. A method according to claim 7, characterized in that said electrical pulses are current pulses, and in particular current pulses of - / + 1 mA. 2 .

9. A method according to any one of claims 3 to 8, characterized in that said first predetermined voltage is 1.1 V, when said third electrode is a silver chloride reference electrode.

10. A method according to any one of claims 3 to 9, characterized in that said third predetermined voltage is -0.7 V, when said third electrode is a silver chloride reference electrode.

11. A method according to any one of claims 1 to 10, characterized in that the voltammetric scanning in step c) uses square wave voltammetry.

12. A method according to any one of claims 1 to 11, characterized in that it comprises, prior to said step b), a step of determining said first voltage, when said third electrode is a pseudo-reference electrode.

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